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Michele ZOLI

Professore Ordinario
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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Pubblicazioni

2023 - Cluster Headache Genomewide Association Study and Meta-Analysis Identifies Eight Loci and Implicates Smoking as Causal Risk Factor [Articolo su rivista]
Winsvold, B. S.; Harder, A. V. E.; Ran, C.; Chalmer, M. A.; Dalmasso, M. C.; Ferkingstad, E.; Tripathi, K. P.; Bacchelli, E.; Børte, S.; Fourier, C.; Petersen, A. S.; Vijfhuizen, L. S.; Magnusson, S. H.; O'Connor, E.; Bjornsdottir, G.; Häppölä, P.; Wang, Y. F.; Callesen, I.; Kelderman, T.; Gallardo, V. J.; de Boer, I.; Olofsgård, F. J.; Heinze, K.; Lund, N.; Thomas, L. F.; Hsu, C. L.; Pirinen, M.; Hautakangas, H.; Ribasés, M.; Guerzoni, S.; Sivakumar, P.; Yip, J.; Heinze, A.; Küçükali, F.; Ostrowski, S. R.; Pedersen, O. B.; Kristoffersen, E. S.; Martinsen, A. E.; Artigas, M. S.; Lagrata, S.; Cainazzo, M. M.; Adebimpe, J.; Quinn, O.; Göbel, C.; Cirkel, A.; Volk, A. E.; Heilmann-Heimbach, S.; Skogholt, A. H.; Gabrielsen, M. E.; Wilbrink, L. A.; Danno, D.; Mehta, D.; Guðbjartsson, D. F.; Rosendaal, F. R.; Willems van Dijk, K.; Fronczek, R.; Wagner, M.; Scherer, M.; Göbel, H.; Sleegers, K.; Sveinsson, O. A.; Pani, L.; Zoli, M.; Ramos-Quiroga, J. A.; Dardiotis, E.; Steinberg, A.; Riedel-Heller, S.; Sjöstrand, C.; Thorgeirsson, T. E.; Stefansson, H.; Southgate, L.; Trembath, R. C.; Vandrovcova, J.; Noordam, R.; Paemeleire, K.; Stefansson, K.; Fann, C. S. J.; Waldenlind, E.; Tronvik, E.; Jensen, R. H.; Chen, S. P.; Houlden, H.; Terwindt, G. M.; Kubisch, C.; Maestrini, E.; Vikelis, M.; Pozo-Rosich, P.; Belin, A. C.; Matharu, M.; van den Maagdenberg, A. M. J. M.; Hansen, T. F.; Ramirez, A.; Zwart, J. A.
abstract

Objective: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights. Methods: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses. Results: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine. Interpretation: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor. ANN NEUROL 2023.

2023 - Evidence for a protective effect of the loss of α4-containing nicotinic acetylcholine receptors on Aβ-related neuropathology in Tg2576 mice [Articolo su rivista]
Vilella, Antonietta; Romoli, Benedetto; Bodria, Martina; Pons, Stéphanie; Maskos, Uwe; Zoli, Michele
abstract

2023 - Hydroxypropyl-β-Cyclodextrin Depletes Membrane Cholesterol and Inhibits SARS-CoV-2 Entry into HEK293T-ACEhi Cells [Articolo su rivista]
Alboni, Silvia; Secco, Valentina; Papotti, Bianca; Vilella, Antonietta; Pia Adorni, Maria; Zimetti, Francesca; Schaeffer, Laurent; Tascedda, Fabio; Zoli, Michele; Leblanc, Pascal; Villa, Erica
abstract

2023 - Melanocortin receptor agonist NDP-α-MSH improves cognitive deficits and microgliosis but not amyloidosis in advanced stages of AD progression in 5XFAD and 3xTg mice [Articolo su rivista]
Daini, E.; Vandini, E.; Bodria, M.; Liao, W.; Baraldi, C.; Secco, V.; Ottani, A.; Zoli, M.; Giuliani, D.; Vilella, A.
abstract

Introduction: Alzheimer's disease (AD) is the most frequent cause of dementia and still lacks effective therapy. Clinical signs of AD include low levels of endogenous melanocortins (MCs) and previous studies have shown that treatment with MC analogs induces neuroprotection in the early stages of AD.Methods: We investigated the neuroprotective role of MCs in two transgenic mouse models of severe AD using 5 and 7 month-old (mo) 5XFAD mice and 9 and 12 mo 3xTg mice. These mice were subjected to a chronic stimulation of MC receptors (MCRs) with MC analogue Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH, 340 mu g/kg, i.p.). Mouse behavior and ex-vivo histological and biochemical analyses were performed after 50 days of treatment.Results:Our analysis demonstrated an improvement in cognitive abilities of AD mice at late stage of AD progression. We also showed that these protective effects are associated with decreased levels of hyperphosphorylated Tau but not with A beta burden, that was unaffected in the hippocampus and in the cortex of AD mice. In addition, an age-dependent NDP effect on glial reactivity was observed only in 3xTg mice whereas a global downregulation of p38 mitogen-activated protein kinase was selectively observed in 7 mo 5XFAD and 14 mo 3xTg mice.Conclusion: Our results suggest that MCR stimulation by NDP-alpha-MSH could represent a promising therapeutic strategy in managing cognitive decline also at late stage of AD, whereas the effects on neuroinflammation may be restricted to specific stages of AD progression.

2022 - Delirium in Older Adults: What a Surgeon Needs to Know [Articolo su rivista]
Melegari, Gabriele; Gaspari, Arianna; Gualdi, Eugenia; Zoli, Michele; Meletti, Stefano; Barbieri, Alberto
abstract

2022 - Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells [Articolo su rivista]
Pucci, S.; Bolchi, C.; Bavo, F.; Pallavicini, M.; De Palma, C.; Renzi, M.; Fucile, S.; Benfante, R.; Di Lascio, S.; Lattuada, D.; Bessereau, J. -L.; D'Alessandro, M.; Risson, V.; Zoli, M.; Clementi, F.; Gotti, C.
abstract

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

2022 - α9-Containing Nicotinic Receptors in Cancer [Articolo su rivista]
Pucci, S.; Zoli, M.; Clementi, F.; Gotti, C.
abstract

Neuronal nicotinic acetylcholine receptors containing the α9 or the α9 and α10 subunits are expressed in various extra-neuronal tissues. Moreover, most cancer cells and tissues highly express α9-containing receptors, and a number of studies have shown that they are powerful regulators of responses that stimulate cancer processes such as proliferation, inhibition of apoptosis, and metastasis. It has also emerged that their modulation is a promising target for drug development. The aim of this review is to summarize recent data showing the involvement of these receptors in controlling the downstream signaling cascades involved in the promotion of cancer.

2021 - A regional and cellular analysis of the early intracellular and extracellular accumulation of Aβ in the brain of 5XFAD mice [Articolo su rivista]
Daini, E.; Secco, V.; Liao, W.; Zoli, M.; Vilella, A.
abstract

Intracellular Aβ (iAβ) expression, extracellular Aβ (eAβ) plaque formation and microglial reactivity are characteristic neuropathological events of Alzheimer's disease (AD) and have been detected in several transgenic mouse models of this disease. In this work we decided to investigate the early (2–7 months of age) development of these phenomena at both regional and cellular levels in 5XFAD mice, a severe transgenic mouse model of AD. We demonstrated that 1) Aβ pathology develops in many but not all brain regions, 2) iAβ is transient and almost always followed by eAβ in grey matter regions, and the respective levels are roughly proportional, and 3) in about 1/3 of the grey matter regions with Aβ pathology and in several white matter regions, eAβ plaques can appear where no iAβ-positive structures were detected. We also showed that male and female mice share a similar regional and cellular pattern of Aβ pathology development that is more prominent in females. Early iAβ is associated to the activation of microglia, while subsequent formation of eAβ plaques is associated with markedly increased density of microglial cells that acquire a characteristic clustered phenotype. Present analysis is relevant to set a reference for pathophysiological studies and to define specific targets for the test of therapeutic interventions in this widely used AD transgenic model.

2021 - Altered mrna levels of stress-related peptides in mouse hippocampus and caudate-putamen in withdrawal after long-term intermittent exposure to tobacco smoke or electronic cigarette vapour [Articolo su rivista]
Carboni, L.; Ponzoni, L.; Braida, D.; Sala, M.; Gotti, C.; Zoli, M.
abstract

Nicotine addiction is a severe public health problem. The aim of this study was to investigate the alterations in key neurotransmissions after 60 days of withdrawal from seven weeks of intermittent cigarette smoke, e-cigarette vapours, or an e-cigarette vehicle. In the nicotine withdrawal groups, increased depressive and anxiety/obsessive–compulsive-like behaviours were demonstrated in the tail suspension, sucrose preference and marble burying tests. Cognitive impairments were detected in the spatial object recognition test. A significant increase in Corticotropin-releasing factor (Crf) and Crf1 mRNA levels was observed, specifically after cigarette withdrawal in the caudate-putamen nucleus (CPu). The nociceptin precursor levels were reduced by cigarette (80%) and e-cigarette (50%) withdrawal in the CPu. The delta opioid receptor showed a significant reduction in the hippocampus driven by the exposure to an e-cigarette solubilisation vehicle, while the mRNA levels doubled in the CPu of mice that had been exposed to e-cigarettes. Withdrawal after exposure to e-cigarette vapour induced a 35% Bdnf mRNA decrease in the hippocampus, whereas Bdnf was augmented by 118% by cigarette withdrawal in the CPu. This study shows that long-term withdrawal-induced affective and cognitive symptoms associated to lasting molecular alterations in peptidergic signalling may determine the impaired neuroplasticity in the hippocampal and striatal circuitry.

2021 - Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors [Articolo su rivista]
Pucci, S.; Fasoli, F.; Moretti, M.; Benfante, R.; Di Lascio, S.; Viani, P.; Daga, A.; Gordon, T. J.; Mcintosh, M.; Zoli, M.; Clementi, F.; Gotti, C.
abstract

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

2021 - Dorsal and ventral striatal neuronal subpopulations differentially disrupt male mouse copulatory behavior [Articolo su rivista]
Detraux, B.; Vilella, A.; De Groote, A.; Schiffmann, S. N.; Zoli, M.; de Kerchove d'Exaerde, A.
abstract

The specific role of the striatum, especially its dorsolateral (DLS) and dorsomedial (DMS) parts, in male copulatory behavior is still debated. In order to clarify their contribution to male sexual behavior, we specifically ablated the major striatal neuronal subpopulations, direct and indirect medium spiny neurons (dMSNs and iMSNs) in DMS or DLS, and dMSNs, iMSNs and cholinergic interneurons in nucleus accumbens (NAc), The main results of this study can be summarized as follows: In DMS, dMSN ablation causes a reduction in the percent of mice that mount a receptive female, and a complex alteration in the parameters of the copulatory performance, that is largely opposite to the alterations induced by iMSN ablation. In DLS, dMSN ablation causes a widespread alteration in the copulatory behavior parameters, that tends to disappear at repetition of the test; iMSN ablation induces minor copulatory behavior alterations that are complementary to those observed after dMSN ablation. In NAc, dMSN ablation causes a marked reduction in the percent of mice that mount a receptive female and a disruption of copulatory behavior, while iMSN ablation induces minor copulatory behavior alterations that are opposite to those observed with dMSN ablation, and cholinergic neuron ablation induces a selective decrease in mount latency. Overall, present data point to a complex region and cell-specific contribution to copulatory behavior of the different neuronal subpopulations of both dorsal and ventral striatum, with a prominent role of the dMSNs of the different subregions.

2021 - Implantable Organic Artificial Synapses Exhibiting Crossover between Depressive and Facilitative Plasticity Response [Articolo su rivista]
Calandra Sebastianella, G.; Di Lauro, M.; Murgia, M.; Bianchi, M.; Carli, S.; Zoli, M.; Fadiga, L.; Biscarini, F.
abstract

Organic neuromorphic devices mimic signal processing features of biological synapses, with short-term plasticity, STP, modulated by the frequency of the input voltage pulses. Here, an artificial synapse, made of intracortical microelectrodes, is demonstrated that exhibits either depressive or facilitative STP. The crossover between the two STP regimes is controlled by the frequency of the input voltage. STP features are described with an equivalent circuit where an inductance component is introduced in parallel with the RC circuit associated with poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT/PSS)||electrolyte interface. The proposed RLC circuit explains the physical origin of the observed STP and its two timescales in terms of charge build up in PEDOT/PSS.

2021 - Mild to severe neurological manifestations of covid-19: Cases reports [Articolo su rivista]
Melegari, G.; Rivi, V.; Zelent, G.; Nasillo, V.; De Santis, E.; Melegari, A.; Bevilacqua, C.; Zoli, M.; Meletti, S.; Barbieri, A.
abstract

The main focus of Coronavirus disease 2019 (COVID-19) infection is pulmonary complications through virus-related neurological manifestations, ranging from mild to severe, such as encephalitis, cerebral thrombosis, neurocognitive (dementia-like) syndrome, and delirium. The hospital screening procedures for quickly recognizing neurological manifestations of COVID-19 are often complicated by other coexisting symptoms and can be obscured by the deep sedation procedures required for critically ill patients. Here, we present two different case-reports of COVID-19 patients, describing neurological complications, diagnostic imaging such as olfactory bulb damage (a mild and unclear underestimated complication) and a severe and sudden thrombotic stroke complicated with hemorrhage with a low-level cytokine storm and respiratory symptom resolution. We discuss the possible mechanisms of virus entrance, together with the causes of COVID-19-related encephalitis, olfactory bulb damage, ischemic stroke, and intracranial hemorrhage.

2021 - Overview of prognostic systems for hepatocellular carcinoma and ITA.LI.CA external validation of MESH and CNLC classifications [Articolo su rivista]
Vitale, A.; Farinati, F.; Finotti, M.; Di Renzo, C.; Brancaccio, G.; Piscaglia, F.; Cabibbo, G.; Caturelli, E.; Missale, G.; Marra, F.; Sacco, R.; Giannini, E. G.; Trevisani, F.; Cillo, U.; Bhoori, S.; Borzio, M.; Burra, P.; Casadei Gardini, A.; Carrai, P.; Conti, F.; Cozzolongo, R.; Cucchetti, A.; D'Ambrosio, R.; Dell'Unto, C.; De Matthaeis, N.; Di Costanzo, G. G.; Di Sandro, S.; Famularo, S.; Foschi, F. G.; Fucilli, F.; Galati, G.; Gambato, M.; Gasbarrini, A.; Giuliante, F.; Ghinolfi, D.; Grieco, A.; Gruttadauria, S.; Guarino, M.; Iavarone, M.; Kostandini, A.; Lai, Q.; Lenci, I.; Levi Sandri, G. V.; Losito, F.; Lupo, L. G.; Marasco, G.; Manzia, T. M.; Mazzocato, S.; Masarone, M.; Melandro, F.; Mescoli, C.; Miele, L.; Morisco, F.; Muley, M.; Nicolini, D.; Pagano, D.; Persico, M.; Pompili, M.; Ponziani, F. R.; Pravisani, R.; Rapaccini, G. L.; Rendina, M.; Renzulli, M.; Romano, F.; Rossi, M.; Rreka, E.; Russo, F. P.; Sangiovanni, A.; Sessa, A.; Simonetti, N.; Sposito, C.; Tortora, R.; Vigano, L.; Vigano, M.; Villa, E.; Vincenzi, V.; Violi, P.; Azzaroli, F.; Brunetto, M. R.; Di Marco, A.; Gasbarrini, A.; Foschi, F. G.; Guarino, M.; Masotto, A.; Mega, A.; Morisco, F.; Nardone, G.; Oliveri, F.; Raimondo, G.; Rapaccini, G. L.; Svegliati Baroni, G.; Vidili, G.; Zoli, M.
abstract

Prognostic assessment in patients with HCC remains an extremely difficult clinical task due to the complexity of this cancer where tumour characteristics interact with degree of liver dysfunction, patient general health status, and a large span of available treatment options. Several prognostic systems have been proposed in the last three decades, both from the Asian and European/North American countries. Prognostic scores, such as the CLIP score and the recent MESH score, have been generated on a solid statistical basis from real life population data, while staging systems, such as the BCLC scheme and the recent CNLC classification, have been created by experts according to recent HCC prognostic evidences from the literature. A third category includes combined prognostic systems that can be used both as prognostic scores and staging systems. A recent example is the ITA.LI.CA prognostic system including either a prognostic score and a simplified staging system. This review focuses first on an overview of the main prognostic systems for HCC classified according to the above three categories, and, second, on a comprehensive description of the methodology required for a correct comparison between different systems in terms of prognostic performance. In this second section the main studies in the literature comparing different prognostic systems are described in detail. Lastly, a formal comparison between the last prognostic systems proposed for each of the above three categories is performed using a large Italian database including 6882 HCC patients in order to concretely apply the comparison rules previously described.

2021 - S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis [Articolo su rivista]
Daini, E.; Hagmeyer, S.; De Benedictis, C. A.; Cristovao, J. S.; Bodria, M.; Ross, A. M.; Raab, A.; Boeckers, T. M.; Feldmann, J.; Gomes, C. M.; Zoli, M.; Vilella, A.; Grabrucker, A. M.
abstract

Autism Spectrum Disorders (ASD) are caused by a combination of genetic predisposition and nongenetic factors. Among the nongenetic factors, maternal immune system activation and zinc deficiency have been proposed. Intriguingly, as a genetic factor, copy-number variations in S100B, a pro-inflammatory damage-associated molecular pattern (DAMP), have been associated with ASD, and increased serum S100B has been found in ASD. Interestingly, it has been shown that increased S100B levels affect zinc homeostasis in vitro. Thus, here, we investigated the influence of increased S100B levels in vitro and in vivo during pregnancy in mice regarding zinc availability, the zinc-sensitive SHANK protein networks associated with ASD, and behavioral outcomes. We observed that S100B affects the synaptic SHANK2 and SHANK3 levels in a zinc-dependent manner, especially early in neuronal development. Animals exposed to high S100B levels in utero similarly show reduced levels of free zinc and SHANK2 in the brain. On the behavioral level, these mice display hyperactivity, increased stereotypic and abnormal social behaviors, and cognitive impairment. Pro-inflammatory factors and zinc-signaling alterations converge on the synaptic level revealing a common pathomechanism that may mechanistically explain a large share of ASD cases.

2020 - Neuromorphic Organic Devices: Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions (Adv. Funct. Mater. 28/2020) [Altro]
Giordani, Martina; Sensi, Matteo; Berto, Marcello; Di Lauro, Michele; Bortolotti, Carlo Augusto; Gomes, Henrique Leonel; Zoli, Michele; Zerbetto, Francesco; Fadiga, Luciano; Biscarini, Fabio
abstract

2020 - Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions [Articolo su rivista]
Giordani, M.; Sensi, M.; Berto, M.; Di Lauro, M.; Bortolotti, C. A.; Gomes, H. L.; Zoli, M.; Zerbetto, F.; Fadiga, L.; Biscarini, F.
abstract

Specific detection of dopamine (DA) is achieved with organic neuromorphic devices with no specific recognition function in an electrolyte solution. The response to voltage pulses consists of amplitude-depressed current spiking mimicking the short-term plasticity (STP) of synapses. An equivalent circuit hints that the STP timescale of the device arises from the capacitance and resistance of the poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) in series with the electrolyte resistance. Both the capacitance and resistance of PEDOT:PSS change with solution compositions. Dose curves are constructed from the STP timescale for each DA metabolite from pM to mM range of concentrations. The STP response of DA is distinctive from the other metabolites even when differences are by one functional group. Both STP and sensitivity to DA are larger across the patho-physiological range with respect to those to DA metabolites. Density functional theory calculations hint to a stronger hydrogen bond pattern of DA ammonium compared to cationic metabolites. The exponential correlation between STP and the binding energy of DA metabolites interacting with PEDOT:PSS indicates that the slow dynamics of ionic species in and out PEDOT:PSS is the origin of the neuromorphic STP. The sensing framework discriminates differences of nonspecific interactions of few kcal mol−1, corresponding to one functional group in the molecule.

2020 - Novel peptide-conjugated nanomedicines for brain targeting: In vivo evidence [Articolo su rivista]
Duskey, J. T.; Ottonelli, I.; Da Ros, F.; Vilella, A.; Zoli, M.; Kovachka, S.; Spyrakis, F.; Vandelli, M. A.; Tosi, G.; Ruozi, B.
abstract

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood–brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models.

2020 - Persistent cognitive and affective alterations at late withdrawal stages after long-term intermittent exposure to tobacco smoke or electronic cigarette vapour: Behavioural changes and their neurochemical correlates [Articolo su rivista]
Ponzoni, L.; Braida, D.; Carboni, L.; Moretti, M.; Viani, P.; Clementi, F.; Zoli, M.; Gotti, C.; Sala, M.
abstract

Smoking cessation induces a withdrawal syndrome associated with anxiety, depression, and impaired neurocognitive functions, but much less is known about the withdrawal of e-cigarettes (e-CIG). We investigated in Balb/c mice the behavioural and neurochemical effects of withdrawal for up to 90 days after seven weeks’ intermittent exposure to e-CIG vapour or cigarette smoke (CIG). The withdrawal of e-CIG and CIG induced early behavioural alterations such as spatial memory deficits (spatial object recognition task), increased anxiety (elevated plus maze test) and compulsive-like behaviour (marble burying test) that persisted for 60–90 days. Notably, attention-related (virtual object recognition task) and depression-like behaviours (tail suspension and sucrose preference tests) appeared only 15–30 days after withdrawal and persisted for as long as up to 90 days. At hippocampal level, the withdrawal-induced changes in the levels of AMPA receptor GluA1 and GluA2/3 subunits, PSD 95 protein, corticotropin-releasing factor (Crf) and Crf receptor 1 (CrfR1) mRNA were biphasic: AMPA receptor subunit and PSD95 protein levels initially remained unchanged and decreased after 60–90 days, whereas Crf/CrfR1 mRNA levels initially increased and then markedly decreased after 60 days. These late reductions correlated with the behavioural impairments, particularly the appearance of depression-like behaviours. Our findings show that major behavioural and neurochemical alterations persist or even first appear late after the withdrawal of chronic CIG smoke or e-CIG vapour exposure, and underline importance of conducting similar studies of humans, including e-CIG vapers.

2020 - PLGA-PEG-ANG-2 Nanoparticles for Blood-Brain Barrier Crossing: Proof-of-Concept Study [Articolo su rivista]
Hoyos-Ceballos, Gina P; Ruozi, Barbara; Ottonelli, Ilaria; Da Ros, Federica; Vandelli, Maria Angela; Forni, Flavio; Daini, Eleonora; Vilella, Antonietta; Zoli, Michele; Tosi, Giovanni; Duskey, Jason T; López-Osorio, Betty L
abstract

The treatment of diseases that affect the central nervous system (CNS) represents a great research challenge due to the restriction imposed by the blood-brain barrier (BBB) to allow the passage of drugs into the brain. However, the use of modified nanomedicines engineered with different ligands that can be recognized by receptors expressed in the BBB offers a favorable alternative for this purpose. In this work, a BBB-penetrating peptide, angiopep-2 (Ang-2), was conjugated to poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles through pre- and post-formulation strategies. Then, their ability to cross the BBB was qualitatively assessed on an animal model. Proof-of-concept studies with fluorescent and confocal microscopy studies highlighted that the brain-targeted PLGA nanoparticles were able to cross the BBB and accumulated in neuronal cells, thus showing a promising brain drug delivery system.

2019 - Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons [Articolo su rivista]
Uberbacher, C.; Obergasteiger, J.; Volta, M.; Venezia, S.; Muller, S.; Pesce, I.; Pizzi, S.; Lamonaca, G.; Picard, A.; Cattelan, G.; Malpeli, G.; Zoli, M.; Beccano-Kelly, D.; Flynn, R.; Wade-Martins, R.; Pramstaller, P. P.; Hicks, A. A.; Cowley, S. A.; Corti, C.
abstract

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase – enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.

2019 - DAT atypical inhibitors as novel antipsychotic drugs [Poster]
Daini, Eleonora; Linciano, Pasquale; Sorbi, Claudia; Grilli, Massimo; Zoli, Michele; Franchini, Silvia; Vilella, Antonietta
abstract

Despite its classification as a psychiatric disease, schizophrenia is both a behavioral and a biological disorder resulting in neurocognitive dysfunction. Social and economic costs of schizophrenia are extremely high compared to its incidence and prevalence, however, due to a heterogeneous pattern of brain pathology and symptoms and to an unknown etiology, developing an effective treatment has been really challenging. Among the many neurochemical hypothesis, the dysregulation of dopaminergic neurotransmission has been considered as a central dogma of schizophrenia over the last few decades. In fact, patients with this pathology exhibit increased dopamine (DA) synthesis and release in the striatum which seems to correlate with positive symptoms and moreover, most of the effective antipsychotic drugs (APDs) are D2-receptor antagonists. Unfortunately, chronic treatment with APDs is associated with the induction of extrapyramidal side effects (EPS). In order to identify new possible APDs with a novel mechanism of action and potentially less EPS we tested 3 different compounds generated from the structural modification of vanoxerine (or GBR12909), a known atypical inhibitor of the presynaptic DA transporter (DAT) with cocaine-like activity but cardiotoxic properties that have precluded its clinical use. Preliminary in vitro studies showed that DAhLIs (DAT atypical inhibitors) are able to bind to DAT and inhibit DA reuptake. Additionally, our in vivo results showed that DAhLI i) have putative central effects, ii), unlike vanoxerine, reduce novelty-induced locomotor activity, and iii) counteract cocaine stimulating effects, suggesting that DAhLI may potentiate DA reuptake via DAT. These compounds may provide a way to reduce DA extracellular levels and DA neurotransmission with a selective action on active DA synapses, thus with reduced EPS typical of D2 antagonists, representing a new promising class of presynaptic APDs.

2019 - Distribution and relative abundance of S100 proteins in the brain of the APP23 Alzheimer’s disease model mice [Articolo su rivista]
Hagmeyer, S; Romão, M. A.; Cristóvão, J. S.; Vilella, A.; Zoli, M.; Gomes, C. M.; Grabrucker A., M
abstract

Increasing evidence links proteins of the S100 family to the pathogenesis of Alzheimer’s disease (AD). S100 proteins are EF-hand calcium-binding proteins with intra- and extracellular functions related to regulation of proliferation, differentiation, apoptosis, and trace metal homeostasis, and are important modulators of inflammatory responses. For example, S100A6, S100A8, and S100B expression levels were found increased in inflammatory diseases, but also neurodegenerative disorders, and S100A8/A9 complexes may provide a mechanistic link between amyloid-beta (Aβ) plaque formation and neuroinflammation. On the other hand, S100B, a proinflammatory protein that is chronically up-regulated in AD and whose elevation precedes plaque formation, was recently shown to suppress Aβ aggregation. Here, we report expression of S100A6 and S100B in astrocytes and less so in neurons, and low level of expression of S100A8 in both neurons and glial cells in vitro. In vivo, S100A8 expression is almost absent in the brain of aged wildtype mice, while S100A6 and S100B are expressed in all brain regions and most prominently in the cortex and cerebellum. S100B seems to be enriched in Purkinje cells of the cerebellum. In contrast, in the brain of APP23 mice, a mouse model for Alzheimer’s disease, S100B, S100A6, and S100A8 show co-localization with Aβ plaques, compatible with astrocyte activation, and the expression level of S100A8 is increased in neural cells. While S100A6 and S100B are enriched in the periphery of plaques where less fibrillar Aβ is found, S100A8 is more intense within the center of the inclusion. In vitro assays show that, similarly to S100B, S100A6, and S100A8 also delay Aβ aggregation suggesting a regulatory action over protein aggregation. We posit that elevated expression levels and overlapping spatial distribution of brain S100 proteins and plaques translates functional relationships between these inflammatory mediators and AD pathophysiology processes that uncover important molecular mechanisms linking the aggregation and neuroinflammation cascades. Copyright © 2019 Hagmeyer, Romão, Cristóvão, Vilella, Zoli, Gomes and Grabrucker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

2019 - Increased sensitivity to Δ 9 -THC-induced rewarding effects after seven-week exposure to electronic and tobacco cigarettes in mice [Articolo su rivista]
Ponzoni, L.; Moretti, M.; Braida, D.; Zoli, M.; Clementi, F.; Viani, P.; Sala, M.; Gotti, C.
abstract

Cigarette (CIG) smoking often precedes the use of illegal drugs. Electronic-cigarettes (e-CIGs) have been promoted as a means of stopping smoking and reducing the harmful effects of CIGs on the population. However, although e-CIGs eliminate some of the morbidity associated with combustible tobacco, they are still nicotine-delivery devices. In order to study whether the nicotine delivered via e-CIG acts as “a gateway drug” to the use of cannabis, we analysed the behavioural and molecular effects of 7 weeks’ pre-exposure to air (AIR), e-CIGs or CIGs on addiction-related conditioned place preference (CPP) in mice using a sub-threshold (0.01 mg/kg) dose of delta-9-tetrahydrocannabinol (Δ 9 -THC), the principal psychoactive constituent of cannabis. After 8 and 66 days of withdrawal, this Δ 9 -THC dose was ineffective in inducing CPP in mice pre-exposed to pump-driven AIR, but very effective in mice pre-exposed to e-CIGs or CIGs. Exposure to e-CIGs or CIGs increases the expression of ΔFosB in the nucleus accumbens (NAc), which remains high during short-term e-CIG or CIG withdrawal and long-term CIG withdrawal and is not influenced by treatment with Δ 9 -THC. At the end of e-CIG or CIG exposure and during withdrawal, the mice also had a higher AMPA receptors GluA1/GluA2-3 ratio in the NAc. Chronic nicotine exposure increases sensitivity to rewarding effects of Δ 9 -THC in mice and produces long-lasting neurobiological changes regardless of the delivery method (CIG vs. e-CIG). The exposure to passive tobacco smoke or e-CIG vapours can similarly increase vulnerability to the effects of cannabis and possibly other drugs of abuse.

2019 - Nanomedicine against Aβ aggregation by β–sheet breaker peptide delivery: In vitro evidence [Articolo su rivista]
Pederzoli, F.; Ruozi, B.; Duskey, J.; Hagmeyer, S.; Sauer, A. K.; Grabrucker, S.; Coelho, R.; Oddone, N.; Ottonelli, I.; Daini, E.; Zoli, M.; Vandelli, M. A.; Tosi, G.; Grabrucker, A. M.
abstract

The accumulation of amyloid β (Aβ) triggers a cascade of toxic events in Alzheimer’s disease (AD). The KLVFF peptide can interfere with Aβ aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood–brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aβ-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD.

2019 - P.1.04 Expression of histone variants H3.3 and H2a.z in the rat brain: Physiopathological and pharmacological implications [Abstract in Rivista]
Radighieri, G.; Benatti, C.; Zoli, M.; Blom, J. M. C.; Brunello, N.; Tascedda, F.
abstract

In the overall context of epigenetic modifications in charge of managing genome plasticity and dynamics [1, 2], the role of nucleosomal loading of histone variants is becoming increasingly captivating. Two replication-independent isoforms of histones H3 and H2A, namely H3.3 and H2A.Z, have caught attention because of their involvement in neuronal plasticity processes, cognitive functions and behavioral outcomes. In fact, their incorporation/eviction in nucleosomes and their turnover in neurons influence chromatin accessibility and therefore transcription. H3.3 enrichment at gene bodies and promoters of genes involved in synaptic plasticity was proved to be positively correlated with their expression, while learning-induced H2A.Z eviction in specific genes promotes gene transcription, intervening in memory consolidation processes. H3.3 is encoded by H3f3a and H3f3b independent genes, generating identical proteins, namely H3.3A and H3.3B. Notably, H3f3b gene, but not H3f3a, was proved responsive to neuronal activating stimuli as well as environmental triggers and stressful procedures [3]. H2A.Z hypervariants H2A.Z.1 and H2A.Z.2, encoded respectively by H2afz and H2afv genes, regulate both basal and stimulus-induced neuronal gene expression of independent gene sets [4]. Starting from this evidence, the purpose of this study was to characterize basal expression levels of all genes encoding for the histones variants above mentioned in rodent hippocampus and prefrontal cortex (PFC), two brain regions closely related to brain plasticity, cognition and behavior. Adult male rats (n=7) were sacrificed, their brains removed and dissected. Total RNA extraction was performed, followed by total RNA reverse transcription and Real Time PCR, where specific forward and reverse primer were used for each gene encoding for H3.3 (H3f3a and H3f3b), H2A.Z (H2afz and H2afv) and endogenous control GAPDH. Statistical analysis was performed by means of one-way ANOVA; p<0.05 was considered as a threshold for statistically significant difference. Molecular analyses revealed that, for both hippocampus and PFC, H3f3a mRNA was more expressed at the steady-state compared to H3f3b (p<0.001), as happens for H2afz mRNA, which displays higher levels than H2afv (p<0.001). Moreover, comparing hippocampal and PFC mRNA levels for each variant, H3f3a and H3f3b expression was increased in the hippocampus with respect to the prefrontal cortex (p<0.001), and a comparable outcome was showed for H2afv (p<0.001) but not for H2afz (p>0.05). Our results suggest that 1) differential basal expression levels of genes encoding for H3.3 and H2A.Z may underlie unique gene responsiveness following different stimuli, as previously hypothesized by others [3,4], and this may be crucial in highly-responsive, pathological- and environment-related tissues like hippocampus and PFC; 2) striking lower steady-state expression of H3f3b and H2afv genes might imply a major sensitivity to neuronal inputs compared to their correspondent counterparts; 3) higher expression levels in the hippocampus with respect to the PFC might underpin brain-region specific expression and function for histone variants and their isoforms. Together, these data clear the way for further studies meant at investigating stimulus-dependent regulation of H3.3 and H2A.Z gene isoforms expression and their putative involvement in the physiopathology of brain and its diseases [5]. References [1] Rigillo, G., Vilella, A., Benatti, C., Schaeffer, L., Brunello, N., Blom, J.M.C., Zoli, M., Tascedda, F., 2018. LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response. Brain Behav Immun. https://doi.org/10.1016/j.bbi.2018.09.019. [2] Ottaviani, E., Accorsi, A., Rigillo, G., Malagoli, D., Blom, J.M., Tascedda, F., 2013. Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challe

2019 - The novel hybrid agonist HyNDA-1 targets the D3R-nAChR heteromeric complex in dopaminergic neurons [Articolo su rivista]
Matera, C.; Bono, F.; Pelucchi, S.; Collo, G.; Bontempi, L.; Gotti, C.; Zoli, M.; De Amici, M.; Missale, C.; Fiorentini, C.; Dallanoce, C.
abstract

In this paper, we designed, synthesized and tested a small set of three new derivatives potentially targeting the D3R-nAChR heteromer, a receptor complex recently identified and characterized as the molecular entity that, in dopaminergic neurons, mediates the neurotrophic effects of nicotine. By means of a partially rigidified spacer of variable length, we incorporated in the new compounds (1a–c) the pharmacophoric substructure of a known β2-subunit-containing nAChR agonist (A-84543) and that of the D2/D3R agonist drug ropinirole. All the compounds retained the ability to bind with high affinity both β2-subunit-containing nAChR and D3R. Compound 1a, renamed HyNDA-1, which is characterized by the shortest linker moiety, was the most interesting ligand. We found, in fact, that HyNDA-1 significantly modulated structural plasticity on both mice and human dopaminergic neurons, an effect strongly prevented by co-incubating this ligand with either nAChR or D3R antagonists. Moreover, the neurotrophic effects of HyNDA-1 were specifically lost by disrupting the complex with specific interfering peptides. Interestingly, by using the Bioluminescence Resonance Energy Transfer 2 (BRET 2 ) assay in HEK-293 transfected cells, we also found that HyNDA-1 has the ability to increase the affinity of interaction between nAChR and D3R. Overall, our results indicate that the neurotrophic effects of HyNDA-1 are mediated by activation of the D3R-nAChR heteromeric complex specifically expressed on dopaminergic neurons.

2018 - Alphα6-containing nicotinic acetylcholine receptors mediate nicotine-induced structural plasticity in mouse and human iPSC-derived dopaminergic neurons [Articolo su rivista]
Collo, Ginetta; Cavalleri, Laura; Zoli, Michele; Maskos, Uwe; Ratti, Emiliangelo; Pich, Emilio Merlo
abstract

Midbrain dopamine (DA) neurons are considered a critical substrate for the reinforcing and sensitizing effects of nicotine and tobacco dependence. While the role of the α4 and β2 subunit containing nicotinic acetylcholine receptors (α4β2*nAChRs) in mediating nicotine effects on DA release and DA neuron activity has been widely explored, less information is available on their role in the morphological adaptation of the DA system to nicotine, eventually leading to dysfunctional behaviors observed in nicotine dependence. In particular, no information is available on the role of α6*nAChRs in nicotine-induced structural plasticity in rodents and no direct evidence exists regarding the occurrence of structural plasticity in human DA neurons exposed to nicotine. To approach this problem, we used two parallel in vitro systems, mouse primary DA neuron cultures from E12.5 embryos and human DA neurons differentiated from induced pluripotent stem cells (iPSCs) of healthy donors, identified using TH+ immunoreactivity. In both systems, nicotine 1-10 μM produced a dose-dependent increase of maximal dendrite length, number of primary dendrites, and soma size when measured after 3 days in culture. These effects were blocked by pretreatments with the α6*nAChR antagonists α-conotoxin MII and α-conotoxin PIA, as well as by the α4β2nAChR antagonist dihydro-β-erythroidine (DHβE) in both mouse and human DA neurons. Nicotine was also ineffective when the primary DA neurons were obtained from null mutant mice for either the α6 subunit or both the α4 and α6 subunits of nAChR. When pregnant mice were exposed to nicotine from gestational day 15, structural plasticity was also observed in the midbrain DA neurons of postnatal day 1 offspring only in wild-type mice and not in both null mutant mice. This study confirmed the critical role of α4α6*nAChRs in mediating nicotine-induced structural plasticity in both mouse and human DA neurons, supporting the translational relevance of neurons differentiated from human iPSCs for pharmacological studies.

2018 - Analysis and quantification of GPCR allosteric receptor–receptor interactions using radioligand binding assays: the a2ar-d2r heteroreceptor complex example [Capitolo/Saggio]
Borroto-Escuela, D. O.; Perez de la Mora, M.; Zoli, M.; Benfenati, F.; Narvaez, M.; Rivera, A.; Diaz-Cabiale, Z.; Beggiato, S.; Ferraro, L.; Tanganelli, S.; Ambrogini, P.; Filip, M.; Liu, F.; Franco, R.; Agnati, L. F.; Fuxe, K.
abstract

There is a large body of biochemical and biophysical experimental evidences which establishes the existence of G protein-coupled receptors (GPCRs) as homo- and heteroreceptor complexes. The results indicate that there are allosteric interactions across the receptor–receptor interface of homo- and heteroreceptor complexes that modulate the binding properties of their receptor protomer components in terms of affinity and density, and thereby change their pharmacology. In the adenosine A2A-dopamine D2 heteroreceptor complexes (A2AR-D2R), the activation of the A2AR protomer by its standard receptor agonist CGS21680 causes a conformational change in the A2AR-D2R heteroreceptor complex. The allosteric wave passes over the receptor interface, invades the orthostatic dopamine binding site of the dopamine D2R protomer, and reduces the affinity of the high but not the low affinity D2R agonist binding site. In view of the complex nature of allosteric mechanisms, the detection, analysis, and quantification of the effects of this phenomenon rely on the use of competition radioligand binding assays to ensure proper demonstration of the high and low affinity D2R agonist binding sites. Outlined in this chapter is simple but useful experimental approaches for measuring the allosteric receptor–receptor interactions at GPCR heteroreceptor complexes. The readers will also find tips and discussion on the pitfalls of these assay and instructions for data analysis.

2018 - Deletion of Maged1 in mice abolishes locomotor and reinforcing effects of cocaine [Articolo su rivista]
De Backer, Jean-François; Monlezun, Stéphanie; Detraux, Bérangère; Gazan, Adeline; Vanopdenbosch, Laura; Cheron, Julian; Cannazza, Giuseppe; Valverde, Sébastien; Cantacorps, Lídia; Nassar, Mérie; Venance, Laurent; Valverde, Olga; Faure, Philippe; Zoli, Michele; De Backer, Olivier; Gall, David; Schiffmann, Serge N; de Kerchove d'Exaerde, Alban
abstract

Melanoma antigen genes (Mage) were first described as tumour markers. However, some of Mage are also expressed in healthy cells where their functions remain poorly understood. Here, we describe an unexpected role for one of these genes, Maged1, in the control of behaviours related to drug addiction. Mice lacking Maged1 are insensitive to the behavioural effects of cocaine as assessed by locomotor sensitization, conditioned place preference (CPP) and drug self-administration. Electrophysiological experiments in brain slices and conditional knockout mice demonstrate that Maged1 is critical for cortico-accumbal neurotransmission. Further, expression of Maged1 in the prefrontal cortex (PFC) and the amygdala, but not in dopaminergic or striatal and other GABAergic neurons, is necessary for cocaine-mediated behavioural sensitization, and its expression in the PFC is also required for cocaine-induced extracellular dopamine (DA) release in the nucleus accumbens (NAc). This work identifies Maged1 as a critical molecule involved in cellular processes and behaviours related to addiction.

2018 - Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD [Articolo su rivista]
Palazzoli, Federica; Citti, Cinzia; Licata, Manuela; Vilella, Antonietta; Manca, Letizia; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe
abstract

The investigation of the possible conversion of cannabidiol (CBD) into Δ 9 -tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50 mg/kg) was administered orally to rats and their blood was collected after 3 and 6 h. A highly sensitive and selective LC–MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6 h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

2018 - Exploiting interfacial phenomena in organic bioelectronics: Conformable devices for bidirectional communication with living systems [Articolo su rivista]
Di Lauro, Michele; Benaglia, Simone; Berto, Marcello; Bortolotti, Carlo A.; Zoli, Michele; Biscarini, Fabio
abstract

A novel fully organic bioelectronic device is presented and validated as electronic transducer and current stimulator for brain implants. The device integrates polymeric electrodes made of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) on paper thin foils, resulting in a high surface-to-volume ratio architecture that exhibits high sensitivity to interfacial ionic transport phenomena. The prototyping technique herein presented yields devices for the bidirectional communication with biological systems whose dimensionality can be controlled according to the desired application. Transduction of ultra-low local-field potentials and delivery of voltage pulse-trains alike those used in deep-brain stimulation are herein assessed, paving the way towards novel theranostic strategies for the treatment of Parkinson's Disease and other severe neurodegenerative and/or traumatic pathologies of the central nervous system.

2018 - Genetic variation in CHRNA7 and CHRFAM7A is associated with nicotine dependence and response to varenicline treatment [Articolo su rivista]
Cameli, Cinzia; Bacchelli, Elena; De Paola, Maria; Giucastro, Giuliano; Cifiello, Stefano; Collo, Ginetta; Cainazzo, Maria Michela; Pini, Luigi Alberto; Maestrini, Elena; Zoli, Michele
abstract

The role of nicotinic acetylcholine receptors (nAChR) in nicotine dependence (ND) is well established; CHRNA7, encoding the α7 subunit, has a still uncertain role in ND, although it is implicated in a wide range of neuropsychiatric conditions. CHRFAM7A, a hybrid gene containing a partial duplication of CHRNA7, is possibly involved in modulating α7 nAChR function. The aim of this study was to investigate the role of CHRNA7 and CHRFAM7A genetic variants in ND and to test the hypothesis that α7 nAChR variation may modulate the efficacy of varenicline treatment in smoking cessation. We assessed CHRNA7 and CHRFAM7A copy number, CHRFAM7A exon 6 ∆2 bp polymorphism, and sequence variants in the CHRNA7 proximal promoter in an Italian sample of 408 treatment-seeking smokers. We conducted case-control and quantitative association analyses using two smoking measures (cigarettes per day, CPD, and Fagerström Test for Nicotine Dependence, FTND). Next, driven by the hypothesis that varenicline may exert some of its therapeutic effects through activation of α7 nAChRs, we restricted the analysis to a subgroup of 142 smokers who received varenicline treatment. The CHRNA7 promoter variant rs28531779 showed association with both smoking quantitative measures (FNTD p = 0.026, β = 0.89, 95% CI 0.11–1.67; CPD p = 0.006, β = 4.82 95% CI 1.42–8.22). Moreover, in the varenicline-treated subgroup we observed association of CHRFAM7A copy number with 6 months smoking abstinence (p = 0.035, OR = 3.18, 95% CI = 1.09–9.30). Thus, our study points to a possible role of genetic variation in CHRNA7 and CHRFAM7A in tobacco addiction mechanisms and response to varenicline treatment.

2018 - Increased expression of CRF and CRF-receptors in dorsal striatum, hippocampus, and prefrontal cortex after the development of nicotine sensitization in rats [Articolo su rivista]
Carboni, Lucia; Romoli, Benedetto; Bate, Simon T.; Romualdi, Patrizia; Zoli, Michele
abstract

Background: Nicotine addiction supports tobacco smoking, a main preventable cause of disease and death in Western countries. It develops through long-term neuroadaptations in the brain reward circuit by modulating intracellular pathways and regulating gene expression. This study assesses the regional expression of the transcripts of the CRF transmission in a nicotine sensitization model, since it is hypothesised that the molecular neuroadaptations that mediate the development of sensitization contribute to the development of addiction. Methods: Rats received intraperitoneal nicotine administrations (0.4 mg/kg) once daily for either 1 day or over 5 days. Locomotor activity was assessed to evaluate the development of sensitization. The mRNA expression of CRF and CRF1 and CRF2 receptors was measured by qPCR in the ventral mesencephalon, ventral striatum, dorsal striatum (DS), prefrontal cortex (PFCx), and hippocampus (Hip). Results: Acute nicotine administration increased locomotor activity in rats. In the sub-chronic group, locomotor activity progressively increased and reached a clear sensitization. Significant effects of sensitization on CRF mRNA levels were detected in the DS (increasing effect). Significantly higher CRF1 and CRF2 receptor levels after sensitization were detected in the Hip. Additionally, CRF2 receptor levels were augmented by sensitization in the PFCx, and treatment and time-induced increases were detected in the DS. Nicotine treatment effects were observed on CRF1R levels in the DS. Conclusions: This study suggests that the CRF transmission, in addition to its role in increasing withdrawal-related anxiety, may be involved in the development of nicotine-habituated behaviours through reduced control of impulses and the aberrant memory plasticity characterising addiction.

2018 - LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response [Articolo su rivista]
Rigillo, Giovanna; Vilella, Antonietta; Benatti, Cristina; Schaeffer, Laurent; Brunello, Nicoletta; Blom, Johanna M. C.; Zoli, Michele; Tascedda, Fabio
abstract

Epigenetic modifications of DNA and histone proteins are emerging as fundamental mechanisms by which neural cells adapt their transcriptional response to environmental cues, such as, immune stimuli or stress. In particular, histone H3 phospho(Ser10)-acetylation(Lys14) (H3S10phK14ac) has been linked to activation of specific gene expression. The purpose of this study was to investigate the role of H3S10phK14ac in a neuroinflammatory condition. Adult male rats received a intraperitoneal injection of lipopolysaccharide (LPS) (830 μg/Kg/i.p., n = 6) or vehicle (saline 1 mL/kg/i.p., n = 6) and were sacrificed 2 or 6 h later. We showed marked region- and time-specific increases in H3S10phK14ac in the hypothalamus and hippocampus, two principal target regions of LPS. These changes were accompanied by a marked transcriptional activation of interleukin (IL) 1β, IL-6, Tumour Necrosis Factor (TNF) α, the inducible nitric oxide synthase (iNOS) and the immediate early gene c-Fos. By means of chromatin immunoprecipitation, we demonstrated an increased region- and time-specific association of H3S10phK14ac with the promoters of IL-6, c-Fos and iNOS genes, suggesting that part of the LPS-induced transcriptional activation of these genes is regulated by H3S10phK14ac. Finally, by means of multiple immunofluorescence approach, we showed that increased H3S10phK14ac is cell type-specific, being neurons and reactive microglia, the principal histological types involved in this response. Present data point to H3S10phK14ac as a principal epigenetic regulator of neural cell response to systemic LPS and underline the importance of distinct time-, region- and cell-specific epigenetic mechanisms that regulate gene transcription to understand the mechanistic complexity of neuroinflammatory response to immune challenges.

2018 - Neuronal and extraneuronal nicotinic acetylcholine receptors [Articolo su rivista]
Zoli, Michele; Pucci, Susanna; Vilella, Antonietta; Gotti, Cecilia
abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) belong to a super-family of Cys-loop ligand-gated ion channels that respond to endogenous acetylcholine (ACh) or other cholinergic ligands. These receptors are also the targets of drugs such as nicotine (the main addictive agent delivered by cigarette smoke) and are involved in a variety of physiological and pathophysiological processes. Numerous studies have shown that the expression and/or function of nAChRs is compromised in many neurological and psychiatric diseases. Furthermore, recent studies have shown that neuronal nAChRs are found in a large number of non neuronal cell types including endothelial cells, glia, immune cells, lung epithelia and cancer cells where they regulate cell differentiation, proliferation and inflammatory responses. The aim of this review is to describe the most recent findings concerning the structure and function of native nAChRs inside and outside the nervous system.

2018 - Reduced plaque size and inflammation in the APP23 mouse model for Alzheimer's disease after chronic application of polymeric nanoparticles for CNS targeted zinc delivery [Articolo su rivista]
Vilella, Antonietta; Belletti, Daniela; Sauer, Ann Katrin; Hagmeyer, Simone; Sarowar, Tasnuva; Masoni, Martina; Stasiak, Natalia; Mulvihill, John J. E; Ruozi, Barbara; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni; Zoli, Michele; Grabrucker, Andreas M.
abstract

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer's disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aβ), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aβ degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD.

2018 - Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol [Articolo su rivista]
Citti, Cinzia; Palazzoli, Federica; Licata, Manuela; Vilella, Antonietta; Leo, Giuseppina; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Pacchetti, Barbara; Cannazza, Giuseppe
abstract

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

2017 - In vivo study of the role of a6-containing nicotinic acetylcholine receptor in retinal function using subtype-specific RDP-MII(E11R) toxin [Articolo su rivista]
Barloscio, Davide; Cerri, Elisa; Domenici, Luciano; Longhi, Renato; Dallanoce, Clelia; Moretti, Milena; Vilella, Antonietta; Zoli, Michele; Gotti, Cecilia; Origlia, Nicola
abstract

Although a6-contaning (a6∗) nicotinic acetylcholine receptors (nAChRs) are densely expressed in the visual system, their role is not well known.We have characterized a family of toxins that are antagonists for a6b2∗ receptors and used one of these [RDP-MII(E11R)] to localize a6∗ nAChRs and investigate their impact on retinal function inadult Long-Evans rats.Thea6∗nAChRsinretinal tissuewere localized using either a fluorescently tagged [RDP-MII(E11R)] or anti-a6-specific antibodies and found to be predominantly at the level of the ganglion cell layer. After intraocular injection of RDP-MII(E11R) in one eye and vehicle or inactiveMII in contralateral eyes as controls, we recorded flash electroretinograms (F-ERGs), pattern ERGs (P-ERGs), and cortical visual-evoked potential (VEPs). There was no significant difference in F-ERG between the RDP-MII(E11R)-treated and control eyes. In contrast, P-ERG response amplitude was significantly reduced in the RDP-MII(E11R)-injected eye. Blocking a6∗ nAChRs at retinal level also decreased the VEP amplitude recorded in the visual cortex contralateral to the injected eye. Because both the cortical and inner retina output were affected by RDP-MII(E11R), whereas photoreceptor output was preserved, we conclude that the reduced visual response was due to an alteration in the function of a6∗ nAChRs present in the ganglion cell layer.-Barloscio, D., Cerri, E., Domenici, L., Longhi, R., Dallanoce, C., Moretti, M., Vilella,A., Zoli,M.,Gotti, C., andOriglia, N. In vivo study of the roleofa6-containing nicotinic acetylcholine receptor in retinal function using subtype-specific RDP-MII(E11R) toxin. FASEB J. 31, 192-202 (2017). www.fasebj.org.

2017 - Serum protein changes in a rat model of chronic pain show a correlation between animal and humans [Articolo su rivista]
Bellei, Elisa; Vilella, Antonietta; Monari, Emanuela; Bergamini, Stefania; Tomasi, Aldo; Cuoghi, Aurora; Guerzoni, Simona; Manca, Letizia; Zoli, Michele; Pini, Luigi Alberto
abstract

In previous works we showed the overexpression of some proteins in biological fluids from patients suffering chronic pain. In this proteomic study we analysed serum from a rat model of neuropathic pain obtained by the chronic constriction injury (CCI) of sciatic nerve, at two time intervals, 2 and 5 weeks after the insult, to find proteins involved in the expression or mediation of pain. Sham-operated and CCI rats were treated with saline or indomethacin. Two weeks after ligation, we identified three serum proteins overexpressed in CCI rats, two of which, alpha-1-macroglobulin and vitamin D-binding protein (VDBP), remained increased 5 weeks post-surgery; at this time interval, we found increased levels of further proteins, namely apolipoprotein A-I (APOA1), apolipoprotein E (APOE), prostaglandin-H2 D-isomerase (PTGDS) and transthyretin (TTR), that overlap the overexpressed proteins found in humans. Indomethacin treatment reversed the effects of ligation. The qPCR analysis showed that transcript levels of APOA1, APOE, PTGDS and VDBP were overexpressed in the lumbar spinal cord (origin of sciatic nerve), but not in the striatum (an unrelated brain region), of CCI rats treated with saline 5 weeks after surgery, demonstrating that the lumbar spinal cord is a possible source of these proteins.

2017 - Specific Dopamine Sensing Based on Short-Term Plasticity Behavior of a Whole Organic Artificial Synapse [Articolo su rivista]
Giordani, Martina; Berto, Marcello; Di Lauro, Michele; Bortolotti, Carlo A.; Zoli, Michele; Biscarini, Fabio
abstract

In this work, we demonstrate the ultrasensitive and selective detection of dopamine by means of a neuro-inspired device platform without the need of a specific recognition moiety. The sensor is a whole organic device featuring two electrodes made of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate - PEDOT:PSS - patterned on a polydymethylsiloxane - PDMS - flexible substrate. One electrode is pulsed with a train of voltage square waves, to mimic the presynaptic neuron behavior, while the other is used to record the displacement current, mimicking the postsynaptic neuron. The current response exhibits the features of synaptic Short-Term Plasticity (STP) with facilitating or depressing response according to the stimulus frequency. We found that the response characteristic time USTPdepends on dopamine (DA) concentration in solution. The dose curve exhibits superexponential sensitivity at the lowest concentrations below 1 nM. The sensor detects [DA] down to 1 pM range. We assess the sensor also in the presence of ascorbic acid (AA) and uric acid (UA). Our sensor does not respond to UA, but responds to AA only at concentration above 100 μM. However, it is still able to detect DA down to 1 pM range in the presence of [AA] = 100 μM and 100 pM in the presence of [UA] = 3 μM, these values for AA and UA being the physiological levels in the cerebrospinal fluid and the striatum, respectively.

2016 - A genome-wide analysis in cluster headache points to neprilysin and PACAP receptor gene variants [Articolo su rivista]
Bacchelli, Elena; Cainazzo, Maria Michela; Cameli, Cinzia; Guerzoni, Simona; Martinelli, Angela; Zoli, Michele; Maestrini, Elena; Pini, Luigi Alberto
abstract

Background: Cluster Headache (CH) is a severe primary headache, with a poorly understood pathophysiology. Complex genetic factors are likely to play a role in CH etiology; however, no confirmed gene associations have been identified. The aim of this study is to identify genetic variants influencing risk to CH and to explore the potential pathogenic mechanisms. Methods: We have performed a genome-wide association study (GWAS) in a clinically well-defined cohort of 99 Italian patients with CH and in a control sample of 360 age-matched sigarette smoking healthy individuals, using the Infinium PsychArray (Illumina), which combines common highly-informative genome-wide tag SNPs and exonic SNPs. Genotype data were used to carry out a genome-wide single marker case-control association analysis using common SNPs, and a gene-based association analysis focussing on rare protein altering variants in 745 candidate genes with a putative role in CH. Results: Although no single variant showed statistically significant association at the genome-wide threshold, we identified an interesting suggestive association (P = 9.1 × 10−6) with a common variant of the PACAP receptor gene (ADCYAP1R1). Furthermore, gene-based analysis provided significant evidence of association (P = 2.5 × 10−5) for a rare potentially damaging missense variant in the MME gene, encoding for the membrane metallo-endopeptidase neprilysin. Conclusions: Our study represents the first genome-wide association study of common SNPs and rare exonic variants influencing risk for CH. The most interesting results implicate ADCYAP1R1 and MME gene variants in CH susceptibility and point to a role for genes involved in pain processing. These findings provide new insights into the pathogenesis of CH that need further investigation and replication in larger CH samples.

2016 - Activity and circadian rhythm influence synaptic Shank3 protein levels in mice [Articolo su rivista]
Sarowar, Tasnuva; Chhabra, Resham; Vilella, Antonietta; Boeckers, Tobias M.; Zoli, Michele; Grabrucker, Andreas M.
abstract

Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan–McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. (Figure presented.) Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively.

2016 - Alterations in alpha5* nicotinic acetylcholine receptors result in midbrain- and hippocampus-dependent behavioural and neural impairments [Articolo su rivista]
Besson, Morgane; Guiducci, Stefania; Granon, Sylvie; Guilloux, Jean Philippe; Guiard, Bruno; Repérant, Christelle; Faure, Philippe; Pons, Stéphanie; Cannazza, Giuseppe; Zoli, Michele; Gardier, Alain M.; Maskos, Uwe
abstract

Rationale: Evidence links alterations in α5-containing nicotinic receptors (α5*-nAChRs) to nicotine addiction. Notably, the rs16969968 polymorphism in the α5 gene (α5SNP) increases the risk for heavy smoking and impairs nicotine-rewarding properties in mice. Additional work is needed to understand how native and polymorphic α5*-nAChRs contribute to processes associated with the risk for nicotine addiction. Objectives: We aimed at understanding the contribution of α5*-nAChRs to endophenotypes like increased responses to novelty and anxiety, known to promote vulnerability to addiction, and to the response of the dopamine and serotonin systems to nicotine. Methods: Behavioural phenotypes were investigated in mice lacking the α5 gene (α5−/−). Nicotine injections were performed to test the consequences of nicotine exposure on the phenotypes identified. Dopamine and serotonin signalling were assessed using in vivo microdialysis and electrophysiology. We used lentiviral vectors to compare the consequences of re-expressing either the α5 wild-type allele or the α5SNP in specific brain areas of α5−/− mice. Results: α5−/− mice did not exhibit high responses to novelty but showed decreased novelty-induced rearing behaviour together with high anxiety. Exposure to high doses of nicotine rescued these phenotypes. We identified altered spontaneous and nicotine-elicited serotonin and dopamine activity in α5−/− mice. Re-expression of α5 in the ventral tegmental area and hippocampus rescued rearing and anxiety levels in α5−/− mice, respectively. When expressing the α5SNP instead, this resulted in a knockout-like phenotype for both behaviours. Conclusions: We propose that altered α5*-nAChR cholinergic signalling contributes to emotional/behavioural impairments that may be alleviated by nicotine consumption.

2016 - Chronic nicotine and withdrawal affect glutamatergic but not nicotinic receptor expression in the mesocorticolimbic pathway in a region-specific manner [Articolo su rivista]
Pistillo, Francesco; Fasoli, Francesca; Moretti, Milena; McClure Begley, Tristan; Zoli, Michele; Marks, Michael J.; Gotti, Cecilia
abstract

Tobacco addiction is a complex form of dependence process that leads high relapse rates in people seeking to stop smoking. Nicotine elicits its primary effects on neuronal nicotinic cholinergic receptors (nAChRs), alters brain reward systems, and induces long-term changes during chronic nicotine use and withdrawal. We analysed the effects of chronic nicotine treatment and withdrawal on the mesocorticolimbic pathway (a brain reward circuit in which addictive drugs induce widespread adaptations) by analysing the expression of nAChRs in the midbrain, striatum and prefrontal cortex (PFC) of mice receiving intravenous infusions of nicotine (4 mg/kg/h) or saline (control) for 14 days and mice sacrified two hours, and one, four and 14 days after treatment withdrawal. We biochemically fractionated whole tissue homogenates in order to obtain crude synaptosomal membranes. Western blotting analyses of these membrane fractions, ligand binding and immunoprecipitation studies, showed that chronic nicotine up-regulates heteromeric β2∗nAChRs in all three mesocorticolimbic areas, and that these receptors are rapidly removed from synapses upon the cessation of nicotine treatment. The extent of nicotine-induced nAChR up-regulation, and the time course of its reversal were comparable in all three areas. We also analysed the expression of glutamate receptor subunits (GluRs) and scaffold proteins, and found that it was altered in an area-specific manner during nicotine exposure and withdrawal. As the functional properties of GluRs are determined by their subunit composition, the observed changes in subunit expression may indicate alterations in the excitability of mesocorticolimbic circuitry, and this may underlie the long-term biochemical and behavioural effects of nicotine dependence.

2016 - "Heart-cut" bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system [Articolo su rivista]
Battisti, Umberto M.; Citti, Cinzia; Larini, Martina; Ciccarella, Giuseppe; Stasiak, Natalia; Troisi, Luigino; Braghiroli, Daniela; Parenti, Carlo; Zoli, Michele; Cannazza, Giuseppe
abstract

A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs.Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

2016 - Heterosynaptic GABAergic plasticity bidirectionally driven by the activity of pre- and postsynaptic NMDA receptors [Articolo su rivista]
Mapelli, Jonathan; Gandolfi, Daniela; Vilella, Antonietta; Zoli, Michele; Bigiani, Albertino
abstract

Dynamic changes of the strength of inhibitory synapses play a crucial role in processing neural information and in balancing network activity. Here, we report that the efficacy of GABAergic connections between Golgi cells and granule cells in the cerebellum is persistently altered by the activity of glutamatergic synapses. This form of plasticity is heterosynaptic and is expressed as an increase (long-term potentiation, LTPGABA) or a decrease (long-term depression, LTDGABA) of neurotransmitter release. LTPGABA is induced by postsynaptic NMDA receptor activation, leading to calcium increase and retrograde diffusion of nitric oxide, whereas LTDGABA depends on presynaptic NMDA receptor opening. The sign of plasticity is determined by the activation state of target granule and Golgi cells during the induction processes. By controlling the timing of spikes emitted by granule cells, this form of bidirectional plasticity provides a dynamic control of the granular layer encoding capacity.

2016 - In vivo chronic nicotine exposure differentially and reversibly affects upregulation and stoichiometry of α4β2 nicotinic receptors in cortex and thalamus [Articolo su rivista]
Fasoli, F.; Moretti, M.; Zoli, Michele; Pistillo, F.; Crespi, A.; Clementi, F.; Mc Clure Begley, T.; Marks, M. J.; Gotti, C.
abstract

Studies with heterologous expression systems have shown that the α4β2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(β2)3] or three [(α4)3(β2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4β2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and β2/α4 subunit ratio of 3H-epibatidine labeled α4β2∗-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the β2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(β2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by 3H-epibatidine binding, an increase in the α4 and β2 protein levels, and an increase in β2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(β2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and β2 protein levels, or changes in β2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4β2 nAChR containing three β2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(β2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(β2)3 nAChR).

2016 - MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks [Articolo su rivista]
Lippi, Giordano; Fernandes, Catarina C; Ewell, Laura A; John, Danielle; Romoli, Benedetto; Curia, Giulia; Taylor, Seth R; Frady, E.  Paxon; Jensen, Anne B; Liu, Jerry C; Chaabane, Melanie M; Belal, Cherine; Nathanson, Jason l; Zoli, Michele; Leutgeb, Jill K; Biagini, Giuseppe; Yeo, Gene W; Berg, Darwin K
abstract

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

2016 - Nicotine inside neurons [Articolo su rivista]
Gotti, Cecilia; Zoli, Michele
abstract

The crucial effects of chronic nicotine exposure are intracellular and include a cell-specific influence on the assembly and stoichiometry of the nAChRs exported to the plasma membrane, that affects the function of surface nAChRs and cholinergic transmission. In this context, it is worthwhile to consider extraneuronal tissues, especially where nicotine may reach much higher concentrations such as the lung epithelia. Many peripheral cell types and tissues express nAChR subunit transcripts, that may play crucial roles in signal transduction underlying tumor initiation or growth

2016 - Repeated nicotine exposure modulates prodynorphin and pronociceptin levels in the reward pathway [Articolo su rivista]
Carboni, Lucia; Romoli, Benedetto; Romualdi, Patrizia; Zoli, Michele
abstract

Background Nicotine dependence is maintained by neurobiological adaptations in the dopaminergic brain reward pathway with the contribution of opioidergic circuits. This study assessed the role of opioid peptides and receptors on the molecular changes associated with nicotine dependence. To this aim we analysed nicotine effects on opioid gene and receptor expression in the reward pathway in a nicotine sensitization model. Methods Sprague-Dawley rats received nicotine administrations for five days and locomotor activity assessment showed the development of sensitization. The mRNA expression of prodynorphin (pdyn), pronociceptin (pnoc) and the respective receptors was measured by quantitative PCR in the ventral midbrain (VM), the nucleus accumbens (NAc), the caudate-putamen (CPu), the pre-frontal cortex (PFCx), and the hippocampus. Results A significant positive effect of sensitization on pdyn mRNA levels was detected in the CPu. This effect was supported by a significant and selective correlation between the two parameters in this region. Moreover, chronic but not acute nicotine treatment significantly decreased pdyn mRNA levels in the NAc and increased expression in the PFCx. Pnoc mRNA was significantly increased in the VM and the PFCx after sub-chronic administration of nicotine, whereas no alterations were observed after acute treatment. No treatment associated changes were detected in κ-opioid receptor or nociceptin receptor mRNAs. Conclusions This experiment revealed an effect of nicotine administration that was distinguishable from the effect of nicotine sensitization. While several pnoc and pdyn changes were associated to nicotine administration, the only significant effect of sensitization was a significant increase in pdyn in the CPu.

2016 - Whole organic electronic synapses for dopamine detection [Relazione in Atti di Convegno]
Giordani, Martina; DI LAURO, Michele; Berto, Marcello; Bortolotti, Carlo Augusto; Vuillaume, Dominique; Gomes, Henrique L.; Zoli, Michele; Biscarini, Fabio
abstract

A whole organic artificial synapse has been fabricated by patterning PEDOT:PSS electrodes on PDMS that are biased in frequency to yield a STP response. The timescale of the STP response is shown to be sensitive to the concentration of dopamine, DA, a neurotransmitter relevant for monitoring the development of Parkinson's disease and potential locoregional therapies. The sensitivity of the sensor towards DA has been validated comparing signal variation in the presence of DA and its principal interfering agent, ascorbic acid, AA. The whole organic synapse is biocompatible, soft and flexible, and is attractive for implantable devices aimed to real-time monitoring of DA concentration in bodily fluids. This may open applications in chronic neurodegenerative diseases such as Parkinson's disease.

2016 - 7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm? [Articolo su rivista]
Citti, Cinzia; Battisti, UMBERTO MARIA; Cannazza, Giuseppe; Jozwiak, Krzysztof; Stasiak, Natalia; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino; Zoli, Michele
abstract

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

2015 - Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo [Articolo su rivista]
Chhabra, Resham; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Mangus, Katharina; Pfaender, Stefanie; Sarowar, Tasnuva; Boeckers, Tobias Maria; Zoli, Michele; Forni, Flavio; Vandelli, Maria Angela; Tosi, Giovanni; Grabrucker, Andreas Martin
abstract

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

2015 - Bifunctional compounds targeting both D2 and non-α7 nACh receptors: Design, synthesis and pharmacological characterization [Articolo su rivista]
Matera, Carlo; Pucci, Luca; Fiorentini, Chiara; Fucile, Sergio; Missale, Cristina; Grazioso, Giovanni; Clementi, Francesco; Zoli, Michele; De Amici, Marco; Gotti, Cecilia; Dallanoce, Clelia
abstract

We designed, prepared and tested a set of structural analogs 1-4 as new hybrid compounds by incorporating, through a common alkyl chain of variable length, the pharmacophoric elements of N-n-alkyl nicotinium salts (non-α7 nicotinic acetylcholine receptors antagonists) and of 7-hydroxy-2-(aminomethyl)chromanes (dopaminergic D2receptor agonists). The target compounds, which were assayed in binding experiments and electrophysiological, functional and Erk1/2 activation tests, essentially combined the pharmacological profiles of their individual receptor ligands. Among the studied derivatives, hybrid 2, one of the shortest homologs, in addition to the antagonist nicotinic profile similar to the other three congeners, behaved as a high affinity ligand at the investigated heteromeric nAChRs and as a low efficacy agonist at D2Rs. These bifunctional derivatives represent novel pharmacological tools in the study of nicotine addiction.

2015 - Different physiological and behavioural effects of e-cigarette vapour and cigarette smoke in mice [Articolo su rivista]
Ponzoni, L; Moretti, M; Sala, M; Fasoli, F; Mucchietto, V; Lucini, V; Cannazza, Giuseppe; Gallesi, G; Castellana, Carmela Nives; Clementi, F; Zoli, Michele; Gotti, C; Braida, D.
abstract

Nicotine is the primary addictive substance in tobacco smoke and electronic cigarette (e-cig) vapour. Methodological limitations have made it difficult to compare the role of the nicotine and non-nicotine constituents of tobacco smoke. The aim of this study was to compare the effects of traditional cigarette smoke and e-cig vapour containing the same amount of nicotine in male BALB/c mice exposed to the smoke of 21 cigarettes or e-cig vapour containing 16.8mg of nicotine delivered by means of a mechanical ventilator for three 30-min sessions/day for seven weeks. One hour after the last session, half of the animals were sacrificed for neurochemical analysis, and the others underwent mecamylamine-precipitated or spontaneous withdrawal for the purposes of behavioural analysis. Chronic intermittent non-contingent, second-hand exposure to cigarette smoke or e-cig vapour led to similar brain cotinine and nicotine levels, similar urine cotinine levels and the similar up-regulation of α4β2 nicotinic acetylcholine receptors in different brain areas, but had different effects on body weight, food intake, and the signs of mecamylamine-precipitated and spontaneous withdrawal episodic memory and emotional responses. The findings of this study demonstrate for the first time that e-cig vapour induces addiction-related neurochemical, physiological and behavioural alterations. The fact that inhaled cigarette smoke and e-cig vapour have partially different dependence-related effects indicates that compounds other than nicotine contribute to tobacco dependence.

2015 - Diversity of native nicotinic receptor subtypes in mammalian brain [Articolo su rivista]
Zoli, Michele; Pistillo, F; Gotti, C.
abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of pentameric ligand-gated cation channels that are expressed throughout the brain and involved in a wide range of physiological and pathophysiological processes. The nAChR subtypes share a common basic structure, but their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding their function in the nervous system and discovering new subtype selective drugs. The development of new technologies and the generation of mice carrying deletions or the expression of gain-of-function nAChR subunits, or GFP-tagged receptor genes has allowed the in vivo identification of complex subtypes and to study the role of individual subtypes in specific cells and complex neurobiological systems but much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions in human brain. We briefly review some recent findings concerning the structure and function of native nAChRs, focussing on the subtypes identified in the rodent habenulo-interpeduncular pathway, a pathway involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the expression of native subtypes in primate brain. This article is part of the Special Issue entitled ‘The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition’.

2015 - Endocytosis of Nanomedicines: The Case of Glycopeptide Engineered PLGA Nanoparticles [Articolo su rivista]
Vilella, Antonietta; Ruozi, Barbara; Belletti, Daniela; Pederzoli, Francesca; Galliani, Marianna; Semeghini, Valentina; Forni, Flavio; Zoli, Michele; Vandelli, Maria Angela; Tosi, Giovanni
abstract

The success of nanomedicine as a new strategy for drug delivery and targeting prompted the interest in developing approaches toward basic and clinical neuroscience. Despite enormous advances on brain research, central nervous system (CNS) disorders remain the world's leading cause of disability, in part due to the inability of the majority of drugs to reach the brain parenchyma. Many attempts to use nanomedicines as CNS drug delivery systems (DDS) were made; among the various non-invasive approaches, nanoparticulate carriers and, particularly, polymeric nanoparticles (NPs) seem to be the most interesting strategies. In particular, the ability of poly-lactide-co-glycolide NPs (PLGA-NPs) specifically engineered with a glycopeptide (g7), conferring to NPs' ability to cross the blood brain barrier (BBB) in rodents at a concentration of up to 10% of the injected dose, was demonstrated in previous studies using different routes of administrations. Most of the evidence on NP uptake mechanisms reported in the literature about intracellular pathways and processes of cell entry is based on in vitro studies. Therefore, beside the particular attention devoted to increasing the knowledge of the rate of in vivo BBB crossing of nanocarriers, the subsequent exocytosis in the brain compartments, their fate and trafficking in the brain surely represent major topics in this field.

2015 - Exploiting Bacterial Pathways for BBB Crossing with PLGA Nanoparticles Modified with a Mutated Form of Diphtheria Toxin (CRM197): In Vivo Experiments [Articolo su rivista]
Tosi, Giovanni; Vilella, Antonietta; Veratti, P; Belletti, Daniela; Pederzoli, F; Ruozi, Barbara; Vandelli, Maria Angela; Zoli, Michele; Forni, Flavio
abstract

Drugs can be targeted to the brain using polymeric nanoparticles (NPs) engineered on their surface with ligands able to allow crossing of the blood-brain barrier (BBB). This article aims to investigate the BBB crossing efficiency of polymeric poly lactide-co-glycolide (PLGA) NPs modified with a mutated form of diphtheria toxin (CRM197) in comparison with the results previously obtained using PLGA NPs modified with a glycopeptide (g7-NPs). Different kinds of NPs, covalently coupled PLGA with different fluorescent probes (DY405, rhodamine-B base and DY675) and different ligands (g7 and CRM197) were tested in vivo to assess their behavior and trafficking. The results highlighted the possibility to distinguish the different kinds of simultaneously administered NPs and to emphasize that CRM-197 modified NPs and g7-NPs can cross the BBB at a similar extent. The analysis of BBB crossing and of the neuronal tropism of CRM197 modified NPs, along with their BBB crossing pathways were also developed. In vivo pharmacological studies performed on CRM197 engineered NPs, loaded with loperamide, underlined their ability as drug carriers to the CNS.

2015 - Nanomedicine in neurodegenerative disorders: Understanding the journey [Relazione in Atti di Convegno]
Tosi, G.; Ruozi, B.; Vilella, A.; Grabrucker, A. M.; Belletti, D.; Vandelli, M. A.; Boeckers, T. M.; Forni, F.; Zoli, M.; Sharma, A.; Muresanu, D. F.; Sharma, H. S.
abstract

Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. We investigated these issues by employing poly-lactide-co- glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7- NPs display increased in vitro uptake in neurons and glial cells, in vivo administration of g7-NPs leads to a region- And cell type-specific enrichment of NPs within the brain. Moreover, g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. Moreover, in order to understand the journey of NPs, we demonstrated that g7-NPs can be transported intra- And intercellularly inside vesicles. Cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration in mice. We also tested Ab-modified g7-NPs both in vitro and in vivo to investigate the possibility of a specific targeting.

2015 - Nicotinic, glutamatergic and dopaminergic synaptic transmission and plasticity in the mesocorticolimbic system: Focus on nicotine effects [Articolo su rivista]
Pistillo, Francesco; Clementi, Francesco; Zoli, Michele; Gotti, Cecilia
abstract

Cigarette smoking is currently the leading cause of preventable deaths and disability throughout the world, being responsible for about five million premature deaths/year. Unfortunately, fewer than 10% of tobacco users who try to stop smoking actually manage to do so. The main addictive agent delivered by cigarette smoke is nicotine, which induces psychostimulation and reward, and reduces stress and anxiety. The use of new technologies (including optogenetics) and the development of mouse models characterised by cell-specific deletions of receptor subtype genes or the expression of gain-of-function nAChR subunits has greatly increased our understanding of the molecular mechanisms and neural substrates of nicotine addiction first revealed by classic electrophysiological, neurochemical and behavioural approaches. It is now becoming clear that various aspects of nicotine dependence are mediated by close interactions of the glutamatergic, dopaminergic and γ-aminobutyric acidergic systems in the mesocorticolimbic system. This review is divided into two parts. The first provides an updated overview of the circuitry of the ventral tegmental area, ventral striatum and prefrontal cortex, the neurotransmitter receptor subtypes expressed in these areas, and their physiological role in the mesocorticolimbic system. The second will focus on the molecular, functional and behavioural mechanisms involved in the acute and chronic effects of nicotine on the mesocorticolimbic system.

2015 - O015. Evaluation of the genetic polymorphism of the α3 (CHRNA3) and α5 (CHRNA5) nicotinic receptor subunits, in patients with cluster headache [Articolo su rivista]
Cainazzo, Maria Michela; Tiraferri, Ilaria; Ciccarese, Michela; Martinelli, Angela; Cameli, Cinzia; Bacchelli, Elena; Zoli, Michele; Pini, Luigi Alberto
abstract

N/A

2015 - PEG-g-chitosan nanoparticles functionalized with the monoclonal antibody OX26 for brain drug targeting [Articolo su rivista]
Monsalve, Yuliana; Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vilella, Antonietta; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; López, Betty L.; Sierra, Ligia
abstract

Aim: Drug targeting to the CNS is challenging due to the presence of blood-brain barrier. We investigated chitosan (Cs) nanoparticles (NPs) as drug transporter system across the blood-brain barrier, based on mAb OX26 modified Cs. Materials & methods: Cs NPs functionalized with PEG, modified and unmodified with OX26 (Cs-PEG-OX26) were prepared and chemico-physically characterized. These NPs were administered (intraperitoneal) in mice to define their ability to reach the brain. Results: Brain uptake of OX26-conjugated NPs is much higher than of unmodified NPs, because: long-circulating abilities (conferred by PEG), interaction between cationic Cs and brain endothelium negative charges and OX26 TfR receptor affinity. Conclusion: Cs-PEG-OX26 NPs are promising drug delivery system to the CNS.

2015 - Proteomic research of proteins involved in pain expression in an animal model of chronic pain [Articolo su rivista]
Bellei, Elisa; Bergamini, Stefania; Monari, Emanuela; Cuoghi, Aurora; Zoli, Michele; Tomasi, Aldo; Cainazzo, Maria Michela; Guerzoni, Simona; Pini, Luigi Alberto
abstract

nd

2014 - Insight on the fate of CNS-targeted nanoparticles. Part I: Rab5-dependent cell-specific uptake and distribution [Articolo su rivista]
Vilella, Antonietta; Tosi, Giovanni; Andreas M., Grabrucker; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Tobias M., Boeckers; Forni, Flavio; Zoli, Michele
abstract

Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. In this paper, we investigated these issues by employing poly-lactide-co-glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glial cells. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs can be promising carriers for the treatment of neuropsychiatric and neurodegenerative diseases.

2014 - Insight on the fate of CNS-targeted nanoparticles. Part II: Intercellular neuronal cell-to-cell transport [Articolo su rivista]
Tosi, Giovanni; Vilella, Antonietta; Resham, Chhabra; Michael J., Schmeisser; Tobias M., Boeckers; Ruozi, Barbara; Vandelli, Maria Angela; Forni, Flavio; Zoli, Michele; Andreas M., Grabrucker
abstract

The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of a specific targeting. Our results show that g7-NPs can be transported intra- and intercellularly inside vesicles. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration in mice.

2014 - The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization [Articolo su rivista]
Moretti, Milena; Zoli, Michele; George, Andrew A; Lukas, Ronald J; Pistillo, Francesco; Maskos, Uwe; Whiteaker, Paul; Gotti, Cecilia
abstract

We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.

2013 - Brain-targeted polymeric nanoparticles: in vivo evidences after different routes of administration in rodents. [Articolo su rivista]
Tosi, Giovanni; Ruozi, Barbara; Belletti, Daniela; Vilella, Antonietta; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio
abstract

The capacity of polymeric nanoparticles (NPs) to reach the target regardless to the administration route is a neglected field of investigation in the pharmaceutical nanotechnology. Therefore, after having demonstrated in previous studies that glycopeptide-engineered NPs (g7-NPs) were able to reach the brain after intravenous administrations in rodents, this paper aimed to evaluate if they can reach the Central Nervous System (CNS) also when administered by different routes. The confocal microphotographs on murine brain sections showed the capability of g7-NPs to reach the target also after intraperitoneal, intranasal and oral administrations. These highlights could open new vistas to a future application of the g7-NPs in the therapeutic treatments of CNS diseases.

2013 - Calcineurin A versus NS5A-TP2/HD Domain Containing 2: A Case Study of Site-directed Low-frequency Random Mutagenesis for Dissecting Target Specificity of Peptide Aptamers [Articolo su rivista]
S., Dibenedetto; D., Cluet; P. N., Stebe; V., Baumle; J., Leault; R., Terreux; M., Bickle; B. D. E., Chassey; I., Mikaelian; P., Colas; M., Spichty; Zoli, Michele; B. B., Rudkin
abstract

We have previously identified a peptide aptamer (named “R5G42”) via functional selection, for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using Apta R5G42 as “bait”, allowed identification of two binding proteins with very different functions: Calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized e.g. for its role in the immune response, and NS5A-TP2/HDDC2, a much less studied protein, induced subsequent to Hepatitis C virus Non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective, in the present study, was to dissect their specificity in order to have tools with which to be able to better characterize the actions of the peptide aptamers towards their individual targets. This was achieved by the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity towards CnA and NS5A-TP2, and analyzing their sequence. An interdisciplinary approach, involving biomolecular computer simulations with integration of the sequence data and yeast two hybrid binding phenotypes of these mutants, yielded two structurally-distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA vs NS5A-TP2-specific peptide aptamers indicated that while both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate nuclear translocation of NFAT, indicative of activation of endogenous CnA. By dissecting target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is the first exogenous molecule reported, capable of directly activating CnA independently of binding to NS5A-TP2, whereas Apta-E1 is the first molecule reported that will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signalling pathways.

2013 - CC4, a dimer of cytisine, is a selective partial agonist at α4β2/α6β2 nAChR with improved selectivity for tobacco smoking cessation [Articolo su rivista]
Sala, M; Braida, D; Pucci, L; Manfredi, I; Marks, Mj; Wageman, Cr; Grady, Sr; Loi, B; Fucile, S; Fasoli, F; Zoli, Michele; Tasso, B; Sparatore, F; Clementi, F; Gotti, C.
abstract

Background And Purpose Many of the addictive and rewarding effects of nicotine are due to its actions on the neuronal nicotinic acetylcholine receptor (nAChR) subtypes expressed in dopaminergic mesocorticolimbic cells. The partial agonists, cytisine and varenicline, are helpful smoking cessation aids. These drugs have a number of side effects that limit their usefulness. The aim of this study was to investigate the pre-clinical pharmacology of the cytisine dimer CC4. Experimental Approach The effects of CC4 on nAChRs were investigated using in vitro assays and animal behaviors. Key Results When electrophysiologically tested using heterologously expressed human subtypes, CC4 was less efficacious than cytisine on neuronal α4β2, α3β4, α7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through α4β2 and α6β2 nAChRs, CC4 is a partial agonist of nAChR-mediated striatal dopamine release and, when co-incubated with nicotine, prevented nicotine's maximal effect on this response. In addition, it had low affinity for, and was less efficacious than nicotine and cytisine on the α3β4 and α7− nAChR subtypes. Like cytisine and nicotine, CC4 induced conditioned place preference (CPP), and its self-administration shows an inverted-U dose-response curve. Pre-treatment with non-reinforcing doses of CC4 significantly reduced nicotine-induced self-administration and CPP without affecting motor functions. Conclusions And Implications Our in vitro and in vivo findings reveal that CC4 selectively reduces behaviours associated with nicotine addiction consistent with the partial agonist selectivity of CC4 for β2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation.

2013 - Nanomedicine in Neuroscience: the potential of targeted nanoparticles in crossing the Blood-Brain Barrier [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; A., Grabrucker; Forni, Flavio; Vandelli, Maria Angela
abstract

Non-invasive strategies for treatment of Central Nervous System (CNS) diseases based on colloidal carriers represent a huge potential to efficiently transport drug across the BBB, since nanocarriers can protect drugs (or gene material) and deliver them to target specific populations of brain cells. The efficacy of the nanotechnological approach for brain targeting has been proved by several papers and widely reviewed. Literature contributions mainly deal with several kind of nanometric carriers such as polymeric nanoparticles (NPs), liposomes, solid-lipid NPs, micelles, nanogels and dendrimers. However, these nanocarriers, target and reach the brain poorly, if not engineered in their surface to take advantage of BBB transport mechanisms. Recent studies demonstrated the efficacy of the medicinal chemistry approach, based on the modification of the physico-chemical properties of drugs and the biological approach, based on the conjugation of molecules with antibodies or ligands targeting the BBB. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate an deliver cholesterol an BDNF to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain, as Rhodamine-123 and Loperamide) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses.

2013 - Nanotechnology and Central Nervous System Drug Delivery [Abstract in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; A., Grabrucker; A., Sharma; H. S., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

In line with the overall increase in knowledge and nanotechnologies, surface engineering of nano-sized carriers is now representing the cutting edge of nanomedicine, leading to the production of selectively targeted therapies based on targeted nanocarriers. In fact, achieving nanocarriers able to be stable in the blood-stream, to protect the drug from metabolism and to promote a long-lasting release of the drug is still a pivotal pre-requisite for nanomedicine, but it is now to be considered as “not enough”. Active targeting to specific pathological cells is now the challenge of pharmaceutical nanotechnologists, who are facing with difficulties in colloidal chemistry and most of all in the characterization of the engineered nanocarriers from a technological and physiological points of view. As an example, the application of nanotechnology to brain-related disorders, called nanoneuromedicine, is surely representing one of the most stimulating challenge as well as one the most difficult due to the presence of biological barriers (BBB) and the great variability in BBB permeability depending on the chosen disease. Encouraging results have been obtained demonstrating the possibility of targeting the CNS up to an important percentage of brain localization. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate some drugs able to rescue from neurodegeneration, to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses. Notwithstanding these outputs, it is our opinion that in order to obtain a real update of neurological disorders’ therapy based on innovative and non invasive protocols (i.e. nanomedicine), a team work is strongly needed. The interdisciplinar competences and skills of all the experts in Neuro-diseases and Nano-Technology (from neurobiologists to neurophysiologist, from nanotechnologists to physicians) must be shared, discussed, considered and applied, thus opening the pave to new vistas in treatments and most of all for the correct development of the research.

2013 - Nicotine-induced structural plasticity in mesencephalic dopaminergic neurons is mediated by dopamine d3 receptors and akt-mtorc1 signaling [Articolo su rivista]
Collo, Ginetta; Bono, Federica; Cavalleri, Laura; Plebani, Laura; Mitola, Stefania; Pich, Emilio Merlo; Millan, Mark J.; Zoli, Michele; Maskos, Uwe; Spano, Pierfranco; Missale, Cristina
abstract

Although long-term exposure to nicotine is highly addictive, one beneficial consequence of chronic tobacco use is a reduced risk for Parkinson's disease. Of interest, these effects both reflect structural and functional plasticity of brain circuits controlling reward and motor behavior and, specifically, recruitment of nicotinic acetylcholine receptors (nAChR) in mesencephalic dopaminergic neurons. Because the underlying cellular mechanisms are poorly understood, we addressed this issue with use of primary cultures of mouse mesencephalic dopaminergic neurons. Exposure to nicotine (1-10 mM) for 72 hours in vitro increased dendritic arborization and soma size in primary cultures. These effects were blocked by mecamylamine and dihydro-b-erythroidine, but not methyllycaconitine. The involvement of a4b2 nAChR was supported by the lack of nicotineinduced structural remodeling in neurons from a4 null mutant mice (KO). Challenge with nicotine triggered phosphorylation of the extracellular signal-regulated kinase (ERK) and the thymoma viral proto-oncogene (Akt), followed by activation of the mammalian target of rapamycin complex 1 (mTORC1)-dependent p70 ribosomal S6 protein kinase. Upstream pathway blockade using the phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)- 8-phenyl-4H-1-benzopyran-4-one hydrochloride] resulted in suppression of nicotine-induced phosphorylations and structural plasticity. These effects were dependent on functional DA D3 receptor (D3R), because nicotine was inactive both in cultures from D3R KO mice and after pharmacologic blockade with D3R antagonist trans-N-4-2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin- 2-yl)ethylcyclohexyl-4-quinolinecarboxamide (SB-277011-A) (50 nM). Finally, exposure to nicotine in utero (5 mg/kg/day for 5 days) resulted in increased soma area of DAergic neurons of newborn mice, effects not observed in D3 receptor null mutant mice mice. These findings indicate that nicotine-induced structural plasticity at mesencephalic dopaminergic neurons involves a4b2 nAChRs together with dopamine D3R-mediated recruitment of ERK/Akt-mTORC1 signaling. © 2013 by The American Society for Pharmacology and Experimental Therapeutics.

2013 - Rab5-dependent cell-specific uptake and distribution of engineered nanoparticles for CNS targeted drug delivery in vivo [Abstract in Atti di Convegno]
Vilella, Antonietta; Tosi, Giovanni; Grabrucker, A. M.; Ruozi, Barbara; Belletti, Daniela; Vandelli, Maria Angela; Boeckers, T. M.; Forni, Flavio; Zoli, Michele
abstract

Employment of brain-targeted nanocarriers as tools for drug delivery to the central nervous system (CNS) represents a pivotal step forward in the development of innovative therapeutic strategies. If nanocarriers are to be translated into the clinic, their distribution within the brain and interaction with CNS cells must be assessed accurately. Here, we investigated these issues by employing polylactide-co-glycolide nanoparticles (NPs) specifically engineered with g7, a glycopeptide conferring the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glia. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain that is not dependent on the presence of the BBB. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs are promising drug carriers for the treatment of neuropsychiatric diseases.

2012 - An improved LC-S/MS method for the quantitation of adenosine concentration in mice brain microdialysates. [Articolo su rivista]
Carrozzo, Mm; Troisi, L; Cannazza, Giuseppe; Cazzato, As; Braghiroli, Daniela; Parenti, Carlo; Guiducci, Stefania; Zoli, Michele
abstract

A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of adenosine concentrations in mouse brain microdialysis samples was developed. High method sensitivity (LLOQ of 1.25 fmol) was achieved by on-line switching column. A C18 was employed as enrichment column and a cyano based (CN-SB) as analytical column. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. It was successfully applied to measure quantitatively adenosine in brain of freely moving mice after different stimuli.

2012 - CHRNA5 polymorphism and nicotine dependence in patients with cluster headache [Abstract in Rivista]
I., Tiraferri; M., Ciccarese; M. M., Cainazzo; Ferrari, Anna; Zoli, Michele; Pini, Luigi Alberto
abstract

Up to 90% of cluster headache (CH) patients have a prolonged history of cigarette smoking prior to the headache onset. A genetic link has been suggested between CH and nicotine addiction and, also, that agents found in cigarette smoke have a direct effect on the hypothalamus, a pivotal area for the pathogenesis of CH . Case-control and genome-wide association studies have reported links between single nucleotide polymorphisms (SNPs) in the alpha-5 nicotinic acetylcholine receptor subunit (CHRNA5) genes and cigarettes smoked per day (CPD). In particular, in vivo studies have demonstrated that 5 subunit is involved in controlling nicotine intake, in mediating nicotine withdrawal symptoms and in affecting anxiety-related behaviour.

2012 - NANOMEDICINE IN NEUROSCIENCE: THE POTENTIAL OF TARGETED NANOPARTICLES IN NEURODEGENERATIVE DISORDERS [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; H. S., Sharma; A., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

Pathologic conditions affecting the brain such as neurodegenerative diseases and neurological disorders (i.e. Parnkison’s disease, Alzheimer’s disease, Huntington disease, multiple sclerosis, brain tumors, etc.) are amongst the most un-treatable syndromes. A major obstacle for the application of therapeutics is that a great number of pharmacologically active molecules (estimated 98%) are not able to reach the Central Nervous System (CNS) and to exert their activity as they cannot cross the Blood-Brain Barrier (BBB). Thus, one of the challenges of pharmaceutical research nowadays is to discover tools enabling an effective and efficacious delivery of drugs into the CNS. Non-invasive techniques based on colloidal carriers (nanomedicine) could represent a huge potential and, in line with the overall increase in knowledge and nanotechnologies, surface engineering of nano-sized carriers is now representing the cutting edge of nanomedicine, leading to the production of selectively targeted therapies based on targeted nanocarriers. In fact, achieving nanocarriers able to be stable in the blood-stream, to protect the drug from metabolism and to promote a long-lasting release of the drug, is still a pivotal pre-requisite for nanomedicine, but it is now to be considered as “not enough”. Active targeting to specific pathological cells is now the challenge of pharmaceutical nanotechnologists, who are facing with difficulties in colloidal chemistry and most of all in the characterization of the engineered nanocarriers from a technological and physiological points of view. As an example, the application of nanotechnology to brain-related disorders, called nanoneuromedicine, is certainly representing one of the most stimulating as well as one of the most difficult challenges, due to the presence of biological barriers (BBB) and the great variability in BBB permeability depending on the chosen disease. Nevertheless, encouraging results have been obtained demonstrating the possibility of targeting the CNS up to reaching a significant percentage in brain localization of nanocarriers. As an example of targeted NPs, new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with glycopeptides (g7-NPs) have been recently demonstrated, by in vivo and in vitro experiments, to be able to trigger brain delivery of active substances (brain accumulation up to 10-15% of the injected dose). Moreover, BBB crossing of g7-NPs was recently assessed by our team, evidencing endocytosis/macropinocytosis pathways as preferential mechanisms for g7-NPs movements and interactions. With this work, we will also show new developments and insights of our research with highlights mainly on g7-NPs in vitro behavior on neurons/glia as well as in vivo (rodents) brain localization and trafficking after different routes of administration. Notwithstanding these results, it is our opinion that in order to obtain a real progress in neurological disorders’ therapy based on innovative and non invasive protocols (i.e. nanomedicine), a team effort is highly desired. The interdisciplinary competences and skills of all the experts in Neuro-diseases and Nano-Technology (from neurobiologists to neurophysiologists, from nanotechnologists to physicians) must be shared, discussed, considered and applied, thus paving the way to new vistas in treatments and most of all for the correct development of this field of research.

2012 - Nanomedicine in Neuroscience: The potential of targeted nanoparticles in neurodegenerative disorders [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; H. S., Sharma; A., Sharma; Forni, Flavio; Vandelli, Maria Angela
abstract

Non-invasive strategies for treatment of Central Nervous System (CNS) diseases based on colloidal carriers represent a huge potential to efficiently transport drug across the BBB, since nanocarriers can protect drugs (or gene material) and deliver them to target specific populations of brain cells. The efficacy of the nanotechnological approach for brain targeting has been proved by several papers and widely reviewed. Literature contributions mainly deal with several kind of nanometric carriers such as polymeric nanoparticles (NPs), liposomes, solid-lipid NPs, micelles, nanogels and dendrimers. However, these nanocarriers, target and reach the brain poorly, if not engineered in their surface to take advantage of BBB transport mechanisms. Recent studies demonstrated the efficacy of the medicinal chemistry approach, based on the modification of the physico-chemical properties of drugs and the biological approach, based on the conjugation of molecules with antibodies or ligands targeting the BBB. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate an deliver cholesterol an BDNF to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain, as Rhodamine-123 and Loperamide) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses.

2012 - Nano-Neuroscience: Targeted nanoparticles For CNS drug delivery [Relazione in Atti di Convegno]
Tosi, Giovanni; Ruozi, Barbara; Vilella, Antonietta; Belletti, Daniela; Veratti, Patrizia; Baraldi, Elisa; Zoli, Michele; M., Schmeisser; A., Grabrucker; Forni, Flavio; Vandelli, Maria Angela; A., Sharma; H. S., Sharma
abstract

Non-invasive strategies for treatment of Central Nervous System (CNS) diseases based on colloidal carriers represent a huge potential to efficiently transport drug across the BBB, since nanocarriers can protect drugs (or gene material) and deliver them to target specific populations of brain cells. The efficacy of the nanotechnological approach for brain targeting has been proved by several papers and widely reviewed. Literature contributions mainly deal with several kind of nanometric carriers such as polymeric nanoparticles (NPs), liposomes, solid-lipid NPs, micelles, nanogels and dendrimers. However, these nanocarriers, target and reach the brain poorly, if not engineered in their surface to take advantage of BBB transport mechanisms. Recent studies demonstrated the efficacy of the medicinal chemistry approach, based on the modification of the physico-chemical properties of drugs and the biological approach, based on the conjugation of molecules with antibodies or ligands targeting the BBB. In this contest, polymeric nanoparticles (NPs) and liposomes (LPs) were formulated and specifically engineered to cross the BBB and arrive to CNS and proposed to encapsulate an deliver cholesterol an BDNF to the CNS. Our attention point on the use of polymeric nanoparticles engineered on surface by a selective ligand able to promote the NPs crossing of BBB. In fact, preliminary studies demonstrated the ability of new targeted polymeric poly-lactide-co-glycolide (PLGA) NPs modified with a short peptide (H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 (g7-NPs) to create BBB interaction and trigger an efficacious BBB crossing delivering of active. In particular, several in vivo biodistribution studies and pharmacological proof-of-evidence of brain delivery of model drugs (not able by themselves to reach the brain, as Rhodamine-123 and Loperamide) demonstrated the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. A total biodistibution of g7-NPs, obtained after i.v. administration in rats, evidenced a strong and significant localization of the g7-NPs into CNS in a quantity about two orders of magnitude greater (10-15%) than that found with the other known NP drug carriers. More recently, the g7-NP BBB crossing mechanism was investigated, pointing out an interaction between g7-NPs and BBB and endocytosis/macropinocytosis pathways for BBB crossing. Same results were pointed out also in vitro on neurons/glia cell coltures, evidencing the endocytotic pathways as g7-NPs cell entrance as well as the assessing of the safety of g7-NPs not creating any damage to cells even at high doses.

2012 - Nicotinic Regulation of Energy Homeostasis [Articolo su rivista]
Zoli, Michele; Picciotto, Mr
abstract

Introduction: The ability of nicotine, the primary psychoactive substance in tobacco smoke, to regulate appetite and body weight is one of the factors cited by smokers that prevents them from quitting and is the primary reason for smoking initiation in teenage girls. The regulation of feeding and metabolism by nicotine is complex, and recent studies have begun to identify nicotinic acetylcholine receptor (nAChR) subtypes and circuits or cell types involved in this regulation. Discussion: We will briefly describe the primary anatomical and functional features of the input, output, and central integration structures of the neuroendocrine systems that regulate energy homeostasis. Then, we will describe the nAChR subtypes expressed in these structures in mammals to identify the possible molecular targets for nicotine. Finally, we will review the effects of nicotine and its withdrawal on feeding and energy metabolism and attribute them to potential central and peripheral cellular targets.

2012 - Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC–ESI-MS/MS [Articolo su rivista]
Cannazza, Giuseppe; Carrozzo, Mm; Cazzato, As; Bretis, Im; Troisi, L; Parenti, Carlo; Braghiroli, Daniela; Guiducci, Stefania; Zoli, Michele
abstract

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous measurement of adenosine (ADE), dopamine (DA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) in mouse brain microdialysates. High method sensitivity (LLOQ of 0.05 nM) was achieved by optimization of chromatographic and mass spectrometric parameters. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. The LC–MS/MS method was successfully applied to evaluate the effect of the systemic administration of cocaine or amphetamine on the extracellular levels of ADE, DA, ACh and 5-HT in the mouse nucleus accumbens by microdialysis.

2011 - Brain targeting by engineered nanoparticles [Relazione in Atti di Convegno]
Tosi, Giovanni; A., Grabrucker; L., Bondioli; Ruozi, Barbara; Zoli, Michele; Vilella, Antonietta; Forni, Flavio; Rivasi, Francesco; Vandelli, Maria Angela
abstract

In the last years, the application of "nanotechnology“ to the field of “medicine” surely represented the most innovative strategy to cope with difficult-to-treat diseases. Thus, nanotech-based drug delivery and targeting are nowadays some of the hottest topics in science and in particular in Neuroscience. The results of our research, based on in vitro and in vivo preclinical tests strongly indicate that specifically engineered nanoparticles, made of poly-lactide-co-glycolide (PLGA) polymer, are able to cross the Blood-brain barrier (BBB) and to deliver a variety of drugs or active molecules inside the Central Nervous System (CNS). A recent report from the World Health Organization (WHO) emphasizes that neurological disorders (brain injuries, neuroinfections, multiple sclerosis, epilepsy, stroke, Alzheimer and Parkinson disease) affect up to one billion people worldwide [World Health Organization, Neurological disorders : public health challenger, Geneva, 2006]. Until now, only 2% of the overall drugs are able to enter the brain as the BBB restricts the diffusion of substances from blood to the brain. Thus, one of the challenges of pharmaceutical research nowadays is to discover tools enabling an effective and efficacious delivery of drugs into the CNS. To improve the efficacy of drugs, a possible answer could be the nanomedicine approach, and its application on neuroscience (neuro-nanomedicine). Thereby, the perspective of introducing a tool, capable of directed delivery of every drug into the brain, is undoubtedly an attractive goal for researchers and practitioners. To that end, neuro -nanomedicine exploits pharmaceutical technology, using well-known nanocarriers such as liposomes and polymeric nanoparticles (NPs). These nanosystems, ranging from 100 nm to 250 nm, are able to protect loaded drugs from being metabolised and eliminated, to assure the controlled release of the embedded drugs and to target specific cell population if specifically engineered.To achieve this goal we planned, create and test specifically engineering the NPs surface able to take advantage of the BBB crossing pathways, such as endocytosis or transcytosis. We applied this approach modifying FDA-approved biodegradable NPs with two different peptides to produce highly selective nanosystems able to enter the brain after i.v. administration in the rodents model. The administration of engineered-NPs allowed a variety of drugs to cross the BBB at a rate of 15-20% of the injected dose. The mechanism of BBB crossing of those NPs were elucidated by means of several in vitro and in vivo experiments as the safety of NPs on neuron cell colture was proven. The potential impact of such nanotech-based innovations relies on the possible changes in treatments and cures of the most difficult-to-treat neurological diseases, opening the pave to a new vista on the future trend in medicine, which should strengthen the relationship between different field of research (from clinician-based to chemistry, nanotechnology, biology and pre-clinical study) becoming more and more translational and interdisciplinar.

2011 - Brain targeting by engineered nanoparticles: in vivo and in vitro evidences [Abstract in Atti di Convegno]
Tosi, Giovanni; A., Grabrucker; L., Bondioli; Ruozi, Barbara; Zoli, Michele; Vilella, Antonietta; Forni, Flavio; Rivasi, Francesco; Vandelli, Maria Angela
abstract

In the last years, the application of "nanotechnology“ to the field of “medicine” surely represented the most innovative strategy to cope with diseases and could be named as nanomedicine, which is mostly applied to difficult-to-treat diseases. In this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug-action. Several results with stimulating findings in preclinical or clinical phases have been obtained using nanocarriers delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. The nano-technological approach consists of the use of nanosystems (colloidal carriers), which could be polymer-based (nanoparticles, Np) or solid lipid material made (solid lipid nanoparticles, SLNp) and lipid-based (liposomes, LP). In fact, the development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, are able to cross the BBB without causing apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, focusing in particular on peptide-decorated nanosystems able to target the CNS.In vitro and in vivo experiments allowed to establish a pathway through which engineered NPs can cross the BBB and showed the possible NPs’ transport from cell to cell inside the CNS and the possible tropism of NPs for specific neuronal cell populations.References• A.M. Grabrucker, C. C. Garner, T.M. Boeckers, L. Bondioli, B. Ruozi, F.Forni, M.A. Vandelli, G.Tosi , Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences. PLOS ONE, 2011, Vol 6, e17851.• G. Tosi, R.A. Fano, L. Badiali, R. Benassi, F. Rivasi, B. Ruozi, F. Forni, M.A., Vandelli. Investigation on the mechanisms for Blood-Brain Barrier crossing of brain-targeted glycopeptides nanoparticles, Nanomedicine UK, 2011, 6(3), 423-436• G. Tosi, AV Vergoni, B. Ruozi, L. Bondioli, L. Badiali, F. Rivasi, L. Costantino, F. Forni, M.A. Vandelli, Sialic-acid and glycopeptides conjugated PLGA nanoparticles for Central Nervous System targeting: in vivo pharmacological evidence and biodistribution, Journal of Controlled Release, 2010,145, 49-57.

2011 - Developmental overfeeding alters hypothalamic neuropeptide mRNA levels and response to a high-fat diet in adult mice [Articolo su rivista]
Ferretti, Silvia; Fornari, Alice; Pedrazzi, Patrizia; Pellegrini, Massimo; Zoli, Michele
abstract

It has been suggested that nutritional manipulations during the first weeks of life can alter the development of the hypothalamic circuits involved in energy homeostasis. We studied the expression of a large number of the hypothalamic neuropeptide mRNAs that control body weight in mice that were overfed during breastfeeding (mice grown in a small litter, SL) and/or during adolescence (adolescent mice fed a high-fat diet, AHF). We also investigated possible alterations in mRNA levels after 50 days of a high-fat diet (high-fat challenge, CHF) at 19 weeks of age. Both SL and AHF conditions caused overweight during the period of developmental overfeeding. During adulthood, all of the mouse groups fed a CHF significantly gained weight in comparison with mice fed a low-fat diet, but the mice that had undergone both breast and adolescent overfeeding (SL-AHF-CHF mice) gained significantly more weight than the control CHF mice. Of the ten neuropeptide mRNAs studied, only neuropeptide Y (NPY) expression was decreased in all of the groups of developmentally overfed adult mice, but CHF during adulthood by itself induced a decrease in NPY, agouti-related protein (AgRP) and orexin (Orx) mRNA levels. Moreover, in the developmentally overfed CHF mice NPY, AgRP, galanin (GAL) and galanin-like peptide (GalP) mRNA levels significantly decreased in comparison with the control CHF mice. These results show that, during adulthood, hypothalamic neuropeptide systems are altered (NPY) and/or abnormally respond to a high-fat diet (NPY, AgRP, GAL and GalP) in mice overfed during critical developmental periods.

2011 - Nanoparticles and the BBB crossing: in vivo and in vitro upcomings [Relazione in Atti di Convegno]
Tosi, Giovanni; Bondioli, Lucia; Ruozi, Barbara; Andreas, Grabrucker; Vilella, Antonietta; Zoli, Michele; Rivasi, Francesco; Vandelli, Maria Angela; Forni, Flavio
abstract

In the last years, the application of "nanotechnology “to the field of “medicine” surely represented the most innovative strategy to cop_20e with diseases and it could be named as nanomedicine applied to difficult-to-treat diseases. As known, in this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug action. Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to targeted pathologies, and among them, it is known that neuro-pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. In fact, the development of new delivery systems (nanoparticles and liposomes) started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, were shown to be able to cross the Blood-Brain Barrier without apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, carried out with all the most popular kinds of nanoparticulate systems, focusing in particular on immune-nanoparticles and peptide-decorated nanosystems able to target the CNS, with in vivo and in vitro evidences investigating the pathway for BBB crossing and CNS localization of engineered nanoparticles. The brain localization and the multi-modal pathways for BBB crossing highlighted the endocytosis as preferential pathway; moveover, in vitro test on hippocampal neurons showed the presence of cell-to-cell transport of nanoparticles.

2011 - Ryanodine receptor-2 upregulation and nicotine-mediated plasticity. [Articolo su rivista]
E., Ziviani; Lippi, Giordano; D., Bano; E., Munarriz; Guiducci, Stefania; Zoli, Michele; Kw, Young; P., Nicotera
abstract

Nicotine, the major psychoactive component of cigarette smoke, modulates neuronal activity to produce Ca2+-dependent changes in gene transcription. However the downstream targets that underlie the long-term effects of nicotine on neuronal function, and hence behaviour, remain to be elucidated. Here we demonstrate that nicotine administration to mice upregulates levels of the type 2 ryanodine receptor (RyR2), a Ca2+ release channel present on the endoplasmic reticulum, in a number of brain areas associated with cognition and addiction, notably the cortex and ventral midbrain. Nicotine-mediated RyR2 upregulation was driven by CREB, and caused a long-lasting reinforcement of Ca2+ signalling via the process of Ca2+-induced Ca2+-release. RyR2 upregulation was itself required for long-term phosphorylation of CREB in a positive-feedback signalling loop. We further demonstrate that inhibition of RyR-activation in vivo abolishes sensitization to nicotine-induced habituated locomotion, a well-characterised model for onset of drug dependence. Our findings therefore demonstrate that gene-dependent reprogramming of Ca2+ signalling is involved in nicotine-stimulated neuronal plasticity.

2011 - Targeting of the Arpc3 actin nucleation factor by miR-29a/b regulates dendritic spine morphology. [Articolo su rivista]
Lippi, Giordano; Steinert, Jr; Marczylo, El; D'Oro, Sabina; Fiore, R; Forsythe, Id; Schratt, G; Zoli, Michele; Nicotera, P; Young, Kw
abstract

Previous studies have demonstrated that microribonucleic acids (miRs) are key regulators of protein expression in the brain and modulate dendritic spine morphology and synaptic activity. To identify novel miRs involved in neuronal plasticity, we exposed adult mice to chronic treatments with nicotine, cocaine, or amphetamine, which are psychoactive drugs that induce well-documented neuroadaptations. We observed brain region- and drug-specific changes in miR expression levels and identified miR-29a/b as regulators of synaptic morphology. In vitro imaging experiments indicated that miR-29a/b reduce mushroom-shaped dendritic spines on hippocampal neurons with a concomitant increase in filopodial-like outgrowths, suggesting an effect on synapse formation via actin cytoskeleton remodeling. We identified Arpc3, a component of the ARP2/3 actin nucleation complex, as a bona fide target for down-regulation by miR-29a/b. This work provides evidence that targeting of Arpc3 by miR-29a/b fine tunes structural plasticity by regulating actin network branching in mature and developing spines.

2010 - A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes. [Articolo su rivista]
M., Moretti; M., Mugnaini; M., Tessari; Zoli, Michele; A., Gaimarri; I., Manfredi; F., Pistillo; F., Clementi; C., Gotti
abstract

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous selfadministration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

2010 - Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area alpha6beta2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement. [Articolo su rivista]
Gotti, C; Guiducci, Stefania; Tedesco, V; Corbioli, S; Zanetti, Lara; Moretti, M; Zanardi, Alessio; Rimondini, R; Mugnaini, M; Clementi, F; Chiamulera, C; Zoli, Michele
abstract

alpha6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of alpha6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of alpha6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of alpha6* nAChR in the mesostriatal system is heterogeneous, with (non-alpha4)alpha6beta2* being predominant in the mesolimbic pathway and alpha4alpha6beta2* in the nigrostriatal pathway. We verified whether alpha6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists alpha-conotoxin MII (CntxMII) (alpha3/alpha6beta2* selective) or alpha-conotoxin PIA (CntxPIA) (alpha6beta2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the alpha6beta2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that alpha6beta2*-selective compounds capable of crossing the blood-brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

2010 - The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia [Articolo su rivista]
Peppoloni, Samuele; S., Ricci; Orsi, Carlotta Francesca; Colombari, Bruna; M. M., De Santi; Messinò, Massimino; G., Fabio; Zanardi, Alessio; Righi, Elena; V., Braione; S., Tripodi; D., Chiavolini; M., Cintorino; Zoli, Michele; M. R., Oggioni; Blasi, Elisabetta; G., Pozzi
abstract

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10(2) CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10(7) CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.

2009 - Can the role of genetic factors in schizophrenia be enlightened by studies of candidate gene mutant mice behaviour? [Articolo su rivista]
Mazzoncini, R; Zoli, Michele; Tosato, S; Lasalvia, A; Ruggeri, M.
abstract

Schizophrenia is one of the most severe psychiatric disorders. Despite the knowledge accumulated over years, aetiology and pathophysiology remain uncertain. Research on families and twins suggests that genetic factors are largely responsible for the disease and implies specific genes as risk factors. Genetic epidemiology indicates a complex transmission mode, compatible with a multi-locus model, with single genes accounting for specific traits rather than for the entire phenotype. To better understand every single gene contribution to schizophrenia, the use of intermediate endophenotypes has been proposed. A straight communication between preclinical and clinical researchers could facilitate research on the association between genes and endophenotypes. Many behavioural tasks are available for humans and animals to measure endophenotypes. Here, firstly, we reviewed the most promising mouse behavioural tests modelling human behavioural tasks altered in schizophrenia. Secondly, we systematically reviewed animal models availability for a selection of candidate genes, derived from linkage and association studies. Thirdly, we systematically reviewed the studies which tested mutant mice in the above behavioural tasks. Results indicate a large mutant mice availability for schizophrenia candidate genes but they have been insufficiently tested in behavioural tasks. On the other hand, multivariate and translational approach should be implemented in several behavioural domains.

2009 - D2R–striatopallidal neurons inhibit both locomotor and drug reward processes [Articolo su rivista]
P., Durieux; B., Bearzatto; Guiducci, Stefania; T., Buch; A., Waisman; Zoli, Michele; Sn, Schiffmann; A., de Kerchove d’Exaerde
abstract

The striatum is involved in motor and motivational control but the specific functions of D2R–striatopallidal (dopamine D2 receptor–positive) neurons remain poorly understood. Using a genetic mouse model, we showed that ablation of D2R–striatopallidal neurons in the entire striatum induces hyperlocomotion while ablation in the ventral striatum increases amphetamine conditioned place preference. These results demonstrate the motor inhibitory role of D2R–striatopallidal neurons and an involvement in limiting the drug–reinforcement.

2009 - Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus [Articolo su rivista]
Grilli, M; Zappettini, S; Zanardi, Alessio; Lagomarsino, F; Pittaluga, A; Zoli, Michele; Marchi, M.
abstract

We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [3H]noradrenaline ([3H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K+. NMDA -evoked [3H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [3H]NA overflow elicited by 100 μM AMPA, 1 μM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn2+ ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.

2009 - Neurosteroids and epileptogenesis in the pilocarpine model: evidence for a relationship between P450scc induction and length of the latent period [Articolo su rivista]
Biagini, Giuseppe; Longo, Daniela; Baldelli, Enrica; Zoli, Michele; M. A., Rogawski; G., Bertazzoni; M., Avoli
abstract

Purpose: Cytochrome P450 cholesterol side-chaincleavage enzyme (P450scc) catalyzes the initialstep in the biosynthesis of neurosteroids withinthe brain. We sought to determine which cellsexpress P450cc and whether neurosteroids play arole in the regulation of epileptogenesis followingpilocarpine-induced status epilepticus (SE).Methods: Rats experienced uninterrupted SE orSE terminated with diazepam at 60, 120, and180 min. P450scc induction in CA3 hippocampuswas determined by double immunolabeling withP450scc antiserum and monoclonal antibodiesagainst GFAP (astrocytes), RIP (oligodendrocytes),or heme oxygenase-1 (microglia).Results: SE was associated with P450scc inductionin many astrocytes and a small number of microgliaand oligodendrocytes in the hippocampal CA3strata radiatum and lacunosum-moleculare. Theextent of P450scc induction increased withincreasing SE duration. Paradoxically, increasedP450scc induction in rats experiencing SE for180 min or more was associated with the delayedonset of spontaneous recurrent seizures. Treatmentwith the 5a-reductase inhibitor finasteride(100 mg/kg/day for 25 days), which inhibits thesynthesis of c-aminobutyric acid (GABA)A receptormodulating neurosteroids such as allopregnanolone,was associated with a significantreduction in time to the onset of spontaneous seizuresin rats exposed to 180-min but not 90-minSE.Discussion: P450scc is induced by SE in a diversepopulation of hippocampal glia. Induction ofP450scc is associated with the delayed onsetof spontaneous seizures. Conversely, inhibitionof neurosteroid synthesis accelerated the onset ofspontaneous seizures, but only in animals exhibitingsignificant increases in P450scc. These findingssuggest that induction of neurosteroid synthesis inreactive glial cells is associated with delayed onsetof spontaneously recurrent seizures.

2009 - Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens [Articolo su rivista]
M., Grilli; S., Zappettini; Zoli, Michele; M., Marchi
abstract

We investigated whether nAChRs and D2 receptors co-exist and interact on the same nerve endings using synaptosomes prelabeled with [3H]DA and exposed to nicotinic anddopaminergic receptor ligands. The nicotinic agonists (-)nicotine or epibatidine provoked [3H]DA release which was inhibited by quinpirole. This effect was blocked by sulpiride and raclopride. The [3H]DA overflow evoked by 4-aminopyridine (4-AP) was markedly inhibited by quinpirole. This inhibitory effect did not change either in absence or in presence of (-)nicotine when the nAChRs were desensitized. The inhibitory effect of quinpirole disappeared after a preincubation with this drug. However, the stimulatory effect of (-)nicotine did not change when the D2 receptors were desensitized. (-)Nicotine and 4-AP were able to stimulate [3H]DA overflow also in mouse synaptosomes and this overflow was strongly inhibited by quinpirole. In the nAChR subunits β2 knockout mice the (-)nicotineevoked [3H]DA overflow did not occur anymore but quinpirole was still able to inhibit the [3H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (-)nicotine evokedrelease can be modulated by D2 autoreceptors coexisting in the same DA terminals and nAChRs function is independent from the activation of D2 autoreceptors which themselves may function independently from the activation of presynaptic nAChRs.

2009 - Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the {alpha}3{beta}4 and {alpha}3{beta}3{beta}4 subtypes mediate acetylcholine release [Articolo su rivista]
Sr, Grady; M., Moretti; Zoli, Michele; Mj, Marks; Zanardi, Alessio; L., Pucci; F., Clementi; C., Gotti
abstract

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo-interpeduncular (Hb-IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat, and wildtype (+/+) and beta2 knockout (-/-) mice to establish that the Hb and IPn contain significant beta2* and beta4* populations of nAChR receptors (each of which is heterogeneous). The beta4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (alpha2beta2*, alpha4beta3beta2* alpha3beta3beta4*, alpha6beta3beta4*). Our studies on IPn synaptosomes obtained from +/+ and alpha2 alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4-/- mice, show that only the alpha3beta4 and alpha3beta3beta4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation and Western blotting studies in beta3-/- mice showed that in the IPn of these mice there is a concomitant reduction of ACh release and alpha3beta4* receptors, while the receptor number remains the same in the Hb. We suggest that in habenular cholinergic neurons the beta3 subunit may be important for transporting the alpha3beta4* subtype from the medial habenula (MHb) to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb-IPn system and identify alpha3beta4 and alpha3beta3beta4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.

2009 - Structural and functional diversity of native brain neuronal nicotinic receptors [Articolo su rivista]
Gotti, C; Clementi, F; Fornari, Alice; Gaimarri, A; Guiducci, Stefania; Manfredi, I; Moretti, M; Pedrazzi, Patrizia; Pucci, L; Zoli, Michele
abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels present in the central and peripheral nervous systems, that are permeable to mono- and divalent cations. They share a common basic structure but their pharmacological and functional properties arise from the wide range of different subunit combinations making up distinctive subtypes.nAChRs are involved in many physiological functions in the central and peripheral nervous systems, and are the targets of the widely used drug of abuse nicotine. In addition to tobacco dependence, changes in their number and/or function are associated with neuropsychiatric disorders, ranging from epilepsy to dementia.Although some of the neural circuits involved in the acute and chronic effects of nicotine have been identified, much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions.We briefly review some recent findings concerning the structure and function of native nAChRs, focusing on the subtypes identified in the mesostriatal and habenulo-interpeduncular pathways, two systems involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the effect of chronic nicotine on the expression of native subtypes.

2008 - Neuroprotection via nAChRs: the role of nAChRs in neurodegenerative disorders such as Alzheimer's and Parkinson's disease [Articolo su rivista]
M. R., Picciotto; Zoli, Michele
abstract

Epidemiological studies have identified a negative correlation between smoking and the development of neurodegenerative disorders such as Parkinson’s disease, and in some studies, Alzheimer’s disease. These findings have been attributed to the ability of nicotine to act as a neuroprotectant. A large number of studies demonstrate that nicotine can protect against neuronal death in vitro and in vivo, and the mechanisms underlying the ability of nicotine to protect against excitotoxicity and amyloid- toxicity are beginning to be elucidated. Despite the compelling evidence that nicotine is neuroprotective, it is clear that nicotine can be toxic under some circumstances. The balance between nicotine neuroprotection and toxicity depends on dose, developmental stage and regimen of administration. Therefore, a full understanding of the molecular and cellular effects of nicotine on signaling pathways relevant to neuronal survival is critical for informed drug discovery of nicotinic compounds to combat human neurodegeneration. This review summarizes recent studies related to the mechanisms underlying nicotine-mediated neuroprotection, and addresses issues that are relevant to use of nicotine as a neuroprotective agent in vivo.

2008 - Regional patterns and clinical correlates of basal ganglia morphology in non-medicated schizophrenia [Articolo su rivista]
M., Ballmaier; F., Schlagenhauf; A. W., Toga; J., Gallinat; M., Koslowski; Zoli, Michele; C., Hojatkashani; K. L., Narr; A., Heinz
abstract

Although structural changes of the basal ganglia are widely implicated in schizophrenia, prior findings in chronically medicated patients show that these changes relate to particular antipsychotic treatments. In unmedicated schizophrenia, local alterations in morphological parameters and their relationships with clinical measures remain unknown. Novel surface-based anatomical modelling methods were applied to magnetic resonance imaging data to examine regional changes in the shape and volume of the caudate, the putamen and the nucleus accumbens in 21 patients (19 males/2 females; mean age=30.7+/-7.3) who were either antipsychotic-naïve or antipsychotic-free for at least 1 year and 21 healthy comparison subjects (19 males/2 females; mean age=31.1+/-8.2). Clinical relationships of striatal morphology were based on exploratory analyses. Left and right global putamen volumes were significantly smaller in patients than controls; no significant global volume effects were observed for the caudate and the nucleus accumbens. However, surface deformation mapping results showed localized volume changes prominent bilaterally in medial/lateral anterior regions of the caudate, as well as in anterior and midposterior regions of the putamen, pronounced on the medial surface. A significant positive correlation was observed between right anterior putamen surface contractions and affective flattening, a core negative symptom of schizophrenia. The diagnostic effects of local surface deformations mostly pronounced in the associative striatum, as well as the correlation between anterior putamen morphology and affective flattening in unmedicated schizophrenia suggest disease-specific neuroanatomical abnormalities and distinct cortical-striatal dysconnectivity patterns relevant to altered executive control, motor planning, along with abnormalities of emotional processing.

2008 - Serum proteomic analysis during nicotine self-administration, extinction and relapse in rats [Articolo su rivista]
D., Cecconi; M., Tessari; D. R., Willé; Zoli, Michele; E., Domenici; P. G., Righetti; L., Carboni
abstract

Nicotine dependence is known to induce long-term neural adaptations in brain. The purpose of this study was to verify whether specific protein patterns related to nicotine self-administration states could also be detected in a peripheral tissue. A serum proteomic analysis was performed by 2D electrophoresis on samples taken at six time-points: N, Naïve; P, Priming; S, Self-administration; W, Withdrawal; E, Extinction; R, Relapse. After image analysis, spot volume values were submitted to a Principal Component Analysis and relevant comparisons were selected. In N versus S; S versus W; E versus R; S versus R; and S versus E comparisons a clear separation between groups could be observed, suggesting that each self-administration state correlates with a specific protein expression pattern. Partial least squares-discriminant analysis was adopted to rank proteins by the contribution to the overall separation. A number of spots were identified; among them, C reactive protein and hemopexin displayed a significant reduction after nicotine administration; two hemopexin isoforms were decreased in the S state and antithrombin III was increased in the E phase. This study showed that specific protein patterns related to the nicotine self-administration states exist in serum. Further development of this approach may provide biomarkers to assess dependence states of drug-taking individuals.

2008 - Synapsin-I- and synapsin-II-null mice display an increased age-dependent cognitive impairment [Articolo su rivista]
A., Corradi; Zanardi, Alessio; C., Giacomini; F., Onofri; F., Valtorta; Zoli, Michele; F., Benfenati
abstract

Synapsins (Syns) I and II are major synaptic vesicle (SV) proteins that function in the regulation of SV availability for release in mature neurons. Syns I/II show high level of sequence similarity and share many functions in vivo, although distinct physiological roles for the two proteins have been proposed. Both SynI-/- and SynII-/- mice have a normal lifespan, but exhibit decreased number of SVs and pronounced depression upon high frequency stimulation. Due to the Syns’ role in synaptic organization and plasticity, we studied the long-lasting effects of Syn deletion on the phenotype of SynI-/- and SynII-/- mice during aging. Both SynI-/- and SynII -/- mice displayed behavioural defects that emerged during aging and involved emotional memory in both mutants, and spatial memory in SynII-/- mice. These abnormalities were associated with neuronal loss and gliosis in the cerebral cortex and hippocampus, which were more pronounced in SynII-/- mice. The data indicate that synaptic dysfunctions associated with Syn mutations negatively modulate cognitive performance and neuronal survival during senescence.

2007 - Cannabinoid receptor antagonists counteract sensorimotor gating deficits in the phencyclidine model of psychosis [Articolo su rivista]
M., Ballmaier; M., Bortolato; C., Rizzetti; Zoli, Michele; G. L., Gessa; A., Heinz; P. F., Spano
abstract

Clinical and laboratory findings suggest that cannabinoids and their receptors are implicated in schizophrenia. The role of cannabinoids in schizophrenia remains however poorly understood, as data are often contradictory. The primary aim of this study was to investigate whether the cannabinoid CB1 receptor antagonists rimonabant and AM251 are able to reverse deficits of sensorimotor gating induced by phencyclidine and to mimic the “atypical” antipsychotic profile of clozapine. The prepulse inhibition of the startle reflex was used to measure deficits of sensorimotor gating. PPI-disruptive effects of phencyclidine and their antagonism by rimonabant, AM251 and clozapine were studied in rats. The effects of rimonabant were carefully examined taking into account dose ranges, vehicle and route of administration. We also examined the ability of rimonabant to reduce the PPI-disruptive effects of dizocilpine and apomorphine. Rimonabant as well as AM251 significantly counteracted the phencyclidine-disruptive model of PPI, comparable to the restoring effect of clozapine; no augmentation effect was observed with rimonabant and clozapine as cotreatment. Rimonabant also significantly attenuated the PPI disruptive effects of dizocilpine and apomorphine. Taken together, our results indicate that CB1 receptor antagonists do produce “atypical” antipsychotic profile mimicking that of clozapine in the phencyclidine disruption of sensorimotor gating. Our findings further suggest that CB1 receptor antagonism may be involved in restoring disturbed interactions between the activity of the endocannabinoid system and glutamate neurotransmitter system implied in schizophrenia.

2007 - Differential effects of nicotinic antagonists perfused into the nucleus accumbens or the ventral tegmental area on cocaine-induced dopamine release in the nucleus accumbens of mice [Articolo su rivista]
Zanetti, Lara; M. R., Picciotto; Zoli, Michele
abstract

Rationale The mesolimbic dopamine (DA) system is considered a principal site for nicotine-cocaine interactions. Objectives and methods The aim of this paper is to study the effects of local perfusions (through the microdialysis cannula) of nicotinic acetylcholine receptor (nAChR) antagonists in the ventral tegmental area (VTA, where mesolimbic DA cell bodies are located) or nucleus accumbens (nAc, where mesolimbic DA nerve terminals project) on cocaine-elicited increase in DA levels in the nAc of mice using intracerebral microdialysis. Results Intra-nAc perfusion of mecamylamine (a nonselective central nicotinic antagonist) or coperfusion of methyllycaconitine (MLA, 10 nM) and dihydro-beta-erythroidine (DH beta E, 10-100 mu M) decreased cocaine-elicited increase in DA perfusate levels. In contrast, intra-nAc perfusion of MLA alone (a relatively selective antagonist of alpha 7 subunit-containing nAChRs) increased, while DH beta E (a relatively selective antagonist of heteromeric nAChR subtypes) did not alter, cocaine-elicited increase in DA perfusate levels. Intra-VTA perfusion of MLA (100 nM) or DH beta E (100 mu M) significantly increased the cocaine-elicited increase of DA levels in the nAc or VTA, whereas DH beta E and MLA coperfusion or mecamylamine perfusion had no significant effect. Conclusions These results show that intra-nAc and intra-VTA perfusion of nAChR antagonists differentially affect cocaine-elicited increase in DA levels in a region and subtype-specific manner. This suggests that multiple cholinergic/nicotinic pathways influence the effects of cocaine on mesolimbic DA neurons in complex, and sometimes opposing, patterns.

2007 - Heterogeneity and complexity of native brain nicotinic receptors [Articolo su rivista]
C., Gotti; M., Moretti; A., Gaimarri; Zanardi, Alessio; F., Clementi; Zoli, Michele
abstract

Neuronal cholinergic nicotinic receptors (nAChRs) are a heterogeneous class of cationic channels that are widely distributed in the nervous system that have specific functional and pharmacological properties. They consist of homologous subunits encoded by a large multigene family, and their opening is physiologically controlled by the acetylcholine neurotransmitter or exogenous ligands such as nicotine. Their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding receptor function in the nervous system and discovering new subtype-selective drugs. We will review rodent brain subtypes by discussing their subunit composition, pharmacology and localisation and, when possible, comparing them with the same subtypes present in the brain of other mammalian species or chick.In particular, we will focus on the nAChRs present in the visual pathway (retina, superior colliculus and nucleus geniculatus lateralis), in which neurons express most, if not all, nAChR subunits. In addition to the major alpha4beta2 and alpha7 nAChR subtypes, the visual pathway selectively expresses subtypes with a complex subunit composition. By means of ligand binding and imunoprecipitation and immunopurification experiments on tissues obtained from control and lesioned rats, and wild-type and nAChR subunit knockout mice, we have qualitatively and quantitatively identified, and pharmacologically characterised, the multiple complex native subtypes containing up to four different subunits.

2007 - Loss of high-affinity nicotinic receptors increases the vulnerability to excitotoxic lesion and decreases the positive effects of an enriched environment [Articolo su rivista]
Zanardi, Alessio; Ferrari, Rosaria; Leo, Giuseppina; U., Maskos; J. P., Changeux; Zoli, Michele
abstract

Pharmacological activation of nicotinic acetylcholine receptors (nAChRs) exerts neuroprotective effects in cultured neurons and the intact animal. Much less is known about a physiological protective role of nAChRs. In order to understand whether endogenous activation of beta2* nAChRs contributes to the maintenance of the functional and morphological integrity of neural tissue, adult beta2-/- mice were subjected to in vivo challenges which cause neurodegeneration and cognitive impairment (intrahippocampal injection of the excitotoxin quinolinic acid), or neuroprotection and cognitive potentiation (2 month-exposure to an enriched environment). The excitotoxic insult caused an increased deficit in the Morris water maze learning curve and increased loss of hippocampal pyramidal cells in beta2-/- mice. Exposure to an enriched environment improved performance in contextual and cued fear conditioning and object recognition tests in beta2+/+ whereas the improvement was absent in beta2-/- mice. In addition, beta2+/+, but not beta2-/-, mice exposed to an enriched environment showed a significant hypertrophy of the CA1/3 regions. Thus, lack of beta2* nAChRs increased susceptibility to an excitotoxic insult and diminished the positive effects of an enriched environment. These results may be relevant to understanding the pathophysiological consequences of the marked decrease in nAChRs that occurs in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.

2007 - Neuroscienze e libero arbitrio [Relazione in Atti di Convegno]
Zoli, Michele
abstract

Nel presente lavoro vengono discussi due insiemi di ricerche di ambito neuroscientifico attinenti al problema del libero arbitrio. La nozione classica di libero arbitrio incorpora due concetti, libertà come autonomia di un agente che controlla le sue azioni (libertas spontaneitatis) e libertà come possibilità di agire altrimenti (libertas indifferentiae). E’ quindi interessante descrivere, da un punto di vista neuroscientifico, come avviene un’azione e, più specificamente, quali processi mentali / cerebrali portino alla scelta di un’azione. Verrà discusso un primo insieme di ricerche su meccanismi e strutture cerebrali che codificano i processi di scelta, ovvero come viene presa una decisione (decision-making) (capitolo 1). Porsi il problema del free will (volizione libera, libero arbitrio) è tipicamente porsi il problema del conscious will (volizione cosciente). Verrà quindi discussa la metafisica che è intrinseca al programma di ricerca neuroscientifico sul decision-making, cercando di esplicitare quale statuto questa metafisica conferisca ai fenomeni mentali coscienti (capitolo 2). Quindi verrà descritto un secondo insieme di ricerche riguardanti la neurobiologia e neuropsicologia della volizione cosciente (capitolo 3). Infine, verranno presentati alcuni spunti per una discussione sul libero arbitrio ricavati dalle ricerche scientifiche precedentemente descritte (capitolo 4).

2007 - Neuroscienze ed etica empirica [Articolo su rivista]
Zoli, Michele
abstract

Le ricerche di psicologia cognitiva e neuroscienze stanno portando all’identificazione delle componenti psicologiche del giudizio etico e dei circuiti neuronali ad esse associati. Questi studi mettono in luce il ruolo cruciale che la componente emozionale opera in campo morale. In parallelo, le ricerche di biologia e psicologia evoluzionistica mostrano i meccanismi evolutivi alla base dei comportamenti animali ed umani di cooperazione altruistica. L’insieme di questi studi sta costruendo un quadro teorico coerente delle basi biologiche dell’etica.

2007 - Nicotine withdrawal increases body weight, neuropeptide Y and Agouti-related protein expression in the hypothalamus and decreases uncoupling protein-3 expression in the brown adipose tissue in high-fat fed mice [Articolo su rivista]
Fornari, Alice; Pedrazzi, Patrizia; Lippi, Giordano; M. R., Picciotto; Zoli, Michele; Zini, Isabella
abstract

Nicotine is known to decrease body weight in normal rodents and human smokers, whereas nicotine withdrawal or smoking cessation can increase body weight. We have found that mice fed a high fat diet do not show the anorectic effect of chronic nicotine treatment, but do increase their body weight following nicotine withdrawal. Nicotine withdrawal is accompanied by increased expression of the orexigenic peptides neuropeptide Y and Agouti-related protein in the hypothalamus, and decreased expression of the metabolic protein uncoupling protein-3 in brown adipose tissue. These data suggest that diet can influence the ability of nicotine to modulate body weight regulation and demonstrate that chronic nicotine exposure results in adaptive changes in central and peripheral molecules which regulate feeding behavior and energy metabolism. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

2007 - Selective disarrangement of the rostral telencephalic cholinergic system in heterozygous reeler mice [Articolo su rivista]
S., Sigala; Zoli, Michele; F., Palazzolo; S., Faccoli; Zanardi, Alessio; N. B., Mercuri; P. F., Spano
abstract

Reelin (RELN) is a key molecule for the regulation of neuronal migration in the developing CNS. The reeler mice, which have spontaneous autosomal recessive mutation in the RELN gene, reveal multiple defects in brain development. Morphological, neurochemical and behavioral alterations have been detected in heterozygous reeler (HR) mice, suggesting that not only the presence, but also the level of RELN influences brain development. Several studies implicate an involvement of RELN in the pathophysiology of neuropsychiatric disorders in which an alteration of the cholinergic cortical pathways is implicated as well. Thus, we decided to investigate whether the basal forebrain (BF) cholinergic system is altered in HR mice by examining cholinergic markers at the level of both cell body and nerve terminals. In septal and rostral, but not caudal, basal forebrain region, HIR mice exhibited a significant reduction in the number of choline acetyltransferase (ChAT) immunoreactive (ir) cell bodies compared with control mice. Instead, an increase in ChAT ir neurons was detected in lateral striaturn. This suggests that an alteration in ChAT ir cell migration which leads to a redistribution of cholinergic neurons in subcortical forebrain regions occurs in HIR mice. The reduction of ChAT ir neurons in the BF was paralleled by an alteration of cortical cholinergic nerve terminals. In particular, the HIR mice presented a marked reduction of acetylcholinesterase (AChE) staining accompanied by a small reduction of cortical thickness in the rostral clorsomedial cortex, while the density of AChE staining was not altered in the lateral and ventral cortices. Present results show that the cholinergic basalo-cortical system is markedly, though selectively, impaired in HIR mice. Rostral sub-regions of the BF and rostro-medial cortical areas show significant decreases of cholinergic neurons and innervation, respectively. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

2006 - Brain nicotinic acetylcholine receptors: native subtypes and their relevance [Articolo su rivista]
C., Gotti; Zoli, Michele; F., Clementi
abstract

Neuronal nicotinic acetylcholine receptors comprise a heterogeneous class of cationic channels that is present throughout the nervous system. These channels are involved both in physiological functions (including cognition, reward, motor activity and analgesia) and in pathological conditions such as Alzheimer's disease, Parkinson's disease, some forms of epilepsy, depression, autism and schizophrenia. They are also the targets of tobacco-smoking effects and addiction. Neuronal nicotinic acetylcholine receptors are pentamers of homomeric or heteromeric combinations of alpha (alpha 2-alpha 10) and beta (beta 2-beta 4) subunits, which have different pharmacological and biophysical properties and locations in the brain. The lack of subtype-specific ligands and the fact that many neuronal cells express multiple subtypes initially hampered the identification of the different native nicotinic acetylcholine receptor subtypes, but the increasing knowledge of subtype composition and roles will be of considerable interest for the development of new and clinically useful nicotinic acetylcholine receptor ligands.

2006 - Inhibition of both alpha 7* and beta 2* nicotinic acetylcholine receptors is necessary to prevent development of sensitization to cocaine-elicited increases in extracellular dopamine levels in the ventral striatum [Articolo su rivista]
Zanetti, Lara; A., DeKerchove D'Exaerde; Zanardi, Alessio; J. P., Changeux; M. R., Picciotto; Zoli, Michele
abstract

Rationale: Several studies have suggested that nicotine treatment can modulate the behavioral and neurochemical responses to other psychostimulants, such as cocaine. Objectives: The current study examined the hypothesis that nicotinic acetylcholine receptor (nAChR) blockade influences the ability of cocaine to elicit increases in extracellular dopamine levels. Materials and Methods: Pharmacological studies using nicotinic antagonists as well as genetic inactivation of beta 2* nAChRs were used to determine the effect of nAChR blockade on dopamine levels in ventral striatum elicited by acute or repeated administrations of cocaine in mice. Results: Administration of mecamylamine (a general nicotinic antagonist that is not highly selective for individual nAChR subtypes) or co-administration of methyllycaconitine (a more selective antagonist of alpha 7* nAChRs) with dihydro-beta-erythroidine (a more selective antagonist of beta 2* nAChRs and other heteromeric nAChR subtypes) prevented sensitization of cocaine-elicited increases in extracellular DA levels in the ventral striatum in wild-type mice. In contrast, neither of the more specific antagonists alone was effective in preventing sensitization. Finally, methyllycaconitine administration prevents sensitization in beta 2-/- mice but not in beta 2+/+ or wild-type mice. Conclusions: These data indicate that inhibition of both alpha 7* and beta 2* nAChRs is necessary to prevent development of sensitization of cocaine-elicited increases in extracellular dopamine levels in the ventral striatum of mice.

2006 - Neurosteroidi ed epilettogenesi: evidenze a favore di una relazione tra induzione dell’enzima P450scc e durata della fase di latenza nel modello della pilocarpina. [Articolo su rivista]
Baldelli, Enrica; Longo, Daniela; Zini, Isabella; Zoli, Michele; M., Avoli; Biagini, Giuseppe
abstract

Neurosteroids are GABAA receptor agonists able to modulate seizure susceptibility. They are mainly synthesizedin astrocytes, a type of glial cells that are activated by neuronal damage. Interestingly, status epilepticus (SE)induces neuronal damage and a chronic epileptic condition that becomes evident following a “latent” period,in which glial cells are highly activated. Investigating glial cell activation in the pilocarpine model, wefound that P450scc, the rate-limiting enzyme in neurosteroid synthesis, is upregulated in hippocampal astrocytesduring the latent period. The induction of P450scc immunoreactivity was stronger with prolonged statusepilepticus and was associated with longer latencies to the development of seizures, suggesting thatenhanced neurosteroid synthesis retards epileptogenesis. Accordingly, the 5α-reductase inhibitor finasteride,which blocks neurosteroid synthesis, reduced the latent period, thus indicating that glia-derived neurosteroidsmay be antiepileptogenic.

2006 - The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningitis [Abstract in Atti di Convegno]
S., Ricci; Peppoloni, Samuele; S., Tripodi; D., Chaivolini; R., Parigi; V., Braione; Zanardi, Alessio; M., Messinò; F., Iannelli; M., De Santi; M., Cintorino; M. R., Oggioni; Zoli, Michele; Blasi, Elisabetta; G., Pozzi
abstract

The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningitis

2005 - Heterogeneity and selective targeting of nAChR subtypes expressed on retinal afferents of the superior colliculus and lateral geniculate nucleus. Identification of a new native nAChR subtype alpha3beta2(alpha5 or beta3) enriched in retinocollicular afferents [Articolo su rivista]
C., Gotti; M., Moretti; Zanardi, Alessio; A., Gaimarri; N., Champtiaux; J. P., Changeux; P., Whiteaker; M. J., Marks; F., Clementi; Zoli, Michele
abstract

The activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been implicated in the activity-dependent development and plasticity of retina and the refinement of retinal projections. Pharmacological and functional studies have also indicated that different presynaptic nAChRs can have a modulatory function in retinotectal synapses. We biochemically and pharmacologically identified the multiple nAChR subtypes expressed on retinal afferents of the superior colliculus (SC) and lateral geniculate nucleus (LGN). We found that the alpha 6 beta 2* and alpha 4(non alpha 6) beta 2* nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of alpha 2 alpha 6 beta 2* and alpha 3 beta 2* subtypes, whereas the SC contains a relatively large population of a new native subtype, the alpha 3 beta 2(alpha 5/beta 3) nAChR. This subtype binds the alpha-conotoxin MII with an affinity 50 times lower than that of the native alpha 6 beta 2* subtype. Studies of tissues obtained from eye-enucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC alpha 6 beta 2*, alpha 4 alpha 6 beta 2*, and alpha 3 beta 2* (approximately 45, 35, and 20%, respectively), in LGN, alpha 4 alpha 6 beta 2*, alpha 6 beta 2*, alpha 4 beta 2*, alpha 2 alpha 6 beta 2*, and alpha 3 beta 2* ( approximately 40, 30, 20, 5, and 5%, respectively). In both regions, more than 50% of nAChRs were not expressed by retinal afferents and belonged to the alpha 4 beta 2* (90%) or alpha 4 alpha 5 beta 2* (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and alpha 4, alpha 6, and beta 3 knockout mice confirmed and extended the data obtained in rat tissue and allowed a comprehensive dissection of the composition of nAChR subtypes present in this retinorecipient area.

2004 - Alteration of hippocampal cell proliferation in mice lacking the beta(2) subunit of the neuronal nicotinic acetylcholine receptor [Articolo su rivista]
A., Harrist; R. D., Beech; S. L., King; Zanardi, Alessio; M. A., Cleary; B. J., Caldarone; A., Eisch; Zoli, Michele; M. R., Picciotto
abstract

Adult hippocampal neurogenesis declines with age in parallel with decreased performance on a variety of hippocampal-dependent tasks. We measured the rate of cellular proliferation in the hippocampus of mice lacking the 02-subunit of the nicotinic acetylcholine receptor (beta2-/- mice) at three ages: young adult (3 months old), fully adult (7-10 months old), and aged (22-24 months old). Consistent with previous studies, we observed an age-related decline in hippocampal proliferation in both groups. However, in fully adult beta2-/- mice a 43% reduction of granule cell proliferation was detected compared to age-matched controls. This was accompanied by a significant decrease in dentate gyrus area/section and the length of the granule cell layer in beta2-/- mice. These alterations were not the result of a change in plasma corticosterone levels or expression of the neurotrophic factor BDNF in the dentate gyrus, two known regulators of hippocampal cell proliferation. Similarly, there was no increase in gliosis, abnormal myelination, or apoptotic cell death in the beta2-/- animals, although there was a significant shift in the location of apoptotic cells in the dentate gyrus indicative of a change in neuronal survival. These results suggest that the beta2-subunit containing nicotinic acetylcholine receptors play an important role in regulating cell proliferation in the hippocampus and that endogenous acetylcholine may act to oppose the negative effects of normal aging and stress on cellular proliferation.

2004 - Nicotinic acetylcholine receptor subtypes expression during rat retina development and their regulation by visual experience [Articolo su rivista]
M., Moretti; S., Vailati; Zoli, Michele; Lippi, Giordano; L., Riganti; R., Longhi; A., Viegi; F., Clementi; C., Gotti
abstract

By acting through retinal nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in the development of both the retina and central visual pathways. Ligand binding and immunoprecipitation studies with subunit-specific antibodies showed that the expression of alphaBungarotoxin (alphaBgtx) and high-affinity epibatidine (Epi) receptors is regulated developmentally and increases until postnatal day 21 (P21). The increase in Epi receptors is caused by a selective increase in the subtypes containing the alpha2, alpha4, alpha6, beta2, and beta3 subunits. Immunopurification studies revealed three major populations of Epi receptors on P21: alpha6* receptors (26%), which contain the alpha6beta3beta2, alpha6alpha4beta3beta2, and alpha6alpha3/alpha2beta3beta2 subtypes; alpha4(non-alpha6)* receptors (60%), which contain the alpha2alpha4beta2 and alpha4beta2 subtypes; and (non-alpha4/non-alpha6)* receptors (14%), which contain the alpha2beta2/beta4 and alpha3beta2/beta4 subtypes. These three populations can be pharmacologically discriminated using alphaconotoxin MII, which binds the alpha6* population with high affinity. In situ hybridization showed that the transcripts for all of the subunits are heterogeneously distributed throughout retinal neurons at P21, with alpha3, alpha6, and beta3 transcripts preferentially concentrated in the ganglion cell layer, alpha5 in the inner nuclear layer, and alpha4 and beta2 distributed rather homogeneously. To investigate whether nAChR expression is affected by visual experience, we also studied dark-reared P21 rats. Visual deprivation had no effect on the expression of alphaBgtx receptors or the developmentally regulated Epi receptors containing the alpha2, alpha6, and/or beta3 subunits but significantly increased the expression of the Epi receptors containing the alpha4 and beta2 subunits. Overall, this study demonstrates that the retina is the rat neural region that expresses the widest array of nAChR subtypes. These receptors have a specific distribution, and their expression is finely regulated during development and by visual experience.

2003 - A2A receptor and striatal cellular functions: Regulation of gene expression, currents and synaptic transmission [Articolo su rivista]
Schiffmann, S. N.; Dassesse, D.; D'Alcantara, P.; Ledent, C.; Swillens, S.; Zoli, Michele
abstract

A2A receptor is highly coexpressed with enkephalin and D2 receptor in striatopallidal neurons. A2A antagonists acutely enhance motor behavior in animal models of Parkinson's disease (PD) and are therefore considered potential PD therapeutic agents. Analysis of gene expression regulation using pharmacologic tools or A2A receptor-deficient mice (A2A-/-) shows that the A2A receptor positively and tonically controls the expression of enkephalin and immediate early genes in striatopallidal neurons. Because this regulation strictly mirrors the effect of D2 receptor, these data strongly support the hypothesis that A2A antagonists reduce the activity of striatopallidal neurons in models of PD. However, analysis of A2A-/- mice suggests additional effects of A2A receptor in the control of striatal physiology. Unexpectedly, these animals exhibited a reduction in exploratory activity and a 50% reduction in substance P expression. This was associated with a 45% decrease in the striatal extracellular dopamine concentration, suggesting that chronic absence of A2A receptor results in a functional hypodopaminergic state in the striatum. The A2A receptor controls inhibitory synaptic transmission negatively in the striatum and positively in the globus pallidus; this further supports the efficacy of A2A antagonists in reducing the activity of striatopallidal neurons in PD. The A2A receptor does not modulate basal alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-mediated excitatory corticoaccumbal synaptic transmission during normal physiologic conditions. However, genetic inactivation or pharmacologic blockade of the A2A receptor significantly reduced long-term potentiation (LTP) at this synapse. Therefore, this receptor is implicated in the induction of corticoaccumbal LTP, an effect that could be related to its involvement in long-term behavioral sensitization to repeated dopaminergic treatment.

2003 - Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors [Articolo su rivista]
Lm, Marubio; Am, Gardier; S., Durier; D., David; R., Klink; MM Arroyo, Jimenez; Jm, Mcintosh; F., Rossi; N., Champtiaux; Zoli, Michele; Jp, Changeux
abstract

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the alpha4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the alpha4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with (125) I alpha-conotoxin MII. These receptors are comprised of the alpha4, beta2 and alpha6 nAChR subunits and non-alpha4, beta2, and alpha6 nAChR subunits. Non-alpha4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and alpha4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that alpha4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.

2003 - Localization of [H-3]nicotine, [H-3]cytisine, [H-3]epibatidine, and [I-125]alpha-bungarotoxin binding sites in the brain of Macaca mulatta [Articolo su rivista]
Z. Y., Han; Zoli, Michele; A., Cardona; J. P., Bourgeois; J. P., Changeux; N., Le Novere
abstract

We determined the localization of [H-3]nicotine, [H-3]cytisine, [H-3]epibatidine, and [I-125]alpha- bungarotoxin binding sites in the brain of rhesus monkey by means of receptor autoradiography. The labelings by [H-3]nicotine, [H-3]cytisine, and [H-3]epibatidine were highly concordant, except for epibatidine. Layer IV of some cortical areas, most thalamic nuclei, and presubiculum displayed high levels of labeling for the three ligands. Moderate levels of binding were detected in the subiculum, the septum, and the mesencephalon. Low levels were present in layers I-II and VI of the cortex, the cornu Ammonis, the dentate gyrus, and the amygdala. In addition, the level of epibatidine labeling was very high in the epithalamic nuclei and the interpeduncular nucleus, whereas labeling by nicotine and cytisine was very weak in the same regions. The distribution of [I-125]alpha-bungarotoxin binding differed from the binding of the three agonists. The labeling was dense in layer I of most cortical areas, dentate gyrus, stratum lacunosum-moleculare of CA1 field, several thalamic nuclei, and medial habenula. A moderate labeling was found in layers V and VI of the prefrontal and frontal cortices, layer IV of primary visual cortex, amygdala, septum, hypothalamus, and some mesencenphalic nuclei. A weak signal was also detected in subiculum, claustrum, stratum oriens, and stratum lucidum of cornu Ammonis and also in some mesencephalic nuclei. The distribution of nicotine, cytisine, and epibatidine bindings corresponds broadly to the patterns observed in rodents, with the marked exception of the epithalamus. However, in monkey, those distributions match the distribution of alpha2 messenger RNA, rather than that of alpha4 transcripts as it exists in rodent brains. The distribution of the binding sites for alpha-bungarotoxin is larger in the brain of rhesus monkeys than in rodent brain, suggesting a more important role of alpha7 receptors in primates.

2003 - Ontogeny and tissue-specific regulation of ghrelin mRNA expression suggest that ghrelin is primarily involved in the control of extraendocrine functions in the rat [Articolo su rivista]
Torsello, A; Scibona, B; Leo, Giuseppina; Bresciani, E; Avallone, R; Bulgarelli, I; Luoni, M; Zoli, Michele; Rindi, G; Cocchi, D; Locatelli, V.
abstract

Ghrelin is a 28-amino-acid gastric peptide that potently stimulates growth hormone (GH) secretion in vivo and in vitro. Ghrelin-expressing cells have been found in the oxyntic region of the stomach and in the arcuate nucleus of the hypothalamus. The aim of this work was to investigate the regional distribution and developmental changes in ghrelin mRNA levels in the pituitary, hypothalamus and gastrointestinal (GI) tract of the rat using a semiquantitative RT-PCR assay. We also describe the effects of ghrelin immunoneutralization in late gestation and those resulting from induction of an isolated GH deficiency in adult rats. Ghrelin mRNA was already expressed in the fetus by embryonic day 12 (E12), by E17 most of ghrelin mRNA was in the trunk. At E17, in situ hybridization did not reveal a clear expression of ghrelin mRNA in fetal stomach but showed high ghrelin mRNA levels in the placenta. In the pituitary gland, levels of ghrelin mRNA were high after birth but declined significantly with puberty, whereas in the hypothalamus they were barely detectable at birth and remained very low at all subsequent time points tested. In the GI tract, ghrelin mRNA levels were high from birth to 270 days of life. Immunoneutralization of ghrelin at E16 had no effect on survival or development. Rats showed normal somatotropic function, ghrelin expression and onset of puberty. In young adult rats, passive immunization against GHRH did not affect ghrelin mRNA levels in the pituitary, hypothalamus and stomach. Only a 72-hour fasting period induced a significant increase in ghrelin mRNA levels in the stomach, but not in the pituitary and hypothalamus. These results strongly indicate that ghrelin is an important GI hormone expressed early in life and primarily sensitive to nutritional status.

2002 - Acute and long-term changes in the mesolimbic dopamine pathway after systemic or local single nicotine injections [Articolo su rivista]
Ferrari, Rosaria; N., Le Novere; M. R., Picciotto; J. P., Changeux; Zoli, Michele
abstract

We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra-accumbens (Acb) infusion or intraventral tegmental area (intra-VTA) microinjection. The present modes of systemic, intra-Acb and intra-VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra-VTA nicotine administration, whereas unilateral or bilateral intra-Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra-VTA, but not systemic or intra-Acb, nicotine administration caused a long-term (at least 24-h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra-VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra-Acb infusion did not modify any of the mRNAs tested. The long-term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra-VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.

2002 - Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues. [Articolo su rivista]
Rindi, G.; Necchi, V.; Savio, A.; Torsello, A.; Zoli, Michele; Locatelli, V.; Raimondo, F.; Cocchi, D.; Solcia, E.
abstract

Ghrelin is a new gastric peptide involved in food intake control and growth hormone release. We aimed to assess its cell localisation in man during adult and fetal life and to clarify present interspecies inconsistencies of gastric endocrine cell types. A specific serum generated against amino acids 13-28 of ghrelin was tested on fetal and adult gastric mucosa and compared with ghrelin in situ hybridisation. Immunogold electron microscopy was performed on normal human, rat and dog adult stomach. Ghrelin cells were detected in developing gut, pancreas and lung from gestational week 10 and in adult human, rat and dog gastric mucosa. By immunogold electron microscopy, gastric ghrelin cells showed distinctive morphology and hormone reactivity in respect to histamine enterochromaffin-like, somatostatin D, glucagon A or serotonin enterochromaffin cells. Ghrelin cells were characterised by round, compact, electron-dense secretory granules of P/D(1) type in man (mean diameter 147+/-30 nm), A-like type in the rat (183+/-37 nm) and X type in the dog (273+/-49 nm). It is concluded that, ghrelin is produced by well-defined cell types, which in the past had been labelled differently in various mammals mostly because of the different size of their secretory granule. In man ghrelin cells develop during early fetal life.

2002 - Coaggregation, cointernalization, and codesensitization of adenosine A(2A) receptors and dopamine D-2 receptors [Articolo su rivista]
J., Hillion; M., Canals; M., Torvinen; V., Casado; R., Scott; A., Terasmaa; A., Hansson; S., Watson; Me, Olah; J., Mallol; Ei, Canela; Zoli, Michele; Agnati, Luigi Francesco; Cf, Ibanez; C., Lluis; R., Franco; S., Ferre; K., Fuxe
abstract

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A A receptors (A(2A)R) and dopamine D-2 receptors (D2R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D2R and in cultured striatal cells. A(2A)R/D2R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D2R-transfected SH-SY5Y cells and from mouse fibroblast Ltk(-) cells stably transfected with human D2R (long form) and transiently cotransfected with the A(2A)R double-tagged with hemagglutinin. Long term exposure to A(2A)R and D2R agonists in D2R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A(2A)R and D2R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D2R are involved.

2002 - Diet-induced changes in hypothalamic pro-opio-melanocortin mRNA in the rat hypothalamus [Articolo su rivista]
C., Torri; Pedrazzi, Patrizia; Leo, Giuseppina; E. E., Muller; D., Cocchi; Agnati, Luigi Francesco; Zoli, Michele
abstract

Hypothalamic mRNA and peptide levels of pro-opio-melanocortin (POMC) and other neuropeptides were studied in rats that either develop obesity (diet-induced obese, DIO), when fed a palatable and hypercaloric diet (cafeteria diet, caf) or do not develop obesity (diet resistant, DR), when fed the same diet. cafDIO rats showed a significant increase in POMC, but not in melanin concentrating hormone, mRNA levels as determined by semiquantitative in situ hybridization. cafDR and cafDIO rats showed no change in POMC-derived peptide levels, whereas neuropeptide Y immunoreactivity was significantly increased in cafDR rats. POW mRNA levels were also studied in high-fat diet-fed rats but no significant change was observed. Altered hypothalamic transmission by POMC-derived peptides may contribute to the susceptibility of cafDIO rats to the weight promoting action of caf diet.

2002 - Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice [Articolo su rivista]
N., Champtiaux; Zy, Han; A., Bessis; Fm, Rossi; Zoli, Michele; L., Marubio; Jm, Mcintosh; Jp, Changeux
abstract

The alpha6 subunit of the nicotinic acetylcholine receptor (nAChR) is expressed at very high levels in dopaminergic (DA) neurons. However, because of the lack of pharmacological tools selective for alpha6-containing nAChRs, the role of this subunit in the etiology of nicotine addiction remains unknown. To provide new tools to investigate this issue, we generated an alpha6 nAChR knock-out mouse. Homozygous null mutants (alpha6-/-) did not exhibit any gross neurological or behavioral deficits. A careful anatomic and molecular examination of alpha6-/- mouse brains demonstrated the absence of developmental alterations in these animals, especially in the visual and dopaminergic pathways, where the alpha6 subunit is normally expressed at the highest levels. On the other hand, receptor autoradiography revealed a decrease in [H-3] nicotine, [H-3] epibatidine, and [H-3] cytisine high-affinity binding in the terminal fields of retinal ganglion cells of alpha6-/- animals, whereas high-affinity [I-125]alpha-conotoxinMII (alphaCtxMII) binding completely disappeared in the brain. Moreover, inhibition of [H-3] epibatidine binding on striatal membranes, using unlabeled alphaCtxMII or cytisine, revealed the absence of alphaCtxMII-sensitive and cytisine-resistant [H-3] epibatidine binding sites in alpha6-/- mice, although the total amount of binding was unchanged. Because alphaCtxMII, a toxin formerly thought to be specific for alpha3beta2-containing nAChRs, is known to partially inhibit nicotine-induced dopamine release, these results support the conclusion that alpha6 rather than alpha3 is the partner of beta2 in the nicotinic modulation of DA neurons. They further show that alpha6-/- mice might be useful tools to understand the mechanisms of nicotine addiction, although some developmental compensation might occur in these mice.

2002 - Ghrelin expression in gut endocrine growths [Articolo su rivista]
Rindi, G.; Savio, A.; Torsello, A.; Zoli, Michele; Locatelli, V.; Cocchi, D.; Paolotti, D.; Solcia, E.
abstract

We aimed to assess the occurrence of ghrelin, a new gut hormone, in endocrine growths of the stomach. In addition, since ghrelin has been detected in other gut derivatives during adult and/or fetal life, we also studied endocrine tumours of the pancreas, intestine and lung. A specific serum generated against amino acids 13-28 of ghrelin was tested on 16 specimens of gastric mucosa with endocrine cell hyperplasia and on 75 endocrine tumours. Ghrelin-immunoreactive cells were moderately represented in normal, atrophic or hypertrophic gastric mucosa, as a rule with no obvious hyperplastic changes even in the presence of concurrent, prominent enterochromaffin-like cell hyperplasia associated with hypergastrinemia. Ghrelin cells were also found in tumour cell fractions of well-differentiated gastric (25 of 33, 76%), pancreatic (6 of 15, 40%) and pulmonary (4 of 8) endocrine tumours. No ghrelin immunoreactivity was detected in 14 intestinal tumours and in five poorly differentiated endocrine carcinomas of the stomach or pancreas. We conclude that ghrelin cells may take part in gut endocrine growths, with special reference to well-differentiated endocrine tumours of the stomach, independently from associated signs of endocrine hyperfunction.

2002 - Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum [Articolo su rivista]
Zoli, Michele; M., Moretti; Zanardi, Alessio; Jm, Mcintosh; F., Clementi; C., Gotti
abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) expressed on mesostriatal dopaminergic neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Using immunoprecipitation and ligand-binding techniques, we have shown that both alpha6beta2* and alpha4(nonalpha6)beta2* nAChRs are expressed in the caudate-putamen and that only alpha6* nAChRs can bind alpha-conotoxin MII and methyllycaconitine with affinities of 1.3 and 40 nM, respectively. Further studies performed on 6-hydroxydopamine-lesioned striatum led to the identification of nAChR subtypes selectively expressed on dopaminergic terminals [alpha4alpha5beta2, alpha4alpha6beta2(beta3), and alpha6beta2(beta3)], nondopaminergic neuronal structures (alpha2alpha4beta2), or both structures (alpha4beta2). The identification of the nAChRs expressed on striatal dopaminergic terminals opens up the possibility of developing selective nAChR ligands active on dopaminergic systems and associated diseases, such as Parkinson's disease.

2002 - Nicotine and neurodegeneration in ageing [Articolo su rivista]
Zanardi, Alessio; Leo, Giuseppina; Biagini, Giuseppe; Zoli, Michele
abstract

Impairment in cholinergic systems is a highly consistent finding in human dementia. Among cholinergic markers, marked decreases in nicotine binding have been most consistently observed in the telencephalic regions of demented patients and are thought to contribute to the cognitive deficits associated with ageing and age-related neurodegenerative diseases. New evidence that the cholinergic system has a specific pathogenic role in the neurodegenerative alterations of aged and, especially, demented patients is fast accumulating. Both in vivo and in culture, nicotine protects striatal, hippocampal and cortical neurons against the neurotoxicity induced by excitotoxic amino acids as well as the toxicity caused by beta-amyloid, the major component of senile plaques. Further support for the implication of nicotinic receptors in brain ageing is come from recent studies on transgenic animals lacking nicotinic receptor subtypes, which shed light on the mechanisms of nicotine neuroprotection and neurotoxicity. (C) 2002 Published by Elsevier Science Ireland Ltd.

2002 - Nicotinic receptors in aging and dementia [Articolo su rivista]
Mr, Picciotto; Zoli, Michele
abstract

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been shown to maintain cognitive function following aging or the development of dementia. Nicotine and nicotinic agonists have been shown to improve cognitive function in aged or impaired subjects. Smoking has also been shown in some epidemiological studies to be protective against the development of neurodegenerative diseases. This is supported by animal studies that have shown nicotine to be neuroprotective both in vivo and in vitro. Treatment with nicotinic agonists may therefore be useful in both slowing the progression of neurodegenerative illnesses, and improving function in patients with the disease. While increased nicotinic function has been shown to be beneficial, loss of cholinergic markers is often seen in patients with dementia, suggesting that decreased cholinergic function could contribute to both the cognitive deficits, and perhaps the neuronal degeneration, associated with dementia. In this article we will review the literature on each of these areas. We will also present hypotheses that might address the mechanisms underlying the ability of nAChR function to protect against neurodegeneration or improve cognition, two potentially distinct actions of nicotine.

2002 - Pharmacological manipulation of brain galaninergic system and sexual behavior in male mice [Articolo su rivista]
Benelli, Augusta; Bertolini, Alfio; Zoli, Michele; Leo, Giuseppina; Filaferro, Monica; S., Saltini; Genedani, Susanna
abstract

Available data suggest a complex role for the brain galaninergic system in male sexual behavior; however, the results so far obtained in animals with either galanin or galanin antagonists are conflicting. Objective: To define the better influence of galanin on male sexual behavior by studying, in mice, (i) the effect of galanin and of the chimeric galanin peptide M40 on the copulatory performance, and (ii) galanin mRNA levels in hypothalamic arcuate and dorso-medial nuclei. Methods: For the behavioral testing, only sexually sluggish male mice were used. Galanin mRNA levels were studied in both sexually potent and impotent mice by means of in situ hybridization. Standard behavioral parameters for sexual behavior were recorded or calculated. Synthetic galanin (0.05, 0.1 or 1 mug/mouse) and M40 (5 or 20 mug/mouse) were intracerebroventricularly (ICV) injected, 15 min before the copulatory test. Galanin mRNA levels were evaluated. Results: In sexually sluggish male mice, both galanin (0.1 and 1 mug/mouse ICV) and M40 (20 mug/mouse ICV), significantly increased intromission frequency and ejaculation latency; M40 also improved copulatory efficacy. On the other hand, in the hypothalamic arcuate and dorso-medial nuclei, the levels of galanin mRNA were not significantly different in sexually potent and impotent male mice. Conclusions.-These results show that in sexually sluggish male mice the ICV injection of either galanin or the chimeric analogue M40 greatly prolongs the duration of the copulation; without a reduction of the sexual drive or of the copulatory performance. On the other hand, the hybridization experiments seem to rule out an important physiological role of the brain galaninergic system in the regulation of male sexual behavior, at least in mice.

2002 - Preferential alterations in the mesolimbic dopamine pathway of heterozygous reeler mice: an emerging animal-based model of schizophrenia [Articolo su rivista]
M., Ballmaier; Zoli, Michele; Leo, Giuseppina; Agnati, Luigi Francesco; P., Spano
abstract

Based on a number of neuroanatomical and behavioural similarities, recent evidence suggests that heterozygous reeler mice, haploinsufficient for reelin expression, represent a useful model of psychosis vulnerability. As brain mesolimbic dopamine pathways have been proposed to be associated with the pathophysiology of psychotic disorders, we thought it would be of interest to examine whether these animals present disturbances in the mesolimbic dopamine system. To this end we studied by immunocytochemical, in situ hybridization procedures and receptor autoradiography, several markers of the mesotelencephalic dopamine pathway in heterozygous reeler mice and controls. We report that heterozygous reeler mice exhibit a reduction in the number of tyrosine hydroxylase-immunoreactive cell bodies and tyrosine hydroxylase mRNA levels in the ventral tegmental area, as well as a reduction of tyrosine hydroxylase and dopamine transporter immunoreactivity in the dopamine terminal fields of the limbic striatum. In these areas we also observed a reduction of dopamine D2 receptor mRNA. Finally, a marked increase in D3 receptor mRNA levels was observed concomitant with a significant increase in D3 binding sites. On the contrary, the nigrostriatal pathway did not show any significant alteration in heterozygous reeler mice with regards to the dopaminergic markers examined in substantia nigra cell bodies and dorsal striatum dopamine terminal fields. These results suggest a specific link between reelin-related neuronal pathology and dopamine involvement in the pathophysiology of psychotic disorders.

2002 - Rivastigmine antagonizes deficits in prepulse inhibition induced by selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis [Articolo su rivista]
M., Ballmaier; F., Casamenti; C., Scali; R., Mazzoncini; Zoli, Michele; G., Pepeu; Pf, Spano
abstract

Impairments of cortical cholinergic inputs from the nucleus basalis magnocellularis fundamentally alter information processing and attentional function, thereby advancing the severity of psychopathology in major neuropsychiatric disorders. It was previously shown in adult rats that bilateral 192 IgG saporin-induced selective immunolesioning of the cholinergic neurons in the nucleus basalis produces pronounced and long-lasting deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex. This behavioral paradigm is considered a valid model of sensorimotor gating deficits in the psychotic spectrum and efforts to analyze the significance of the cholinergic basal forebrain in this context are of great interest. In the present study the predictive value of the selective cholinergic immunolesioning model was tested by examining the ability of the cholinesterase inhibitor rivastigmine to restore prepulse inhibition in immunolesioned rats. We report here a pronounced restoring effect of acute (0.75 or 1.5 mg/kg s.c.) as well as repeated (0.75 mg/kg s.c. b.i.d., for 10 days) treatment with rivastigmine in this model of disrupted prepulse inhibition. Intra-nucleus basalis magnocellularis infusions of 192 IgG saporin resulted in extensive loss of basal-cortical cholinergic neurons as shown by the marked decrease in basal telencephalic choline acetyltransferase immunopositive neurons and cortical choline acetyltransferase activity. In this condition, rivastigmine was found to significantly increase cortical acetylcholine extracellular levels in lesioned animals measured by in vivo microdialysis. Taken together, our results strengthen the proposal that the nucleus basalis represents a critical station of the startle gating circuitry. In addition, our findings strongly indicate that even after dramatic decrease of cholinergic neurons, inhibition of acetylcholinesterase restores the cholinergic synaptic function to a point approaching normalization of experimentally induced psychopathology.

2002 - Selective activation of central subtypes of the nicotinic acetylcholine receptor has opposite effects on neonatal excitotoxic brain injuries [Articolo su rivista]
Laudenbach, V.; Medja, F.; Zoli, Michele; Rossi, F. M.; Evrard, P.; Changeux, J. P.; Gressens, P.
abstract

The incidence of neurological disabilities ascribable to perinatal injury is rising in Western countries, raising ethical and financial problems. No curative treatments are available. The pathophysiology of brain lesions of hypoxic-ischemic or inflammatory origin involves various neurotransmitters or neuromodulators. Among these, glutamate plays a key role. By overactivating N-methyl-D-aspartate receptors, it triggers the excitotoxic cascade. Although addictive, nicotine prevents excitotoxic neuronal death in adult animals. Its potential neuroprotective effects have not been evaluated in neonates. We found that nicotine is neuroprotective in vivo, in a murine model of neonatal excitotoxic brain injury, and in vitro, in primary cultures of cortical neurons. We investigated the respective roles in nicotine-related neuroprotection of the two dominant nicotinic acetylcholine receptor (nAChR) isoforms, namely, alpha4beta2 (heteropentameric) and alpha7 (homopentameric). Inhibition of alpha4beta2, either pharmacological (i.e., an alpha4beta2 nAChR antagonist) or molecular (beta2-/- knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of alpha4beta2 nAChR in neonatal nicotine-related neuroprotection. In contrast, activation of alpha7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (alpha7-/- knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.

2001 - Adenosine A2A agonist CGS 21680 decreases the affinity of dopamine D2 receptors for dopamine in human striatum [Articolo su rivista]
Diaz Cabiale, Z; Hurd, Y; Guidolin, D; Finnman, Ub; Zoli, Michele; Agnati, Luigi Francesco; Vanderhaeghen, Jj; Fuxe, K; Ferre, S.
abstract

Adenosine A2A receptors (A2AR) and dopamine D2 receptors (D2R) are highly concentrated in the striatum, where they are co-localized and exert reciprocal antagonistic interactions. It has been suggested that the A2R/D2R interactions might provide a therapeutic approach for basal ganglia disorders, such as Parkinson´s disease, and schizophrenia. In the present work evidence is presented for the existence of an A2AR/D2R interaction in human brain by using quantitative autoradiography. The areas analyzed were the dorsal caudate nucleus and putamen. Parallel studies were performed in rat striatal sections. The A2AR agonist CGS 21680 was found to significantly increase IC50 values of competitive inhibition curves of the D2R/D3R antagonist [I-125]iodosulpiride vs dopamine both in rat striatal and human striatal brain sections.

2001 - Combined alpha(2)-adrenergic/D-2 dopamine receptor blockade fails to reproduce the ability of clozapine to reverse phencyclidine-induced deficits in prepulse inhibition of startle [Articolo su rivista]
M., Ballmaier; Zoli, Michele; R., Mazzoncini; M., Gennarelli; P. F., Spano
abstract

Rationale: The combination of idazoxan, a specific alpha(2)-adrenoceptor antagonist with raclopride, a selective D-2/D-3 receptor antagonist, has been recently proposed to produce an atypical antipsychotic profile comparable to that of clozapine, based on an animal study which analysed dopamine efflux in the medial prefrontal cortex and the preclinical test of conditioned avoidance response (CAR) for evaluation of antipsychotic potential. Accordingly, the combination of a typical antipsychotic with idazoxan has been proposed as an augmentation strategy in treatment-resistant schizophrenia, although its therapeutic potential remains difficult to predict. Objectives: Given the momentum stimulated by these reports, the present study investigated whether the combination of idazoxan with raclopride is indeed sufficient to mimic the ability of clozapine to reverse prepulse inhibition (PPI) deficits in rats, a behavioral paradigm that models PPI deficits observed in the schizophrenia spectrum, and currently the only test which reliably appears to distinguish between typical antipsychotics and compounds with atypical antipsychotic potential. Methods: The effects of the combination idazoxan/raclopride were examined in two PPI paradigms: 1) phencyclidine (PCP)-induced disruption of PPI, which has been shown to be preferentially reversed by atypical antipsychotics; 2) apomorphine-induced disruption of PPI which can be reversed by either typical high-potency D-2 dopamine antagonists or atypical antipsychotics. Results: In contrast to clozapine, combining idazoxan with raclopride failed to reverse PCP-induced deficits in PPI In addition, there was no evidence of an enhancing effect of idazoxan on the blockade of apomorphine-induced disruption of PPI by raclopride. Conclusion: The present results challenge the hypothesis that simple alpha(2)/D-2 blockade is sufficient to produce clozapine-like atypical antipsychotic activities, and support the consensus that the PPI paradigm represents the most sophisticated behavioral preclinical test for detecting selective atypical profile of antipsychotics.

2001 - Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors [Articolo su rivista]
Dassesse, D; Massie, A; Ferrari, Rosaria; Ledent, C; Parmentier, M; Arckens, L; Zoli, Michele; Schiffmann, Sn
abstract

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R-/-). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus, Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D-1 and D-2 dopamine receptors expression but not with changes in ionotropic glutamate receptors, The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R-/- and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.

2001 - Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei [Articolo su rivista]
R., Klink; Ad, D'Exaerde; Zoli, Michele; Jp, Changeux
abstract

Nicotinic acetylcholine receptors (nAChRs) on dopaminergic (DA) and GABAergic (Gaba) projection neurons of the substantia nigra (SN) and ventral tegmental area (VTA) are characterized by single-cell RT-PCR and patch-clamp recordings in slices of rat and wild-type, beta2-/-, alpha4-/-, and alpha7-/- mice. The eight nAChR subunits expressed in these nuclei, alpha3-7 and beta2-4, contribute to four different types of nAChR-mediated currents. Most DA neurons in the SN and VTA express two nAChR subtypes. One is inhibited by dihydro-beta -erythroidine (2 muM), alpha -conotoxin MII (10 nM), and methyllycaconitine (1 nM) but does not contain the alpha7 subunit; it possesses a putative alpha4 alpha6 alpha5(beta2)(2) composition. The other subtype is inhibited by dihydro-beta -erythroidine (2 muM) and has a putative alpha4 alpha5(beta2)(2) composition. Gaba neurons in the VTA exhibit a third subtype with a putative (alpha4)(2)(beta2)(3) composition, whereas Gaba neurons in the SN have either the putative (alpha4)(2)(beta2)(3) oligomer or the putative alpha4 alpha6 alpha5(beta2)(2) oligomer. The fourth subtype, a putative (alpha7)(5) homomer, is encountered in less than half of DA and Gaba neurons, in the SN as well as in the VTA. Neurons in the DA nuclei thus exhibit a diversity of nAChRs that might differentially modulate reinforcement and motor behavior.

2001 - Selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis impairs prepulse inhibition of acoustic startle [Articolo su rivista]
M., Ballmaier; F., Casamenti; Zoli, Michele; G., Pepeu; P., Spano
abstract

Information processing and attentional abnormalities are prominent in neuro psychiatric disorders. Since the cholinergic neurons located in the nucleus basalis magnocellularis have been shown to be involved in attentional performance and information processing, recent efforts to analyze the significance of the basal forebrain in the context of schizophrenia have focused on this nucleus and its projections to the cerebral cortex. We report here that bilateral selective immunolesioning of the cholinergic neurons in the nucleus basalis magnocellularis is followed by significant deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex in adult rats. This behavioral approach is used in both humans and rodents and has been proposed as a valuable model contributing to the understanding of the neurobiological substrates of schizophrenia. The disruption of prepulse inhibition persisted over repeated testing. The selective lesions were induced by bilateral intraparenchymal infusions of 192 IgG saporin at a concentration having minimal diffusion into adjacent nuclei of the basal forebrain. The infusions were followed by extensive loss of choline acetyltransferase-immunopositive neurons. Our results show that the cholinergic neurons of the nucleus basalis magnocellularis represent a critical station of the startle gating circuitry and suggest that dysfunction of these neurons may result in impaired sensorimotor gating characteristic of schizophrenia. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

2000 - Degenerazione e rigenerazione nel sistema nervoso centrale: rilevanza dei fattori trofici e della trasmissione neuronale colinergica nel trofismo del sistema nervoso centrale. [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; Zanardi, Alessio; Zini, Isabella; Agnati, Luigi Francesco
abstract

Contrary to a diffuse belief lasting until the end of the past century, recent data demonstrate that even the mammalian central nervous system possesses regenerative capabilities. The recovery that follows neuronal lesions could be morphological and/or functional. In both cases, the recovery is dependent on chemical signals (i.e., trophic factors and neurotransmitters) that influence neuronal cell survival and capability of producing structural changes. The synthesis and release of these chemical signals could be modulated by drugs acting on glial and neuronal systems. We showed that it is possible to maintain, at least in part, the anatomo-functional integrity of damaged brain areas by modulating polyamine synthesis. These molecules are synthesized in neurons belonging to damaged areas and can stimulate astroglial reactivity upon their release, leading to astrocyte hypertrophy and hyperplasia and neurotrophic factor synthesis. Trophic factor synthesis can be modulated also by specific drugs acting on glial and/or neuronal metabolism. We found that selegilin is able to induce basic fibroblast growth factor (bFGF), and that certain antiepileptic drugs can induce brain-derived neurotrophic factor (BDNF) by acting, respectively, on glial and neuronal cells.

2000 - Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes [Articolo su rivista]
S., Ginés; J., Hillion; M., Torvinen; S., Le Crom; V., Casadó; Ei, Canela; S., Rondin; Jy, Lew; S., Watson; Zoli, Michele; Agnati, Luigi Francesco; P., Vernier; C., Lluis; S., Ferré; K., Fuxe; R., Franco
abstract

The possible molecular basis for the previously described antagonistic interactions between adenosine A1 receptors (A1R) and dopamine D1 receptors (D1R) in the brain have been studied in mouse fibroblast Ltk cells cotransfected with human A1R and D1R cDNAs or with human A1R and dopamine D2 receptor (long-form) (D2R) cDNAs and in cortical neurons in culture. A1R and D1R, but not A1R and D2R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A1R/D1R heteromerization disappeared after pretreatment with the D1R agonist, but not after combined pretreatment with D1R and A1R agonists. A high degree of A1R and D1R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A1R and D2R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A1R agonist caused coclustering (coaggregation) of A1R and D1R, which was blocked by combined pretreatment with the D1R and A1R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D1R and A1R agonists, but not with either one alone, substantially reduced the D1R agonist-induced accumulation of cAMP. The A1R/D1R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A1R of D1R receptor signaling in the brain. The persistence of A1R/D1R heteromerization seems to be essential for the blockade of A1R agonist-induced A1R/D1R coclustering and for the desensitization of the D1R agonist-induced cAMP accumulation seen on combined pretreatment with D1R and A1R agonists, which indicates a potential role of A1R/D1R heteromers also in desensitization mechanisms and receptor trafficking.

2000 - Evidence for adenosine/dopamine receptor interactions: Indications for heteromerization [Articolo su rivista]
Franco, R.; Ferré, S.; Agnati, Luigi Francesco; Torvinen, M.; Gines, S.; Hillion, J.; Casado, V.; Lledo, P. M.; Zoli, Michele; Lluis, C.; Fuxe, K.
abstract

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

2000 - Evidence for the existence of pulses of dopamine in the extracellular space of the rat striatum [Relazione in Atti di Convegno]
Agnati, Luigi Francesco; Zoli, Michele; Ferrari, Renata; L., Di Paola; C., Torri; K., Fuxe; Zini, Isabella
abstract

2000 - Localization of nAChR subunit mRNAs in the brain of Macaca mulatta. [Articolo su rivista]
Han, Z. Y.; LE NOVERE, N.; Zoli, Michele; Hill, J. A.; Champtiaux, N.; Changeux, J. P.
abstract

We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both alpha4 and beta2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both alpha4 and beta2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of alpha4 signal. alpha6 and beta3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for alpha3 or beta4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific alpha3 or beta4 signals in mesencephalic dopaminergic nuclei. alpha5 and alpha7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than alpha4 and beta2. The distribution of alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, alpha2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of alpha4 and beta2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides alpha4beta2*, alpha2beta2* nAChRs constitute a main nAChR isoform in primate brain.

2000 - Protective effects of delapril combined with indapamide or hydrochlorothiazide in spontaneously hypertensive stroke-prone rats: a comparative dose-response analysis [Articolo su rivista]
S., Boschi; G., Vantaggiato; C., Torri; Zini, Isabella; Agnati, Luigi Francesco; Zoli, Michele; Biagini, Giuseppe
abstract

In previous articles, we have shown that the combination of the angiotensin-converting enzyme (ACE) inhibitor delapril (12 mg/kg/day) and the diuretic indapamide (1 mg/kg/day) was able to prolong the life span significantly in salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Because this finding was partly dependent on the antagonism of salt-loading effects by pharmacologic induction of diuresis, which prevented any increase in blood pressure values, we decided to evaluate whether lower doses of the combination could be equally protective without changing the progression of hypertension. Thus, we studied several treatments with progressively lower doses of delapril (6, 3, or 1.5 mg/kg/day) combined with indapamide (0.5, 0.25, or 0.125 mg/kg/day) or hydrochlorothiazide (2.5, 1.25, or 0.625 mg/kg/day) in salt-loaded SHRsp. Salt-loaded untreated animals were considered to be the control group. In agreement with previous experiments, control rats reached 50% mortality similar to 7 weeks after the beginning of salt loading. The combination of delapril and hydrochlorothiazide at the two lowest doses was nor able to delay animal death significantly, whereas treatment with april and indapamide at the lowest dose was effective (50% survival rate, 15 weeks). The groups treated with the highest dose of delapril and hydrochlorothiazide or with the intermediate or highest dose of delapril and indapamide did not reach 50% mortality by the end of the experiment, at 44 weeks of treatment (i.e., when animals reached age 1 year). Only the highest delapril and indapamide doses were able to increase diuresis, but for a relatively short period. None of the treatments was able to lower or control blood pressure levels adequately. Therefore, blood pressure levels by themselves were not predictive of rat mortality. In contrast, the maximal value of proteinuria in the weeks preceding death was inversely correlated with the survival time. In conclusion, this study shows that low doses of an ACE inhibitor in combination with a diuretic can be effectively protective in a model of severe hypertension, independent of any change in blood pressure levels.

1999 - Changes in nicotinic acetylcholine receptor subunit mRNAs and nicotinic binding in spontaneously hypertensive stroke prone rats [Articolo su rivista]
R., Ferrari; A., Frasoldati; Leo, Giuseppina; C., Torri; Zini, Isabella; Agnati, Luigi Francesco; Zoli, Michele
abstract

We have studied nicotinic acetylcholine receptors in spontaneously hypertensive 'stroke prone' (SHsp) rats. We found a significant decrease in I-125-alpha-bungarotoxin binding and alpha 7 subunit mRNA levels in cortical areas of the SHsp rats with respect to Wistar Kyoto (WKy) normotensive rats. Antihypertensive drug treatment counteracted these changes in cerebral cortex but not in hippocampus. No significant change was instead found in [H-3]-epibatidine binding and alpha 4 and beta 2 subunit mRNA levels. SHsp rats showed decreased latency at the active avoidance test and transiently increased threshold at both hot-plate and tail-flick tests in comparison with WKy rats. None of these behavioral parameters was correlated with I-125-alpha-bungarotoxin binding in cortical areas. In conclusion, present data show a preferential impairment of alpha-bungarotoxin sensitive nAChRs in SHsp rats. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

1999 - Effects of nitric oxide inhibition on the spread of biotinylated dextran and on extracellular space parameters in the neostriatum of the male rat [Articolo su rivista]
Jansson, A; Mazel, T; Andbjer, B; Rosen, L; Guidolin, D; Zoli, Michele; Sykova, E; Agnati, Luigi Francesco; Fuxe, K.
abstract

Volume transmission in the brain is mediated by the diffusion of neurotransmitters, modulators and other neuroactive substances in the extracellular space. The effects of nitric oxide synthase inhibition on extracellular space diffusion properties were studied using two different approaches, the histological dextran method and the real-time iontophoretic tetramethylammonium method. The spread of biotinylated dextran (mol, wt 3000) in the extracellular space was measured morphometrically following microinjection into the neostriatum of male rats. Two parameters were used to describe the spread of biotinylated dextran in brain tissue, namely, total volume of spread and the mean grey value. The nonspecific nitric oxide synthase inhibitors N-G-nitro-L-aginine methyl ester (10-100 mg/kg) and N-G-monomethyl-L-arginine acetate (30-200 mg/kg) decreased the total volume of spread of dextran in a dose dependent manner. 7-Nitroindazole monosodium salt (50-100 mg/kg), a specific neuronal nitric oxide synthase inhibitor, did not change the total volume of spread of dextran. Using the tetramethylammonium method, the extracellular space diffusion properties can be described by the volume fraction (alpha = extra-cellular space volume/total tissue volume), tortuosity lambda (lambda(2) = free diffusion coefficient/apparent diffusion coefficient in tissue), and non-specific uptake k´ [Nicholson C. and Sykova E. (1998) Trends Neurosci. 21, 207-215]. Nitric oxide synthase inhibition by N-G-nitro-L-arginine methyl ester (50 mg/kg) had relatively little effect on volume fraction and tortuosity, and no changes were observed after N-G-monomethyl-L-arginine acetate (20 mg/kg) or 7-nitroindazole monosodium salt (100 mg/kg) treatment. A substantial increase was found only in non-specific uptake, by 13% after N-G-nitro-L-arginine methyl ester and by 16% after N-G-monomethyl-L-arginine acetate, which correlates with the decreased total volume of spread of dextran observed with the dextran method. N-G-Nitro-L-arginine methyl ester treatment (100 mg/kg) decreased striatal blood flow and increased mean arterial blood pressure. The changes in dextran spread and non-specific uptake can be explained by an increased capillary clearance following the inhibition of endothelial nitric oxide synthase, as neuronal nitric oxide synthase inhibition had no effect. The observed changes after non-specific nitric oxide synthase inhibition may affect the extracellular space concentration of neurotransmitters and modulators, and influence volume transmission pathways in the central nervous system by increased capillary and/or cellular clearance rather than by changes in extracellular space diffusion.

1999 - Increased neurodegeneration during ageing in mice lacking high-affinity nicotine receptors [Articolo su rivista]
Zoli, Michele; Mr, Picciotto; Ferrari, Renata; D., Cocchi; Jp, Changeux
abstract

We have examined neuroanatomical, biochemical and endocrine parameters and spatial learning in mice lacking the beta 2 subunit of the nicotinic acetylcholine receptor (nAChR) during ageing. Aged beta 2(-/-) mutant mice showed region-specific alterations in cortical regions, including neocortical hypotrophy, loss of hippocampal pyramidal neurons, astro- and microgliosis and elevation of serum corticosterone levels. Whereas adult mutant and control animals performed well in the Morris maze, 22- to 24-month-old beta 2(-/-) mice were significantly impaired in spatial learning. These data show that beta 2 subunit-containing nAChRs can contribute to both neuronal survival and maintenance of cognitive performance during ageing. beta 2(-/-) mice may thus serve as one possible animal model for some of the cognitive deficits and degenerative processes which take place during physiological ageing and in Alzheimer's disease, particularly those associated with dysfunction of the cholinergic system.

1999 - Involvement of alpha 6 nicotinic receptor subunit in nicotine-elicited locomotion, demonstrated by in vivo antisense oligonucleotide infusion [Articolo su rivista]
Le Novere, N; Zoli, Michele; Lena, C; Ferrari, Renata; Picciotto, Mr; Merlo Pich, E; Changeux, Jp
abstract

ENHANCED locomotion in a habituated environment is a well documented effect of nicotine mediated by the mesotelencephalic dopaminergic system. The nicotinic receptor subunit alpha 6 is, among other subunits, strongly expressed in the dopaminergic neurons of the mesencephalon. To examine the functional role of this subunit, we inhibited its expression in vivo using antisense oligonucleotides. lit vitro treatments of embryonic mesencephalic neuron cultures demonstrated that the alpha 6 antisense oligonucleotides caused a marked decrease in the level of alpha 6 subunit protein. In vivo, 1 week infusion of alpha 6 antisense oligonucleotides by osmotic mini-pump reduced the effect of nicotine on locomotor activity in habituated environment by 70%. These data support the notion that the effects of nicotine on the dopaminergic system involve alpha 6 subunit containing nAChRs.

1999 - On the distribution patterns of D-1, D-2, tyrosine hydroxylase and dopamine transporter immunoreactivities in the ventral striatum of the rat [Articolo su rivista]
Jansson, A; Goldstein, M; Tinner, B; Zoli, Michele; Meador Woodruff, Jh; Lew, Jy; Levey, Ai; Watson, S; Agnati, Luigi Francesco; Fuxe, K.
abstract

The distribution of dopamine D-1, and D-2 receptor immunoreactivities in the nucleus accumbens and the olfactory tubercle of adult and postnatal male rats were compared with the distribution of tyrosine hydroxylase and dopamine transporter immunoreactivities. An overall co-distribution of D-1 and D-2 receptor immunoreactivities with tyrosine hydroxylase immunoreactivity was found in the nucleus accumbens and the olfactory tubercle. However, the major finding in this study was, following a more detailed analysis in coronal sections of the shell part of the nucleus accumbens, the existence of nerve cell patches of strong D-1 receptor immunoreactivity associated with low D-2 receptor, dopamine transporter and tyrosine hydroxylase immunoreactivities. These patches were mainly surrounded by areas of strong D-2 receptor, tyrosine hydroxylase and dopamine transporter immunoreactivities and could be found also in the olfactory tubercle. Similar observations were made in postnatal rats. Serial reconstructions of the patches of strong D-1 receptor immunoreactivity in the rostrocaudal direction were made. The patches formed a continuous tubular nerve cell system in the shell part of the nucleus accumbens. Since this nerve cell system was found to be surrounded by a high density of dopamine terminals, it may represent a compartment where dopamine transmission mainly acts on D-1 receptors via local diffusion (i.e. via volume transmission). However, it must be noted that the D-1 receptor rich patches constitute only a small fraction of the nucleus accumbens and the overall density of tyrosine hydroxylase immunoreactive terminals correlates with the density of both D-1 and D-2 receptors in the nucleus accumbens. In conclusion, the present paper gives new aspects on the chemical microarchitecture of the nucleus accumbens.

1999 - Subunit and region-specific decreases in nicotinic acetylcholine receptor mRNA in the aged rat brain [Articolo su rivista]
Ferrari, Rosaria; Pedrazzi, Patrizia; Algeri, S; Zoli, Michele; L. F., Agnatia
abstract

We have investigated possible changes in the mRNA levels for several alpha and beta subunits of the nicotinic acetylcholine receptor (nAChR) and the level of binding for nicotinic ligands in 7- to 32-month-old rats. Alpha4 and beta2, and to a lesser extent alpha6 and beta3, mRNA levels showed decreases between 20 and 30% at 29 months of age which in some areas reached 50% at 32 months of age. Alpha7 showed a small increase from 7 to 14 months and then a progressive decrease from 14 to 32 months down to the 7-month levels. 3H-epibatidine binding did not significantly change from 7 to 32 months of age in rat tel- and diencephalon. Binding in the substantia nigra was exceptional in that it showed a significant decrease starting from 23 months of age. 125I-alpha-bungarotoxin binding showed a pattern of change which roughly paralleled that of alpha7 mRNA. These findings show that an alteration in some steps of nAChR biosynthesis takes place during aging, which may be related to functional changes in nicotinic transmission.

1999 - Use of knock-out mice to determine the molecular basis for the actions of nicotine [Articolo su rivista]
Picciotto, M. R.; Zoli, M.; Changeux, J. -P.
abstract

Recombinant DNA techniques have been used to identify the family of molecules that mediate nicotine's effects on the brain. Nicotine binds and activates nicotinic acetylcholine receptors (nAChRs) which are made up of combinations of individual nicotinic subunits. It is important to determine which of the many possible subunit combinations are responsible for the physiological and behavioral effects of nicotine that lead to addiction. Molecular genetic tools such as antisense strategies have been useful in elucidating the electrophysiological properties of nAChRs in different tissues. Use of knock-out mice lacking individual nAChR subunits has also begun to elucidate how nicotine exerts its actions from the molecular level to the behavioral level. Experiments using mice lacking the β2 subunit of the nAChR have shown that binding of nicotine to receptors containing this subunit is the first step in a pathway leading to increased dopamine levels in the mesolimbic dopamine system, and ultimately to the behavioral effects of nicotine in a test of nicotine reinforcement. Mice deficient in various α subunits of the nAChR will identify the partners of β2 mediating the addictive properties of nicotine. In addition, more data needs to be gathered on the electrophysiological properties of different subunit combinations, the effects of nicotine on different neurotransmitter systems and the links between the molecular biology of nicotine receptors, their physiology and the ultimate role of individual receptor subtypes in complex behaviors. Multidisciplinary approaches to nAChR function will be essential to answering these questions. © 1999 Society for Research on Nicotine and Tobacco.

1999 - Volume transmission in the CNS and its relevance for neuropsychopharmacology [Articolo su rivista]
Zoli, Michele; A., Jansson; E., Sykova; Agnati, Luigi Francesco; K., Fuxe
abstract

The terms 'wiring' and 'volume' transmission (WT and VT) have been introduced to provide a systematic categorization of intercellular communication in the brain. WT is one-to-one transmission and includes classical synapses, gap junctions and membrane juxtapositions, whereas VT is a one-to-many transmission and includes paracrine and endocrine-like transmissions in the brain extracellular space and cerebrospinal fluids. Any brain cell can participate in WT and VT and any kind of substance (e.g. ions, classical transmitters, peptides, neurosteroids) can be a signal in WT and VT. These concepts are relevant for the pharmacokinetics and actions of neuropsychoactive drugs. These drugs can be regarded as exogenous VT signals in that they diffuse in the cerebral extracellular space and are constrained there by the same factors that influence migration of endogenous VT signals. In addition, neuropsychoactive drugs can better mimic and more effectively interact with the relatively unconstrained VT-type transmissions than with the rigidly constrained WT mechanisms, such as synaptic transmission.

1998 - Acetylcholine receptors containing b2 subunits are involved in the reinforcing properties of nicotine. [Articolo su rivista]
Picciotto, M. R.; Zoli, Michele; Rimondini, R.; Lena, C.; Marubio, L.; MERLO PICH, E.; Fuxe, K.; Changeux, J. P.
abstract

Release of the neurotransmitter dopamine in the mesolimbic system of the brain mediates the reinforcing properties of several drugs of abuse, including nicotine. Here we investigate the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the beta2 subunit of this receptor. We found that nicotine stimulates dopamine release in the ventral striatum of wild-type mice but not in the ventral striatum of beta2-mutant mice. Using patch-clamp recording, we show that mesencephalic dopaminergic neurons from mice without the beta2 subunit no longer respond to nicotine, and that self-administration of nicotine is attenuated in these mutant mice. Our results strongly support the idea that the beta2-containing neuronal nicotinic acetylcholine receptor is involved in mediating the reinforcing properties of nicotine.

1998 - Brain nicotinic receptors: structure and regulation, role in learning and reinforcement. [Articolo su rivista]
Changeux, J. P.; Bertrand, D.; Corringer, P. J.; Dehaene, S.; Edelstein, S.; Lena, C.; LE NOVERE, N.; Marubio, L.; Picciotto, M.; Zoli, Michele
abstract

The introduction, in the late sixties, of the concepts and methods of molecular biology to the study of the nervous system had a profound impact on the field, primarily through the identification of its basic molecular components. These structures include, for example, the elementary units of the synapse: neurotransmitters, neuropeptides and their receptors, but also ionic channels, intracellular second messengers and the relevant enzymes, cell surface adhesion molecules, or growth and trophic factors [21,78,81, 52,79]. Attempts to establish appropriate causal relationships between these molecular components, the actual organisation of neural networks, and a defined behavior, nevertheless, still must overcome many difficulties. A first problem is the recognition of the minimum levels of organisation, from the molecular, cellular, or multicellular (circuit) to the higher cognitive levels, that determine the given physiological and/or behavioral performance under investigation. A common difficulty (and potential source of errors of interpretation) is to relate a cognitive function to a network organization which does not possess the required structural complexity and vice-versa. Another problem is to distinguish, among the components of the system, those which are actually necessary and those which, taken together, suffice for a given behavior to take place. Identification of such a minimal set of building blocks may receive decisive insights from the elaboration of neurally plausible formal models that bring together, within a single and coherent 'artificial organism', the neuronal network, the circulating activity, and the behavior they determine (see [42,43,45,72,30]). In this communication, we shall attempt, still in a preliminary fashion, to bring together: (1) our recent knowledge on the molecular biology of brain nicotinic receptors (nAChRs) and their allosteric properties and (2) integrated behaviors, such as cognitive learning, investigated for instance with delayed-response or passive avoidance tasks that are likely to involve nAChRs in particular at the level of reinforcement (or reward) mechanisms (see [18,29,135]).

1998 - Hypothalamic neuropeptide Y and galanin in overweight rats fed a cafeteria diet [Articolo su rivista]
Pedrazzi, Patrizia; L., Cattaneo; L., Valeriani; S., Boschi; D., Cocchi; Zoli, Michele
abstract

We evaluated neuropeptide Y (NPY) and galanin (GAL) immunoreactivity (IR) and mRNA in the paraventricular and arcuate nucleus, respectively, in rats that became overweight (Ov) or not (NOv) when fed a cafeteria diet. After 2 months of diet, NOv rats showed a significant increase in NPY IR, whereas Ov rats showed a significant increase in GAL mRNA levels. None of these changes was present in rats overfed for 6.5 months. These differential changes in hypothalamic GAL and NPY transmissions may contribute to the different susceptibility of the two rat subpopulations to the weight-promoting effects of the hypercaloric diet.

1998 - Identification of four classes of brain nicotinic receptors using beta 2 mutant mice [Articolo su rivista]
Zoli, Michele; C., Lena; Mr, Picciotto; Jp, Changeux
abstract

Although the expression patterns of the neuronal nicotinic acetylcholine receptor (nAChR) subunits thus far described are known, the subunit composition of functional receptors in different brain areas is an ongoing question. Mice lacking the beta 2 subunit of the nAChR were used for receptor autoradiography studies and patch-clamp recording in thin brain slices. Four distinct types of nAChRs were identified, expanding on an existing classification [Alkondon M, Albuquerque EX (1993) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J Pharmacol Exp Ther 265:1455-1473.], and tentatively identifying the subunit composition of nAChRs in different brain regions. Type 1 nAChRs bind alpha-bungarotoxin, are not altered in beta 2 -/- mice, and contain the alpha 7 subunit, Type 2 nAChRs contain the beta 2 subunit because they are absent in beta 2 -/- mice, bind all nicotinic agonists used with high affinity (excluding alpha-bungarotoxin), have an order of potency for nicotine >> cytisine in electrophysiological experiments, and are likely to be composed of alpha 4 beta 2 in most brain regions, with other alpha subunits contributing in specific areas. Type 3 nAChRs bind epibatidine with high affinity in equilibrium binding experiments and show that cytisine is as effective as nicotine in electrophysiological experiments; their distribution and persistence in beta 2 -/- mice strongly suggest a subunit composition of alpha 3 beta 4. Type 4 nAChRs bind cytisine and epibatidine with high affinity in equilibrium binding experiments and persist in beta 2 -/- mice; cytisine = nicotine in electrophysiological experiments. Type 4 nAChRs also exhibit faster desensitization than type 3 nAChRs at high doses of nicotine. Knock-out animals lacking individual alpha subunits should allow a further dissection of nAChR subclasses.

1998 - Integrated events in central dopamine transmission as analyzed at multiple levels. Evidence for intramembrane adenosine A(2A) dopamine D-2 and adenosine A(1) dopamine D-1 receptor interactions in the basal ganglia [Articolo su rivista]
Fuxe, K; Ferre, S; Zoli, Michele; Agnati, Luigi Francesco
abstract

An analysis at the network and membrane level has provided evidence that antagonistic interactions between adenosine A(2A)/dopamine D-2 and adenosine A(1)/dopamine D-1 receptors in the ventral and dorsal striatum are at least in part responsible for the motor stimulant effects of adenosine receptor antagonists like caffeine and for the motor depressant actions of adenosine receptor agonists. The results obtained in stably cotransfected cells also underline the hypothesis that the intramembrane A(2A)/D-2 and A(1)/D-1 receptor interactions represent functionally important mechanisms that may be the major mechanism for the demonstrated antagonistic A(2A)/D-2 and A(1)/D-1 receptor interactions found in vivo in behavioural studies and in studies on in vivo microdialysis of the striopallidal and strioentopeduncular GABAergic pathways. A major mechanism for the direct intramembrane A(2A)/D-2 and A(1)/D-1 receptor interactions may involve formation of A(2A)/D-2 and A(1)/D-1 heterodimers leading to allosteric changes that will alter the affinity as well as the G protein coupling and thus the efficacy to control the target proteins in the membranes. This is the first molecular network to cellular integration in the nerve cell membrane and may be well suited for a number of integrated tasks and can be performed in a short-time scale, in comparison with the very long-time scale observed when receptor heteroregulation involves phosphorylation or receptor resynthesis. Multiple receptor-receptor interactions within the membranes through formation of receptor clusters may lead to the storage of information within the membranes. Such molecular circuits can represent hidden layers within the membranes that substantially increase the computational potential of neuronal networks. These molecular circuits are biased and may therefore represent part of the molecular mechanism for the storage of memory traces (engrams) in the membranes. (C) 1998 Elsevier Science B.V. All rights reserved.

1998 - Promoter analysis of the neuronal nicotinic acetylcholine receptor a4 gene: methylation and expression of the transgene. [Articolo su rivista]
Watanabe, A.; Zoli, Michele; Changeux, J. P.
abstract

Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compose a family of genes. The major isoform of nAChR in the brain is made up of the alpha4 and beta2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nAChR alpha4 gene expression in mouse, its genomic DNA was cloned and characterized. The transcription initiation site was mapped by primer extension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5' flanking region of this gene did not have typical TATA box but GC-rich sequences were found around the initiation site. Methylation analysis of this region revealed that genomic DNAs from liver and muscle are partially methylated, whereas little methylation was observed in genomic DNA from brain. To characterize the cis-acting elements driving cell-specific expression of the alpha4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the alpha4 gene fused with bacterial lacZ as a reporter gene. An 11.5-kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the first intron was found to confer an expression pattern which coincides rather well with the endogenous gene expression pattern at early embryonic stages, suggesting that the elements necessary for the onset of alpha4 gene expression are located in this region. A DNA fragment containing the 1.8-kb upstream sequence and the first intron drove expression of lacZ in a limited subset of alpha4 expressing cells, whereas the 1.8-kb upstream sequence alone did not elicit any significant expression. These results show that both upstream and intronic sequences are important for cell-specific expression of the nAChR alpha4 gene.

1998 - The emergence of the volume transmission concept [Articolo su rivista]
Zoli, Michele; C., Torri; Ferrari, Renata; A., Jansson; Zini, Isabella; K., Fuxe; Agnati, Luigi Francesco
abstract

Interneuronal communication in the central nervous system (CNS) have always been of basic importance for theories on the cerebral morphofunctional architecture. Our group has proposed that intercellular communication in the brain can be grouped into 2 broad classes based on some general features of the transmission: wiring (WT) and volume (VT) transmission. WT occurs via a relatively constrained cellular chain (wire), while VT consists of 3-dimensional diffusion of signals in the extracellular fluid (ECF) for distances larger than the synaptic cleft. Both morphological and functional evidence indicates that dopamine (DA) synapses in striatum are 'open' synapses, i.e., synapses which favor diffusion of the transmitter into the surrounding ECF and observations are compatible with the view that DA varicosities can synthesize, store and release DA for VT. The DAergic mesostriatal transmission has, therefore, been examined by several groups to give experimental support to VT. Moreover, due to its minor structural requirements, VT may become prevalent under some pathological conditions, e, g. Parkinson's disease. In animal models of DAergic pathway degeneration, it has been shown that a compensatory activation of surviving DA terminals may lead to a preferential potentiation of VT. WT and VT favor different and complementary types of computation. VT is markedly slower and less safe than WT, but has minor spatial constraints and allows the reach of a large number of targets. Models of neuronal systems integrating classical neuronal circuits and diffusible signals begin to show how WT and VT may interact in the neural tissue.

1997 - Contribution of nicotinic acetylcholine receptors containing the beta 2-subunit to the behavioural effects of nicotine [Articolo su rivista]
Picciotto, Mr; Zoli, Michele; Zachariou, V; Changeux, Jp
abstract

1997 - Hypothalamo-pituitary-IGF-1 axis in female rats made obese by overfeeding [Articolo su rivista]
L., Cattaneo; V. D., Colonna; Zoli, Michele; E. E., Muller; D., Cocchi
abstract

A gender-related impairment of the somatotrophic axis is present in obese Zucker rats, female rats being better preserved than males. We showed that another animal model of obesity, male rats made obese by feeding a hypercaloric diet had a reduced function of somatotrophic axis which was likely related to impairment of gonadal function. Aim of this work was that of studying the function of somatotrophic axis in female overfed rats and comparing it to that of male rats of the previous study. Sprague-Dawley female rats were fed an energy-rich palatable diet for seven months. At the end of overfeeding, according to the degree of overweight, rats were divided into overtly obese (Obese), overweight (Overweight) and Non-Obese, i.e. rats whose weights were similar to those of controls. Rats fed ad libitum with the standard pellet chow served as controls (Controls) Acute administration of a supramaximal dose of GHRH (2 mu g/rat, iv) elicited a plasma GH rise similar to that of Controls in all the groups, except in Obese which had a lower GH response. Growth hormone responses after GHRH administration were inversely related to plasma levels of free fatty acids (FFA) Pituitary GH content and gene expression as well as hypothalamic GHRH and SS mRNA content, were similar in all experimental groups and in Controls and the same was true for plasma concentrations of free IGF-I These results indicate that, similarly to obese female Zucker rats, also overfed female rats had a better preservation of the somatotrophic axis than their male counterparts. In diet-induced obese rats, also the etiology of the impairment of somatotrophic axis seems to be gender-related i.e. due to a reduction of gonadal function in males and to an elevation of FFA in females.

1997 - Neuron-glia cross talk in rat striatum after transient forebrain ischemia [Capitolo/Saggio]
Zoli, M.; Biagini, G.; Ferrari, R.; Pedrazzi, P.; Agnati, L. F.
abstract

Striatum is highly vulnerable to transient forebrain ischemia induced by the 4 vessel occlusion (4V0) method (Brierley 1976. Pulsinelli et al. 1982, Zini et al. 1990a). Massive degeneration and loss of Nissl-stained neurons occur within 24 hr from an ischemia of long duration (30 min) (Pulsinelli et al. 1982). Neuronal loss is mainly restricted to the lateral part of caudate-putamen (Pulsinelli et al. 1982, Zini et al. 1990a). Cellular alterations include loss of medium-size spiny projection neurons (Pulsinelli et al. 1982, Francis and Pulsinelli 1982), largely corresponding to dopaminoceptive neurons (Benfenati et al. 1989, Zoli et al. 1989), and increase in reactive astrocytes (Pulsinelli et al. 1982, Grimaldi et al. 1990) and microglia (Gehrmann et al. 1982). On the other hand, large cholinergie (Francis and Pulsinelli 1982) and medium-size aspiny somatostatin (SS)/neuropeptide Y (NPY)-containing interneurons are resistant to the ischemic insult (Pulsinelli et al. 1982, Grimaldi et al. 1990). In a few instances, such as in the case of SS and NPY immunoreactivity (IR), the initial loss is followed by full recovery within 7 (SS) or 40 (NPY) days post-ischemia (Grimaldi et al. 1990). However, it is not known whether some kind of recovery is present for the bulk of medium-size spiny projections neurons after the first days post-ischemia.

1997 - Protective effects of delapril, indapamide and their combination chronically administered to stroke-prone spontaneously hypertensive rats fed a high-sodium diet [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; C., Torri; S., Boschi; G., Vantaggiato; M., Ballestri; A., Baraldi; Agnati, Luigi Francesco
abstract

1. Stroke-prone spontaneously hypertensive rats (SHRsp) have been used widely to test agents putatively capable of vascular protection. These animals present an accelerated time course of hypertension and a reduced life-span. When fed a high-sodium diet from the eighth week of life, a further acceleration in blood pressure increase is obtained, and rats start to die after 5 weeks of diet as a consequence of cerebral haemorrhage. In this model, angiotensin-converting enzyme (ACE) inhibitors were repeatedly proved to prevent vascular lesions and death. Notably, this effect was independent of any hypotensive effect. On the contrary, diuretics were shown not to be equally effective. A combination of ACE inhibitors and diuretics, although known to have synergistic effects in the therapy of hypertension, has never previously been tested. 2. Our aim was to study the effects of long-term treatment with the ACE inhibitor delapril (12 mg day(-1) kg(-1)), the thiazide-like diuretic indapamide (1 mg day(-1) kg(-1)), and their combination (12 and 1 mg day(-1) kg(-1) respectively), on the survival of SHRsp rats fed a high-sodium diet from the eighth week of life onwards. The effects of the treatments on blood pressure, body weight, food and fluid intake, diuresis, proteinuria and the appearance of lesion signs and death were assessed weekly When control rats reached 50% mortality, they were killed, together with some drug-treated rats, to compare lesions in brain and kidney. The other drug-treated rats continued treatments until 50% mortality was reached in two treatment groups. 3. All drug treatments were able to delay death significantly when compared with control rats, which reached 50% mortality after 6 weeks of salt loading. This event was preceded by a highly significant increase in proteinuria, diuresis and fluid intake that took place 3 weeks after the increase in blood pressure over the initial range. In delapril-or indapamide-treated SHRsp these changes were never seen, even when animals started to die. In the combination-treated group, a significant increase (P<0.01) in fluid intake and diuresis, but not proteinuria, was observed from the third week of treatment onwards. 4. Treatment with delapril or indapamide did not block the progressive increase in blood pressure as observed in control animals. However, the increase in blood pressure was markedly retarded with respect to control rats. At variance with this, in combination-treated animals blood pressure levels were maintained until the end of the experiment within the 99% confidence interval initially observed in control animals. 5. Infarctual and haemorrhagic cerebral lesions were observed in 38% of control rats; no lesions were noted in brains of age-matched rats receiving a drug treatment. Kidneys from control animals presented major degenerative lesions of glomeruli and arteries, characterized by fibrinoid necrosis. This condition was absent in drug-treated animals, which presented minor signs of ischaemic lesion. Heart hypertrophy when heart weight was expressed as a percentage of body weight, was similar in saline-, delapril-or indapamide-treated rats. At variance with this, in combination-treated animals the heart weight to body weight ratio was significantly (P<0.01) lower than in the other groups. 6. In conclusion, the diuretic indapamide showed similar protective effects as the ACE inhibitor delapril on acute vascular lesions and survival of SHRsp. Moreover, their combination synergized in preventing heart hypertrophy consequent to long-term hypertension. This result is probably related to the enhanced diuresis and the better control of blood pressure levels selectively found in combination-treated animals.

1997 - Short- and long-term changes in striatal neurons and astroglia after transient forebrain ischemia in rats [Articolo su rivista]
Zoli, Michele; Grimaldi, R.; Ferrari, Rosaria; Zini, Isabella; Agnati, Luigi Francesco
abstract

BACKGROUND AND PURPOSE:The striatum is one of the regions most sensitive to transient forebrain ischemia. After 30-minute ischemia, areas of massive neuronal degeneration are clearly detectable a few hours after the insult and attain their maximal extension 24 hours after the insult. However, for most cellular and neurochemical parameters it is not known whether some recovery occurs at later times. We examined certain cell populations in the caudate putamen at different times after transient ischemia.METHODS:Adult male Sprague-Dawley rats were subjected to 30-minute forebrain ischemia (four-vessel occlusion model). Six experimental groups were considered: control animals and ischemic animals killed 4 hours, 1 day, 7 days, 40 days, and 8 months after reperfusion. Three striatal cell populations were examined by means of immunocytochemistry coupled to computer-assisted image analysis: vulnerable medium spiny neurons, resistant aspiny neurons, and reactive astrocytes, labeled for their content of dopamine- and cAMP-regulated phosphoprotein mr32 (DARPP-32), somatostatin and neuropeptide Y, and glial fibrillary acidic protein, respectively.RESULTS:(1) The area containing DARPP-32 immunoreactive neurons was markedly decreased (15% to 20% of control caudate putamen area) at 1 day after reperfusion and partially recovered at the following times (40% to 50% at 7 days and 50% to 60% at 40 days and 8 months after reperfusion). (2) The appearance of reactive astrocytes was precocious (4 hours to 1 day after ischemia) in the medial caudate putamen, the region in which DARPP-32 recovered within 40 days after ischemia, and late (7 to 40 days after ischemia) in the lateral caudate putamen, where no DARPP-32 recovery was detected. (3) Neuropeptide Y/somatostatin-containing neurons resisted the ischemic insult and could be detected in areas devoid of DARPP-32 immunoreactive neurons as long as 8 months after reperfusion.CONCLUSIONS:The present results show a marked recovery of DARPP-32-positive neurons within 40 days after 30-minute forebrain ischemia in the medial, but not the lateral, caudate putamen. Medial caudate putamen also contains a high density of reactive astrocytes on the first day after ischemia, suggesting that astrocytic support has an important role in the spontaneous recovery of ischemic neurons.

1996 - Characterization of the hypothalamo-pituitary-IGF-I axis in rats made obese by overfeeding [Articolo su rivista]
L., Cattaneo; Vd, Colonna; Zoli, Michele; Ee, Muller; D., Cocchi
abstract

Obesity is coupled to several disturbances of the endocrine axes. It has previously been shown that genetically obese Zucker male rats have an impaired secretion of growth hormone (GH), probably originating from a primary reduction of hypothalamic GH-releasing hormone (GHRH) function and resulting in a decrease of GH gene expression and release. We sought to evaluate the somatotropic function in another model of experimental obesity. Normal male Sprague-Dawley rats were fed an energy-rich highly palatable diet for 7 months until they reached body weights overlapping those reported for obese rats. They were then evaluated for different of the hypothalamo-pituitary-somatomedin-C (IGF-I) axis. At the end of the overfeeding period, rats were divided into overtly obese (obese group) and overweight (overweight group) rats according to the degree of overweight and the Obesity Lee Index, while rats fed dd libitum with the standard pellet chow served as controls. Acute administration of a supramaximal dose of GHRH (2 mu g/rat i.v.) elicited a significantly (at least P < 0.05) lower plasma GH rise in the overweight and obese groups compared with the controls although no difference was seen in the pituitary GH content and gene expression and plasma concentrations of free IGF-I in the two experimental groups vs the controls. In addition, evaluation of hypothalamic GHRH and somatostatin mRNAs (slot-blot hybridization) did not show any significant differences between the three groups. Of the different metabolic indices investigated, plasma glucose and insulin concentrations were significantly (P < 0.01) higher in the obese than in the overweight and control groups. A sharp decrease in plasma testosterone levels, together with a reduction in testis weight, was seen in both groups of rats fed the palatable diet compared with the controls. These findings underline the 'peripheral' feature of the hyposomatotropinism of rats chronically fed an energy-rich diet, and may account for the reversibility of the GH impairment in many obese subjects once a normal body weight has been restored. Moreover, the peripherally-driven hyposomatotropinism of these rats is in sharp contrast with the hypothalamic-driven GH secretory impairment of the obese Zucker rats.

1996 - Computer-assisted mapping of basic fibroblast growth factor immunoreactive nerve cell populations in the rat brain [Articolo su rivista]
K., Fuxe; B., Tinner; Zoli, Michele; Rf, Pettersson; A., Baird; Biagini, Giuseppe; G., Chadi; Agnati, Luigi Francesco
abstract

We have performed a mapping of basic fibroblast growth factor (bFGF) immunoreactive (ir) glial and nerve cell populations in the male rat brain using a rabbit antibody raised against a synthetic peptide of bovine bFGF. Regional morphometric and microdensitometric analysis of the bFGF ir neuronal profiles in coronal brain sections was carried out by means of an automatic image analyser. The density and intensity of the bFGF ir glial profiles were subjectively evaluated. The bFGF immunoreactivity (IR) was detected within the cytoplasm of neurons, except within the pyramidal neurons of hippocampal CA2 region, the fasciola cinerea and the indusium griseum, where bFGF IR was present in the nucleus. In contrast, in glial cells bFGF IR was always found in the nucleus, Neuronal and glial IR was no longer observed after absorption of the bFGF antiserum with recombinant bFGF. Basic FGF IR was found in neuronal and glial cell populations throughout the brain as well as in the choroid plexus and in the ependymal cells lining the ventricles, Basic FGF ir nerve cells were found in all layers of both the neocortex and allocortex. Within the caudate putamen and the nucleus accumbens a low density of weak bFGF ir neuronal profiles was detected, The majority of the thalamic nuclei showed medium to high densities of moderate to strong bFGF ir neuronal profiles. All the hypothalamic nuclei, with the exception of the anterior and lateral hypothalamic area and of the ventral hypothalamic nucleus, contained a high density of bFGF ir profiles, The pens and the medulla oblongata were characterized by the presence of a large number of nuclei containing moderate to high densities of strong bFGF ir profiles. The Purkinje cell layer of the cerebellar cortex contained a high density of moderately bFGF ir profiles. A moderate density of strong bFGF ir nerve cell profiles was observed within all the laminae of the spinal cord, except within the II and III laminae where a high density of strongly ir profiles was found. Histogram analysis of total immunoreactivity showed that the distribution of bFGF ir profiles within the telencephalon and mesencephalon tend to be similar with regard to the central tendency and spread. Using Kendall's tau, a significant correlation between intensity and density values was obtained only in the diencephalon. The cytoplasmic bFGF IR found in distinct nerve cell populations all over the rat brain and spinal cord may represent forms of bFGF which can be released from the nerve cells via non-exocytotic mechanisms in view of the absence of an intracellular signal peptide in bFGF. The presence of nuclear bFGF IR within the glial cells all over the central nervous system (CNS) suggests an intracellular function of bFGF, such as the promotion of mitogenesis and/or participation in the transcriptional regulation of various genes.

1996 - Effects of single and short-term administration of clonidine on hypothalamic-pituitary somatotropic function of the adult male rat: An in situ hybridization study [Articolo su rivista]
Colonna, Vd; Zoli, Michele; Settembrini, Bp; Ciceri, S; Demarco, A; Cella, Sg; Agnati, Luigi Francesco; Muller, Ee
abstract

The effects of the alpha-2 adrenoceptor agonist clonidine (CLO) on the growth hormone (GH) regulatory neuronal systems, growth hormone-releasing hormone (GHRH) and somatostatin (SS), were studied in adult male rats given a single or a shortterm administration (1, 3 and 6 days) of the drug. Acute administration of CLO significantly decreased hypothalamic GHRH content [leaving unaltered GHRH messenger RNA (mRNA) levels] and increased plasma GH levels; hypothalamic SS content/ mRNA levels and pituitary GH content/mRNA levels remained unchanged. In 1- and 3-day CLO-treated rats, by contrast, decreased hypothalamic GHRH content was coupled with a significant reduction in GHRH mRNA levels. In these rats, pituitary GH content and mRNA levels were also significantly increased, whereas hypothalamic SS content and mRNA levels remained unaltered, In 6-day CLO-treated rats, hypothalamic GHRH content and mRNA levels were still significantly reduced, plasma GH levels were increased, but to a lesser extent than in 1- and 3-day CLO-treated rats, and pituitary GH content and mRNA reverted to control levels. Hypothalamic SS content and mRNA levels remained unaltered. These results indicate that 1)functional activation of alpha-2 adrenergic receptors by CLO increases GHRH release from the hypothalamus, 2) CLO, via GHRH, increases GH secretion and biosynthesis, which in turn feeds back in the hypothalamus to reduce GHRH biosynthesis, and 3) reduction of hypothalamic GH-stimulatory activity tones down the initial pituitary somatotropic hyperfunction. Unaltered hypothalamic SS content and mRNA levels in all CLO-treated rats suggests that the somatostatinergic system is less sensitive than the GHRH system to changes in circulating GH levels.

1996 - Neuronal nicotinic receptor alpha6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. [Articolo su rivista]
Le Novere, N.; Zoli, Michele; Changeux, J. P.
abstract

Although the neuronal nicotinic receptor alpha 6 subunit was cloned several years ago, its functional significance remains to be investigated. Here we describe an in situ hybridization study of the mRNA for this subunit in the adult rat central nervous system using oligonucleotide probes. Specific alpha 6 mRNA labelling was restricted to a few nuclei throughout the brain; it was particularly high in several catecholaminergic nuclei [the locus coeruleus (A6), the ventral tegmental area (A10) and the substantia nigra (A9)] at levels significantly higher than those found for any other known nicotinic receptor subunit mRNA. Labelling for alpha 6 mRNA was also detected at lower levels in the reticular thalamic nucleus, the supramammillary nucleus and the mesencephalic V nucleus. Some cells of the medial habenula (medioventral part) and of the interpeduncular nucleus (central and lateral parts) were also labelled. The distribution of alpha 6 mRNA was compared with the distribution of the other known nicotinic acetylcholine receptor subunit mRNAs. In several nuclei, the expression of alpha 6 was complementary to those of other alpha subunits. Moreover, some of the cell groups (such as the substantia nigra, the ventral tegmental area and the locus coeruleus) previously thought to contain mainly alpha 3 mRNA in fact were found to contain high levels of alpha 6 mRNA. Finally, we found extensive colocalization of alpha 6 and beta 3, indicating the possible existence of nicotinic receptor hetero-oligomers containing both subunits. The present results show that alpha 6 is the major nicotinic acetylcholine receptor alpha subunit expressed in dopaminergic cell groups of the mesencephalon and noradrenergic cells of the locus coeruleus. This suggests the involvement of the alpha 6 subunit in some of the major functions of central nicotinic circuits, including the modulation of locomotor behaviour and reward.

1996 - Nicotinic receptors and brain plasticity [Relazione in Atti di Convegno]
Changeux, J. -P.; Bessis, A.; Bourgeois, J. -P.; Corringer, P. -J.; Devillers-Thiery, A.; Eisele, J. -L.; Kerszberg, M.; Lena, C.; Le Novere, N.; Picciotto, M.; Zoli, M.
abstract

1996 - Regional and cellular distribution of spermidine/spermine N-1-acetyltransferase (SSAT) mRNA in the rat central nervous system [Articolo su rivista]
Zoli, Michele; Pedrazzi, Patrizia; Agnati, Luigi Francesco
abstract

Spermidine/spermine N-1-acetyltransferase (SSAT) is the key enzyme responsible for polyamine interconversion. SSAT mRNA (visualized by in situ hybridization histochemistry) was shown to have a wide but heterogeneous distribution in the central nervous system (CNS) at both regional and cellular levels. The highest labelling was observed in hippocampus (pyramidal and polymorph neurons) and olfactory bulb. Present data suggest that polyamine metabolism in the CNS is not homogeneous but rather that the preferential production of a polyamine species is region- and cell type-specific.

1996 - Spermidine/spermine N-1-acetyltransferase mRNA levels show marked and region-specific changes in the early phase after transient forebrain ischemia [Articolo su rivista]
Zoli, Michele; Pedrazzi, Patrizia; Zini, Isabella; Agnati, Luigi Francesco
abstract

Considerable evidence points to an involvement of natural polyamines (putrescine, spermidine and spermine) in trophic regulation of brain tissue. Spermidine/spermine N-1-acetyltransferase is the key enzyme in the interconversion pathway which leads to the formation of spermidine and putrescine from spermine and spermidine, respectively. In the present paper we have studied using in situ hybridization histochemistry the levels of spermidine/spermine N-1-acetyltransferase mRNA in the rat central nervous system after transient forebrain ischemia. In the first hours after the insult, a modest increase in spermidine/spermine N-1-acetyltransferase mRNA levels was observed in ependymal cells and other non-neuronal cells of all telencephalic and diencephalic regions. In addition, major increases in spermidine/spermine N-1-acetyltransferase mRNA levels were observed in regions selectively vulnerable to the ischemic insult, such as striatum, hippocampus and cerebral cortex, during the first day post-reperfusion. The time course and extent of labelling increase were subregion- and cell-specific. At the cellular level, the labelling appeared markedly increased in neurons (8-10 fold in ventromedial striatum and CA1 region) and, to a lesser extent, in non-neuronal cells. The increase in SSAT mRNA levels was not directly related to cell degeneration, as it was detected in both some vulnerable and some resistant cell populations. However, the peak increase of SSAT labelling was precocious in resistant neurons (such as those of ventromedial striatum and dentate gyrus granular layer) and delayed or very limited in vulnerable neurons (such as those of CA1 pyramidal layer and dorsolateral striatum). The increase in spermidine/spermine N-1-acetyltransferase may contribute to the increase in putrescine and decrease in spermidine levels observed after ischemia and gives further support to the notion that polyamine metabolism in the early phase after lesion is oriented towards putrescine production. This phenomenon could be relevant in determining the prevalence of neurotrophic vs. neurotoxic effects of polyamines.

1996 - The receptor mosaic hypothesis of the engram: Possible relevance of Boolean network modeling [Articolo su rivista]
Zoli, Michele; Guidolin, D; Fuxe, K; Agnati, Luigi Francesco
abstract

In the past 15 years, several lines of evidence have shown that receptors for chemical signals can interact in domains of the plasma membrane and possibly form molecular circuits encoding logical operators. In this frame, the receptor mosaic hypothesis of the engram was advanced. According to this proposal, aggregates of different receptor species (mosaics) may form in neuronal membranes (typically synapses) and constitute a memory trace (engram) of its activity. In the present paper, we present an attempt to model the functioning of aggregates of interacting receptors in membrane domains by means of random Boolean networks.

1996 - Wiring and volume transmission in the central nervous system: The concept of closed and open synapses [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco
abstract

During the past two decades, several revisions of the concepts underlying interneuronal communication in the central nervous system (CNS) have been advanced. Our group has proposed to classify intercellular communication in the CNS under two general frames: 'wiring' (WT) and 'volume' transmission (VT). WT is characterized by a single 'transmission channel' made by cellular (neuronal or glial) structures and with a region of discontinuity not larger than a synaptic cleft. VT is characterized by the diffusion from a cell source (neuronal or glial) of chemical and electrical signals in the extracellular fluid (ECF) for a distance larger than the synaptic cleft Based on morphological and functional characteristics, and in light of the distinction proposed, six main modes of intercellular communication can be recognized in the CNS: gap-junction, membrane juxtaposition, and closed synapse (which represent WT-type modes of communication); open synapse, paracrine transmission and endocrine-like transmission (which represent VT-type modes of communication). Closed and open synapses are distinguished on the basis of the sealing of the signal within or the leakage of the signal outside the synapse Intra-synaptic restriction or extra-synaptic diffusion of transmitters are insured by a number of anatomical arrangements (e.g. glial ensheathment of synapse, size of the synaptic cleft) and functional mechanisms (e.g. density and location of transmitter re-uptake sites and metabolic enzymes). Some central synapses can switch from closed to open state and vice versa, e.g. by changing the amount of transmitter released. Finally, a synapse containing several transmitters can work as an open synapse for one transmitter and as a closed synapse for another.

1995 - A single (-)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra. [Articolo su rivista]
Li, Xm; Zoli, Michele; Finnman, Ub; Le Novère, N; Changeux, Jp; Fuxe, K.
abstract

The in vitro and in vivo effects of (-)-nicotine on dopamine D2 receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (-)-nicotine resulted in a reduction of the KD value of the D2 antagonist [3H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in the Bmax value. This action of (-)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, the KD and the Bmax values of the D2 agonist [3H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5-48 h after a single i.p. injection with 1 mg/kg of (-)-nicotine. No significant change in neostriatal D2 receptor mRNA levels was observed at any time interval after the (-)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4-24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 microM) concentrations of (-)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [3H]raclopride or [3H]NPA binding. These results indicate that a single (-)-nicotine injection can produce a selective and delayed increase in the affinity of D2 receptors for the antagonist, but not for the agonist without modifying the levels of D2 receptor mRNA, probably via the activation of central nicotinic receptors.

1995 - Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain [Articolo su rivista]
Picciotto, Mr; Zoli, Michele; Lena, C; Bessis, A; Lallemand, Y; LE NOVERE, N; Vincent, P; MERLO PICH, E; Brulet, P; Changeux, Jp
abstract

Nicotine affects many aspects of behaviour including learning and memory through its interaction with neuronal nicotinic acetylcholine receptors (nAChR). Functional nAChRs are pentameric proteins containing at least one type of alpha-subunit and one type of beta-subunit. The involvement of a particular neuronal nicotinic subunit in pharmacology and behaviour was examined using gene targeting to mutate beta 2, the most widely expressed nAChR subunit in the central nervous system. We report here that high-affinity binding sites for nicotine are absent from the brains of mice homozygous for the beta 2-subunit mutation. Further, electrophysiological recording from brain slices reveals that thalamic neurons from these mice do not respond to nicotine application. Finally, behavioural tests demonstrate that nicotine no longer augments the performance of beta 2-1- mice on passive avoidance, a test of associative memory. Paradoxically, mutant mice are able to perform better than their non-mutant siblings on this task.

1995 - Age-related alterations in tanycytes of the mediobasal hypothalamus of the male rat [Articolo su rivista]
Zoli, Michele; F., Ferraguti; A., Frasoldati; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

By means of semiquantitative immunocytochemistry, possible age-related changes in dopamine and cyclic AMP-regulated phosphoprotein mr 32 (DARPP-32) and glial fibrillary acidic protein (GFAP) immunoreactivities (IR) were investigated in tanycytes of the arcuate nucleus. These two markers showed opposite changes during aging. DARPP-32 IR decreased by around 70%, whereas GFAP IR increased by around 300% in 24-month-old vs. 3-month-old rats. These changes were accompanied by a progressive loss in the number of tanycytes, measured by counting of their long processes in the arcuate nucleus. No significant age-related change was observed either in GFAP IR in astrocytic populations of the mediobasal hypothalamus or in tyrosine hydroxylase IR in dopaminergic neurons of the dorsal arcuate nucleus. These observations indicate that the tanycytic population of the arcuate nucleus undergoes important modifications during aging, which include cell loss, impairment in the intracellular signalling cascade linked to DARPP-32, and hypertrophy. These changes may be related to the alterations in the neuroendocrine systems known to occur during aging.

1995 - Developmental regulation of nicotinic receptor subunit mRNAs in the rat central and peripheral nervous systems. [Articolo su rivista]
Zoli, Michele; LE NOVERE, N.; HILL J. A., Jr; Changeux, J. P.
abstract

In the present study we have investigated the anatomical distribution pattern of nAChR alpha 3, alpha 4, beta 2, and beta 4 subunit mRNAs during prenatal and perinatal development of the rat CNS and PNS. Three main developmental patterns have been recognized. (1) In the majority of cases studied (caudal brain, spinal cord, dorsal root ganglia, trigeminal and geniculate ganglia) all four subunit mRNAs are initially (E11-13) detected but, during subsequent prenatal development, the level of some of these subunit mRNAs (alpha 3 and beta 4 in the brain and spinal cord, alpha 4 and beta 4 in the dorsal root ganglia, alpha 4 in the visceral sensory ganglia, and alpha 3, alpha 4, and beta 4 in the somatic sensory ganglia) become undetectable. (2) In the case of the cerebral cortex a pair of subunit mRNAs (alpha 3-beta 2) is initially (E12-13) expressed followed by a repression of the alpha 3 subunit (E15) and the subsequent (E17-19) induction of the alpha 4 subunit. (3) Only some subunit mRNAs are initially (E13-15) expressed in the retina (alpha 3-alpha 4-beta 2-beta 4), parasympathetic or sympathetic motor ganglia (alpha 3-beta 2-beta 4), and vestibulo-cochlear ganglia (alpha 4-beta 2) and their level remains stable throughout prenatal and early postnatal development. Overall, in most central and peripheral structures the appearance of nAChR subunit mRNAs is precocious and temporally related to the timing of neuronal differentiation. In addition, in several structures the expression of certain subunits (alpha 3, alpha 4 or beta 4) is transient, although not beta 2. Finally, the comparison of the different regional distribution patterns suggests that a limited number of structure-specific receptor isoforms are functional during development of CNS and PNS.

1995 - IMMUNOCHEMICAL LOCALIZATION OF CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-I [Articolo su rivista]
Picciotto, Mr; Zoli, Michele; Bertuzzi, G; Nairn, Ac
abstract

Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was originally identified in rat brain based on its ability to phosphorylate site 1 of synapsin I. Recently a cDNA for the rat brain enzyme has been cloned and the primary structure elucidated [Picciotto et al. (1993), J. Biol. Chem., 268:26512-26521]. The rat cDNA encoded a protein of 374 amino acids with a calculated M(r) of 41,636. Antibodies have now been raised against the recombinant kinase expressed in E. coli as a glutathione-S-transferase fusion protein. Immunoblot analysis of rat cortex lysates revealed two major immunoreactive bands of similar to M(r) 38,000 and 42,000. Minor immunoreactive species of slightly lower M(r) were also detected. Two distinct CaM kinase I activities were partially purified from rat brain and shown to correspond to the two major immunoreactive species. A variety of immunoreactive species of M(r) 35-43,000 were detected in ´´brain´´ tissue from cow, zebra finch, goldfish, Xenopus, lamprey, and Drosophila. In rat brain, immunocytochemistry revealed strong staining in cortex, hippocampus, amygdala, hypothalamus, brain stem, and choroid plexus. The labelling was mainly observed in neuropil but clusters of intensely labelled neuronal cell bodies were also detected all along the neuraxis. Neuronal nuclei and glial cells did not appear to be stained. Subcellular fractionation studies confirmed the cytosolic localization of the kinase in the brain. In various rat non-neuronal tissues and in a number of cell lines, immunoreactive species of similar to M(r) 38,000 and similar to 42,000 were detected at lower levels than that detected in brain. The M(r) 38,000 and 42,000 species were also found in different ratios and at different levels in the non-neuronal tissues. These results support a role for CaM kinase I in the regulation of multiple neuronal processes. Furthermore, the widespread cell and tissue distribution suggests that CaM kinase I may function as a ubiquitous multi-functional protein kinase. Finally, the multiple immunoreactive species may represent isoforms of CaM kinase I.

1995 - Intercellular communication in the brain: Wiring versus volume transmission [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; I., Stromberg; K., Fuxe
abstract

During the past two decades several revisions of the concepts underlying interneuronal communication in the central nervous system have been advanced. We propose here to classify communicational phenomena between cells of the central neural tissue under two general frames: ''wiring'' and ''volume'' transmission. ''Wiring'' transmission is defined as intercellular communication occurring through a well-defined connecting structure. Thus, wiring transmission Is characterized by the presence of physically identifiable communication channels within the neuronal and/or glial cell network. It includes synaptic transmission but also other types of intercellular communication through a connecting structure (e.g., gap junctions). ''Volume'' transmission is characterized by signal diffusion in a three-dimensional fashion within the brain extracellular fluid. Thus, multiple, structurally often not well characterized extracellular pathways connect intercommunicating cells. Volume transmission includes short- (but larger than synaptic cleft, i.e. about 20 nm) and long-distance diffusion of signals through the extracellular and cerebrospinal fluid. It must be underlined that the definitions of wiring and volume transmission focus on the modality of transmission and are neutral with respect to the source and target of the transmission, as well as type of informational substance transmitted. Therefore, any cell present in the neural tissue (neurons, astroglia, microglia, ependyma, tanycytes, etc.) can be a source or a target of wiring and volume transmission. In this paper we discuss the basic definitions and some distinctive characteristics of the two types of transmission. In addition, we review the evidence for different types of intercellular communication besides synaptic transmission in the central nervous system during phylogeny, and in vertebrates in physiological and pathological conditions.

1995 - LONG-DISTANCE PATHWAYS OF DIFFUSION FOR DEXTRAN ALONG FIBER-BUNDLES IN BRAIN - RELEVANCE FOR VOLUME TRANSMISSION [Articolo su rivista]
Bjelke, B; England, R; Nicholson, C; Rice, Me; Lindberg, J; Zoli, Michele; Agnati, Luigi Francesco; Fuxe, K.
abstract

TEXAS Red-labelled dextran with a mol. wt of 3000 g mol(-1), a marker for the extracellular space, was injected unilaterally into the neostriatum of adult rats (0.3-30 mu g mu(-1)) and its distribution evaluated 1 min to 5 h later. Diffusion in the neuropil was observed with clearance starting after 30 min. After 10-15 min strong labelling along the myelinated fibre bundles was observed in the entire neostriatum. After about 20 min the labelling along the fibres reached into the corpus callosum and the overlaying deep layers of the cerebral cortex. A marked cellular uptake and accumulation of labelled dextran was found in putative perivascular pericytes. Thus, in the living brain preferential extracellular fluid pathways for diffusion exist, especially along fibre bundles, which allow the exchange of chemical signals between two distant regions. These may represent extracellular fluid pathways for volume transmission.

1995 - Promoter elements conferring neuron specific expression of the b2 subunit of the neuronal nicotinic acetylcholine receptor studied in vitro and in transgenic mice. [Articolo su rivista]
Bessis, A.; Salmon, A. M.; Zoli, Michele; LE NOVERE, N.; Picciotto, M. R.; Changeux, J. P.
abstract

Several genes encoding subunits of the neuronal nicotinic acetylcholine receptors have been cloned and regulatory elements involved in the transcription of the alpha 2 and alpha 7-subunit genes have been described. Yet, the detailed mechanisms governing the neuron-specific transcription and the spatio-temporal expression pattern of these genes remain largely uninvestigated. The beta 2-subunit is the most widely expressed neuronal nicotinic receptor subunit in the nervous system. We have studied the structural and regulatory properties of the 5' sequence of this gene. A fragment of 1163 bp of upstream sequence is sufficient to drive the cell-specific transcription of a reporter gene in both transient transfection assays and in transgenic mice. Deletion analysis and site-directed mutagenesis of this promoter reveal two negative elements and one positive element. The positively-acting sequence includes one functional E-box. One of the repressor elements is located in the transcribed region and is the NRSE/RE1 sequence already described in promoters of neuronal genes. In this paper, we describe the neuron-specific promoter of the gene encoding the neuronal nicotinic acetylcholine receptor beta 2-subunit.

1995 - TEMPORAL CHANGES IN SULFATED GLYCOPROTEIN-2 (CLUSTERIN) AND ORNITHINE DECARBOXYLASE MESSENGER-RNA LEVELS IN THE RAT TESTIS AFTER ETHANE-DIMETHANE SULFONATE-INDUCED DEGENERATION OF LEYDIG-CELLS [Articolo su rivista]
A., Frasoldati; Zoli, Michele; F. F. G., Rommerts; Biagini, Giuseppe; M. F., Fustini; Carani, Cesare; Agnati, Luigi Francesco; Marrama, Paolo
abstract

Short- (3-24 h) and long-term (4-50 days) changes in sulphated glycoprotein-2 (SGP3) and ornithine decarboxylase (ODC) mRNA levels in the adult rat testis were studied following a single dose of ethane-dimethane sulphonate (EDS), to destroy the Leydig cells. Distribution patterns of SGP-2 and ODC labelling were consistent with prevailing expression of the two transcripts in Sertoli cells and germ cells, respectively This pattern did not show appreciable changes following EDS administration. No labelling of SGP-2 mRNA was noted in the interstitium of control and EDS-treated rats. This finding indicates that Leydig cell death induced by EDS is not associated with increased SGP-2 mRNA levels, a phenomenon related to apoptotic cell death in many tissues. Semi-quantitative densitometric analysis of the preparations demonstrated differential changes in SGP-2 and ODC mRNA levels in:the tubular compartment following EDS treatment. At 6, but not at 3 and 12, h following EDS administration, SGP-2 mRNA levels showed a significant increase, possibly secondary to a direct effect of the alkylating agent on Sertoli cells. A significant decrease in ODC mRNA levels was observed from day 7 to day 28, matching degenerative changes in the seminiferous epithelium. In contrast, a decrease in SGP-2 transcript levels was observed from days 21-35 after treatment. In conclusion, our findings demonstrate that SGP3 mRNA, a putative marker of apoptosis, is not altered in the testicular interstitium during EDS-induced degeneration of Leydig cells. In the tubular compartment, the content of both ODC and SGP-2 mRNAs following EDS administration appears to be dependent mostly on the integrity of the germ cell complement and not to be influenced directly by changes in testosterone levels.

1994 - Effects of nicotine injections on messenger RNAs encoding proteins involved in transmission in the mesostriatum dopaminergic system [Articolo su rivista]
Le Novere, N.; Zoli, M.; Changeux, J. P.
abstract

1994 - MAPPING AND COMPUTER-ASSISTED MORPHOMETRY AND MICRODENSITOMETRY OF GLUCOCORTICOID RECEPTOR IMMUNOREACTIVE NEURONS AND GLIAL-CELLS IN THE RAT CENTRAL-NERVOUS-SYSTEM [Articolo su rivista]
Cintra, A; Zoli, Michele; Rosen, L; Agnati, Luigi Francesco; Okret, S; Wikstrom, Ac; Gustafsson, Ja; Fuxe, K.
abstract

By means of a monoclonal mouse immunoglobulin G2a antibody against the rat liver glucocorticoid receptor and the indirect immunoperoxidase technique, the distribution of glucocorticoid receptors in neuronal and glial cell populations was mapped in the central nervous system of the male rat. The mapping was complemented by computer-assisted morphometric and microdensitometric evaluation of glucocorticoid receptor immunoreactivity in many brain regions. The quantitative analysis allowed us to achieve for the first time an objective characterization of glucocorticoid receptor distribution in the CNS, thus avoiding the ambiguities of previous mapping studies based on subjective evaluations. In addition, a taxonomic analysis of central nervous system regions containing glucocorticoid receptor immunoreactivity was carried out utilizing the quantitative parameters obtained in the morphometric evaluation. Nuclei of neuronal and glial cells containing glucocorticoid receptor immunoreactivity were detected in a widespread, but still highly heterogeneous, fashion in the central nervous system, underlining the view that glucocorticoids can control a large number of central nervous system target cells via effects on gene expression. Many nerve cell populations have been shown to contain substantial amounts of nuclear glucocorticoid receptor immunoreactivity, whereas only a low density of glial cells, in both gray and white matter, show nuclear glucocorticoid receptor immunoreactivity. Thus, in most brain areas, the major target for glucocorticoids appears to be the nerve cells. Interestingly, an inverse correlation was found in the regional density of glucocorticoid receptor-immunoreactive nerve and glial cells, suggesting that glucocorticoids may influence a brain area either via glial cells or, more frequently, via nerve cells. The results on mapping highlight the impact of glucocorticoids in areas both traditionally and not traditionally involved in stress responses. The distribution of glucocorticoid receptor immunoreactivity also emphasizes a role of glucocorticoids in the regulation of the afferent regions of the basal ganglia and the cerebellar cortex, and of both afferent and efferent layers of the cerebral cortex. Glucocorticoid receptor immunoreactivity is widely distributed over the thalamus, probably leading to modulation of activity in the various thalamocortical pathways transmitting inter alia specific sensory information to the cerebral cortex. Many unspecific afferents to the cerebral cortex are potentially regulated by glucocortoid receptors such as the noradrenaline and 5-hydroxytryptamine afferents, since their nerve cells of origin contain strong glucocorticoid receptor immunoreactivity. Eight brain regions involving sensory, motor and limbic areas were shown to have a similarity with regard to glucocorticoid receptor-immunoreactive parameters at the level of 95%. The density of glucocorticoid receptor-immunoreactive nerve cells appeared to be the main factor in determining such a very high level of similarity. Overall, our results emphasize that glucocorticoids may appropriately tune networks of different areas to obtain optimal integration and in this way improve survival of the animal under challenging conditions.

1993 - Chicken neuronal acetylcholine receptor alpha 2-subunit gene exhibits neuron-specific expression in the brain and spinal cord of transgenic mice. [Articolo su rivista]
Daubas, P; Salmon, Am; Zoli, Michele; Geoffroy, B; Devillers Thiéry, A; Bessis, A; Médevielle, F; Changeux, J. P.
abstract

Transgenic mice carrying the complete structural gene of the alpha 2 subunit of the chicken neuronal nicotinic acetylcholine receptor (nAChR) and 7 kilobase pairs (kbp) of 5' upstream and 3 kbp of 3' downstream sequences have been generated. The transgene was stably integrated in transgenic lines and transmitted to their progeny. Avian transgene expression was predominant in the central nervous system as detected by specific alpha 2-subunit cDNA amplification. Moreover, in at least two independent mouse lines, its expression appeared to be neuron-specific and reproducibly restricted to subregions in the brain and spinal cord, as revealed by in situ hybridization histochemistry. Most cranial motor nuclei were positive, and several of the alpha 2-subunit transgene-expressing structures corresponded to cholinergic areas in rodents. This study reveals that regulatory mechanisms giving rise to neuronal-specific gene expression have been conserved at least in part between birds and mammals.

1993 - CORTICOSTERONE INCREASES FGF-2 (BFGF) IMMUNOREACTIVITY IN THE SUBSTANTIA-NIGRA OF THE RAT [Articolo su rivista]
Chadi, G; Rosen, L; Cintra, A; Tinner, B; Zoli, Michele; Pettersson, Rf; Fuxe, K.
abstract

THE effects of acute and subchronic (7 days) administrations of the adrenocortical hormone corticosterone on basic fibroblast growth factor (bFGF, FGF-2) immunoreactivity were studied in the substantia nigra of the rat by semiquantitative immunocytochemistry coupled with image analysis. Corticosterone was able to increase FGF-2 immunoreactivity in different nigral subregions and cell types (astrocytes and neurones) depending on the duration of the treatment. These results open up the possibility that stress hormones can modulate the trophic state of the substantia nigra through an action on FGF-2.

1993 - DISTRIBUTION OF DOPAMINE-IMMUNOREACTIVE NEURONS AND THEIR RELATIONSHIPS TO TRANSMITTER AND HYPOTHALAMIC HORMONE-IMMUNOREACTIVE NEURONAL SYSTEMS IN THE RAT MEDIOBASAL HYPOTHALAMUS - A MORPHOMETRIC AND MICRODENSITOMETRIC ANALYSIS [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; Tinner, B; Steinbusch, Hwm; Fuxe, K.
abstract

A morphometric and microdensitometric characterization of the dopamine neurons of the mediobasal hypothalamus and their relationships with several other chemically identified systems, including putative tyrosine hydroxylase-positive/dopamine-negative neurons, was carried out after visualization of dopamine content by both immunocytochemistry and the Falck-Hillarp technique. Quantitative assessment of co-existence demonstrated that more than 95% of dopamine-immunoreactive neurons also contained tyrosine hydroxylase immunoreactivity and more than 90% of growth hormone-releasing factor-immunoreactive neurons also contained tyrosine hydroxylase immunoreactivity. Morphometric and densitometric analysis of dopamine, tyrosine hydroxylase and growth hormone-releasing factor-immunoreactive neurons in the arcuate nucleus showed that dopamine/tyrosine hydroxylase-containing and growth hormone-releasing factor/tyrosine hydroxylase-containing neuronal populations are two largely segregated cell groups with specific localization in the arcuate region, rostrocaudal extension and tyrosine hydroxylase-immunoreactivity content. Morphometric characteristics of dopamine-immunoreactive neurons were shown to be equivalent to those of catecholamine fluorescent cell bodies in the arcuate region. In addition, a cell group lacking detectable catecholamine fluorescence in normal animals but accumulating L-DOPA after peripheral loading was identified and characterized from a morphometric standpoint in the ventral premammillary nucleus. Quantitative analysis of nerve terminal co-distribution in the median eminence revealed significant correlations between dopamine and other transmitter or neurohormone systems, such as gamma-aminobutyric acid, galanin, luteinizing hormone-releasing hormone, in specific subregions of the palissade zone. These data point to discrete subregions of the median eminence, which have been called 'medianosomes', as main sites of interactions between transmitter-identified nerve terminal systems in the control of hypothalamic hormone release.

1993 - Effects of polyamine synthesis blockade on neuronal loss and astroglial reaction after transient forebrain ischemia [Articolo su rivista]
Zoli, Michele; Zini, Isabella; R., Grimaldi; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylomithine, a suicide inhibitor of omithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylomithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hT, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylomithine can reduce the increase of glial fibrillary acidic protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylomithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylomithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that omithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylomithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.

1993 - Immunocytochemical localization of a neuronal nicotinic receptor: the beta2 subunit. [Articolo su rivista]
Hill, J. A.; Zoli, Michele; Bourgeois, J. P.; Changeux, J. P.
abstract

We have characterized in adult rat the tissue-specific expression of the nicotinic ACh receptor (AChR) beta 2-subunit using antisera raised against fusion protein constructs. Immunohistochemical localization revealed immunoreactivity distributed throughout the neuraxis. Overall, beta 2-like immunoreactivity (beta 2-LI) correlated well with in situ localization of beta 2 transcript in neuronal cell bodies. Particularly strong labeling was detected in the thalamus, and scattered other regions, whereas relatively weak staining was observed in the hypothalamus and amygdala. At the cellular level, beta 2-LI appeared to be exclusively neuronal and concentrated predominantly in perikarya, although strongly positive dendrites (cerebral cortical pyramidal neurons, cerebellar Purkinje cells) and axon terminals (e.g., striatum) were detected. At the ultrastructural level, beta 2-LI was membrane associated, with strong staining observed in endoplasmic reticulum and cytoplasmic transport vesicles. beta 2-LI was rarely detected at synapses. The widespread distribution of beta 2 suggests it may serve as a common subunit in different AChR combinations in various brain regions. Regulation of the expression of beta 2-subunit appears to be relatively unrestrained, with an apparent excess of protein synthesized in the cytoplasm relative to that which ultimately arrives at functional targets in the plasma membrane.

1993 - INCREASES IN SULFATED GLYCOPROTEIN-2 MESSENGER-RNA LEVELS IN THE RAT-BRAIN AFTER TRANSIENT FOREBRAIN ISCHEMIA OR PARTIAL MESODIENCEPHALIC HEMITRANSECTION [Articolo su rivista]
Zoli, Michele; Ferraguti, F; Zini, Isabella; Bettuzzi, Saverio; Agnati, Luigi Francesco
abstract

Sulphated glycoprotein-2, thought to be involved in programmed cell death in peripheral organs, has been detected at increased levels in brain degenerative states. In this paper we have investigated the regional and cellular expression of this protein during development of brain lesion. With this aim sulphated glycoprotein-2 mRNA levels were studied in models of ischemic (transient forebrain ischemia) or mechanical (partial mesodiencephalic hemitransection) brain injuries using in situ hybridization histochemistry. Marked increases in sulphated glycoprotein-2 mRNA were observed in lesioned brains in both models. In addition, we report a shift in the regional distribution of positive cells in both lesion models 1-7 days post-lesion. After transient forebrain ischemia, sulphated glycoprotein-2 mRNA increases were always localized in selectively vulnerable regions (caudate-putamen, hippocampal formation), showing a temporal change in the pattern of intraregional distribution from less to more lesioned parts. In the case of mechanical lesion at 1 day, increased labelling had a widespread distribution on the lesioned side and was also observed on the intact side near the midline. In contrast, at 7 days increased labelling was restricted to regions directly lesioned (either areas whose input and/or output connections were severed by the transection or areas which were directly affected by the mechanical lesion). Analysis at the cellular level revealed that at both time intervals and in both lesion models most cell bodies overlain by dense clusters of specific grains were non-neuronal cells. The distribution patterns and their change over time suggest that at least some of these cells are inflammatory and phagocytic cells. The majority of degenerating neuronal cells after ischemia did not show increased levels of sulphated glycoprotein-2 mRNA. However, seven days after hemitransection and at all time intervals after transient ischemia, some cells clearly identifiable as neurones exhibited increased sulphated glycoprotein-2 mRNA levels.

1993 - Indole-pyruvic acid treatment reduces damage in striatum but not in hippocampus after transient forebrain ischemia in the rat [Articolo su rivista]
Zoli, Michele; MERLO PICH, Emilio; F., Ferraguti; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

The effects of treatment with indole-pyruvic acid, an endogenous metabolite of tryptophan converted into kynurenic acid in the brain, were studied in rats after transient forebrain ischemia induced by the 4-vessel occlusion procedure. The histological analysis showed a significant protective effect of indole pyruvic acid treatment on striatal ischemic lesions assessed by the extent of regional atrophy and the area of neuronal disappearance 14 days after ischemia. Striatal neurons were labelled by dopamine and adenosine 3':5'monophosphate regulated phosphoprotein-32 immunoreactivity. Conversely, increased neuronal loss, regional atrophy and glial fibrillary acidic protein immunoreactivity, an index of post-injury astroglial activation, were observed in the hippocampal formation, especially the CA3 field, of indole-pyruvic acid-treated rats when compared with vehicle-treated ischemic rats. The treatment with indole-pyruvic acid did not produce any improving effects in a test assessing short-term impairments after transient ischemia (motor test score at 24 h and 48 h post-ischemia). Furthermore, no significant effects of indole-pyruvic acid treatment were found on performance in water T-maze studied at 7 and 14 days post-ischemia. The opposite effects of indole pyruvic acid on ischemic lesion in different brain regions may be related to its multiple neurochemical actions in the brain. The protective effect of indole pyruvic acid on ischemic damage in striatum may be due to its conversion into kynurenic acid, a broad spectrum glutamate receptor antagonist. At hippocampal level, where glutamate receptor antagonists have been proved ineffective in the present lesion model, indole-pyruvic acid-induced changes in monoamine availability may lead to a worsening of neuronal damage.

1993 - L-deprenyl increases GFAP immunoreactivity selectively in activated astrocytes in rat brain [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; K., Fuxe; Agnati, Luigi Francesco
abstract

L-DEPRENYL is a selective inhibitor of monoamine oxidase (MAO) B, an enzyme predominantly localized in astrocytes. We have investigated the effect of treatment with L-deprenyl (0.25 mg kg-1 day-1) on glial fibrillary acidic protein (GFAP) immunoreactivity (IR) after lesioning the rat striatum with an injection cannula. No effect of drug treatment on GFAP IR was found in unlesioned striata. A significant increase (p < 0.01 vs saline treated rats) in GFAP IR was found in the tissue surrounding the lesion in striata of rats treated with L-deprenyl for 4 days after the lesion. When post-treated for 42 days, however, L-deprenyl no longer increased GFAP IR although reactive astrocytes were still present in the lesioned area. These results suggest that L-deprenyl can enhance the activation of astrocytes during a critical time-period following a striatal injury.

1993 - NEUROCHEMICAL ALTERATIONS BUT NOT NERVE-CELL LOSS IN AGED RAT NEOSTRIATUM [Articolo su rivista]
Zoli, Michele; Ferraguti, F; Toffano, G; Fuxe, K; Agnati, Luigi Francesco
abstract

Numerical changes in the overall neostriatal neuronal population have been investigated by morphometric analysis of Nissl-stained and glucocorticoid receptor-immunoreactive neurons. Number and staining intensity of various chemically-identified nerve cell populations were analysed by means of immunocytochemistry coupled with computer-assisted image analysis. Three- and 24-month-old male Sprague-Dawley rats were used. No change in the number of Nissl-stained, glucocorticoid receptor-, dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein- and enkephalin-immunoreactive neurons and a 50% decrease of neuropeptide Y-immunoreactive neurons were observed in the aged rat. In our preparations. the glucocorticoid receptor antibody stains around 90% of the neostriatal neurons, the dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein and enkephalin antibodies label 25-35% and the neuropeptide Y- antibody stains only 1% of neostriatal neurons. In the same preparations a significant decrease in the intensity of immunostaining was observed for enkephalin-, dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein- and neuropeptide Y-immunoreactive neuronal cell bodies and tyrosine hydroxylase-immunoreactive nerve terminals in the aged rat. In the case of neuropeptide Y-, and dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein-immunoreactive neurons, the changes in the intensity of immunostaining were differentially compartmentalized within neostriatum, suggesting selective vulnerability of striatal subregions to ageing processes. In conclusion, these data indicate that no significant age-related neuronal cell loss occurs in neostriatum. On the other hand, a generalized decrease in the levels of peptide transmitters and molecules related to dopamine transmission is observed in aged rat neostriatum, possibly resulting in the known age-related deficits of neostriatally-controlled behaviours.

1993 - Neuronal nicotinic acetylcholine receptor: From gene to smoking addiction [Articolo su rivista]
Le Novere, N.; Bessis, A.; Lena, C.; Piccioto, M.; Zoli, M.
abstract

The nicotinic acetylcholine receptor present in muscle is an allosteric pentameric protein and has been used as a model for the structure and function of the superfamily of ligand gated ion channels. A related family of receptors is localized in the nervous system. These receptors, which are composed of two alpha subunits that are chiefly responsible for the binding of acetylcholine and three non-alpha subunits, mediate neuronal nicotinic neurotransmission. To date, ten genes have been isolated encoding subunits of the neuronal nicotinic receptor expressed in various brain regions. The electrophysiological and pharmacological properties of the neuronal nicotinic receptors are dependent upon the subunit composition of the receptor, and can be modified by innervation as well as by agents such as calcium and cAMP. The expression of the receptor can be altered as a result of diverse neuronal pathologies.

1993 - Neuropeptide Y (NPY) and adrenaline (A) interactions in the central control of arterial blood pressure (ABP) in the male rat [Articolo su rivista]
Agnati, L. F.; Zoli, M.; Aguirre, J. A.; Fuxe, K.; Benfenati, F.
abstract

1993 - RECEPTOR-RECEPTOR INTERACTIONS AN INTEGRATIVE MECHANISM IN NERVE-CELLS [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; Hedlund, Pb; Li, Xm; Ferre, S; Fuxe, K.
abstract

Several lines of evidence indicate that interactions among transmission lines can take place at the level of the cell membrane via interactions among macromolecules, integral or associated to the cell membrane, involved in signal recognition and transduction. The present view will focus on this last subject, i.e., on the interactions between receptors for chemical signals at the level of the neuronal membrane (receptor-receptor interaction). By receptor-receptor interaction we mean that a neurotransmitter or modulator, by binding to its receptor, modifies the characteristics of the receptor for another transmitter or modulator. Four types of interactions among transmission Lines may be considered, but mainly intramembrane receptor-receptor interactions have been dealt with in this article, exemplified by the heteroregulation of D2 receptors via neuropeptide receptors and A2 receptors. The role of receptor-receptor interactions in the integration of signals is discussed, especially in terms of filtration of incoming signals, of integration of coincident signals, and of neuronal plasticity.

1992 - Central mechanisms subserving the impaired growth hormone secretion induced by persistent blockade of NMDA receptors in immature male rats. [Articolo su rivista]
L., Cocilovo; V., DE GENNARO COLONNA; Zoli, Michele; Biagini, Giuseppe; B. P., Settembrini; E. E., Muller; D., Cocchi
abstract

Recently, we have reported in immature female rats that short-term blockade of glutamate receptors of the N-methyl-D-aspartic acid (NMDA) subtype by the noncompetitive antagonist MK-801 induced a reduction of growth rate, basal and stimulated growth hormone (GH) release and plasma somatomedin C levels. In the present study, we investigated in immature male rats the mechanism(s) through which agonists and antagonists of glutamate receptors affect GH secretion. In 21-day-old male rats, administration of MK-801 (0.2 mg/kg i.p. b.i.d.) for 10 days induced a significant impairment of growth rate, which was unrelated to a significant reduction of food intake. GH secretion from anterior pituitary fragments of MK-801-treated rats was not significantly reduced under basal conditions but was significantly less under stimulation by 40 mM K+. Incubation of dispersed pituitary cells of 31-day-old rats with N-methyl-aspartic acid (1 and 100-mu-M), alone or associated with MK-801 (1-mu-M) did not change GH secretion. Semi quantitative densitometric analysis of hypothalami of MK-801-treated rats evidenced a clearcut decrease in the intensity of GHRH-like immuno-reactivity (LI) staining in the median eminence (ME), whereas no differnece was observed in the ME-somatostatin (SS)-LI. Finally, GHRH mRNA but not SS-mRNA, evaluated by slot-blot hybridization, was reduced in the hypothalamus of MK-801-treated rats. These and our previous data would demonstrate that NMDA glutamate receptors play an important role in the neuroendocrine control of GH secretion in the rat, and suggest an action mediated by GHRH-secreting neurons.

1992 - Feeding and drinking responses to neuropeptide Y injections in the paraventricular hypothalamic nucleus of aged rats [Articolo su rivista]
MERLO PICH, Emilio; B., Messori; Zoli, Michele; F., Ferraguti; Marrama, Paolo; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

Neuropeptide Y (NPY), a peptide of the pancreatic polypeptide family, exerts a potent stimulatory action on eating when injected into the paraventricular hypothalamic nucleus (PVN) in rats. Several NPY-containing systems are altered with advancing age, and aged rodents develop anorexia and a modified daily cycle pattern of feeding. These findings suggest that a relationship may exist between the aging-related anorexia and the reduced function of NPY-containing systems projecting to the PVN. In the present study eating and drinking behavior in satiated or fasted young (3 months) and aged (24 months) rats have been investigated over 22 h after NPY injection into the PVN. The levels of NPY immunoreactivity (IR) in PVN were also evaluated by means of semiquantitative immunocytochemistry. NPY injections into PVN increased food and water consumption in both young and aged satiated rats 30, 90 and 240 min after injection. However, the feeding and drinking responses elicited by 0.05, 0.10 and 1.0 nmol of NPY were significantly attenuated in the aged rats when compared to young rats. In aged rats, 24 h of food and water deprivation produced significant increase of food consumption measured at 30, 90 min and 22 h, which was equivalent to that induced by 1:0 nmol NPY injection. Administration of 1.0 nmol NPY in PVN did not further increase the 24 h deprivation effect on feeding in both groups of rats, but enhanced drinking in deprived young rats. This effect was not present in aged rats. In addition, aged rats showed a stronger response to 24 h deprivation than to 1.0 nmol NPY administration. These results and the severe reduction of NPY IR levels in PVN nerve terminals of aged rats suggest that NPY deficiency may be a factor responsible for anorexia in the aged.

1992 - MORPHOMETRIC EVALUATION OF POPULATIONS OF NEURONAL PROFILES (CELL-BODIES, DENDRITES, AND NERVE-TERMINALS) IN THE CENTRAL-NERVOUS-SYSTEM [Articolo su rivista]
Zoli, Michele; Guidolin, D; Agnati, Luigi Francesco
abstract

Morphometric techniques have been developed to quantitatively characterize groups of transmitter-identified neuronal profiles, such as cell groups, dendrite and nerve terminal fields. These morphometric techniques will be illustrated by introducing some general tools for image analysis which can be considered as a background for the present specific applications. The following methods have been included: (1) methods to identify and quantitatively characterize, from both numerical and geometrical standpoints, groups of profiles in a two- and three-dimensional frame; (2) methods to evaluate the evenness of a certain distribution of profiles in the plane; (3) methods to identify subgroups of profiles based on their different spatial or optical density; and (4) methods to compare the distributions of two or more groups of profiles. The applications of these general tools to some neuroanatomical problems, such as cell group definition and description, have been illustrated. Practical examples performed on immunocytochemical preparations of neuronal profile populations are also given. Finally, the potentiality of numerical classification to classify and compare morphometric data has been shown. As an example, numerical classification methods have been applied to the morphometric and microdensitometric analysis of adrenaline/neuropeptide Y costoring neuronal systems of the brainstem in adult and aged rats.

1992 - NEURONAL PLASTICITY AND AGING PROCESSES IN THE FRAME OF THE RED QUEEN THEORY [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Biagini, Giuseppe; Fuxe, K.
abstract

On the basis of the morphofunctional evidence obtained in old brains of humans and mammals the present hypothesis has been introduced. This hypothesis states that neuronal plasticity can be used either to compensate for neuronal degeneration or to store new information. Thus, in pathological ageing the marked rate of degeneration has fully exhausted the already reduced plasticity capability of neural networks. In this way marked impairments of memory trace formation take place in pathological ageing conditions such as Alzheimer's disease. The essence of this hypothesis is that a competition for the available plasticity exists between the compensatory responses to ageing-induced degeneration and the processes necessary for memory trace formation. We have called this hypothesis the 'Red Queen Theory', an analogy borrowed from Lewis Carroll's book Through the Looking Glass. Thus, in ageing, processes responsible for plasticity must be forced to run at the highest possible rate to maintain the morphofunctional substrate of the existing networks as well as to allow the formation of new memory traces.

1992 - NEUROPEPTIDES, EXCITATORY AMINO-ACID AND ADENOSINE A2 RECEPTORS REGULATE D2 RECEPTORS VIA INTRAMEMBRANE RECEPTOR-RECEPTOR INTERACTIONS - RELEVANCE FOR PARKINSONS-DISEASE AND SCHIZOPHRENIA [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Voneuler, G; Tanganelli, S; Oconnor, Wt; Ferre, S; Hedlund, P; Zoli, Michele
abstract

1992 - REGIONAL AND CELLULAR-DISTRIBUTION WITHIN THE RAT PROSTATE OF 2 MESSENGER-RNA SPECIES UNDERGOING OPPOSITE REGULATION BY ANDROGENS [Articolo su rivista]
Bettuzzi, Saverio; Zoli, Michele; Ferraguti, F; Ingletti, Mc; Agnati, Luigi Francesco; Corti, Arnaldo
abstract

We have investigated, by in-situ hybridization histochemistry, the distribution within the rat ventral prostate of the mRNAs for sulphated glycoprotein-2 (SGP-2) and ornithine decarboxylase, two proteins that appear to be inversely regulated by androgens in this organ, in that the level of SGP-2 mRNA is lowered, while the activity of ODC is enhanced by the latter hormones. Low-magnification autoradiograms of whole ventral prostate sections showed that, in intact animals, the SGP-2 transcript was only detectable in restricted areas and not diffused evenly throughout the section. Reciprocally, the ODC transcript was not detectable in areas where the SGP-2 transcript was detected, but appeared distributed uniformly in the remaining parts of the section. The effect of castration on the levels of the two mRNAs was evaluated by a semiquantitative analysis of autoradiograms from whole ventral prostate sections using an autoimmune image analyser. Four days after castration, SGP-2 mRNA increased by about 11-fold, while ODC mRNA decreased by fivefold. The distribution of the two mRNAs among the different cell types, studied by treating the slides with photographic emulsion and counterstaining, showed that both were expressed exclusively in the epithelial luminal cells of the ducts. Furthermore, each of the two mRNAs preferentially accumulated in a cell population which was morphologically distinct while accumulation of the other was negligible. SGP-2 mRNA was mostly found in cuboidal epithelial cells and ODC mRNA in columnar epithelial cells. Castration caused a dramatic accumulation of SGP-2 and a decrease in ODC mRNAs in the cells of the columnar epithelium 4 days later. These data suggest that, in the ventral prostate of intact animals, SGP-2 is expressed in the proximal ducts whose luminal epithelium consists of cuboidal cells undergoing apoptotic phenomena and not in the intermediate and distal ducts characterized by columnar epithelia with active protein synthesis and cell multiplication. In the intermediate and distal parts of the ductal system the ODC gene would be expressed while being turned off in the proximal ducts. Castration, resulting in apoptosis of the epithelial cells of the intermediate and distal ducts, caused SGP-2 to be actively expressed and ODC to be repressed

1992 - Siagoside selectively attenuates morphological and functional striatal impairments induced by transient forebrain ischemia in rats [Articolo su rivista]
MERLO PICH, Emilio; R., Grimaldi; Zoli, Michele; Biagini, Giuseppe; V., Solfrini; G., Toffano; K., Fuxe; Agnati, Luigi Francesco
abstract

Background and Purpose: Transient forebrain ischemia induced in rats by the four-vessel occlusion method is known to produce severe neural damage in the hippocampus and striatum and a behavioral syndrome the major symptom of which is a working memory deficit. Recent evidence suggests that monosialogangliosides can ameliorate postischemic symptoms. Our purpose was to study the effect of siagoside, the inner ester of GM1 ganglioside, on some behavioral and morphological impairments induced by four-vessel occlusion in rats. Methods: Rats were injected daily with 5 mg/kg i.p. siagoside starting 4 hours after the cerebral ischemia. After 14 days the rats were tested for working memory in a water T maze or scored for apomorphine-induced stereotypy. The rats were killed 21 days after the cerebral ischemia. Histological and computer-assisted morphometric analyses were performed on cresyl violet-stained brain sections, which were graded according to a neuropathologic score, and on sections stained with a monoclonal antiserum against dopamine and cyclic adenosine-3',5'-monophosphate-regulated phosphoprotein, a marker for striatal dopaminoceptive neurons. Results: Siagoside treatment reduced the stereotypy score induced by low doses of apomorphine and the extent of striatal lesions but did not affect the working memory deficit or the extent of hippocampal lesions. Conclusion: Daily siagoside treatment after acute cerebral ischemia attenuates some morphological and functional deficits related to striatal damage. These effects can be interpreted as a selective protective action on striatal neural populations or as a modulatory action on neural systems involved in striatal control. These data are consistent with preliminary clinical reports showing that monosialogangliosides enhance motor recovery after acute ischemic stroke.

1991 - A NEW MODEL OF FOCAL BRAIN ISCHEMIA BASED ON THE INTRACEREBRAL INJECTION OF ENDOTHELIN-1 [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Kurosawa, M; Benfenati, Fabio; Biagini, Giuseppe; Zini, Isabella; Hallstrom, A; Ungerstedt, U; Toffano, G; Fuxe, K.
abstract

Endothelin-1 and its receptors are widely distributed in the brain of rodents and humans. In view of its potent and long-lasting vasoconstrictor activity, a role of endothelin-1 has been proposed in brain ischemia. In the present paper, the local ijnection of endothelin-1 was utilized to induce ischemia in rat striatum. An evaluation of the rostrocaudal extension of the lesion is reported. By using intracerebral microdialysis, a marked increase of lactate and dopamine, but not glutamate, was observed in this region upon endothelin-1 administration. Moreover, preliminary data reported show a protective effect of ganglioside treatment on endothelin-1 lesion of rat striatum. The characteristics of the present model of brain ischemia are discussed in comparison with well characterized models, such as the Pulsinelli's four vessel occlusion and the middle cerebral artery occlusion.

1991 - AGE-RELATED DECREASE OF GALANIN IMMUNOREACTIVITY IN DISCRETE FOREBRAIN AREAS OF THE MALE-RAT ASSESSED BY SEMIQUANTITATIVE IMMUNOCYTOCHEMISTRY [Articolo su rivista]
Ferraguti, F; Zoli, Michele; Fuxe, K; Agnati, Luigi Francesco
abstract

By means of semiquantitative immunocytochemistry the distribution of galanin like-immunoreactivity (LI) in various diencephalic and subcortical telencephalic areas of young (3 months), adult (12 months) and aged (24 months) rats was evaluated. A significant decrease in galanin LI was observed in all the analysed areas of the aged rats when compared with the young ones. In the adult animals a decrease in the microdensitometrical values with respect to the young rats was present only in the periventricular hypothalamic nucleus. A disappearance of nerve cell bodies was also observed in the central amygdaloid nucleus of the aged animals. The present data show a widespread age-related decrease of galanin LI in discrete diencephalic and subcortical telencephalic areas and suggest an involvement of this peptide in the multiple impairments of neuroendocrine and autonomic functions related to aging.

1991 - Central integration of chemical signals involved in the control of prolactin and LH secretion. Focus on the medianosomes [Relazione in Atti di Convegno]
Fuxe, K.; Agnati, L. F.; Bjelke, B.; Zoli, M.; Aguirre, J. A.; Cintra, A.; Hedlund, P.; Von Euler, G.; Andersson, K.; Eneroth, P.
abstract

1991 - CHANGES IN STRIATAL MU-OPIOID AND DELTA-OPIOID RECEPTORS AFTER TRANSIENT FOREBRAIN ISCHEMIA - A QUANTITATIVE AUTORADIOGRAPHIC STUDY [Articolo su rivista]
Benfenati, Fabio; MERLO PICH, Emilio; Zoli, Michele; Grimaldi, R; Fuxe, K; Agnati, Luigi Francesco
abstract

Transient forebrain ischemia induces specific changes in several neurochemical markers in the dorsolateral striatum. In the present paper, the density and distribution of mu and sigma-opioid receptors were analyzed in rat striatum 7 days after 30 min forebrain ischemia using the 4-vessel occlusion model. A marked (about 70%) decrease in the density of both opioid receptor subtypes was found in the dorsolateral striatum overlapping the areas of histological damage and of D1 dopamine receptor disappearance. Moreover, the density of sigma-opioid receptors and of the diffuse mu-opioid receptors was also affected (30% decrease) in the ventromedial striatum, an area which is substantially spared by the ischemic lesion. In contrast, the striatal patches of mu-opioid receptors were not affected in the ventromedial striatum and were preserved to a large extent in the area of lesion, although their area and receptor density resulted markedly reduced. The impairment of both opioid receptor subtypes suggests that opiate systems, like dopaminergic systems, are involved in the neurochemical changes observed in the striatum after transient forebrain ischemia.

1991 - Corticosterone treatment counteracts lesions induced by neonatal treatment with monosodium glutamate in the mediobasal hypothalamus of the male rat [Articolo su rivista]
Zoli, Michele; F., Ferraguti; Biagini, Giuseppe; A., Cintra; K., Fuxe; Agnati, Luigi Francesco
abstract

The effects of glucocorticoids on monosodium glutamate-induced neurotoxicity in the neonatal basal hypothalamus were studied by means of semiquantitative immunocytochemistry for tyrosine hydroxylase, growth hormone releasing factor and luteinizing hormone releasing hormone. Neonatal monosodium glutamate treatment induced a marked decrease in tyrosine hydroxylase immunoreactive neurons in the arcuate nucleus and growth hormone releasing factor immunoreactive nerve terminals in the median eminence. These effects were significantly antagonized by the coadministration of corticosterone. Corticosterone alone had no effect on the parameters studied. No significant change in luteinizing hormone releasing hormone immunoreactivity in the median eminence was detected after any treatment. These results demonstrate that corticosterone, possibly acting via type II corticosterone receptors which are highly enriched in the arcuate neurons, can exert a protective action on glutamate-induced neurotoxicity.

1991 - DECREASE IN MESSENGER-RNA LEVELS BUT NOT IN THE DENSITY OF D2 DOPAMINE-RECEPTORS IN RAT STRIATUM AFTER TRANSIENT FOREBRAIN ISCHEMIA [Articolo su rivista]
Benfenati, Fabio; Cimino, M; Zoli, Michele; Grimaldi, R; Zini, Isabella; Agnati, Luigi Francesco
abstract

D2 dopamine receptor mRNA was analyzed by in situ hybridization histochemistry in rat striatum 7 days after transient forebrain ischemia. A patchy disappearance of the D2 receptor mRNA was observed in the dorsolateral striatum. In the same area, a disappearance of D1 binding sites occurred in the absence of significant changes in D2 receptor density. These results suggest that, although D2 receptors seem to be apparently unaffected after forebrain ischemia, a long-lasting impairment of their neosynthesis may be present in striatal D2 dopaminoceptive neurons.

1991 - DIFFERENTIAL ONTOGENIC EXPRESSION AND REGULATION OF PROENKEPHALIN AND PREPROSOMATOSTATIN MESSENGER-RNAS IN RAT CAUDATE-PUTAMEN AS STUDIED BY INSITU HYBRIDIZATION HISTOCHEMISTRY [Articolo su rivista]
Cimino, M; Zoli, Michele; Weiss, B.
abstract

Specific oligonucleotide probes and in situ hybridization histochemistry were used to study the ontogeny and regulation of the mRNAs for proenkephalin A and preprosomatostatin in rat brain. In adult brain the most intense hybridization signal for the proenkephalin A mRNA was in caudate putamen, nucleus accumbens and olfactory tubercle. By contrast, the hybridization signal for preprosomatostatin mRNA was more diffusely scattered throughout the brain, with high signals in the neocortex, olfactory bulb and hippocampal formation. Studies of the ontogeny of these mRNAs revealed a different pattern of ontogenetic expression and differential regulation by dopaminergic input. The mRNA for preprosomatostatin reached the highest level within the first postnatal week, whereas proenkephalin A mRNA progressively increased throughout the entire period studied. In addition the proenkephalin A mRNA showed a medial to lateral gradient in 2-day-old rat striatum which disappeared with increasing age, whereas preprosomatostatin mRNA increased in most brain areas in a fairly uniform fashion with increasing age. Treatment of newborn rats with 6-hydroxydopamine increased the expression of proenkephalin A mRNA by 1.6 fold but had no effect on the expression of preprosomatostatin mRNA. The 6-hydroxydopamine-induced change in proenkephalin A mRNA expression was not observed until postnatal day 32, indicating that enkephalin-containing neurons of the developing striatum are relatively insensitive to dopamine input and that they cannot compensate for the neonatal lesion, despite the fact that the insult was given in a period of high plasticity of the neural tissue.

1991 - REGIONAL INCREASES IN ORNITHINE DECARBOXYLASE MESSENGER-RNA LEVELS IN THE RAT-BRAIN AFTER PARTIAL MESODIENCEPHALIC HEMITRANSECTION AS REVEALED BY INSITU HYBRIDIZATION HISTOCHEMISTRY [Articolo su rivista]
Zoli, Michele; Bettuzzi, Saverio; Ferraguti, F; Ingletti, Mc; Zini, Isabella; Fuxe, K; Agnati, Luigi Francesco; Corti, Arnaldo
abstract

Changes in the level of ornithine decarboxylase mRNA after partial mesodiencephalic hemitransection were evaluated in various regions of the rat brain by means of in situ hybridization histochemistry coupled with computer-assisted image analysis. On days 1 and 2 after the lesion, increased accumulation of ornithine decarboxylase mRNA was observed on the lesioned side in various telencephalic regions (e.g. neostriatum and frontoparietal cortex), and in the pars compacta of the substantia nigra. Both in the frontoparietal cortex and substantia nigra a decreasing gradient of ornithine decarboxylase mRNA activation was observed going far from the site of the lesion. Seven days after the operation, ornithine decarboxylase mRNA levels returned to control values on the lesioned side but increased in some regions, such as the frontoparietal cortex, on the intact side. The present results demonstrate that the parent cell body biosynthetic machinery is activated by the mechanical lesion of the axons at the level of ornithine decarboxylase gene expression. The increase of ornithine decarboxylase mRNA is not as large as the enhancement in ornithine decarboxylase activity previously shown, suggesting that the response to the lesion may also involve changes in the rate of translation of ornithine decarboxylase mRNA and/or in the rate of degradation of ornithine decarboxylase protein.

1991 - SELECTIVE REDUCTION OF GLUCOCORTICOID RECEPTOR IMMUNOREACTIVITY IN THE HIPPOCAMPAL-FORMATION AND CENTRAL AMYGDALOID NUCLEUS OF THE AGED RAT [Articolo su rivista]
Zoli, Michele; Ferraguti, F; Gustafsson, Ja; Toffano, G; Fuxe, K; Agnati, Luigi Francesco
abstract

Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n =5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of age rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat. However, only in the aged hippocampal formation GR decrease seems to be due to a loss of GR-containing neurons. The absence of age-related changes in the number of GR target neurons in all the other areas analyzed (even neurons which contain high levels of GR) suggests that the activation of CR, which are selectively concentrated in the hippocampal formation, or possibly the combined activation of CR and GR, may be responsible for corticosterone cytotoxicity.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL NERVOUS SYSTEM .5. STUDIES ON A MODEL OF TRANSIENT FOREBRAIN ISCHEMIA IN MALE SPRAGUE-DAWLEY RATS [Articolo su rivista]
ZINI, Isabella; GRIMALDI, R; MERLO PICH, Emilio; ZOLI, Michele; FUXE, K; AGNATI, Luigi Francesco
abstract

A morphological and functional characterization of the four-vessel occlusion model of transient (30 min) forebrain ischemia has been carried out. The rats were classified as fully ischemic when an isoelectric pattern of electroencephalographic activity was present within 5 min of the occlusion of carotid arteries. Otherwise they were considered as partially ischemic rats. The modifications of cerebral blood content during and after the ischemic insult were assessed by a histochemical method which visualizes red blood cells in cerebral vessels. The periods of increase and decrease of red blood cell content were found to correspond to previous reports of post-ischemic hyper- and hypoperfusion. Neuronal damage was assessed by a quantitative analysis of Nissl stained preparations of cingulate cortex, dorsal hippocampus and striatum. The signs of morphological damage were quantified by means of computer-assisted image analysis of Nissl preparations. The highest vulnerability to the ischemic insult was demonstrated in the pyramidal layer of the hippocampal CA1 field and in the lateral striatum. Arterial blood pressure measurements were performed during the ischemic and post-ischemic periods, demonstrating a peak increase of arterial blood pressure within 2 min after carotid artery occlusion, followed by a slow decrease towards basal levels during the ischemic period and a full recovery within 15 min of reperfusion. Ischemic rats were tested in a neurological test battery and in a passive avoidance task. While a full recovery of the relatively simple tasks of the neurological test battery was attained within 14 days of reperfusion, a highly significant impairment of passive avoidance behavior was still present 15 days after the ischemic insult. Finally, a discriminant analysis was applied to separate, on the basis of non-invasive techniques (neurological tests and hot plate), the group of completely ischemic rats from that of partially ischemic rats.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL NERVOUS SYSTEM .7. THEORETICAL ASPECTS OF BRAIN COMMUNICATION AND COMPUTATION [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; MERLO PICH, Emilio; Benfenati, Fabio; Fuxe, K.
abstract

Some aspects of the communicational and computational features of the central nervous system are discussed. The existence in the central nervous system of two main types of interneuronal communication, the wiring (i.e. the classical type of synaptic transmission) and the volume (i.e. a humoral type of non-synaptic transmission) transmission, has been proposed. Some features of these types of transmission are discussed, with special reference to the informational properties of peptide transmitters. With respect to the computational aspects of neural function, the identification of putative computational structures at the macroscopic (network) and microscopic (local circuit, synapse) levels suggests the existence of a computational hierarchical organization. In this context, the existence of a compartmental organization of various cerebral regions is discussed. It is hypothesized that membrane domains, made by patches of membrane in which preselected molecular movements are possible resulting in molecular interactions, can have an important role in the integrative capabilities of neural tissue. The coexistence of multiple neuroactive substances in central synapses is analyzed in the framework of information transfer processes at this level. The presence of putative homeostatic, heterostatic and mnestic mechanisms in the synapse is also discussed.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL-NERVOUS-SYSTEM .1. COMPUTER-ASSISTED IMAGE-ANALYSIS METHODS [Articolo su rivista]
Zoli, Michele; Zini, Isabella; Agnati, Luigi Francesco; Guidolin, D; Ferraguti, F; Fuxe, K.
abstract

Microdensitometric and morphometric techniques have been developed to quantitatively characterize cell groups and terminal populations of transmitter-identified neuronal systems. Various microdensitometric methods implemented on the image analyzer or on the scanning microdensitometer were introduced and compared. On this basis a technique to assess the half-life of dopamine stores determined by quantitative immunocytochemistry has been developed. The problem of relative and absolute quantification of microdensitometric analysis of immunocytochemical preparations is discussed here. A method has been developed for the study, both in 2- and 3-dimensions, of the overall features of the profile distribution in a defined neuroanatomical area. An approach to determine the degree of uniformity of a certain profile distribution is also proposed. Furthermore, methods for the evaluation of the codistribution of two or more different types of profiles and to characterize the morphometric features of patches of profiles in a certain region are presented. All these quantitative morphological approaches were tested in relevant preparations of the central nervous system.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL-NERVOUS-SYSTEM .2. METHODOLOGICAL STUDIES ON THE MICRODIALYSIS TECHNIQUE [Articolo su rivista]
Ruggeri, Mirella; Zoli, Michele; Grimaldi, R; Ungerstedt, U; Eliasson, A; Agnati, Luigi Francesco; Fuxe, K.
abstract

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL-NERVOUS-SYSTEM .2. NUMERICAL CLASSIFICATION IN NEUROANATOMY [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Benfenati, Fabio; MERLO PICH, Emilio; Grimaldi, R; Fuxe, K.
abstract

The possibility of using taxonomic techniques to classify neuronal populations was explored. In particular, coefficients of similarity such as the Canberra metric and the Shannon diversity index were examined. The theoretical work in the field of numerical classification was adapted to the aim of characterizing various brain areas in classes according to their transmitter contents. The study of neuropeptide distribution in 15 brain areas clearly demonstrated that, of these, the hypothalamus is particularly noteworthy due to its higher neuropeptide content.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL-NERVOUS-SYSTEM .4. CHEMICAL ANATOMICAL STUDIES ON THE AGING BRAIN [Articolo su rivista]
ZOLI, Michele; BENFENATI, Fabio; MERLO PICH, Emilio; TOFFANO, G; FUXE, K; AGNATI, Luigi Francesco
abstract

Some effects of aging processes on the neurochemical features of central transmitter-identified neuronal populations have been investigated by means of immunocytochemistry and receptor autoradiographic techniques coupled with image analysis. A selective decrease of tyrosine hydroxylase immunoreactivity in the ventrolateral region of the arcuate nucleus in aged rats was observed. The level and turnover (recovery after irreversible blockade of monoamine receptors with the peptide coupling agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) of alpha2-adrenergic ([(3)H]paraaminoclonidine binding) and D2 dopamine ([(3)H]spiperone binding) receptors were reduced in most regions of the rat brain. Peptide receptors showed a more complex pattern of change, since while mu opiate receptors (preferentially labeled with [(3)H]etorphine binding) were reduced in the old animals, delta opiate ([(3)H]DSTLenkephalin binding) receptors were affected only in certain areas. The effect of irreversible blockade of monoamine receptors on mu and delta opiate receptors was also studied in young adult and aged rats. A mu but not delta opiate receptor up-regulation was observed after monoamine receptor blockade in the young adult animals. This effect was greatly reduced in the n. caudatus-putamen, n. accumbens and tuberculum olfactorium of the old animals.

1990 - ASPECTS OF NEURAL PLASTICITY IN THE CENTRAL-NERVOUS-SYSTEM .6. STUDIES OF THE EFFECTS OF GANGLIOSIDES ON BRAIN METABOLIC LESIONS [Articolo su rivista]
Zoli, Michele; Ruggeri, Mirella; Zini, Isabella; Grimaldi, R; MERLO PICH, Emilio; Toffano, G; Fuxe, K; Agnati, Luigi Francesco
abstract

The effects of gangliosides have been studied in two models of metabolic insult (insulin-induced hypoglycemia and transient forebrain ischemia) of the central nervous system. In the severe hypoglycemia experiments lactate extracellular fluid levels were evaluated by means of a microdialysis probe implanted in the frontoparietal cortex. Ganglioside GM1, given either peripherally (10 mg/kg, i.p.) or intracerebrally (2 x 10(?4) M, via the microdialysis probe) 2 h before insulin injection, was able to reduce the decay of the perfusate levels of lactate induced by the insulin injection. In the same animal model peripheral, but not central, administration of GM1 reduced the hypoglycemia-induced increase of cerebral blood flow and increased the survival time observed after the insulin injection. In the experiments on transient forebrain ischemia, a GM1 derivative, AGF2 (5 mg/kg/day, i.p.), was administered chronically, starting 5 days before or the day after the ischemic insult. With both treatment schedules a similar protective effect was observed in a neurological test battery and in the step-through latency in a passive avoidance test.

1990 - Cellular and synaptic alterations in the aging brain. [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Grimaldi, R; Fuxe, K; Toffano, G; Zini, Isabella
abstract

The morphological and functional impairments observed in the aging brain are discussed in the framework of theoretical concepts, such as the existence of different modalities of intercellular communication and of specific trophic features in the central nervous system. The relevance of changes at the cellular level (disappearance of neuronal cell bodies and proliferation of astroglial cells) and at the synaptic level (alterations in neurotransmitter and receptor levels) is discussed. Two, non-mutually exclusive hypotheses are advanced to explain the frequent absence of correlation between neuropathological findings and functional deficits in aged patients. According to the first, the physiological reshaping of brain circuits during aging may lead to "wrong" readjustments of neural networks (e.g. due to less effective endogenous and exogenous orienting signals) causing minor morphological alterations but marked functional deficits. The second hypothesis maintains that the absence of correlation between neuropathological and functional deficits is due to the impairment of restricted neuronal populations ("pacemaker and command neurons") which play a special role in the hierarchical organization of neuronal networks. These neurons (inter alia, peptidergic neurons) may also be involved in volume transmission (diffusion of electrical and chemical signals in the extracellular fluid to reach distant targets). Moreover, the relevance of glial cells, not only as regulators of the extracellular medium but also on the basis of their trophic links with neurons, is considered. Finally, the interplay between trophic factors and therapeutical experience for the maintenance and/or recovery of an impaired function in elderly patients is discussed.

1990 - CHRONIC BUT NOT ACUTE URIDINE TREATMENT REDUCES HALOPERIDOL-INDUCED DOPAMINE RELEASE IN NEOSTRIATUM AS STUDIED BY INTRACEREBRAL MICRODIALYSIS [Articolo su rivista]
Ruggeri, M; Zoli, Michele; Ungerstedt, U; Peruzzi, G; Agnati, Luigi Francesco; Fuxe, K.
abstract

1990 - DISTRIBUTION OF GLUTAMIC-ACID DECARBOXYLASE MESSENGER RNA-CONTAINING NERVE-CELL POPULATIONS OF THE MALE-RAT BRAIN [Articolo su rivista]
Ferraguti, F; Zoli, Michele; Aronsson, M; Agnati, Luigi Francesco; Goldstein, M; Filer, D; Fuxe, K.
abstract

The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number of GAD-immunoreactive cell bodies which express a low level of GAD mRNA. The opposite seems to be true for other nuclei, such as the globus pallidus, the zona reticulata of the substantia nigra and the inferior collicle, where few GAD-immunoreactive cell bodies contain high levels of GAD mRNA. In conclusion, the present study gives a low magnification map of GAD mRNA levels in the adult male rat brain. Marked biochemical heterogeneities may be present among GABA neuronal populations based on their expression of GAD mRNA. The comparison between the present in situ hybridization and previous immunocytochemical studies suggests that there may exist at least two populations of GABA neurons in the brain, having high and low levels respectively of both GAD mRNA and GAD enzyme.

1990 - EFFECTS OF TRANSIENT FOREBRAIN ISCHEMIA ON PEPTIDERGIC NEURONS AND ASTROGLIAL CELLS - EVIDENCE FOR RECOVERY OF PEPTIDE IMMUNOREACTIVITIES IN NEOCORTEX AND STRIATUM BUT NOT HIPPOCAMPAL-FORMATION [Articolo su rivista]
Grimaldi, R; Zoli, Michele; Agnati, Luigi Francesco; Ferraguti, F; Fuxe, K; Toffano, G; Zini, Isabella
abstract

The effects of transient (30') forebrain ischemia (4 vessel occlusion model) on peptidergic neurons and astroglial cells in various diencephalic and telencephalic areas have been analyzed. The study was performed at various time intervals of reperfusion, i.e. 4 h, 1, 7 and 40 days. Neuropeptide Y (NPY), somatostatin (SRIF), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP) and arginine-vasopressin (AVP) immunoreactive (IR) neuronal systems and glial fibrillary acidic protein (GFAP)-IR glial cells have been visualized by means of the indirect immunoperoxidase procedure using the avidin-biotin technique. The analysis was performed by means of computer assisted microdensitometry and manual cell counting. At the hippocampal level a huge reduction of neuropeptide (CCK, SRIF, VIP) IR cell bodies was observed, still present 40 days after reperfusion. On the contrary, in the frontoparietal cortex the number of the neuropeptide (CCK, SRIF, VIP, NPY) IR neurons showed a decrease at 4 h, 1 and 7 days after reperfusion followed by a complete recovery at 40 days. A rapid reduction followed by an almost complete recovery (7 days after reperfusion) was also observed at striatal level where SRIF- and NPY-IR neurons were detected. A marked decrease of NPY-IR terminals was observed in the paraventricular and periventricular hypothalamic nuclei and in the paraventricular thalamic nucleus. AVP-IR was markedly reduced in the magnocellular part of the paraventricular nucleus throughout the analyzed period (7 days after reperfusion). GFAP-IR was increased in the hippocampal formation and neostriatum while a not consistent increase was observed at neocortical level. These data point to a differential recovery of peptide-IR and to a different astroglial response in the various brain areas after transient forebrain ischemia. Region-specific factors rather than factors related to neuronal chemical coding seems to play a major role in determining the vulnerability of neuronal populations to transient ischemia.

1990 - Evidence for a role of neosynthetized putrescine in the increase of glial fibrillary acid protein immunoreactivity induced by a mechanical lesion in the rat brain [Articolo su rivista]
Zini, Isabella; Zoli, Michele; R., Grimaldi; MERLO PICH, Emilio; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

The effect of alpha-difluoromethylornithine (alpha-DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) the rate limiting enzyme of polyamine biosynthesis, was studied on the astroglial reaction in a model of mechanical brain injury. alpha-DFMO markedly decreased the astroglial activation induced by the microdialysis probe implantation in the striatum of the male rat, as studied by glial fibrillary acidic protein (GFAP) immunocytochemistry. This response was restored by putrescine (20 nmol/ml) administered via the microdialysis probe. These results suggest that the astroglial reaction and the polyamine biosynthesis activation induced by a localized mechanical lesion are causally linked phenomena.

1990 - INCREASE IN THE DENSITY OF NEUROPEPTIDE-Y IMMUNOREACTIVE CELL-BODIES SELECTIVELY IN THE FUNDUS STRIATI OF RATS PRENATALLY EXPOSED TO METHYLAZOXYMETHANOL ACETATE [Articolo su rivista]
Zoli, Michele; Peruzzi, G; Cimino, M.
abstract

1990 - Long-lasting reduction of glucocorticoid receptor immunoreactivity in the hippocampal field CA1 but not in the dentate gyrus after neonatal treatment with corticosterone in the rat [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; K., Fuxe; F., Ferraguti; Biagini, Giuseppe; A., Cintra; Ja, Gustafsson
abstract

The aim of the present immunohistochemical study was to investigate the effects of neonatal treatment with corticosterone on the long-term regulation of glucocorticoid receptor in the hippocampus.

1990 - Morphometrical and microdensitometrical studies on peptide- and tyrosine hydroxylase-like immunoreactivities in the forebrain of rats prenatally exposed to methylazoxymethanol acetate. [Articolo su rivista]
Zoli, Michele; MERLO PICH, Emilio; Cimino, M; Lombardelli, G; Peruzzi, G; Fuxe, K; Agnati, Luigi Francesco; Cattabeni, F.
abstract

Methylazoxymethanol acetate (MAM Ac) injected into pregnant rats at a dose of 25 mg/kg at gestational day 15 causes microcephaly due to an atrophy of various telencephalic areas, mainly neocortex, hippocampus and basal ganglia. Previous studies demonstrated alterations in various neurochemical markers of classical transmitter systems in these regions. The present paper deals with changes in peptide and tyrosine hydroxylase (TH)-containing neurons in MAM Ac-induced microcephaly using immunocytochemistry coupled with computer-assisted morphometry and microdensitometry. No change in the number of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in the neocortex and neuropeptide Y (NPY)-immunoreactive neurons in the nucleus caudatus-putamen was found whereas cholecystokinin (CCK)-and NPY-immunoreactive neurons in the neocortex and CCK- and VIP-immunoreactive neurons in the hippocampus were decreased. The reduction of the latter peptide containing neuronal populations led to a maintained density of cells in MAM Ac-exposed rats, due to the parallel reduction of the overall mass of these regions. TH immunoreactivity was found to be unchanged in the basal ganglia, and increased in the cerebral cortex in agreement with previous reports on noradrenaline cortical system after MAM Ac exposure. The present results show a heterogenous vulnerability of different peptide immunoreactive neuronal populations to MAM Ac exposure. The sparing of VIP- and NPY-immunoreactive neurons may be due to their late development in the neocortex and striatum, respectively. The hypothesis is introduced that cortical VIP interneurons can develop independent of marked alterations in the intrinsic circuitry of the cortical region.

1990 - NERVE-CELL CLUSTERS IN DORSAL STRIATUM AND NUCLEUS-ACCUMBENS OF THE MALE-RAT DEMONSTRATED BY GLUCOCORTICOID RECEPTOR IMMUNOREACTIVITY [Articolo su rivista]
Zoli, Michele; Cintra, A; Zini, Isabella; Hersh, Lb; Gustafsson, Ja; Fuxe, K; Agnati, Luigi Francesco
abstract

Glucocorticoid receptor-immunoreactive nerve cells have been analysed in the dorsal striatum and nucleus accumbens of the rat by means of a monoclonal antibody against rat liver glucocorticoid receptor. Glucocorticoid receptor immunoreactivity was present in the nuclei of the vast majority of the striatal nerve cells. The analysis of sections stained with glucocorticoid receptor antibody and cresyl violet showed that around 90% of the entire striatal neuronal population contained glucocorticoid receptor immunoreactivity. By means of the double immunoperoxidase technique evidence was provided that somatostatin- and choline acetyltransferase-immunoreactive nerve cells in the striatum do not contain glucocorticoid receptor immunoreactivity. The density of glucocorticoid receptor-immunoreactive nerve cells in the grey matter and the presence of clusters of glucocorticoid receptor-immunoreactive nerve cells have been investigated in three fields located in the medial and central dorsal striatum and nucleus accumbens at the coronal level A 8620 microns according to the König and Klippel atlas using computer-assisted image analysis. Every aggregate containing three or more glucocorticoid receptor-immunoreactive nerve cells, which had an intercenter distance less than the mean diameter (10-11 microns) of the striatal cells, was considered an island. A higher density of both glucocorticoid receptor-immunoreactive nerve cell nuclei and islands was found in the nucleus accumbens with respect to dorsal striatal areas. The most frequent island formed consisted of three to ten nerve cells both in dorsal striatum and nucleus accumbens. Furthermore, some nucleus accumbens islands contained up to 100 nerve cells, whereas in the dorsal striatum the maximum number of glucocorticoid receptor-immunoreactive nerve cells per island ranged from 50 to 60. The present procedure proved to be a sensitive method to reveal clusters of chemically identified structures and provided evidence for a basic cytoarchitectonic organization of the dorsal striatum and nucleus accumbens of the rat. This paper also demonstrated that the vast majority, but not all, striatal nerve cells contained glucocorticoid receptor immunoreactivity, and thus may be under the control of circulating glucocorticoids. In fact, only small transmitter-identified neuronal populations, such as somatostatin- and choline acetyltransferase-immunoreactive nerve cells, were devoid of glucocorticoid receptor immunoreactivity.

1990 - NEUROPEPTIDE-Y AND CENTRAL CARDIOVASCULAR REGULATION - FOCUS ON ITS ROLE AS A COTRANSMITTER IN CARDIOVASCULAR ADRENERGIC-NEURONS [Articolo su rivista]
Fuxe, K; Aguirre, Ja; Agnati, Luigi Francesco; Voneuler, G; Hedlund, P; Covenas, R; Zoli, Michele; Bjelke, B; Eneroth, P.
abstract

1990 - ON THE ROLE OF NEUROPEPTIDE Y IN INFORMATION HANDLING IN THE CENTRAL-NERVOUS-SYSTEM IN NORMAL AND PHYSIOPATHOLOGICAL STATES - FOCUS ON VOLUME TRANSMISSION AND NEUROPEPTIDE Y DELTA-2 RECEPTOR INTERACTIONS [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Harfstrand, A; Zoli, Michele; Voneuler, G; Grimaldi, R; Pich, Em; Bjelke, B; Eneroth, P; Benfenati, F; Cintra, A; Zini, Isabella; Martire, M.
abstract

1990 - REPEATED ELECTROCONVULSIVE SHOCK INCREASES GLIAL FIBRILLARY ACIDIC PROTEIN, ORNITHINE DECARBOXYLASE, SOMATOSTATIN AND CHOLECYSTOKININ IMMUNOREACTIVITIES IN THE HIPPOCAMPAL-FORMATION OF THE RAT [Articolo su rivista]
Orzi, F; Zoli, Michele; Passarelli, F; Ferraguti, F; Fieschi, C; Agnati, Luigi Francesco
abstract

Rats were submitted to single or repeated (7 days, one session for each day) sessions of electroconvulsive shock. A computer-assisted morphometric and microdensitometric analysis of glial fibrillary acidic protein-, ornithine decarboxylase-, somatostatin- and cholecystokinin-like immunoreactivities was performed in the hippocampal formation and other brain areas. The results of the study showed a significant increase of the intensity of the immunostaining for glial fibrillary acidic protein, ornithine decarboxylase, somatostatin and cholecystokinin in the hippocampal formation and distinctively in the dentate gyrus following repeated, but not single, electroconvulsive shock. No significant change was found in the number of somatostatin- and cholecystokinin-like immunoreactive cell bodies in any hippocampal subregion and in the number of glial cells in the hilus of dentate gyrus in rats treated with single or repeated electroconvulsive shock. It is a distinct possibility that the observed increase in the content of the neuropeptides in the hippocampal formation reflects a compensatory response of the brain to seizure-inducing stimuli and that such an increase may play a role in the therapeutic effect of electroconvulsive shock.

1989 - Demonstration of NPY transmitter receptor mismatches in the central nervous system of the male rat [Articolo su rivista]
Zoli, M.; Agnati, L. F.; Fuxe, K.; Bjelke, B.
abstract

1989 - Effects of transient forebrain ischaemia on vasoactive intestinal polypeptide‐immunoreactive neuronal populations in the frontoparietal cortex and hippocampal formation of the male rat [Articolo su rivista]
Grimaldi, R.; Zini, I.; Ferraguti, F.; Cortelli, P.; Fuxe, K.; Agnati, L. F.; Zoli, M.
abstract

1989 - Integration of humoral and neuronal signals in neuroendocrine regulation. Focus on interaction of glucocorticoids with central neuropeptide and monoamine neurotransmission [Relazione in Atti di Convegno]
Fuxe, K.; Cintra, A.; Tanganelli, S.; Von Euler, G.; Finnman, E. -B.; Lundgren, K.; Aguirre-Gomez, J.; Bjelke, B.; Zoli, M.; Eneroth, P.; Agnati, L. F.
abstract

1989 - Morphometrical and microdensitometrical studies on peptide immunoreactivities in methylazoxymethanol prenatal treated rats [Articolo su rivista]
Zoli, M.; Cimino, M.; Cattabeni, F.; Agnati, L. F.
abstract

1989 - Neurohistochemical studies on striatal lesions induced by transient forebrain ischemia. Evidence for protective effects of the ganglioside analogue AGF2 [Articolo su rivista]
Zoli, M.; Grimaldi, R.; Agnati, L. F.; Zini, I.; Merlo Pich, E.; Toffano, G.; Fuxe, K.
abstract

1989 - Reduced growth hormone releasing factor (GHRF)-like immunoreactivity and GHRF gene expression in the hypothalamus of aged rats. [Articolo su rivista]
DeGennaro Colonna, V; Zoli, Michele; Cocchi, D; Maggi, A; Marrama, Paolo; Agnati, Luigi Francesco; Müller, E. E.
abstract

Growth hormone releasing factor-like immunoreactivity (GHRF-LI) and GHRF mRNA levels were evaluated in the hypothalamus of aged (24 months) and young (3 months) rats by semiquantitative immunocytochemistry and slot blot hybridization technique, respectively. Simultaneous detection of reduced GHRF-LI and GHRF mRNA levels in aged rats as compared to young counterparts demonstrates the existence in aged rats of an impaired function of GHRF-producing neurons.

1989 - Some aspects of the communicational and computational organization of the brain. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Zoli, Michele; Bjelke, B; Zini, Isabella
abstract

Some features of the morphofunctional organization of the CNS have been analysed. Different types of hierarchical organization have been recognized, each of which could deeply affect the circulation (communicational aspect) and elaboration (computational aspect) of information. These two aspects have been discussed on the basis of the existence of two types of electrochemical transmission in the CNS: wiring and volume transmission. By evaluating the CNS operations at different levels of analysis a 'computational hierarchical organization' has been delineated. This concept is very relevant to the understanding of the 'computational power' of the brain (Agnati & Fuxe 1984, Conrad 1985a). In fact, it leads to the distinction between horizontal and vertical elaboration of information. The hypothesis is introduced that in the vertical elaboration of information a central role may be played by the neuronal membrane. In fact, this structure can not only be influenced by the extra- and intracellular signals, but also effectively interconvert the electrical coding into the chemical coding of information. These aspects are discussed in the frame of the possible organization of the membrane into 'domains', each domain being a patch of membrane in which pre-selected molecular movements are possible, resulting in molecular interactions. The movement of a molecule outside its domain is considered energetically unfavourable. The possible formal treatment of this hypothesis is mentioned in Conrad's work (1985b).

1989 - Transient forebrain ischemia produces multiple deficits in dopamine D1 transmission in the lateral neostriatum of the rat. [Articolo su rivista]
Benfenati, Fabio; MERLO PICH, Emilio; Grimaldi, R; Zoli, Michele; Fuxe, K; Toffano, G; Agnati, Luigi Francesco
abstract

Striatal dopamine D1 transmission was studied in rats 7 days after transient (30 min) forebrain ischemia using the 4-vessel occlusion model. The striatal distribution of dopamine D1 ([3H]SCH 23390 binding sites) and D2 ([3H]sulpiride binding sites) receptors as well as the distribution of adenylate cyclase ( [3H]forskolin binding sites) and of the intracytoplasmic dopamine and cAMP-regulated phosphoprotein DARPP-32 related to D1 transmission were analyzed. While the distribution of D2 receptors was unaffected 7 days after the ischemic insult, all the other markers showed a patchy disappearance in the dorsolateral part of the neostriatum. These findings underline the existence of selective multiple deficits in D1 transmission after transient forebrain ischemia in rat striatum.

1988 - D1 receptor mechanisms in the median eminence and their inhibitory regulation of LHRH release. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Andersson, K; Cintra, A; Härfstrand, A; Zoli, Michele; Eneroth, P; Goldstein, M.
abstract

D1 receptor mechanisms in the median eminence have been studied by means of immunocytochemistry using antisera against dopamine and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) and tyrosine hydroxylase (TH) and by autoradiography using the iodinated analogue of the D1 receptor antagonist SCH-23390. The co-distribution of DARPP-32 and TH immunoreactivity (IR) and of DARPP-32 and luteinizing hormone releasing hormone (LHRH) IR was analysed in the median eminence by means of computer-assisted morphometry and microdensitometry. Functional analysis involved studies on the role of D1 receptors in the regulation of LH serum levels in rats treated with nicotine in the absence and presence of the D1 receptor antagonist. LH serum levels were measured by means of radioimmunoassay procedures. The results on the co-distribution of TH and DARPP-32 IR in the median eminence which were obtained both by analysis of adjacent sections and by two-colour immunocytochemistry on the same section, demonstrated a high degree of overlap of TH and DARPP-32 IR nerve terminals and tanycytes within the medial and lateral palisade zone. Furthermore, studies on LHRH and DARPP-32 IR nerve terminals and tanycytes in the median eminence with the same methodologies demonstrated preferential overlaps within the lateral palisade zone. The overlap area was about 50% of the LHRH or DARPP-32 immunoreactive area in this region. Density maps were also obtained on the distribution of LHRH and DARPP-32 immunoreactive profiles at various rostrocaudal levels. Correlation studies demonstrated a significant and positive co-distribution of LHRH and DARPP-32 immunoreactive terminals and tanycytes within the lateral palisade zone and the subependymal layer (when all DARPP-32 positive squares were considered) of the median eminence. Instead within the medial palisade zone a significant negative correlation coefficient was found, when all the LHRH positive squares were considered. In the receptor autoradiographical analysis a weak-to-moderate labelling was obtained of the part outside the mediobasal hypothalamus using the D1 receptor radioligand [(125)I]SCH-23982, while hardly any labelling was found within the median eminence and the arcuate nucleus. SCH-23390 was found to counteract, in a dose-related way, the inhibitory effects of intermittent nicotine treatment on serum LH levels. The D2 receptor antagonist raclopride in a dose of 1 mg/kg did not counteract the inhibitory effects of nicotine on serum LH levels. The present immunocytochemical, autoradiographic and functional studies suggest the existence of a D1 receptor in the median eminence which can be blocked by the D1 receptor antagonist SCH-23390 in behaviourally relevant doses and which is masked under basal conditions in the male rat. It is proposed that one type of median eminence D1 receptor is located on the axon terminals, not linked to DARPP-32, and which may make possible a rapid regulation of hypothalamic hormone release, e.g. LHRH release from the nerve terminals in the lateral palisade zone as indicated in the present morphological and functional experiments. The other type of median eminence D1 receptor may be located on the tanycytes and linked to DARPP-32. It is suggested that this D1 receptor is responsible for a long-term regulation of hypothalamic hormone release inter alia LHRH release from the terminal and preterminal parts of the LHRH axons in the lateral palisade zone and subependymal layer, respectively.

1988 - Effects of chronic uridine treatment on regional neuropeptide and tyrosine hydroxylase-like immunoreactivities in the brain of 12 month-old male rats. [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; Fuxe, K; Cintra, A; Grimaldi, R; Vanderhaeghen, Jj; Eneroth, P; Goldstein, M.
abstract

Uridine was administered in the drinking water (0.5 mg/ml) in adult 6 month-old rats for 6 months. The mean daily dose of uridine was 12.5 mg/rat. The effects of this treatment on tyrosine hydroxylase, galanin, somatostatin, neuropeptide Y and cholecystokinin-like immunoreactivities were studied by means of semiquantitative immunocytochemistry using the peroxidase-antiperoxidase procedure in combination with image analysis. A decrease of somatostatin, cholecystokinin and galanin-like immunoreactivities in nerve terminals was observed in various brain areas of 12 month-old animals compared with 3 month-old animals, while the levels of tyrosine hydroxylase-like immunoreactivity were unchanged. Uridine-treated animals showed a decrease of galanin, neuropeptide Y and cholecystokinin-like immunoreactivities in nerve terminals of some diencephalic areas and an increase of cholecystokinin-like immunoreactivity in nerve terminals of most of the telencephalic brain areas in comparison with vehicle treated animals of the same age. It is suggested that the pyrimidine nucleoside uridine can affect the synthesis and/or degradation of mRNAs involved in the synthesis of neuropeptides via direct nuclear actions and/or indirect actions involving effects on receptor activated phosphoinositide metabolism. Uridine offers a new way to modulate central peptide synapses.

1988 - Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminals and on glucocorticoid receptor-immunoreactive nerve cell nuclei in the paraventricular hypothalamic nucleus in adult and old male rats. [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; Fuxe, K; Zini, Isabella; MERLO PICH, Emilio; Grimaldi, R; Härfstrand, A; Goldstein, M; Wikström, Ac; Gustafsson, J. A.
abstract

The phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminal profiles and the glucocorticoid receptor-immunoreactive nuclear profiles have been characterized in the parvocellular part of the paraventricular hypothalamic nucleus of the adult (3 month) and the old (24 month) male rat. The phenylethanolamine-N-methyltransferase-, neuropeptide Y- and glucocorticoid receptor-immunoreactive structures have been demonstrated by means of the indirect immunoperoxidase procedure and analysed in a quantitative way by means of morphometrical and microdensitometrical approaches using both semiautomatic and automatic image analysis. During aging there is (a) a marked reduction in the number of neuropeptide Y-immunoreactive profiles, a moderate reduction of phenylethanolamine-N-methyltransferase-immunoreactive profiles and a small reduction in the number of glucocorticoid receptor-immunoreactive profiles without a significant change in the evenness of distribution of such profiles as evaluated by means of Gini's index; (b) a loss of the significant correlation in the distribution of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at the two most caudal levels analysed (A5150 and A5270 micron) while a significant correlation developed between these two distributions at a more rostral level (A5400 micron); (c) a substantial decline in the overlap area of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at four out of five rostrocaudal levels analysed; (d) a marked reduction in the density-intensity of the neuropeptide Y-immunoreactive profiles and a small significant reduction in the density-intensity of the phenylethanolamine-N-methyltransferase-immunoreactive profiles without any associated changes in the intensity of the glucocorticoid receptor-immunoreactive profiles. Furthermore, three-dimensional reconstructions of the overall distribution of the glucocorticoid receptor-, phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive structures have been made in the paraventricular hypothalamic nucleus of the adult male rat. The present results indicate a reduction of neuropeptide Y- and phenylethanolamine-N-methyltransferase-immunoreactive nerve terminal profiles in the parvocellular part of the paraventricular hypothalamic nucleus during aging. These results may in part reflect a loss of neuropeptide Y-like peptides in phenylethanolamine-N-methyltransferase-immunoreactive nerve terminals of the paraventricular hypothalamic nucleus, favouring our view that during aging the modulatory peptides may be lost.

1988 - Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive neurons in the rostral medulla oblongata of the adult and old male rat. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zoli, Michele; Zini, Isabella; Härfstrand, A; Toffano, G; Goldstein, M.
abstract

In the present paper the neuronal systems of the medulla oblongata containing phenylethanolamine-N-methyltransferase- and neuropeptide Y-like immunoreactivity have been characterized in adult (3-month-old) and old (24-month-old) male rats. The phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive neurons have been visualized by means of immunocytochemistry (peroxidase-antiperoxidase technique) and analysed in a quantitative fashion by means of morphometrical (phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell groups) and microdensitometrical (phenylethanolamine-N-methyltransferase-immunoreactive cell groups) approaches developed on the IBAS II image analyser (Zeiss-Kontron). During aging there is (a) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive neuropil for both the C1 and C2 adrenaline cell groups; (b) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive cell bodies, which is highly significant only for the C2 cell group; (c) a decrease in the area covered by the phenylethanolamine-N-methyltransferase-positive cell cluster for both C1 and C2 cell groups; (d) a decrease in the degree of phenylethanolamine-N-methyltransferase immunoreactivity present in the C1 and C2 cell groups; (e) a decay of neuropeptide Y immunoreactivity in the C1 and C2 groups, while the C3 group is unaffected by aging as evaluated by number of phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell body profiles. These results indicate heterogeneities in the responses of the adrenaline-neuropeptide Y cell groups to the aging process. The possible functional consequences of aging-induced changes in the cardiovascular adrenergic neurons are discussed, especially in relation to development of hypertension.

1988 - Morphometrical evidence for a complex organization of tyrosine hydroxylase-, enkephalin- and DARPP-32-like immunoreactive patches and their codistribution at three rostrocaudal levels in the rat neostriatum. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zoli, Michele; Ferraguti, F; Benfenati, Fabio; Ouimet, Cc; Walaas, Si; Hemmings HC, Jr; Goldstein, M; Greengard, P.
abstract

Tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein (Mr = 32,000)-like and enkephalin-like immunoreactive profiles and their codistribution have been evaluated at three rostrocaudal levels of the rat neostriatum by means of a computer-assisted morphometrical method, which allows an objective definition of high density/intensity patches using specific antibodies in combination with the peroxidase-antiperoxidase technique. Our results show that both tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein-like and enkephalin-like profiles are organized in patches in the rat neostriatum. In the marginal zone, the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches both occupied a large part of the total area. Moreover, in this zone, these putative markers for pre- and postsynaptic elements of dopaminergic synapses also showed a complete spatial overlap. In contrast, the enkephalin-like immunoreactive patches in the marginal zone occupied a smaller area, and showed only an incomplete, albeit significant overlap with the tyrosine hydroxylase-like immunoreactive/dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive system. In the central zone, tyrosine hydroxylase-like immunoreactive, dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive and enkephalin-like immunoreactive patches occupied a much smaller part of the total area than did those in the marginal zone. Within the central zone, enkephalin-like immunoreactive patches occupied a significantly larger area than did the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches. No consistent pattern of overlap between the three different staining patterns could be seen in the central zone, probably due to the small, inconsistent size of the patches. Trend analysis showed a consistent trend of more tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the dorsal than in the ventral striatum, and a trend of more enkephalin-like immunoreactive patches in the rostral than in the caudal striatum. Our data thus demonstrate that, by using computer-assisted morphometrical techniques, it is possible to describe a non-homogenous but overlapping distribution of tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the rat neostriatum.

1988 - Polyamines, ornithine decarboxylase, and diamine oxidase in the substantia nigra and striatum of the male rat after hemitransection. [Articolo su rivista]
Desiderio, Ma; Zini, Isabella; Davalli, P; Zoli, Michele; Corti, Arnaldo; Fuxe, K; Agnati, Luigi Francesco
abstract

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosynthetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.

1988 - Regeneration in the central nervous system: concepts and facts [Articolo su rivista]
Agnati, L. F.; Zini, I.; Zoli, M.; Fuxe, K.; Merlo Pich, E.; Grimaldi, R.; Toffano, G.; Goldstein, M.
abstract

1987 - Central Glucocorticoid Receptor Immunoreactive Neurons: New Insights into the Endocrine Regulation of the Brain [Articolo su rivista]
Fuxe, K.; Cintra, A.; Harfstrand, A.; Agnati, L. F.; Kalia, M.; Zoli, M.; Wikstrom, A. -C.; Okret, S.; Aronsson, M.; Gustafsson, J. -A.
abstract

1987 - Evidence for discrete alterations in central cardiovascular catecholamine and neuropeptide Y immunoreactive neurons in aged male rats and in genetically hypertensive male rats of the Lyon strain. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Kitayama, I; Zoli, Michele; Janson, Am; Härfstrand, A; Vincent, M; Kalia, M; Goldstein, M; Sassard, J.
abstract

A computer-assisted morphometrical and microdensitometrical analysis has been performed on cardiovascular noradrenaline (NA), adrenaline (A) and neuropeptide (Y (NPY) neurons in adult and 24-month-old male rats and on hypotensive (LL), normotensive (LN) and hypertensive (LH) male rats of the Lyon strain using the indirect immunoperoxidase procedures. It was found that in NPY/phenylethanolamine-N-methyltransferase (PNMT) costoring neurons of the CI area of the rostral medulla oblongata NPY-like immunoreactivity showed a more marked reduction than the PNMT immunoreactivity. Furthermore, within the parvocellular part of the paraventricular hypothalamic nucleus. NPY immunoreactive nerve terminal profiles were much more affected than the PNMT immunoreactive profiles during aging as revealed by a marked reduction in the number of profiles and by a marked reduction of absorbency values in the microdensitometrical analysis. Thus, in the NPY/PNMT costoring neurons of the A C1 group of the ventrolateral medulla projecting, for example, to the hypothalamus, the peptide transmission line may have a special vulnerability to the aging processes which may contribute to the development of hypertension in old people in view of a vasodepressor role of many central NPY/PNMT neurons. An extensive morphometrical and microdensitometrical analysis of the various catecholamine (CA) cell groups of the medulla oblongata of the LL, LN and LH rats of the Lyon strain was performed. In a comparison between LL and LH rats the A2 cell group of the LH strain showed a trend for an increase in the mean tyrosine hydroxylase (TH) immunoreactive cell body area and the C3 group showed a significant increase in the number of PNMT immunoreactive profiles.

1987 - Evidence for the existence of ornithine decarboxylase-immunoreactive neurons in the rat brain. [Articolo su rivista]
Cintra, A; Fuxe, K; Agnati, Luigi Francesco; Persson, L; Härfstrand, A; Zoli, Michele; Eneroth, P; Zini, Isabella
abstract

By means of polyclonal antibodies against purified mouse kidney ornithine decarboxylase (ODC) and using the biotin-avidin immunoperoxidase procedure in combination with image analysis the cellular localization and distribution of ODC immunoreactivity (ODC IR) have been demonstrated in the rat brain. ODC IR was located in a large number of neuronal populations. A nuclear ODC IR was always present but in many neurons also a cytoplasmic localization of ODC IR was demonstrated (dendrites, axons and putative terminals). Based on the present findings central neurons may be mapped out not only on the basis of their transmitter contents but also on the basis of their contents of trophic factors.

1987 - Studies on the cellular localization and distribution of glucocorticoid receptor and estrogen receptor immunoreactivity in the central nervous system of the rat and their relationship to the monoaminergic and peptidergic neurons of the brain. [Articolo su rivista]
Fuxe, K; Cintra, A; Agnati, Luigi Francesco; Härfstrand, A; Wikstrom, Ac; Okret, S; Zoli, Michele; Miller, Ls; Greene, Jl; Gustafsson, Ja
abstract

By means of monoclonal antibodies against the rat liver glucocorticoid receptor (GR) and the human estrogen receptor (ER), in combination with an immunocytochemical analysis, it has been possible to map out GR and ER immunoreactive (IR) neurons in the rat central nervous system and GR IR glial cells in the white matter. The GR IR is located in the cytoplasm and especially in the nucleus while the ER IR is only demonstrated in the nuclei of the neurons. Upon adrenalectomy the GR IR appears to be present exclusively in the cytoplasm, while after castration the ER IR is still exclusively present in the nuclei. Upon corticosterone treatment of the adrenalectomized rat the GR IR is again predominantly found in the nuclei of the neurons. These results indicate that the occupied GR and the unoccupied and occupied ER are located in the nuclei and the unoccupied GR in the cytoplasm. Evidence has been presented that large numbers of monoamine and peptide nerve cell bodies contain GR IR. Furthermore, neuronal GR IR is found in neuronal populations all over the central nervous system, especially in the cerebral cortex, the thalamus and the hypothalamus, indicating a major role of GR in regulating the metabolic and synaptic functions of the brain. The ER IR is instead limited to certain neuronal populations, mainly those of the preoptic area, the bed nucleus of the striae terminalis and the arcuate nucleus, suggesting a specific role in control of LHRH secretion and reproductive behaviour.

1986 - A correlation analysis of the regional distribution of central enkephalin and beta-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zoli, Michele; Zini, Isabella; Toffano, G; Ferraguti, F.
abstract

By means of semiquantitative immunocytochemistry and quantitative receptor autoradiography a correlation analysis has been performed on the pre- and post-synaptic features of enkephalin and beta-endorphin immunoreactive neuron systems of the 3- and 24-month-old male rat. A parallel disappearance of enkephalin- and beta-endorphin-like immunoreactivity and of the density of mu and delta opiate receptors is shown during ageing. Furthermore, the lack of an overall correlation between the amount of pre- and post-synaptic components of the enkephalin and beta-endorphin synapses give evidence for the existence of a volume type of transmission in such systems in the telencephalic, diencephalic and mesencephalic areas analysed.

1986 - Intravenous uridine treatment antagonizes hypoglycaemia-induced reduction in brain somatostatin-like immunoreactivity. [Articolo su rivista]
AGNATI, Luigi Francesco; Fuxe, K; Eneroth, P; ZINI, Isabella; Härfstrand, A; Grimaldi, R; ZOLI, Michele
abstract

By means of radioimmunoassay procedures, cholecystokinin-(CCK) and somatostatin-(SRIF) like immunoreactivity have been studied in the dorsal hippocampal formation and in the frontoparietal cortex of the male rat in insulin-induced hypoglycaemia, leading to an isoelectric EEG pattern. It has been demonstrated that severe hypoglycaemia of 40-min-duration produces a disappearance of SRIF but not of CCK-like immunoreactivity in both cortical regions. It was found that an i.v. injection of uridine but not of saline could significantly counteract the disappearance of SRIF-like immunoreactivity induced by severe hypoglycaemia in both cortical areas. Uridine did not by itself change plasma glucose levels. It is suggested that uridine may prevent release and/or increase synthesis of cortical SRIF peptides in severe hypoglycaemia, possibly due to an action on the metabolism (e.g. by enhancing the resynthesis of phosphatidyl inositol) within the tissue of the cerebral cortex and/or on putative pyrimidine binding sites in the brain controlling SRIF synthesis and/or release. It is possible that uridine in this way may improve recovery of neuronal function within SRIF-immunoreactive neurons of the cerebral cortex after severe hypoglycaemia (which also may be true in other states of reduced metabolic support). These findings suggest a possibility to use uridine in the treatment of Alzheimer's disease and Status epilepticus.

1986 - Studies on neuropeptide Y-catecholamine interactions in the hypothalamus and in the forebrain of the male rat. Relationship to neuroendocrine function. [Articolo su rivista]
Härfstrand, A; Fuxe, K; Agnati, Luigi Francesco; Eneroth, P; Zini, Isabella; Zoli, Michele; Andersson, K; von Euler, G; Terenius, L; Mutt, V; Goldstein, M.
abstract

Neuropeptide Y-catecholamine interactions have been analyzed within the hypothalamus and in the forebrain of male rats by means of immunocytochemistry in combination with morphometry, quantitative histofluorimetry on catecholamine fluorescence in discrete catecholamine nerve terminal systems, biochemical analysis of catecholamines as well as by studies on serum levels of adenohypophyseal hormones vasopressin, adrenocortical hormones and angiotensin II using radioimmunoassay determinations. (1) Morphologic and morphometrical evidence indicates the existence of separate populations of neuropeptide Y and tyrosine hydroxylase immunoreactive nerve cell bodies in the parvo- and magnocellular components of the arcuate nucleus respectively. In addition, a significant codistribution of NPY immunoreactive nerve terminals and tyrosine hydroxylase immunoreactive nerve cell bodies were demonstrated in the ventrolateral part of the magnocellular component of the arcuate nucleus. (2) Immunocytochemical studies on the distribution of tyrosine hydroxylase, phenyl ethanolamine-N-methyltransferase and neuropeptide Y immunoreactive nerve terminal networks in the peri- and paraventricular hypothalamic nucleus indicated that these types of immunoreactive nerve terminals densely innervate the medial and anterior parvocellular part of the paraventricular hypothalamic nucleus and anterior periventricular hypothalamic nucleus. From studies on the pattern of terminal distribution results have been obtained compatible with the view that neuropeptide Y or a neuropeptide Y related peptide can be a comodulator in noradrenaline and adrenaline nerve terminal networks of these regions. (3) Acute intraventricular injections of neuropeptide Y (1.25 nmol) do not change dopamine and noradrenaline levels in any hypothalamic and telencephalic dopamine and noradrenaline nerve terminal system analyzed with the exception of the anteromedial frontal cortex, in which area a significant increase in the dopamine levels was observed as revealed biochemically. (4) By means of the tyrosine hydroxylase inhibition method it was possible to show that acute intraventricular injection of NPY (1.25 nmol) increased dopamine utilization in the medial and lateral palisade zone of the median eminence and in the anteromedial frontal cortex and reduced noradrenaline utilization in the parvocellular part of the paraventricular hypothalamic nucleus, while dopamine utilization was not influenced in the nucleus caudatus putamen, nucleus accumbens or in the tuberculum olfactorium. (5) In the intraventricular experiments reported above neuropeptide Y (1.25 nmol, 1 h) reduced the serum levels of thyreotropin stimulating hormone, prolactin and luteinizing hormone and increased serum corticosterone, adrenocorticotrophin, vasopressin, angiotensin II and aldosterone levels. The presence of the tyrosine hydroxylase inhibitor by itself, increased corticosterone, adrenocorticotrophin and aldosterone serum levels and reduced serum luteinizing hormone levels. Neuropeptide Y together with the tyrosine hydroxylase inhibitor further enhanced the adrenocorticotrophin, angiotensin II and aldosterone serum levels seen with the inhibitor, but could no longer produce its excitatory and inhibitory effects on serum corticosterone and luteinizing hormone levels, respectively. Vasopressin serum levels were increased to the same extent in the absence or presence of tyrosine hydroxylase inhibition. The present morphological, neurochemical and functional studies indicate that neuropeptide Y given intraventricularly inhibit the secretion of prolactin, luteinizing and thyreotropin stimulating hormones probably by activation mainly of neuropeptide Y receptors located in the somadendritic region of the arcuate DA cell bodies, leading to increased activity in inhibitory tubero-infundibular dopamine neurons. In addition, it is suggested that the ability of neuropeptide Y to increase adrenocorticotrophin and corticosterone s

1986 - Studies on peptide comodulator transmission. New perspective on the treatment of disorders of the central nervous system [Capitolo/Saggio]
Fuxe, K; Agnati, Lf; Härfstrand, A; Andersson, K; Mascagni, F; Zoli, Michele; Kalia, M; Battistini, Nino Carlo; Benfenati, F; Hökfelt, T.
abstract

Studies on peptide comodulator transmission. New perspective on the treatment of disorders of the central nervous system.

1985 - Effects of ganglioside GM1 treatment on striatal glucose metabolism, blood flow, and protein phosphorylation of the rat. [Articolo su rivista]
AGNATI, Luigi Francesco; Fuxe, K; BENFENATI, Fabio; ZOLI, Michele; Owman, C; Diemer, NH; Kåhrström, J; Toffano, G; Cimino, M.
abstract

Effects of ganglioside GM1 administration have been studied in unilaterally partially hemitransected rats on striatal energy metabolism, using the radioactive deoxyglucose (DG) technique, on striatal blood flow, using radiolabelled iodoantipyrine (IAP) as tracer, and on cyclic AMP (cAMP) and Ca2+ induced protein phosphorylation in striatal membranes (P2 fraction). Ganglioside GM1 treatment counteracted the imbalance in striatal energy metabolism, in striatal blood flow, as well as in protein phosphorylation found between the striata of the lesioned and unlesioned side, possibly due to excitatory effects on the lesioned side and inhibitory effects on the unlesioned side. In intact animals, GM1 treatment produced a reduction in cAMP and Ca2+ induced striatal protein phosphorylation. Facilitatory actions of the ganglioside GM1 dominate following a lesion, probably due to its possible function as a modulator of receptors for neuronotrophic factors, leading to restoration of metabolic rate and of cAMP and Ca2+ induced protein phosphorylation in the striatum of the lesioned side. The results emphasize that ganglioside GM1 treatment can restore the metabolism of a partially innervated striatum towards normal, as evaluated both at the level of the entire striatal structure by means of the DG and IAP techniques and at the molecular level by means of studies on the cAMP and Ca2+ induced protein phosphorylation.

1985 - Effects of lesions and ganglioside GM1 treatment on striatal polyamine levels and nigral DA neurons. A role of putrescine in the neurotropic activity of gangliosides. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zini, Isabella; Davalli, Pierpaola; Corti, Arnaldo; Calza, L; Toffano, G; Zoli, Michele; Piccinini, G; Goldstein, M.
abstract

The effects of a partial hemitransection at the meso-diencephalic level, with or without chronic ganglioside GMI treatment, have been evaluated on striatal polyamine levels, 7, 14 and 21 days after lesion, as well as on the ability of the polyamine synthesis inhibitor alpha-difluoromethylornithine (alpha-DFMO) to modulate the protective effects of chronic ganglioside GMI treatment against retrograde degeneration of the nigral dopamine (DA) nerve cell bodies (14 day time interval). The striatal polyamine levels were measured by high pressure liquid chromatography after dansylation of the polyamines. The nigral DA nerve cells were studied by means of tyrosine hydroxylase (TH) immunocytochemistry using the indirect immunoperoxidase technique. Quantitation was performed by means of morphometrical evaluation of the TH immunoreactive area of the substantia nigra. Seven days after partial hemitransection there is a marked increase (above 350%) in striatal putrescine levels, which is not modulated by chronic GMI treatment. This marked increase could, to a large extent, be counteracted by simultaneous treatment with alpha-DFMO, which blocks mainly the synthesis of putrescine. Twenty-one days after lesion chronic GMI treatment could produce an increase in striatal putrescine levels on the intact side and also after this time-interval prevent the reduction of striatal spermine levels. It was also found that simultaneous treatment with alpha-DFMO prevents the development of the protective action of chronic ganglioside GMI treatment against retrograde degeneration of the nigral DA neurons.

1985 - Effects of neurotoxic and mechanical lesions of the mesostriatal dopamine pathway on striatal polyamine levels in the rat: modulation by chronic ganglioside GM1 treatment. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zoli, Michele; Davalli, Pierpaola; Corti, Arnaldo; Zini, Isabella; Toffano, G.
abstract

In male rats, partial hemitransections but not 6-hydroxydopamine (6-OHDA)-induced lesions of the mesostriatal dopamine (DA) pathway produce after 7 days a marked and a modest increase of striatal putrescine and spermidine levels, respectively, on the lesioned side. Following chronic ganglioside GM1 treatment of partially hemitransected rats, an increase of striatal polyamine levels was observed also on the intact side. It is suggested that retrograde cell body changes produced by hemitransection may induce striatal ornithine decarboxylase activity and in this way increase striatal putrescine levels, favoring regenerative mechanisms. The increase of striatal polyamine levels by GM1 treatment on the intact side of both 6-OHDA and mechanically lesioned rats compared with intact unoperated rats may also reflect an increased synthesis of striatal polyamines.

1985 - Evidence for cholecystokinin-dopamine receptor interactions in the central nervous system of the adult and old rat. Studies on their functional meaning. [Articolo su rivista]
Agnati, Lf; Fuxe, K; Giardino, L; Calza, L; Zoli, Michele; Battistini, Nino Carlo; Benfenati, F; Vanderhaeghen, Jj; Guidolin, D; Ruggeri, M.
abstract

Evidence has been presented for the existence of interactions between CCK and DA receptors both in striatal and limbic membranes. A similar type of modulation by CCK-8 of DA receptors also exists after chronic neuroleptic treatment indicating that supersensitive DA receptors are also modulated by this peptide. As seen from simulation curves, CCK-8 increases the binding of [3H]DA agonists and reduces the binding of [3H]DA antagonists in striatal membranes, suggesting that CCK-8 may increase striatal DA transmission. Results of this type may underlie some of the non-neuroleptic effects of CCK-8. In the aged brain, the ability of CCK-8 to modulate DA antagonist binding sites is changed such that the binding of [3H]DA antagonists is increased. Thus, in the aged brain, receptor-receptor interactions may be altered, leading to a derangement of heterostatic mechanisms (mechanisms changing chemical transmission without interfering with synaptic homeostasis). It was also demonstrated that during aging there is a preferential disappearance of CCK-like immunoreactivity versus TH immunoreactivity in the nigral DA neurons, especially in the medially located nigral DA cells; furthermore, co-existence in the TH/CCK co-storing terminals in the nucleus accumbens was reduced during aging. Such alterations should also lead to changes in heterostatic regulation because the CCK co-modulation line controlling the DA receptors may be preferentially affected.

1985 - Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. [Articolo su rivista]
Fuxe, K; Wikström, Ac; Okret, S; Agnati, Luigi Francesco; Härfstrand, A; Yu, Zy; Granholm, L; Zoli, Michele; Vale, W; Gustafsson, J. A.
abstract

By means of a monoclonal antibody against the rat liver glucocorticoid receptor (GR) in combination with the indirect immunoperoxidase technique it has been possible to demonstrate GR-immunoreactive nerve and glial cell nuclei all over the tel- and diencephalon of the male rat. Strongly GR-immunoreactive nerve cell nuclei were only present in the parvocellular part of the paraventricular hypothalamic nucleus, in the anterior periventricular hypothalamic nucleus, in the ventral part of the mediobasal hypothalamus, and in the CA1 and CA2 subregion of the hippocampal formation. Within the paraventricular hypothalamic nucleus a substantial overlap exists between the GR-immunoreactive area and the CRF-immunoreactive area. Medium to high densities of moderately GR-immunoreactive nerve cell nuclei were present all over the cortical hemispheres. Medium densities of moderately GR-immunoreactive nerve cells were demonstrated in many thalamic nuclei and in the central amygdaloid nucleus. After adrenalectomy the GR immunoreactivity was predominantly located in the pericaryon. Upon acute corticosterone treatment of adrenalectomized male rats, the GR immunoreactivity was again mainly demonstrated in the nerve cell nuclei indicating that corticosterone can translocate GR from the cytoplasm to the cell nuclei. It is suggested that the hypothalamic GR may be involved in the regulation of especially CRF secretion but also in the secretion of other anterior pituitary hormones such as TRH and somatostatin.

1985 - Morphometrical analysis of the distribution of corticotrophin releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase immunoreactive structures in the paraventricular hypothalamic nucleus of the rat. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Yu, Zy; Härfstrand, A; Okret, S; Wikström, Ac; Goldstein, M; Zoli, Michele; Vale, W; Gustafsson, J. A.
abstract

By means of the indirect immunoperoxidase technique the corticotrophin releasing factor (CRF) and glucocorticoid receptor (GR) immunoreactive nerve cell bodies and the phenylethanolamine-N-methyltransferase (PNMT) immunoreactive nerve terminals in the paraventricular hypothalamic nucleus of the rat have been mapped out in adjacent vibratome sections (30 micron thick). By means of morphometrical analysis using a semiautomatic image analyser, it was possible to obtain density maps of CRF, GR and PNMT immunoreactive structures within the paraventricular hypothalamic nucleus. The statistical analysis by the use of correlation coefficients gives evidence that the PNMT immunoreactive nerve terminals innervate the majority of the CRF immunoreactive nerve cell bodies and that GR are located in the majority of the CRF immunoreactive neurons.

1985 - Striatal ornithine decarboxylase activity following neurotoxic and mechanical lesions of the mesostriatal dopamine system of the male rat [Articolo su rivista]
Agnati, L. F.; Fuxe, K.; Davalli, P.; Zini, I.; Corti, A.; Zoli, M.
abstract

1984 - Further studies on the effects of the GM1 ganglioside on the degenerative and regenerative features of mesostriatal dopamine neurons. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Calza, L; Goldstein, M; Toffano, G; Giardino, L; Zoli, Michele
abstract

By means of computer assisted morphometry and microdensitometry the effects of chronic GM-1 ganglioside treatment have been further evaluated on the degenerative and regenerative features of mesostriatal DA neurons in the rat brain. In this study mainly a rostrocaudal morphometrical analysis was performed in the substantia nigra of the lesioned side. The specificity of the action of the GM-1 ganglioside on the substantia nigra DA cells was evaluated by a comparison with antiinflammatory drugs such as betametazon and acetylsalicylic acid. In the rostrocaudal analysis it was demonstrated that chronic GM-1 treatment preferentially protected the caudally located dopamine nerve cells from degeneration after partial hemitransection, while instead this chronic GM-1 treatment increased tyrosine hydroxylase immunoreactivity mainly within the rostrally located DA nerve cells present close to the site of the lesion. Furthermore, the specificity of the GM-1 action was demonstrated by the absence of protective effects of chronic treatment with betametazon and acetylsalicylic acid on the dopamine nerve cells of the lesioned side. These results open up the possibility that chronic GM-1 treatment, by exerting a stimulatory metabolic action on the DA nerve cells located close to the lesion, can enhance the production of neurotrophic factors in these cells, which in turn can diffuse out to increase the survival of the less severely lesioned DA nerve cells located in the caudal part of the substantia nigra. These results indicate that chronic GM-1 treatment may be beneficial in the treatment of neurons undergoing degeneration, which takes place e.g. in Parkinson's disease and after mechanical injury to the brain due to accidents or neurosurgical operations.

1984 - l-Glutamate reduces the affinity of [3H]N-propylnorapomorphine binding sites in striatal membranes. [Articolo su rivista]
Fuxe, K; Celani, Mf; Martire, M; Zini, Isabella; Zoli, Michele; Agnati, Luigi Francesco
abstract

l-Glutamate but not methyl-D-aspartate (NMDA) or quisqualate ( Quis ) (10(-6 M) in vitro with or without preincubation increased significantly the KD value of the [3H]N-propylnorapomorphine ( [3H]NPA) binding sites by 21 and 36% respectively in striatal membranes of rat without influencing the striatal [3H]spiperone binding sites. The number of striatal [3H]NPA binding sites was not changed by l-glutamate (10(-6) and 10(-5) M) in vitro. There may thus exist interactions between striatal glutamate receptors -- not related to excitatory amino-acid receptors of the NMDA or the QUIS type -- and high affinity striatal DA receptors.

1984 - Studies on neurotensin catecholamine interactions in the hypothalamus and in the forebrain of the male rat. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Andersson, K; Eneroth, P; Härfstrand, A; Goldstein, M; Zoli, Michele
abstract

Neurotensin (NT)-catecholamine (CA) interactions have been characterized at the pre- and post synaptic level in the hypothalamus and the forebrain by a combined morphometrical, receptor autoradiographical, biochemical and quantitative microfluorimetrical analysis as well as by radioimmunoassay determinations of serum levels of adenohypophyseal hormones. |

1983 - Computer-assisted morphometry and microdensitometry of transmitter- identified neurons with special reference to the mesostriatal dopamine pathway. Methodological aspects. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Benfenati, Fabio; Zini, Isabella; Zoli, Michele; Fabbri, L; Härfstrand, A.
abstract

New morphometrical and microdensitometrical approaches for evaluation of transmitter-identified neurons in the central nervous system have been developed. These rely at the presynaptic level on the use of immunocytochemistry and at the postsynaptic level on the use of receptor autoradiography. The immunocytochemical analysis involves the indirect immunofluorescence method and the indirect immunoperoxidase method utilizing cryostat and vibratome sections, respectively. In the postsynaptic analysis cryostat sections and tritium-sensitive film were employed. A block diagram representation of the system of the image analyzer used and its connection with its host computer is given. Furthermore, flow charts of the original software developed by our group in presented. The morphometrical analysis has been performed on coronal sections of rat brain resulting in determinations of cell body and cell group parameters. Based on this information, objective criteria have been introduced to assess the existence of a cell group of transmitter-identified neurons in a three-dimensional frame and to give a morphometrical description of this group in the space. Moreover, new quantitative approaches to describe the dendritic and terminal fields have been introduced and for the first time in this type of morphometrical analysis, the Lorenz curves and the Gini index have been utilized in the description of the pattern of dendritic and terminal networks. By means of these morphometrical approaches it became possible to analyze topological and biochemical heterogeneities within cell groups defined in the rostrocaudal frame. In particular, it has been possible to develop a quantitative method for the evaluation of coexistence in nerve cell bodies. This method has been called the overlap method and allows an analysis cell by cell of the possible coexistence of two or more antigens.

1983 - Evidence for the existence of a dopamine receptor of the D-1 type in the rat median eminence. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Benfenati, Fabio; Andersson, K; Camurri, M; Zoli, Michele
abstract

By means of receptor autoradiography using the dopamine (DA) receptor radioligands [3H]cis(z)-flupenthixol ([3H]FLU), [3H]spiperone, [3H]N-propyl-norapomorphine and amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphtalene-2-(5,8[3H]) in combination with a microdensitometrical analysis indications have been obtained for the existence of a DA receptor of the D-1 type in the median eminence of the male rat. Thus, only [3H]FLU (10-20 nM) strongly labeled both the nuc. caudatus putamen and the median eminence and the labeling was markedly prevented by (+)-butaclamol in both regions. Furthermore, a DA receptor agonist of the D-1 type preferentially displaced [3H]FLU from the median eminence. Thus, a DA receptor of the low affinity type may regulate the secretion of hypothalamic hormones from the median eminence.

1983 - Evidence for the existence of receptor-receptor interactions in the central nervous system. Studies on the regulation of monoamine receptors by neuropeptides. [Articolo su rivista]
Fuxe, K; Agnati, Luigi Francesco; Benfenati, Fabio; Celani, M; Zini, Isabella; Zoli, Michele; Mutt, V.
abstract

Substance P (SP) (10(-8) M) can rapidly reduce the affinity and increase the density of 3H-5-HT binding sites in spinal cord membranes. CCK-8 and CCK-4 (10(-8) M) can rapidly and differentially change the characteristics of 3H-spiperone striatal binding sites linked to DA receptors of the D2 type. CCK-4 increase and CCK-8 reduce the number of striatal binding sites for 3H-spiperone, indicating for the first time separate CCK-4 binding sites. CCK-4 (10(-8) M) but not CCK-8 (10(-8) M) can rapidly reduce the affinity and increase the number of the 3H-spiperone binding sites linked to 5-HT receptors of the dorsal cerebral cortex of rats. CCK-8 (10(-8) M) only produces a trend for a small increase in the Bmax values of these receptors. These results again imply the existence of separate CCK-4 binding sites in this case in the cerebral cortex. Glutamate (10(-6) M), but not N-methyl-D-aspartate (10(-6) M) can rapidly change the characteristics of the 3H-N-propylnorapomorphine (3H-NPA) binding sites in striatal membranes of rats. Glutamate (10(-6) M) increases the density and especially reduces the affinity of the 3H-NPA binding sites, which label D2 and D3 types of DA receptors. Taken together the present findings give evidence that neuropeptide receptors and glutamate receptors can in vitro rapidly modulate the characteristics of different types of DA and 5-HT receptors by way of receptor--receptor interactions at the comodulate level or at the local circuit level. It is hypothesized that these receptor--receptor interactions are of importance for the encoding of short-term memory.

1982 - A new approach to quantitate the density and antigen contents of high densities of transmitter-identified terminals. Immunocytochemical studies on different types of tyrosine hydroxylase immunoreactive nerve terminals in nucleus caudatus putamen of the rat. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zini, Isabella; Calza, L; Benfenati, Fabio; Zoli, Michele; Hökfelt, T; Goldstein, M.
abstract

By means of a semi-automatic image analyzer plugged into an Apple II computer and suitable computer programs it is possible to analyze transmitter-identified nerve terminals. Thus, a densitometric approach is applied on the original photograph followed by systematic sampling which is carried out by means of a grating of circles. This procedure allows the study quantitatively of density and intensities of different types of densely packed tyrosine hydroxylase (TH) immunoreactive nerve terminals of the nuc. caudatus putamen. It is shown that the islandic TH immunoreactive nerve terminals have a higher density, but a TH content similar to the diffuse types of TH immunoreactive nerve terminals in the nuc. caudatus putamen.

1982 - New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. [Articolo su rivista]
Agnati, Luigi Francesco; Fuxe, K; Zoli, Michele; Rondanini, C; Ogren, So
abstract

The concepts of coexistence of transmitters and of receptor-receptor interactions have increased our understanding of the integrative processes regulating synaptic homeostasis and synaptic plasticity. Depending upon the ionotropic or metabotropic characteristics of the cotransmitter, it may be mainly involved in synaptic homeostasis or synaptic plasticity, respectively. A chemical trace of the postsynaptic activity can be obtained because of the plasticity of the receptor molecules. Thus, the heuristic hypothesis is introduced that islands of receptors located on postsynaptic membranes of local circuits can be formed by means of receptor-receptor interactions favouring ordered electrotonic sequences in the local circuits. This hypothesis has been named the receptor mosaic hypothesis of the engram. The islands or clusters of receptors can then store specific and complex information and when activated by the transmitters they may induce unique changes in ion permeability and cell metabolism which, at the local circuit level, can mimic exactly a previous electrotonic sequence. They can therefore represent at least part of the engram. This hypothesis is introduced against the background of the possible existence of different types of encodings of memory.