Giulia PUJA - personale UniMoRe

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Giulia PUJA

Professore Associato
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

Pubblicazioni

- “Nuovi modulatori allosterici positivi delrecettore AMPA” [Brevetto]
U., Battisti; Cannazza, Giuseppe; Puja, Giulia; Carozzo, M.; Braghiroli, Daniela; Parenti, Carlo; Troisi, L.; Jozwiak, K.
abstract

brevetto

2022 - BENZOTHIADIAZINES DERIVATIVES AS NOVEL ALLOSTERIC MODULATORS OF KAINIC ACID RECEPTORS [Articolo su rivista]
Puja, G.; Ravazzini, F.; Losi, G.; Bardoni, R.; Battisti, U. M.; Citti, C.; Cannazza, G.
abstract

The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.

2022 - Modulation of NMDA receptor activity by CR4056, an imidazoline-2 receptor ligand with analgesic properties [Articolo su rivista]
Puja, Giulia; Losi, Gabriele; Rovati, Lucio; Lanza, Marco; Caselli, Gianfranco; Bardoni, Rita
abstract

CR4056 is an imidazoline-2 receptor ligand having potent analgesic activity and synergistic effect with opioids. Very recently it has been found that CR4056 can revert the cognitive impairment in animal models of Alzheimer's disease (AD). Since several lines of evidence highlight the importance of NMDAR modulators in nociceptive signaling and in AD progression, we considered as important to investigate the effects of CR4056 on NMDAR activity. In primary culture of cortical neurons, application of NMDA and glycine elicits a current that is decreased in a dose-dependent fashion by CR4056 (IC50 5.3 ± 0.1 µM). CR4056 antagonism is reversible, not competitive and voltage-independent and it is not blocked by pertussis toxin. CR4056 interacts with the co-agonist glycine site in a competitive way, indeed high glycine concentrations diminish its effect. Fibroblasts expressing different recombinant NMDA receptors are differently modulated by CR4056: the potency and the efficacy of the compound are higher in GluN1- GluN2B than in GluN1-GluN2A containing receptors. In lamina II neurons of spinal cord slices, single stimulation of afferent fibers evokes an NMDA-mediated current that is inhibited by 10 µM CR4056. Repetitive stimulation of the dorsal root at high frequency and high intensity produces a firing activity that is significatively depressed by CR4056. Taken together, our results broad the understanding of the molecular mechanisms of CR4056 analgesic activity, involving the modulation of NMDAR activity. Therefore, we propose that the analgesic action of CR4056 and the neuroprotective effects in AD models may be mediated also by NMDAR inhibition.

2021 - IS PREGNENOLONE SULFATE AN ENDOGENOUS MODULATOR OF NICOTINIC ACH RECEPTOR? [Abstract in Atti di Convegno]
Puja, Giulia; Ravazzini, Federica; Balleza, Daniel; Alessandrini, Andrea
abstract

Pregnenolone sulfate (PregS) is an important endogenous modulator of brain activity. PregS reduces the inhibitory activity of GABAA receptors, increases excitatory neurotransmission potentiating NMDA receptors function, trafficking and glutamate release. These latter actions are mediated by PregS at the level of sigma I receptor, calcium and TRP channels [1]. The peleiotropic mechanism of action of PregS could explain some of the pharmacological effects of the steroid: in preclinical studies PregS is neuroprotective, it potentiates synaptic LTP and improve spatial cognitive performance in rats eventhough the pro-mnestic effect of PregS likely depends on the mode of administration and behavioral model being tested [2]. Because cholinergic transmission is fundamental for cognitive functions such as learning and memory we studied the effect of PregS on the activity of nicotinic Ach receptors (nAchR). Using the patch-clamp technique in the whole-cell configuration we tested PregS on the currents mediated by nAChRs expressed in SH-SY5Y cells and in neurons grown in primary cultures. In SH-SY5Y application of PregS reduced NIC (10 μM) - evoked currents with an IC50 in the low micromolar range (14+9 μM) and a maximal effect of 58+10 %. To investigate the mechanism of PregS antagonism we applied the same concentration (10 μM) of the neurosteroid together with different concentrations of NIC (from 5 to 100 μM). PregS effect was not dependent on agonist concentration suggesting a non competitive mechanism of action. The analysis of the dose-response curves of PregS effect at 10 and 100 μM NIC evidentiates that potency and efficacy of the steroid is unchanged. PregS reduces nAchR-mediated currents by increasing receptor desensitization and this effect is more pronounced at high agonist concentrations. Experiments in primary cultures of cortical and cerebellar neurons have confirmed the antagonistic effect of PregS on NIC-evoked current. The inhibitory effect of PregS of nAchR- mediated current cannot support its activity as cognitivie enhancer. To find a rationale it has to be taken into account an additional complexity resulting from the activity of PregS on a neuronal network: it is possible that the reduction of cholinergic activity be limited to specific inhibitory circuits and that the overall effect will be an improved excitatory neurotransmission. It is also conceivable that PregS effect could be served as a compensatory mechanism

2021 - Mechanisms of Peripheral and Central Pain Sensitization: Focus on Ocular Pain [Articolo su rivista]
Puja, G.; Sonkodi, B.; Bardoni, R.
abstract

Persistent ocular pain caused by corneal inflammation and/or nerve injury is accompanied by significant alterations along the pain axis. Both primary sensory neurons in the trigeminal nerves and secondary neurons in the spinal trigeminal nucleus are subjected to profound morphological and functional changes, leading to peripheral and central pain sensitization. Several studies using animal models of inflammatory and neuropathic ocular pain have provided insight about the mechanisms involved in these maladaptive changes. Recently, the advent of new techniques such as optogenetics or genetic neuronal labelling has allowed the investigation of identified circuits involved in nociception, both at the spinal and trigeminal level. In this review, we will describe some of the mechanisms that contribute to the perception of ocular pain at the periphery and at the spinal trigeminal nucleus. Recent advances in the discovery of molecular and cellular mechanisms contributing to peripheral and central pain sensitization of the trigeminal pathways will be also presented.

2020 - BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level [Articolo su rivista]
Avallone, Rossella; Lucchi, Chiara; Puja, Giulia; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases.

2020 - In vitro biocompatibility screening of biopolymers for the development of neuromorphic medical devices [Abstract in Atti di Convegno]
Ren, E; Mcglynn, E; Das, R; Heidari, H; Puja, G; Curia, G
abstract

2020 - Novel Dithiolane-Based Ligands Combining Sigma and NMDA Receptor Interactions as Potential Neuroprotective Agents [Articolo su rivista]
Franchini, S.; Linciano, P.; Puja, G.; Tait, A.; Borsari, C.; Denora, N.; Iacobazzi, R. M.; Brasili, L.; Sorbi, C.
abstract

Sigma receptors (SRs) are recognized as valuable targets for the treatment of neurodegenerative disorders. A series of novel SRs ligands were designed by combining key pharmacophoric amines (i.e., benzylpiperidine or benzylpiperazine) with new 1,3-dithiolane-based heterocycles and their bioisosters. The new compounds exhibited a low nanomolar affinity for sigma-1 and sigma-2 receptors. Five selected compounds were evaluated for their neuroprotective capacity on SH-SY5Y neuroblastoma cell line. They were able to counteract the neurotoxicity induced by rotenone, oligomycin and NMDA. Competition studies with PB212, a S1R antagonist, confirmed the involvement of S1R in neuroprotection from the oxidative stress induced by rotenone. Electrophysiological experiments performed on cortical neurons in culture highlighted the compounds ability to reduce NMDA-evoked currents, suggesting a negative allosteric modulator activity toward the NMDA receptor. Altogether these results qualify our novel dithiolane derivatives as potential agents for fighting neurodegeneration.

2020 - Thyroid hormones reduce nicotinic receptor mediated currents in SH-SY5Y neuroblastoma cells [Articolo su rivista]
Puia, G.; Ravazzini, F.
abstract

Background: Thyroid hormones (THs) are crucial for maturation and functioning of mammalian CNS. THs “classical” signaling involves nuclear receptors binding but also their non genomic actions, as rapid modulators of cell activity, are widely recognized. Since THs imbalance affects cognition and the cholinergic system is deeply involved in learning and memory processes we have studied THs effects at the level of the nicotinic acetylcholine receptors (nAchR). Methods: We used the patch-clamp technique to analyze T3 and T4 modulation of nicotine (NIC)-mediated current in SH-SY5Y neuroblastoma cells. Results: Both hormones decreased NIC-evoked current in a dose dependent fashion. The antagonism was reversible, not competitive and not blocked by Tetrac, an integrin αVβ3 receptor antagonist. A similar effect was detected with the endogenous agonist Acetylcholine. THs potencies were higher at 100 μM NIC (IC50 = 4.6 ± 2 μM for T3 and 4.8 ± 2 μM for T4) compared to those measured at 10 μM NIC (IC50 = 10 ± 4 μM for T3 and 8 ± 4 μM for T4). Furthermore, the efficacy of THs reached almost 90% at 100 μM NIC while was about 30 % at 10 μM NIC. THs inhibited nAchR-mediated currents by enhancing receptor desensitization and this effect was more pronounced at high agonist concentrations. Conclusions: Our results make light on a new non genomic activity of THs at the level of nAchR. This mechanism of action of THs can provide a new explanation for the cognitive deficits associated with tyroid dysfunction.

2017 - EFFECT OF NEUROSTEROIDS ON ARTIFICIAL AND NATIVE PLASMA MEMBRANES : role for the lipid bilayer in determining their mechanism of action. [Abstract in Atti di Convegno]
Puja, Giulia
abstract

2016 - An unexpected reversal in the pharmacological stereoselectivity of benzothiadiazine AMPA positive allosteric modulators [Articolo su rivista]
Battisti, UMBERTO MARIA; Citti, Cinzia; Rastelli, Giulio; Pinzi, Luca; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Cannazza, Giuseppe
abstract

Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure-activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD.

2016 - Erratum: Pro-cognitive activity in rats of 3-furan-2-yl-Np-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor (British Journal of Pharmacology (2015) 172 (5123-5135) DOI: 10.1111/bph.13277) [Articolo su rivista]
Potasiewicz, A.; Kos, T.; Ravazzini, F.; Puia, G.; Arias, H. R.; Popik, P.; Nikiforuk, A.
abstract

2016 - Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content [Articolo su rivista]
Arias, Hugo R; Ravazzini, Federica; Targowska Duda, Katarzyna M.; Kaczor, Agnieszka A.; Feuerbach, Dominik; Boffi, Juan C.; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M.; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puja, Giulia
abstract

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50 = 26 ± 6 μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

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 - Editorial on "New perspectives in neurosteroids action: a special player allopregnanolone" [Articolo su rivista]
Magnaghi, Valerio; Puja, Giulia
abstract

Editorial

2015 - Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study [Articolo su rivista]
Sacchi, Mattia; Puja, Giulia; Vena, Giulia; Alessandrini, Andrea; Facci, Paolo; Balleza, Daniel
abstract

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happenswhen the cholesterol concentration is decreased. Interestingly, an isoform of Allo, Isoallopregnanolone (3β,5α -tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

2015 - Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study [Articolo su rivista]
Balleza, Daniel; Sacchi, Mattia; Vena, Giulia; Galloni, Debora; Puja, Giulia; Facci, Paolo; Alessandrini, Andrea
abstract

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

2015 - Modulatory effects of neurosteroids and thyroid hormones on GABA-evoked currents in cultured dorsal root ganglion cells [Abstract in Atti di Convegno]
Puja, Giulia; Ravegnani, Laura; Ravazzini, Federica; Avallone, Rossella; Bardoni, Rita
abstract

Neurosteroids (NSs) and Thyroid hormones (THs, T3, triiodothyronine and T4, thyroxine) are important endogenous modulators of GABAA receptor (GABAAR) function (Puia and Losi, 2011). The involvement of NSs in several physiological and pathological processes has been largely acknowledged, among them pain transmission. Several studies revealed the antinociceptive properties of some NSs and demonstrated that they can induce a potent peripheral analgesia via a direct GABAAR allosteric modulation (Poisbeau et al., 2014). Very little is known instead of how THs affect synaptic transmission in structures devoted to pain transmission. Pain sensitivity is related to the thyroid status, indeed hyperthyroidism confers greater sensitivity to thermal noxious stimuli (Edmondson et al., 1990) and alters the nociceptive responses in rats (Bruno et al., 2005). Dorsal root ganglion (DRG) cells are primary sensory neurons playing important roles in pain transmission between periphery and CNS. By using the patch clamp technique in the whole cell configuration we analyzed the effect of THs and of some NSs (Pregnenolone Sulfate, PS, and Allopregnenolone, ALLO) on GABA-evoked currents in rat DRG cells grown in primary cultures T3, T4 and PS (from 500 nM to 50 µM) reduce GABA-evoked currents with an IC50 of 0.8±0.3μM for T3 (eff max =-42±9%), of 1.4±0.7 μM for T4 (eff max =-41±6%) and of 4.3±1.2μM (eff max =-60±8%) for PS. ALLO potentiates GABA-evoked current in DRG neurons with an IC50 of 1.3±0.8 μM and a maximal effect of 110±20%. To investigate the mechanism of action of THs and PS we applied increasing concentrations of GABA (5, 10 and 50 µM) to the same concentration of modulator (10 µM). The effect of T3, T4 and PS was not dependent on the GABA concentration used suggesting that they act in a non-competitive way. The modulatory activity of THs and PS on sIPSCs amplitude and frequency measured in lamina II neurons of acutely dissociated spinal cord slices was also investigated. In conclusion, since DRG neurosteroidogenesis is a physiologically relevant process (Schaeffer et al., 2010), our findings suggest that NSs modulation of GABAAR in this cells could play an important role in pain transmission from periphery to spinal cord. Furthermore the decreased GABAAR activity induced by T3 and T4 result in a reduced inhibitory neurotransmission that could contribute to the increased pain sensitivity detected in hyperthyroid animals.

2015 - PKCε and allopregnanolone: functional cross-talk at the GABAA receptor level [Articolo su rivista]
Puja, Giulia; Ravazzini, Federica; Castelnovo, Luca Franco; Magnaghi, Valerio
abstract

Changes in GABAergic inhibition occur during physiological processes, during response to drugs and in various pathologies. These changes can be achieved through direct allosteric modifications at the γ-amino butyric acid (GABA) type A (GABAA) receptor protein level, or by altering the synthesis, trafficking and stability of the receptor. Neurosteroids (NSs) and protein kinase C (PKC) are potent modulators of GABAA receptors and their effects are presumably intermingled, even though evidence for this hypothesis is only partially explored. However, several PKC isoforms are able to phosphorylate the GABAA receptor, producing different functional effects. We focused on the ε isoform, that has been correlated to the sensitivity of the GABAA receptor to allosteric modulators and whose expression may be regulated in peripheral sensory neurons by NSs. The cross-talk between PKC-ε and NSs, leading to changes in GABAA receptor functionality, is considered and discussed in this perspective.

2015 - Pro-cognitive activity in rats of 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor [Articolo su rivista]
Potasiewicz, A; Kos, T; Ravazzini, F; Puja, Giulia; Arias, H. R; Popik, P; Nikiforuk, A.
abstract

BACKGROUND AND PURPOSE: α7 nicotinic acetylcholine receptors (α7 nAChRs) may represent useful targets for cognitive improvement. The aim of this study is to compare the pro-cognitive activity of selective α7-nAChR ligands, including the partial agonists, DMXBA and A-582941, as well as the positive allosteric modulator, 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). EXPERIMENTAL APPROACH: The attentional set-shifting task (ASST) and the novel object recognition task (NORT) in rats, were used to evaluate the pro-cognitive activity of each ligand [i.e., PAM-2 (0.5, 1.0, and 2.0 mg·kg(-1) ), DMXBA and A-582941 (0.3 and 1.0 mg·kg(-1) )], in the absence and presence of methyllycaconitine (MLA), a selective competitive antagonist. To determine potential drug interactions, an inactive dose of PAM-2 (0.5 mg·kg(-1) ) was co-injected with inactive doses of either agonist - DMXBA: 0.1 (NORT); 0.3 mg·kg(-1) (ASST) or A-582941: 0.1 mg·kg(-1) . KEY RESULTS: PAM-2, DMXBA, and A-582941 improved cognition in a MLA-dependent manner, indicating that the observed activities are mediated by α7 nAChRs. Interestingly, the co-injection of inactive doses of PAM-2 and DMXBA or A-582941 also improved cognition, suggesting drug interactions. Moreover, PAM-2 reversed the scopolamine-induced NORT deficit. The electrophysiological results also support the view that PAM-2 potentiates the α7 nAChR currents elicited by a fixed concentration (3 μM) of DMXBA with apparent EC50 = 34 ± 3 μM and Emax = 225 ± 5 %. CONCLUSIONS AND IMPLICATIONS: Our results support the view that α7 nAChRs are involved in cognition processes and that PAM-2 is a novel promising candidate for the treatment of cognitive disorders.

2014 - A Novel class of positive allosteric modulators of KA receptors [Abstract in Atti di Convegno]
Puja, Giulia
abstract

KA-receptors (KARs) are widely expressed in the central nervous system and play an important role in short and long term plasticity (Andrade-Talavera et al., 2012). Because KARs are involved in presynaptic modulation of neurotransmitter release (Sihra and Moreno, 2013) and control both GABA and glutamate release (Jane et al., 2009) they represent a potential target for cognition and memory enhancement drugs. Compounds such as cyclothiazide and diazoxide, acting on AMPARs, potentiate glutamate currents through a removal of receptors desensitization (Yamada and Tang, 2003). More recently benzothiazides derivatives, such as 7-chloro-3- methyl-3, 4- dihydro - 2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21) and 8-chloro-2,3,5,6- tetrahydro - 3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (FUR) were studied because of their ability to cross more efficiently the blood brain barrier and their capability to potentiate AMPA receptor activity (Bertolino et al.1993; Battisti et al 2012). The modulation of these compounds of KAR activity was not investigated so far. By using the patch clamp technique in the whole cell configuration we studied IDRA21 and FUR on primary culture of cerebellum granule cells and in HEK293 transiently transfected with different subunits of KARs. KA-evoked currents were potentiated by both IDRA 21 and FUR, even though the latter compound was more potent (EC50IDRA21 191±58 μΜ; EC50RAC 7±2 μM). Co-application of KA and GYKI 53655, an antagonist of AMPAR, elicits a fast desensitizing current of low amplitude that was augmented in a dose-dependent manner by IDRA21 (EC50 = 538±240μM) and FUR (EC50 20±4μM). To investigate whether a selectivity of the effect for certain KARs subunit exists, we analyzed the effect of both IDRA21 and FUR in HEK293 cells transiently transfected with GluR5, GluR6 and GluR5-KA1 subunits. IDRA21 potentiates Glutamate-evoked currents in a dose-dependent fashion with EC50 of 595±167μM, 653±273μM and 668±271 in GluR5, GluR6 and GluR5-KA1, respectively. The potency of RAC was different in the diverse subunit assembly being higher in GluR5 expressing cells ( EC50=113±50μM, 188±95μM and 550±145μM for GluR5, GluR6 and GluR5-KA1, respectively). The efficacy of RAC was always higher than that of IDRA21 in all the subunit combination OASIS Helpdesk Powered by OASIS, The Online Abstract Submission and Invitation System SM © 1996 - 2014 Coe-Truman Technologies, Inc. All rights reserved. tested. Our data evidentiate that IDRA21 and FUR not only modulate AMPAR but also KAR activity. Considered the importance of KA receptors in memory procesess we think that these drugs exert their pharmacological effect also because of their capability of modulate this receptor activity. :

2014 - Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide [Articolo su rivista]
Carrozzo, Marina Maria; Battisti, UMBERTO MARIA; Cannazza, Giuseppe; Puja, Giulia; Ravazzini, Federica; Falchicchio, Aurelia; Perrone, Serena; Citti, Cinzia; Jozwiak, Krzysztof; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino
abstract

Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.

2013 - MODULATORY EFFECT OF THYROID HORMONES AND NEUROSTEROIDS OF GABA –EVOKED CURRENTS IN CULTURE DRGs. [Abstract in Atti di Convegno]
Puja, Giulia
abstract

Thyroid hormones (THs) and neurosteroids (NSs) are endogenous modulators of GABA-A receptors in the central and peripheral nervous system. Recent studies reported an increase in NSs levels after injury in rats ( Di Michele et al., 2000), and NSs administration reduces inflammatory and neuropathic pain (Charlet et al., 2008) suggesting their involvement in the physiology and pathology of pain transmission. Very little informations are available instead on the participation of THs in pain mechanisms (Bruno et al.2005). The aim of our study was to analyze the effect of THs (3-iodothyronine ,T3, thyroxine, T4) and of the NSs (Pregenolone Sulfate , PS, and allopregnenolone, ALLO) on GABA -evoked current in rat dorsal root ganglia cells in culture. The experiments were performed using the whole cell patch clamp technique. To highlight the nociceptors, at least one sub-population, we used capsaicin, a marker for pain cells expressing TRPV1, a specific nociceptive receptor. Our results show a reduction of GABA current in capsaicin positive cells after applications of THs (both at 10 μM ) and PS (10 uM) of 29,3% ± 8,2 for T3, 23,8%± 5,9 for T4 and 63,8%± 19,6 for PS. ALLO (10 μM) potentiated GABA current of 20 % ±3,5 and applied by itself elicited an inward current. Our results suggest a possible involvement of THs at the level of nociceptive peripheral transmission and open a new view on the complex influences existing among hormones and nociception.

2013 - Neurosteroid modulation of cortical network excitability [Abstract in Atti di Convegno]
Puja, Giulia
abstract

Neurosteroids (NSs) such as allopregnanolone (ALLO) and tetrahydrodeoxy-corticosterone (THDOC ) are well known modulators of GABAA receptor function. While a large amount of data are available at the single cell level, their “global modulation” of network firing activity was not investigated in detail. NSs levels vary in the diverse brain regions and may change under different physiological and pathological conditions [1-3]. The NSs synthesizing enzymes, 5α-reductase type I and 3α-hydroxysteroid dehydrogenase have also different expression profiles, i.e. in the neocortex they co-localize in glutamatergic but not in GABAergic neurons [4]. Therefore these endogenous substances in excitatory neurons that co-express GABAA receptors and NSs synthetic enzymes can act in an autocrine manner [5] while in inhibitory cells their modulation is different. The goal of our study was to investigate the effects of physiological NSs concentrations on the activity of networks formed by acutely dissociated mouse neocortical neurons. By using the multi-electrode arrays (MEA) technique the spontaneous reverberating activity of excitatory and inhibitory neurons was simultaneously recorded and analyzed in control and after perfusion with NSs. Their effects were analyzed, by using statistical methods [6], on physiological variables such as excitability (spikes-per neuron) and number of neurons engaged in bursts. THDOC, at physiological concentrations, selectively decreased inhibitory interneuron activity, whereas at concentrations higher than 100 nM it inhibited both excitatory and inhibitory clusters. The analysis of the network activity after long time wash-out (8 hrs) highlight that the NS effect on inhibitory clusters persisted for hours producing a sort of long-term depression (LTD) in their excitability. To further clarify this point we applied THDOC twice (after a 2h wash-out). The first application induced, as expected, a persistent depression of inhibitory neurons but after the second administration of THDOC no further LTD was observed. This experiment suggests that the first application of the NS produces some stable modifications, a sort of “memory” in the network that could be relevant also in vivo. THDOC and ALLO, at low concentrations, are allosteric modulators of GABAergic neurotransmission but in the μM concentration range they also act as GABAA receptor agonists [7]. In agreement with these properties, our analysis of the global excitability of the network showed a sharp increase in NSs inhibitory effects at concentrations between 100 and 1000 nM, probably due to a direct agonistic activity at GABAA receptors. Many single-cell studies of the effects of ALLO and THDOC have been performed, but no differences were reported between their effects. We show here that THDOC and ALLO, although consistently producing network inhibition at high concentrations, at low concentrations (10-100 nM) have different effects on excitatory and inhibitory clusters; moreover, the network recovered more easily from ALLO than from THDOC effect. Previous studies suggested that tonic GABAergic currents are highly sensitive to NSs [8]. To investigate whether this happens also in our experimental mode we applied Gabazine (GBZ), a GABAA receptor antagonist, at concentrations that only block the phasic GABAergic current. GBZ 100 nM increased the activity of inhibitory neurons, leaving almost unaffected the excitatory neuron excitability suggesting that interneurons are controlling each other mainly through a phasic inhibition. In these conditions the excitability of the network was reduced but also the sensitivity to THDOC was unexpectedly decreased by approximately one order of magnitude compared to control. A detailed analysis [6, 9] of the burst properties showed that in the presence of NSs, the neuronal activity changed and became heterogeneous because of the appearance of different states with oc

2013 - NON GENOMIC EFFECTS OF THYROID HORMONES ON NICOTINIC NEUROTRANSMISSION [Abstract in Atti di Convegno]
Puja, Giulia
abstract

Thyroid hormones (THs) play a crucial role for the correct functioning of the mammalian central nervous system. Their genomic actions are well known and recently the attention has been focused on their novel non-genomic effects as modulators of several neurotransmitter systems (Losi et al. 2008, Puia et. al 2011). Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the brain and play important roles in regulating neuronal activity in several brain areas. By using the patch clamp technique in the whole cell configuration, we tested the effect of THs on nAChRs expressed in SHSY5Y neuroblastoma cells, in primary cultures of cortical neurons and in 293 HEK cell lines. In SHSY5Y, THs showed a dose-dependent reduction of nicotine (NIC) (10μM) current ( IC50 T3 10+/- 2 μM; IC50 T4 7+/- 2.5 μM). Their effect was not competitive and more pronounced at high agonist concentrations. Similar results were obtained using Acetylcholine (Ach) as agonist. THs reduced NIC current in cortical neurons even though their modulatory effects were variable and less marked. To test possible subunit specificity for T3 and T4 modulation, we analyze their effect in HEK293 cells expressing α4β2 nAChRs. T3 (1μM) reduced Ach (1μM)-evoked current of 32 +/- 9 % and T4 of 26 +/- 6 %. It's well known that several cognitive impairments have been resulted from THs deficiency and since “nicotinic circuits” are deeply implicated in learning and memory processes, is possible that also the reported non genomic effect of these hormones could regulate brain function.

2013 - Simultaneous determination of pregnenolone sulphate, dehydroepiandrosterone and allopregnanolone in rat brain areas by liquid chromatography-electrospray tandem mass spectrometry [Articolo su rivista]
Rustichelli, Cecilia; Pinetti, Diego; Lucchi, Chiara; Ravazzini, Federica; Puja, Giulia
abstract

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain.

2013 - Synthesis and biological evaluation of new 2-amino-6-(trifluoromethoxy)benzoxazole derivatives, analogues of riluzole [Articolo su rivista]
Calabrò, M. L.; Caputo, R.; Ettari, R.; Puja, Giulia; Ravazzini, F.; Zappalà, M.; Micale, N.
abstract

This manuscript describes the synthesis of a new series of 2-amino-6-(trifluoromethoxy)benzoxazole derivatives (1–5) and a benzothiazine derivative (6) structurally related to riluzole, the only neuroprotective drug currently approved for the treatment of the amyotrophic lateral sclerosis. Preliminary results indicate that the synthesized compounds, in particular benzamide derivatives 3 and 4, are able to antagonize voltage-dependent Na+ channel currents, and therefore they could be exploited as new inhibitors of these channels. Moreover, all compounds possess low binding affinity for GABA and NMDA receptors.

2012 - Glia-neuron interactions: neurosteroids and epileptogenesis. [Capitolo/Saggio]
Biagini, Giuseppe; C., Marinelli; G., Panuccio; Puja, Giulia; M., Avoli
abstract

Glia can influence the outcome of an epileptogenic insult by controlling the recovery of neuronalnetworks and functions. In particular, glia may facilitate the establishment of epilepsy by impairedremoval of glutamate from synapses or by releasing inflammatory cytokines and excitatoryneurotransmitters, such as interleukin-1β or, respectively, glutamate, aspartate and D-serine.Opposed to these pro-excitatory/pro-epileptogenic mediators, glia can also release molecules thatrestrain neuronal excitability such as neurosteroids, which are potent modulators of inhibitorycurrents dependent on γ-aminobutyric acid (GABA) type A receptors. In normal conditions,neurosteroids are mainly synthesized in neurons by conversion of cholesterol to pregnenolone, a stepcatalyzed by the cytochrome P450 cholesterol-side chain cleavage enzyme (P450scc). Following anepileptogenic insult, astrocytes transform into reactive cells and express high levels of P450scc, thusbecoming major players in neurosteroid synthesis. In this context, we found that the degree ofP450scc expression in astrocytes dictates the duration of the latent period. In line with this view,inhibition of neurosteroid synthesis anticipates the establishment of chronic epilepsy only when theP450scc induction is intense and long lasting. Thus, we hypothesize that reactive astrocytes maydampen neuronal excitability in the course of epileptogenesis through neurosteroid-mediatedmechanisms that likely enhance GABAergic neurotransmission.

2012 - IN VITRO TOXICITY OF METAL NANOPARTICLES IN NEURONAL AND GLIAL CELLS [Poster]
Corsi, Lorenzo; Puja, Giulia; Artoni, Erica
abstract

Several in vitro studies have indicated the potential toxicity of NPs to various types of neuronal and glial cells. SH-SY5Y neuroblastoma cell line has already been used to assess NP-induced neurotoxicity. Exposure of SH-SY5Y cells to Fe2O3, CuO and ZnO NPs was found to decrease cell viability showing a dose-dependent toxic response [3]. Similarly human glioblastoma U87MG cell line was studied to evaluate the effect of silicon dioxide nanoparticles [4]. The results showed a decreased mitochondrial energy production and an altered cell survival/proliferation signaling. At present little is known concerning the potential adverse effects of the NPs on neuronal cells, thus it seems urgent to investigate a possible NPs role in neurotoxicity. U87MG cells showed a decrease in cell viability following treatment with NPs of iron, cobalt oxide and cerium oxide at both 48 and 72h. Cobalt NPs reduced the cell viability of approximately 21% after 48 hours of exposure and the effect remained the same even after 72h. NPs of cerium oxide showed a decrease in cell viability comparable to that of cobalt NPs (21%) but only at the longer incubation time (72h). On the contrary, gold particles did not show any modulation of cell viability. In SH-SY5Y cells all the NPs tested showed a decrease of cell viability after 48 and 72 hours of exposure. Indeed, after 72h the NPs of gold and iron oxide showed a decrease of cell viability by over 16% relative to the control, whereas cobalt and of cerium oxide NPs reduced the cell viability by approximately the 32%. Rat primary cells showed no significant decline in cell viability after 72h of exposure to gold and iron oxide NPs in both sub population (glial cells and neurons). Instead cobalt and cerium oxide NPs led to a decrease of cell viability in glial cells and neurons of 56% and 23% respectively after 72h. Interestingly glial cells, as verified by ESEM imaging, internalized most of the particles but the cell mitochondrial metabolism seemed to be less affected than the neuronal one.

2012 - Novel modulatory effects of neurosteroids and benzodiazepines on excitatory and inhibitory neurons excitability: a multi-electrode array (MEA) recording study. [Articolo su rivista]
Puja, Giulia; F., Gullo; E., Dossi; M., Lecchi; E., Wanke
abstract

The dynamic equilibrium between glutamate- and GABA-mediated synaptic neurotransmission in the brain is fundamental to the control of nervous system function. Such a balance is regulated by the ‘tonic’ release of a variety of neurotransmitters and endogenous factors that influence synaptic function. One such important group of modulatory molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in-vivo physiologically relevant concentrations, the effects of these two classes of GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel method of analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect novel drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 µM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after washout, the excitability of inhibitory neurons continued to be depressed, leading to a network long term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex-vivo neuronal networks show a sensitivity to drugs comparable to that expressed in vivo, but also provide a new global in-vitro description of the physiological mode of action of NSs and BDZs that can help in understanding their activity in more complex systems.

2012 - 5-Arylbenzothiadiazine Type Compounds as Positive Allosteric Modulators of AMPA/Kainate Receptors [Articolo su rivista]
Umberto M., Battisti; Krzysztof, Jozwiak; Cannazza, Giuseppe; Puja, Giulia; Gabriella, Stocca; Braghiroli, Daniela; Parenti, Carlo; Brasili, Livio; Marina M., Carrozzo; Cinzia, Citti; Luigino, Troisi
abstract

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood–brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

2011 - Molecular modeling studies, synthesis, configurational stability and biological activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide [Articolo su rivista]
Battisti, UMBERTO MARIA; Carrozzo, Marina Maria; Cannazza, Giuseppe; Puja, Giulia; Giulia, ; L., Troisi; Braghiroli, Daniela; Parenti, Carlo; K., Jozwiak
abstract

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPArs) has led to a search for new AMPAr positive modulators. Among them, 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (1) has attracted particular attention, because it is one of the most active benzothiadiazine–derived positive modulators of the AMPA receptor. It possesses two stereogenic centers, C3 and C6, thus it can exist as four stereoisomers. In this work, preliminary in silico studies suggested that 1 interacts stereoselectively with AMPArs. Single stereoisomers of 1 were prepared in order to evaluate their biological activity. However, studies regarding the configurational stability of the investigated compounds suggested a rapid epimerization at C3 in aqueous solvents, and we can expect the same reaction in vivo. Thus, electrophysiological experiments were performed on the two epimeric mixtures, (3∗,6R)- and (3∗,6S)- 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide, in order to evaluate their activities as positive allosteric modulators of AMPArs. The obtained data suggest that the (3∗,6S) epimeric mixture is the most active in positively modulating AMPArs, confirming in silico results.

2011 - Neurosteroids modulation of mouse neocortical network: a multielectrode based study [Abstract in Atti di Convegno]
Puja, Giulia
abstract

2011 - “Non genomic mechanisms of thyroid hormones in Nongenomic effects of thyroid hormones.” [Articolo su rivista]
Puja, Giulia
abstract

Ligand gated ionotropic receptors are responsible for the fast neurotransmission in the brain and are the target of widely used drugs, such as anxiolytics, anticonvulsant and antidepressant, and of endogenously produced substances, e.g. hormones. In this review, the fast regulatory effects of thyroid hormones (THs, T3 and T4) on ligand gated ion channels will be analyzed and discussed. More precisely, the focus will be on receptors that mediate the majority of inhibitory and excitatory neurotransmission in the brain, respectively γ-aminobutyric acid (GABA) and glutamate ionotropic receptors.Brain is an important target of THs, as proved by profound alterations in its functionality related to hormonal imbalance. Indeed, dysthyroidism is often associated with several neuropsychiatric disorders that derive also from a dysfunction of GABAergic and glutamatergic neurotransmission. The molecular mechanisms responsible for these changes are not completely clarified yet. THs, through the binding of nuclear receptors, can modulate the expression of proteins directly or indirectly involved in neurotransmission; in addition, their non genomic fast modulation of ionotropic receptors mediating excitatory and inhibitory neurotransmission could also play an important role.THs modulate recombinant and native receptors activated by exogenous GABA or glutamate as well synaptic and extrasynaptic excitatory and inhibitory neurotransmission. These non genomic effects do not depend on protein phosphorylation or on the membrane integrin receptor αvβ3 activation.Keeping in mind that a local regulation of THs levels can occur in different brain regions the fast modulation of THs of synaptic activity could provide a rapid and efficacious control of the function of several brain circuitries.

2011 - Thyroid hormones modulate GABA(A) receptor-mediated currents in hippocampal neurons( 2011) . [Articolo su rivista]
Puja, Giulia; Losi, G.
abstract

Thyroid hormones (THs) play a crucial role in the maturation and functioning of mammalian central nervous system. Thyroxine (T4) and 3, 3', 5-L-triiodothyronine (T3) are well known for their genomic effects, but recently attention has been focused on their non genomic actions as modulators of neuronal activity. In the present study we report that T4 and T3 reduce, in a non competitive manner, GABA-evoked currents in rat hippocampal cultures with IC₅₀s of 13±4μM and 12±3μM, respectively. The genomically inactive compound rev-T3 was also able to inhibit the currents elicited by GABA. Blocking PKC or PKA activity, chelating intracellular calcium, or antagonizing the integrin receptor αVβ3 with TETRAC did not affect THs modulation of GABA-evoked currents. THs affect also synaptic activity in hippocampal and cortical cultured neurons. T3 and T4 reduced to approximately 50% the amplitude and frequency of spontaneous inhibitory synaptic currents (sIPSCs), without altering their decay kinetic. Tonic currents evoked by low GABA concentrations were also reduced by T3 (40±5%, n=14), but not by T4. Similarly, T3 decreased currents elicited by low concentrations of THIP, a low affinity GABAA receptor agonist that preferentially activates extrasynaptic receptors, whereas T4 was ineffective. Thus, our data demonstrate that T3 and T4 selectively affect GABAergic phasic and tonic neurotransmission. Since THs concentrations can be regulated at the level of the synapses these data suggest that the network activity of the whole brain could be differently modulated depending on the relative amount of these two hormones.

2010 - Evidence that isopropylthioxanthone (ITX) is devoid of anxiolytic and sedative effect [Articolo su rivista]
Campioli, Enrico; Zavatti, Manuela; Avallone, Rossella; Puja, Giulia; Losi, Gabriele; Baraldi, Mario
abstract

Isopropylthioxanthone (ITX) is a well-known photo-initiator in ultraviolet light-cured inks frequently used in milk packaging materials, yoghurt, ready-to-feed infant formula, and other drinks. Traces of ITX have beenfound in milk and, as a consequence, there was considerable interest in studying the biological activity of this molecule and its potential hazard for the human health. Although the ITX genotoxic effects have been excluded 10 by the European Food Safety Authority (EFSA), the US Environmental Protection Agency (USEPA) is still examining its possible toxic potential depending on a dose–effect ratio. Little is known about the ITX activity on the function of the central nervous system and cerebral neurotransmitters. Using behavioural, biochemical, andelectrophysiological tests, the authors have found that: (1) ITX did not exert an in vivo anxiolytic or sedativeeffect when administered orally to rats; (2) ITX did not affect the binding characteristics of central and peripheral15 benzodiazepine receptors studied in vitro; and (3) ITX did not influence the ability of GABA to increase thechloride channel permeability studied by patch clamp technique in a single neuron of cultured cerebellargranule cells.

2009 - A novel class of allosteric modulators of AMPA/Kainate receptors [Articolo su rivista]
Cannazza, Giuseppe; Krzysztof, Jozwiak; Parenti, Carlo; Braghiroli, Daniela; Carrozzo, Marina Maria; Puja, Giulia; Losi, Gabriele; Baraldi, Mario; Wolfgang, Lindner; Irving W., Wainer
abstract

The rapid hydrolysis in vivo of IDRA21 to 2-amino-5-chlorobenzensulfonamide has been demonstrated by microdialysis experiments. The IDRA21 metabolite possess in vitro a biological activity similar to that of IDRA21 itself. Taking 2-amino-5-chlorobenzensulfonamide as lead compound, a novel class of AMPAR positive allosteric modulators has been prepared.

2008 - Non genomic regulation of glutamatergic neurotransmission in hyppocampus by thyroid hormones [Articolo su rivista]
Losi, Gabriele; Garzon, Giorgio; Puja, Giulia
abstract

Thyroid hormones (THs) are well known for their genomic effects but recently attention has focused also on their nongenomic actions as rapid modulators of membrane receptors. Here we show that thyroxine (T4) and 3,3',5'-l-triiodothyronine (T3) rapidly decrease N-methyl-d-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore THs' effect on NMDA receptors is independent of voltage and of subunit composition. The mechanism of THs' antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs' modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude and decay. In cultured rat cortical neurons THs prevented glutamate-induced neuronal death at concentrations similar to those effective on glutamatergic receptors. Taken together our data show for the first time that THs can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain function in physiological and pathological states.

2008 - NONGENOMIC REGULATION OF GLUTAMATERGIC NEUROTRANSMISSION IN HIPPOCAMPUS BY THYROID HORMONES [Articolo su rivista]
Losi, Gabriele; Garzon, Giorgio; Puja, Giulia
abstract

Thyroid hormones (THs) are well known for their genomic effects but recently attention has focused also on their nongenomic actions as rapid modulators of membrane receptors. Here we show that thyroxine (T4) and 3,3′,5′-l-triiodothyronine (T3) rapidly decrease N-methyl-d-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore THs’ effect on NMDA receptors is independent of voltage and of subunit composition. The mechanism of THs’ antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs’ modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude and decay.In cultured rat cortical neurons THs prevented glutamate-induced neuronal death at concentrations similar to those effective on glutamatergic receptors. Taken together our data show for the first time that THs can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain function in physiological and pathological states.

2007 - Evidence that the β-acid fraction of hops reduces central GABAergic neurotransmission [Articolo su rivista]
Zanoli, Paola; Zavatti, Manuela; Rivasi, Marianna; F., Brusiani; Losi, Gabriele; Puja, Giulia; Avallone, Rossella; Baraldi, Mario
abstract

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop β-acids on central nervous system function. Oral administration of β-acids (5–10 mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10 mg/kg, β-acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the β-acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [3H]-Ro 15-1788 binding assay. In conclusion, the behavioral effects of β-acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems

2007 - GABAA receptor neurotransmission dysfunction in a mouse model of social isolation-induced stress: Possible insights into a non-serotonergic mechanism of action of SSRIs in mood and anxiety disorders [Articolo su rivista]
K., Matsumoto; Puja, Giulia; E., Dong; G., Pinna
abstract

Protracted social isolation in laboratory animals causes stress, which induces a variety of behavioral abnormalities including increased aggressiveness, anxiety-related behaviors, cognitive deficits and hyper locomotion. Many of these disorders are similar to the symptoms found in psychiatric disorders, such as depression, anxiety, premenstrual dysphoria and posttraumatic stress disorders (PTSD). Recent studies have demonstrated that male mice that have been socially isolated for more than 4 weeks show: (a) reduced responsiveness of GABAA receptors (GABAA-R) to the administrations of GABA mimetic drugs at GABAA-R; (b) downregulated biosynthesis of 3,5-tetrahydroprogesterone (3,5-THP) (allopregnanolone: ALLO), a neurosteroid with a potent positive allosteric modulatory effect on the action of GABA on GABAA-R; and (c) alterations in the expression of GABAA-R subunits (i.e. a decrease of 1/2 and γ2 subunits and an increase of 4 and 5 subunits). The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX) and its congener norfluoxetine (Nor-FLX), when administered systemically at nmol/kg doses, normalize the reduced content of brain ALLO and the reduced responsiveness of GABAA-R to GABA mimetic drugs (i.e. pentobarbital) and also attenuate aggressive behavior in socially isolated mice in a stereospecific manner. Although these compounds inhibit ex vivo serotonin reuptake into brain tissue, their SSRI activities require high μmol/kg dose ranges and are not stereospecific. These studies suggest that in socially isolated mice, abnormalities of GABAA-R signal transduction are attributable to the downregulation of ALLO production and to a switch in heteropentameric GABAA-R subunit assembly composition. Hence, the normalization of ALLO biosynthesis may be a new target for the development of drugs effective for psychiatric disorders related to neurosteroid biosynthesis downregulation.

2006 - Functional in vitro characterization of CR 3394: A novel voltage dependent N-methyl-D-aspartate (NMDA) receptor antagonist [Articolo su rivista]
Losi, Gabriele; M., Lanza; F., Makovec; R., Artusi; G., Caselli; Puja, Giulia
abstract

Using the patch-clamp technique, we studied the effect of two novel adamantane derivatives, N-[2-(3,5-dimethyl-1-adamantyl)ethyl] guanidine (CR 3391) and N-[2-(3,5-dimethyl-1-adamantyl) ethyl] acetamidine (CR 3394), on NMDA receptors expressed in cortical neuron cultures. Our data show that CR 3391 and CR 3394 reduce NMDA-evoked currents (IC50 = 1.7 +/- 0.6 mu M and 6.7 +/- 1.5 mu M, respectively). This antagonism is non-competitive and is completely reversible. The effect of CR 3394, like that of memantine, was strongly voltage dependent. HEK293 cells expressing NR1a/NR2B recombinant NMDA receptors and immature neurons (DIV 8-9) were more sensitive to CR 3394 antagonism than NR1a/NR2A expressing cells and DIV 15 neurons. CR 3394 also reduced the duration and amplitude of miniature excitatory post-synaptic currents mediated exclusively by NMDA receptors (NMDA-mEPSCs). Both memantine and CR 3394 inhibited NMDA-evoked [H-3]norepinephrine release from rat hippocampal slices in a concentration-dependent manner with similar potency. CR 3394, but not memantine, increased cathecholamine resting release at low micromolar concentrations. Moreover, in an in vitro model of neurotoxicity, CR 3394 strongly reduced glutamate- and NMDA-induced neuronal death. Taken together, our data highlight pharmacological features of CR 3394 in vitro that prompt us to further evaluate it as a candidate for the treatment of neurodegenerative disorders.

2005 - Social isolation stress-induced aggression in mice: A model to study the pharmacology of neurosteroidogenesis [Articolo su rivista]
K., Matsumoto; G., Pinna; Puja, Giulia; A., Guidotti; Costa, E.
abstract

Long-term social isolation of laboratory animals is a model to study the behavioral and neurochemical consequences of the absence of social interaction in rodents. Many of the symptoms induced by isolation resemble depression and anxiety disorder symptomatology. Our studies have revealed that male mice socially isolated for more than 4 weeks, exhibit increased aggressiveness, a reduced responsiveness to GABAA receptor acting drugs, and a downregulation of brain levels of 3,5-tetrahydroprogesterone (allopregnanolone: 3,5-THP), a neurosteroid endowed with potent positive allosteric modulatory activity of the action of GABA at various GABAA receptor subtypes. This downregulation of 3,5-THP appeared to be associated with the reduction of brain type I 5-reductase mRNA and protein expression. Systemic administration of the selective serotonin reuptake inhibitor fluoxetine and its metabolite norfluoxetine normalized brain 3,5-THP content and reduced responsiveness to GABAA mimetic drugs in a stereospecific manner. These drugs in nanomolar doses also reduced social isolation-induced aggressiveness with the same stereospecificity as detected in their action on 3,5-THP brain content, while their ex vivo inhibition of serotonin reuptake occurred at high μmolar doses and lacked stereospecificity. From these results we infer that the brain 3,5-THP content physiologically upregulates GABAA receptor responsiveness to GABA and that social isolation induces a reduction of brain 3,5-THP content that is probably causally related to the onset of aggression

2005 - Thyroid hormone (L-T3) directly modulates GABA and NMDA receptors in hippocampal cultures and slices. [Abstract in Atti di Convegno]
Losi, Gabriele; Garzon, Giorgio; Bardoni, Rita; Puja, Giulia
abstract

Not available

2004 - Apigenin modulates GABAergic and glutamatergic transmission in cultured cortical neurons [Articolo su rivista]
Losi, Gabriele; Puja, Giulia; Garzon, Giorgio; M. C., de Vuono; Baraldi, Mario
abstract

Using the patch-clamp technique, we studied the modulation of ionotropic g-aminobutyric acid (GABA) and glutamate neurotransmissionby apigenin, a flavonoid with sedative and antidepressant activity. Apigenin reversibly reduced GABA-evoked currents mediated by a1h2g2receptors expressed in HEK293 cells. Amplitude and frequency of spontaneous postsynaptic inhibitory currents (sIPSCs) mediated byGABAA receptors were also decreased by apigenin in cultured cortical neurons. The flavonoid was almost inactive on a-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) mediated currents while it reduced N-methyl-d-aspartate (NMDA) receptor mediated responses witha half maximal inhibiting concentration (IC50) of 10 AM. The flavonoid inhibited also peak amplitude and frequency of spontaneouspostsynaptic excitatory currents (sEPSCs). Finally, apigenin is neuroprotective against glutamate-induced neurotoxicity in cerebellar andcortical neurons in culture. Our data reveal the antagonistic effect of apigenin on GABA and NMDA channels. While the inhibition onGABA receptor cannot explain the effects of the drug in vivo our data on NMDA channels reveal a new target of apigenin. A reduction of thenetwork excitability could thus account for the sedative effects. Furthermore, our data suggest a potential neuroprotective activity of apigenin.D 2004 Elsevier B.V. All rights reserved.

2004 - Design of 1-substituted 2-arylmethyl-4,5-methylenedioxybenzene derivatives as antiseizure agents [Articolo su rivista]
Micale, N.; De Sarro, G.; Ferreri, G.; Zappala, M.; Grasso, S.; Puia, G.; De Micheli, C.
abstract

A series of 1-substituted 2-[(4-aryl)-methyl]-4,5-methylenedioxybenzene derivatives (13-25), structurally related to model compound 5 (2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5- methylenedioxyphenylacetic acid methyl ester), were synthesized and tested as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds possess anticonvulsant properties lower than those of prototype 5 but, in some instances, comparable to that of GYKI 52466, a well-known noncompetitive AMPA receptor antagonist. © 2004 Elsevier Ltd. All rights reserved.

2004 - IDRA-21, a positive AMPA receptor modulator, inhibits synaptic and extrasynaptic NMDA receptor mediated events in cultured cerebellar granule cells [Articolo su rivista]
Losi, Gabriele; Puja, Giulia; Braghiroli, Daniela; Baraldi, Mario
abstract

IDRA-21 (7-chloro-3-methyl-3,4-dihydro-2H-1-,2,4-benzothiadiazine 1,1-dioxide) reduces alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptors desensitisation in vitro and restores learning and cognitive impairment in vivo. In this study, we show that in cerebellar granule cells (CGCs) in culture IDRA-21 reduces N-methyl-D-aspartate receptor (NMDAR) whole-cell currents. The effect is neither competitive nor voltage-dependent. The reduction of NMDA currents is stronger at low glycine concentrations suggesting an interaction with this site. IDRA-21 shortens miniature excitatory postsynaptic currents mediated by NMDARs (NMDA-mEPSCs) in CGCs grown in low potassium with no effect oil peak amplitudes. By using fast glutamate application onto CGCs nucleated patches, we found that IDRA-21 decreases both decay time constant and amplitude of the current. Experiments performed oil recombinant NMDAR expressed in HEK 293 cells showed that IDRA-21 was more effective on NR1a-NR2B than NR1a-NR2A receptors highlighting a subunit selectivity of the drug. Our findings make light oil a novel target for IDRA-21: NMDA receptors function is negatively modulated and the different action at the level of extrasynaptic and synaptic receptors could be ascribed to a partial selectivity for NR2B subunits.

2003 - On the putative physiological role of allopregnanolone on GABAA receptor function [Articolo su rivista]
Puia, G.; Mienville, J. -M.; Matsumoto, K.; Takahata, H.; Watanabe, H.; Costa, E.; Guidotti, A.
abstract

To obtain definitive evidence for a physiological allosteric modulatory role for endogenous brain ALLO on GABAA receptor function, we studied GABAA receptor activity under conditions in which the concentration of endogenous brain ALLO was decreased by about 80% for longer than 5 h following the administration of SKF 105111- 17β-17-[bis (1methylethyl) amino carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF), a potent inhibitor of 5α-reductases Type I and II. We used the in situ patch-clamp technique to record GABA-evoked currents and spontaneous inhibitory postsynaptic currents (sIPSCs) from pyramidal neurons in neocortical slices of vehicle- or SKF-treated mice. The potency, but not the efficacy, of exogenously applied GABA was decreased in slices from mice treated with SKF. When neocortical slices were treated in vitro for 3 h with 10μM SKF, ALLO was also reduced (25-30%) and in addition, the GABA dose-response curve was shifted to the right; however this shift was not as marked as the shift in the slices obtained from mice treated with SKF, in keeping with the smaller decrease of the ALLO content in these slices. Furthermore, direct application of ALLO to these slices shifted the dose-response curve of GABA back toward a non-SKF treated profile. We then analyzed GABAergic sIPSCs in neocortical slices obtained from vehicle or SKF-treated mice. Mean decay time and charge transfer were significantly reduced by SKF treatment. The decay of sIPSCs was best fitted by two exponentials, but only the fast component was decreased in the SKF group. Direct application of ALLO (100nM) normalizes the sIPSC kinetics in slices from ALLO depleted mice. No changes were detected in the amplitude or frequency of sIPSCs. These data demonstrate that endogenous ALLO physiologically regulates spontaneously induced Cl- current by acting on a specific recognition site, which is probably located on GABAA receptors (a receptor on a receptor), thereby prolonging inhibitory currents by facilitating conformational transition of the GABA-gated Cl- channel to an open state. © 2003 Elsevier Science Ltd. All rights reserved.

2003 - Presenza di composti ad attivita’ ansiolitico-sedativa nella filera di produzione del parmigiano reggiano. [Abstract in Atti di Convegno]
Avallone, Rossella; Corsi, Lorenzo; Zanoli, Paola; Puja, Giulia; Losi, Gabriele; Baraldi, Mario
abstract

Da tempo è stata descritta la presenza di sostanze ad attività sedativa nel latte tuttavia la loro struttura chimica è ancora oggetto di studio. Recentemente è stato isolato dall’s1-caseina un peptide denominato s1-CN-(f91-100) o s1-casozepina [1] mentre il nostro gruppo ha identificato nel latte altre sostanze non peptidiche ad attività benzodiazepino-simile [2]. Non è ancora noto, comunque, quali e quante sostanze presenti nel latte si ritrovano nei vari formaggi dopo i processi di produzione. Nel presente lavoro è stata valutata la presenza di composti ad attività benzodiazepino-simile nel parmigiano reggiano. In particolare è stata analizzata la filiera di produzione del parmigiano reggiano prelevando i seguenti campioni presso un’unica azienda in modo da escludere variabilità legate a fattori di alimentazione, ambiente, procedimento di produzione: fieno, latte intero, latte magro, siero, siero innesto, panna, cagliata, tosone e parmigiano reggiano. I campioni estratti con metanolo sono stati purificati mediante analisi HPLC a 0.8 ml/min utilizzando una colonna LiChrospher 100 RP-18 column (250x4.0 mm; 5 m) equilibrata con 80% di acqua/0.1% TFA e 20% di acetonitrile. I campioni sono stati analizzati a gradiente (0.5%) di acqua/0.1% TFA and acetonitrile dal 20 al 58% di acetonitrile. Per ogni campione sono state raccolte 75 frazioni, liofilizzate e testate per valutare la loro capacità di legarsi specificatamente al sito di riconoscimento centrale benzodiazepinico mediante dosaggio radioisotopici utilizzando come ligando marcato il [3H]RO 15-1788, antagonista benzodiazepinco. La concentrazione ottenuta per estrapolazione da una curva di competizione generata con autentico diazepam, viene espressa in ng diazepam equivalenti /g di campione. Nel tosone e nel parmigiano reggiano sono state rilevate piccole quantità di sostanze BDZ-simili (0,78 ± 0,12 e 0,86 ± 0,17 ng DE/g rispettivamente) mentre nel latte e nel siero la loro concentrazione risulta significativamente maggiore (117,75 ± 9,2 e 215 ± 15,7 ng DE/g rispettivamente). E’ interessante sottolineare che la concentrazione di tali sostanze nel fieno (14,8 ± 1,64 DE ng/g) è inferiore rispetto al latte, considerando che l’alimentazione della mucca è esclusivamente basata sul fieno si può ipotizzare che il contenuto che si ritrova nel latte sia anche il contributo di una sintesi endogena. L’attività ansiolitico-sedativa delle frazione isolate dal siero di latte è stata valutata sia in vitro su neuroni corticali con la tecnica elettrofisiologia del patch-clamp che in vivo.Bibliografia1. L. Miclo, E. Perrin, A. Driou, V. Papadopoulos et al. Characterization of a-casozepina, a tryptic peptide from bovine as1-caseina with benzodiazepine-like activity. FASEB J: 15, 1780-1782, 20012. R. Avallone, L. Corsi, M.L. Zeneroli and M. Baraldi. Presence of benzodiazepine-like molecules in food and their implication in the nutrition of cirrhotic patients. Innovative Food Science and Technologies: 2/3, 193-198, 2001

2002 - Novel potent AMPA/kainate receptor antagonists: Synthesis and anticonvulsant activity of a series of 2-[(4-alkylsemicarbazono)-(4-amino-phenyl)methyl]-4, 5-methylenedioxyphenylacetic Acid alkyl esters [Articolo su rivista]
Micale, N.; Zappala, M.; Grasso, S.; Puja, G.; De Sarro, G.; Ferreri, G.; De Sarro, A.; Toma, L.; De Micheli, C.
abstract

In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemicarbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1 (compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylene-tetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of AMPA or kainate, (KA). Using the patch-clamp technique in primary cultures of granule neurons we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives reduced KA and ATPA currents to a larger extent than that shown by reference compound 1. Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application of KA (100 muM).

2002 - Synaptic versus extrasynaptic NMDA receptor signaling: CREB makes the difference [Articolo su rivista]
Puja, G.
abstract

Commentary on NMDA receptor signaling

2002 - Synthesis of 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/Kainate receptors [Articolo su rivista]
Braghiroli, Daniela; Puja, Giulia; Cannazza, Giuseppe; Tait, A; Parenti, Carlo; Losi, G; Baraldi, M.
abstract

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

2002 - The inhibitory action of an excitatory neurotransmitter [Articolo su rivista]
Puia, G.
abstract

2001 - Diazepam pharmacology under 'dissection' [Articolo su rivista]
Puia, G.
abstract

2001 - "Glial-neuronal interactions: a crescendo in the degree of CNS complexity" [Articolo su rivista]
Puja, Giulia
abstract

Commentary on the importance of the interaction between neurons and glial cells

2001 - Neurosteroids on our minds [Relazione in Atti di Convegno]
Puia, G.; Belelli, D.
abstract

2001 - Synthesis and anticonvulsant activity of novel and potent 1-aryl-7,8-methylenedioxy-1,2,3,5-tetrahydro-4h-2,3-benzodiazepin-4-ones [Articolo su rivista]
Grasso, S; De Sarro, G; De Sarro, A; Micale, N; Polimeni, S; Zappala, M; Puja, Giulia; Baraldi, Mario; De Micheli, C.
abstract

The synthesis and anticonvulsant activity of 1-aryl-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-2,3-benzodiazepin-4-(thi)ones (4a-d) and their 3-N-alkylcarbamoyl derivatives (4e-h) are reported. The new compounds possess marked anticonvulsant properties, comparable to those of the dehydro analogues 3 and higher than that of GYKI 52466 (1). Noteworthy, compound 4c shows a longer-lasting anticonvulsant activity. Electrophysiological experiments show that derivative 4c is less effective than 1 and 3c to reduce the KA-evoked currents in cerebellar granule neurons. (C) 2001 Elsevier Science Ltd. All rights reserved.

2000 - Benzodiazepines outside the CNS [Articolo su rivista]
Puja, Giulia
abstract

Commentary on an article deling with the importance of benzodiazepines outside the CNS

2000 - Brain allopregnanolone regulates the potency of the GABA(A) receptor agonist muscimol [Articolo su rivista]
Pinna, G.; Uzunova, V.; Matsumoto, K.; Puia, G.; Mienville, J. -M.; Costa, E.; Guidotti, A.
abstract

Allopregnanolone (ALLO), a potent positive-allosteric modulator of the action of GABA at GABA(A) receptors, is synthesized in the brain from progesterone by the sequential action of two enzymes: 5α-reductase and 3 α-hydroxysteroidoxidoreductase. The concentration of ALLO in various parts of the mouse brain varies substantially, from 15 pmol/g in the olfactory bulb, to approximately 6 pmol/g in the frontoparietal cortex, and 2.7 pmol/g in the cerebellum. The systemic administration of 48 μmol/kg of the Type I and Type II 5α-reductase inhibitor, (17β)-17-[bis (1-methylethyl) amino carbonyl)] androsta-3, 5-diene-3-carboxylic acid (SKF 105,111), reduced brain ALLO content by 80-90% in 30 min; the rate constant (k) of ALLO decrease in each brain area can be utilized to establish the rate of ALLO biosynthesis, which is higher in the olfactory bulb (62 pmol/g/h) than in the frontoparietal cortex (24 pmol/g/h) or cerebellum (11 pmol/g/h). The duration of the righting reflex loss elicited by the potent GABA(A) receptor agonist muscimol was reduced in SKF 105,111-treated ALLO-depleted mice. SKF 105,111 treatment had no effect on muscimol metabolism or on brain levels of pregnenolone and progesterone; however, the brain levels of 5α-DHP, the precursor of ALLO, were also decreased. Administration of ALLO at a dose of 15 μmol/kg ip by itself did not alter the muscimol-induced loss of the righting reflex; but it completely blocked the effect of SKF 105,111. To elucidate the possible molecular mechanism by which a decrease of brain ALLO content can shorten the duration of the righting reflex loss elicited by muscimol, we patch-clamped neocortical pyramidal neurons of mice pretreated with SKF 105,111 or vehicle, and studied the efficiency of muscimol in eliciting Cl- currents. The current amplitude was significantly smaller in neurons from SKF 105,111-treated mice, especially at lower doses (0.1-1 μM) of muscimol, and the muscimol dose-response (0.1-10 μM) relationship displayed cooperativity (n(H)=1.4). These data suggest that ALLO synthesized in brain plays an important physiological permissive role in the modulation of GABA-gated Cl- channel function. Copyright (C) 2000 Elsevier Science Ltd.

2000 - Evidence that total extract of hypericum perforatum affects exploratory behavior and exerts anxiolytic effects in rats [Articolo su rivista]
Vandenbogaerde, A.; Zanoli, P.; Puia, G.; Truzzi, C.; Kamuhabwa, A.; De Witte, P.; Merlevede, W.; Baraldi, M.
abstract

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.

2000 - Modulation of kainate-activated currents by diazoxide and cyclothiazide analogues (IDRA) in cerebellar granule neurons [Articolo su rivista]
Puja, Giulia; G., Losi; G., Razzini; Braghiroli, Daniela; M., Di Bella; Baraldi, Mario
abstract

1. Patch-clamp technique was used in primary cultures of cerebellar granule neurons to study the modulation of the cyclothiazide analogue(IDRA21) and of the diazoxide derivative (IDRA 5) on KA-evoked currents. 2. The dose-response of kainic acid (KA) reveals an EC50=90 mu M and an Hill coefficient of 1.3. IDRA 21 and cyclothiazide potentiate KA-evoked current in a dose-dependent way, being cyclothiazide more potent but less efficacious than IDRA 21. Conversely IDRA 5 acts as a negative modulator of KA evoked -current. 3. Application of IDRA 21 and cyclothiazide results in a current potentiation of 125+/-18 % and 80 +/- 12 % respectively, while IDRA 5 decreases KA-current (-21+/-5 %). Coapplication of cyclothiazide and IDRA 21 produces a potentiation of 110+/- 17 %, suggesting a competition of the two drugs for the same site. 4. In the same experimental model we studied the ability of IDRA compounds of promoting toxicity through AMPA-receptor activation. Under basal conditions AMPA treatment (50 mu M for 1 hour) results in a negligible excitotoxicity. 5. In contrast similar treatment with AMPA + IDRA 21 (1 mM) or + IDRA 5 (1 mM) or + cyclothiazide (100 mu M) induces citotoxicity. The neurotoxic damage induced by IDRA 21 and cyclothiazide is blocked by GYKI 53655 (50 mu M) and by NBQX (10 mu M). Interestingly GYKI and NBQX are ineffective in reducing IDRA 5 toxicity.

2000 - NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine [Articolo su rivista]
R., Diaz-trelles; A., Novelli; Puja, Giulia; Baraldi, Mario; Mt, Fernandez-sanchez
abstract

Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission.

2000 - Pharmacological profile of apigenin, a flavonoid isolated from Matricaria chamomilla [Articolo su rivista]
Avallone, R.; Zanoli, P.; Puia, G.; Kleinschnitz, M.; Schreier, P.; Baraldi, M.
abstract

Dried flowers of Matricaria chamomilla L. are largely used to provide sedative as well as spasmolytic effects. In the present study, we examined in particular the pharmacological property of a fraction isolated from a methanolic extract of M. chamomilla, which was identified by HPLC-MS-MS analysis as apigenin. By radioreceptor binding assays, we demonstrated the ability of the flavone to displace a specific radioligand, [3H]Ro 15-1788, from the central benzodiazepine binding site. Electrophysiological studies performed on cultured cerebellar granule cells showed that apigenin reduced GABA (gamma-aminobutyric acid)-activated Cl-currents in a dose-dependent fashion. The effect was blocked by co-application of Ro 15-1788, a specific benzodiazepine receptor antagonist. Accordingly, apigenin reduced the latency in the onset of picrotoxin-induced convulsions. Moreover, apigenin injected i.p. in rats reduced locomotor activity, but did not demonstrate anxiolytic, myorelaxant, or anticonvulsant activities. The present results seem to suggest that the inhibitory activity of apigenin on locomotor behaviour in rats cannot be ascribed to an interaction with GABA(A)-benzodiazepine receptor but to other neurotransmission systems, since it is not blocked by Ro 15-1788. (C) 2000 Elsevier Science Inc.

2000 - Synthesis and anticonvulsant activity of novel and potent 6,7- methylenedioxyphthalazin-1(2H)-ones [Articolo su rivista]
Grasso, S.; De Sarro, G.; De Sarro, A.; Micale, N.; Zappala, M.; Puja, G.; Baraldi, M.; De Micheli, C.
abstract

In this paper, we describe the synthesis of a series of novel substituted 4-aryl-6,7-methylenedioxyphthalazin-1(2H)-ones. The anticonvulsant activity of these compounds against audiogenic seizures was evaluated in DBA/2 mice after intraperitoneal (ip) injection. Most of these derivatives are more active than 1-(4-aminophenyl)-4-methyl-7,8-methylene,2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA receptor antagonist. As deduced by the rotarod test, all the compounds exhibit a toxicity lower than that of 1. Within the series of derivatives submitted to investigation, 4-(4-aminophenyl)-2-butylcarbamoyl-6,7-methylenedioxyphthalazin-1(2H)-one (21) proved to be the most active compound and is 11-fold more potent than 1 (i.e., ED50 3.25 mu mol/kg for 21 versus ED50 35.8 mu mol/kg for 1). When compared to 1, compound 21 as well as its analogue 4-(4-aminophenyl)-6,7-methylenedioxyphthalazin-1(2H)-one (16) show a longer lasting anticonvulsant activity. Compound 21 also effectively suppresses seizures induced in Swiss mice by maximal electroshock (MES) and pentylenetetrazole (PTZ). Furthermore, it antagonizes in vivo seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), 2-amino-3-(3-hydroxy-5-tert-butyl-isoxazol-4-yl)propionic acid (ATPA), and kainate (KA), and its anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 16 and 21 to antagonize KA-evoked currents in primary cultures of granule neurons was tested. They behaved as antagonists, but they proved to be less effective than 1 and 1-(4-aminophenyl)-3, 4-dihydro-4-methyl-3-N-methylcarbamoyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine (2, GYKI 53655) to reduce the KA-evoked currents.

1999 - Evidence that total extract of Hypericum Perforatum affects exploratory behaviour and exerts anxioloyic effect in rats [Articolo su rivista]
A., Vandenbogaerde; P., Zanoli; Puja, Giulia; C., Truzzi; A., Kamuhabwa; P., DE WITTE; W., Merlevede; M., Baraldi
abstract

1999 - Synthesis and anticonvulsant activity of novel and potent 2,3- benzodiazepine AMPA/kainate receptor antagonists [Articolo su rivista]
Grasso, S.; De Sarro, G.; De Sarro, A.; Micale, N.; Zappala, M.; Puia, G.; Baraldi, M.; De Micheli, C.
abstract

We have previously shown that 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) acid(AMPA) in analogy to the structurally related 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI: 52466), a well-known noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7,8-methylenedioxy-4H (4a-h) and 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H (5a-c). The activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and compared to that of 1 and 3b,c. Compounds 4a,b and 5a-c antagonize seizures induced by AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared with that of the parent compounds 1 and 1-(4-aminophenyl)-3,4-dihydro-4-methyl-3-methylcarbamoyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (2, GYKI 53655).

1999 - Terfenadine prevents NMDA receptor-dependent and -independent toxicity following sodium channel activation [Articolo su rivista]
Diaz-Trelles, R.; Novelli, A.; Puia, G.; Fernandez-Sanchez, M. T.
abstract

Exposure of cultured cerebellar neurons to terfenadine prevented the N- methyl-D-aspartate (NMDA) receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. Delayed neurotoxicity by veratridine (24 h) occurring independently from NMDA receptor activation was also prevented by terfenadine. Terfenadine did not protect from excitotoxicity following direct exposure of neurons to glutamate. Our results suggest that terfenadine may modulate endogenous glutamate release following activation of VSSCs.

1995 - The density and distribution of six GABAA receptor subunits in primary cultures of rat cerebellar granule cells [Articolo su rivista]
Caruncho, H. J.; Puia, G.; Mohler, H.; Costa, E.
abstract

In cultured cerebellar granule neurons (seven daysin vitro) the expression of GABAA receptor subunits was quantified by using freeze-fracture immunocytochemical techniques with antibodies that specifically recognize the α1, α6, β2-3, γ2 and δ subunits of the GABAA receptor. In some experiments we have also used a less specific antibody that recognizes several α receptor subunits (α-total). The specificity of these antibodies was verified in human embryonic kidney cell line no. 293 cells transfected with complementary DNAs codifying for various GABAA receptor subunits. The most abundant labeling in granule cells was generated by the antibody against the β2-3 subunits (∼44 colloidal gold particles/μm2), while the specific antibodies against α1 and α6 subunits show a labeling of about 16 colloidal gold particles/μm2. The α-total antibody shows a labeling of ∼37 gold particles/μm2. Both the γ2 and δ antibodies show a labeling of about 10 gold particles/μm2. In granule cells, the relative proportion of the label density revealed with antibodies against α-total, β2-3, γ2 and δ subunits is approximately 4:4:1:1. Assuming that one molecular form of the α subunit is assembled in a GABAA receptor, it can be estimated that in granule cells about 50% of receptors include the α1 subunit. A similar relative abundance can be estimated for the α6 subunit. The proportion of GABAA receptors containing the γ2 or δ subunits can be estimated to be about 50% in each case. Cerebellar granule cells express various abundances of GABAA receptor subunits which can be estimated by freeze-fracture immunocytochemistry. Fifty to sixty percent of these subunits form small receptor clusters, which appear to be associated with neuronal cytoskeleton proteins. © 1995 IBRO.

1994 - Changes in γ-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro [Articolo su rivista]
Zheng, T. M.; Zhu, W. J.; Puia, G.; Vicini, S.; Grayson, D. R.; Costa, E.; Caruncho, H. J.
abstract

The amounts of mRNAs encoding α1, α6, β2, β3, γ2, and δ subunits of γ-aminobutyrate type A (GABA(A)) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABA(A) receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of γ2 and δ subunit expression was smaller than the expression of the α1 or α6 or β2/β3 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABA(A) receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABA(A) receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABA(A) receptors including the α6 subunit and/or to the expression, during granule cell maturation in vitro, of GABA(A) receptors devoid of γ2 subunits.

1994 - Functional diversity of GABA activated Cl- currents in Purkinje versus granule neurons in rat cerebellar slices [Articolo su rivista]
Puja, Giulia; Costa, E. AND VICINI S.
abstract

n rat cerebellar slices, we compared whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) with Cl- currents resulting from pulses of GABA (1 mM, < 2 ms) to outside-out patches from Purkinje and granule neurons. sIPSCs in Purkinje cells decayed with a single fast exponential, as previously reported, whereas in granule cells sIPSC decay was best described by the sum of a fast and a slow exponential curve, with a variable contribution of the slow component to the peak current. GABA pulses to nucleated patches from granule cells elicited Cl- currents with decays similar to sIPSC decays, whereas in patches from Purkinje neurons GABA pulses produced Cl- currents decaying largely with a fast component, but often followed by a slower exponential. GABA concentration steps produced rapidly desensitizing currents in patches from both cerebellar neurons. In distinct cerebellar neurons, specific functional properties of GABAA receptors may relate to the presence of distinct receptor subtypes.

1994 - “Mechanistic and pharmacological implications in the partial allosteric modulation of GABAA receptor by imidazenil.” [Capitolo/Saggio]
Costa, E; Puja, Giulia; Giusti, P; Auta, J; Ducic, I; Vicini, S; AND GUIDOTTI, A.
abstract

The allosteric modulation of GABA responses at GABAA receptors elicited by benzodiazepines dpends on heterotropic mechanisms, with molecular modalities of operation that are still not well understood. In this chapter the effects of varoious benzodiazepines are considered in relationship to their affinity for the diffrent GABAA receptor assembies

1993 - "Does neurosteroid modulatory efficacy depend on GABAA receptor subunit composition?" [Articolo su rivista]
Puja, Giulia; Ducic, I; Vicini, S; AND COSTA, E.
abstract

The modulation of GABA activity by 3 alpha-OH-DHP (allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one) and PS (pregnenolone sulfate) has been studied in native GABAA receptors of rat cortical neurons in primary cultures and in structurally different recombinant GABAA receptors of rat cortical neurons in primary cultures and in structurally different recombinant GABAA receptors expressed in the 293 human embryonic kidney cell line (HEK 293). In cortical neurons 3 alpha-OH-DHP positively modulates GABA elicited Cl- currents while PS at 10 microM negatively modulates (50% decrease) this GABA response, but at 10 nM PS positively modulates the GABA current (40% increase). Both neurosteroids are equally active on various types of recombinant GABAA receptors, except for alpha 6 beta 1 gamma 2 receptors which are less sensitive to the positive allosteric modulation by 3 alpha-OH-DHP. In contrast the presence of the gamma 1 subunit doubles the efficacy of 3 alpha-OH-DHP. The negative modulation of PS is similar in recombinant GABAA receptors including various molecular forms of alpha or gamma units. A direct activation of Cl- current by 3 alpha-OH-DHP was observed in native and recombinant receptors but its efficacy on the various molecular forms of GABAA receptor tested was always smaller than that of identical concentrations (10 microM) of GABA.

1993 - Freeze-fracture immunocytochemical study of the expression of native and recombinant GABAA receptors [Articolo su rivista]
Caruncho, H. J.; Puia, G.; Slobodyansky, E.; da Silva, P. P.; Costa, E.
abstract

To assess the density and distribution of native and recombinant GABAA receptors we used label-fracture and fracture-flip technologies combined with immunocytochemistry using monoclonal and polyclonal Abs directed against the extracellular domain of the GABAA receptor protein located in the freeze-fracture replicas. In cortical neurons there is a high density of GABAA receptors on both soma and dendrites with some areas were the density of receptors is higher, but there are no well defined clusters. In cerebellar granule cells most of the receptors are distributed in round clusters both in neurites and soma. In astroglial cells the receptor density is lower than in neurons and only occasionally they appear in clusters. In cells transfected with cDNAs encoding for various molecular forms of GABAA receptor subunits, the receptor density is moderate when cDNAs for α, β and γ subnits are cotransfected; however, on cells cotransfected with cDNAs for β and γ subunits the receptor density is significantly lower. Recombinant receptors appear randomly distributed and occasionally they aggregate in small groups. © 1993.

1993 - )"Imidazenil : a new partial positive allosteric modulator of GABA action at GABAA receptors." [Articolo su rivista]
Giusti, P; Ducic, I; Puja, Giulia; Arbam, R; Walser, A; Guidotti, A; AND COSTA, E.
abstract

Positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors, including benzodiazepines and congeners, can be classified into three categories: 1) full allosteric modulators (i.e., triazolam and alprazolam) that act with high potency and efficacy at many GABAA receptors; 2) selective allosteric modulators (i.e., diazepam) that act with high potency and high efficacy at selected GABAA receptors; and 3) partial allosteric modulators (i.e., bretazenil) that act with high potency but low efficacy at many GABAA receptors. Imidazenil, an imidazobenzodiazepine carboxamide, has been characterized as a novel representative of the partial allosteric modulator class. When tested on a broad spectrum (native and recombinant) of GABAA receptors, imidazenil positively modulates the GABA-elicited Cl- currents with a 4- to 5-fold higher potency but an efficacy (30-50%) lower than that of diazepam, and it antagonizes the effects of the latter drug. Imidazenil in vitro (Ki = 5 x 10(-10) M) and in vivo (ID50 = 0.2 mumol/kg i.v.) displaces [3H]flumazenil from its brain binding sites and in vivo it possesses a marked anticonflict profile in the rat Vogel conflict-punishment test and is 10 times more potent than bretazenil and 100 times more potent than diazepam or alprazolam in antagonizing bicuculline- and pentylenetetrazol-induced seizures. Unlike diazepam and alprazolam, which induce sedation and ataxia and potentiate the effects of ethanol and thiopental at doses similar to those that produce anticonflict effects and occupy 50% of brain flumazenil binding sites, imidazenil does not produce ataxia or sedation in rats nor does it potentiate the effects of ethanol or thiopental in doses 30- to 50-fold higher than those required for the anticonflict effect and for 100% occupancy of brain flumazenil binding sites. Furthermore, when administered with diazepam, imidazenil blocks in a dose-related fashion the sedative, ataxic effects of this drug and thus acts on these unwanted responses as an antagonist (i.e., like flumazenil). In all tests, imidazenil has the pharmacological profile of a partial allosteric modulator, but is more potent than bretazenil, has a longer biological half-life and, in rodents, is virtually unable to cause sedation, ataxia or to potentiate ethanol toxicity.

1993 - Mitochondrial DBI receptor (MDR) regulation and pharmacology of basic steroidogenesis." [Abstract in Atti di Convegno]
Costa, E.; Cavallaro, S.; Korneyev, B.; Puja, Giulia; Romeo, E.; Zivkovic, I.; Guidotti, A.
abstract

Importance of the activity of the peripheral benzodiazepine receptor in regulating steroidogenesis in the brain and modulation of GABAA receptor by endogenous steroids

1993 - Triazolam is more efficacious than diazepam in a broad spectrum of recombinant GABAA receptors [Articolo su rivista]
Ducic, I.; Puia, G.; Vicini, S.; Costa, E.
abstract

Benzodiazepine-induced modifications of GABA (γ-aminobutyric acid) activated Cl- currents were studied in native GABAA receptors expressed in neonatal rat brain cortical neurons in primary cultures and in recombinant GABAA receptors expressed in transformed human embryonic kidney cells (293) after a transient transfection with cDNAs encoding for different molecular forms of α, β, and γ subunits of GABAA receptors. The efficacy of triazolam in cortical neurons was higher than that of diazepam. In transfected cells, triazolam showed a greater efficacy as a positive modulator of GABA-elicited Cl- currents in α1β1γ1, α1β1γ2, α1β1γ3, α6β1γ2 and α1β3γ2 receptors than diazepam, except in α3β1γ2 receptors where diazepam was more efficacious. When triazolam and diazepam were applied together to GABAA receptors assembled by transfecting cDNAs encoding for α1β1γ1 subunits, the action of triazolam was curtailed in a manner related to the dose of diazepam. In recombinant receptors assembled with α1β1γ1 receptors, maximally active doses of triazolam were more efficacious than those of clonazepam, alpidem, zolpidem, diazepam or bretazenil. © 1993.

1992 - A third γ subunit of the gamma-aminobutyric acid type A receptor family." [Articolo su rivista]
Herb, A; Wisden, W; Luddens, H; Puja, Giulia; Vicini, S; AND SEEBURG, P. H.
abstract

Cloned cDNAs encoding a member of the gamma-aminobutyric acid type A receptor gamma-subunit class were isolated from rat-brain-mRNA-derived libraries. The gamma 3 mRNA is present in cortex, claustrum, caudate putamen, and some thalamic nuclei, particularly the medial geniculate nucleus, where it is the predominant gamma-subunit transcript. The gamma 3 gene is expressed at very low levels in cerebellum and hippocampus. In coexpression experiments with the alpha 1 and beta 2 subunits, gamma 3 imparts benzodiazepine binding to gamma-aminobutyric acid type A receptors and forms gamma-aminobutyric acid-gated benzodiazepine-modulated chloride channels that exhibit a larger conductance than alpha 1 beta 2 receptor channels. Furthermore, the presence of gamma 3 in place of gamma 2 in alpha 1 beta 2 gamma x receptors generates a marked decrease in the affinity of agonists while leaving the affinity of antagonists or negative modulators largely unaffected.

1992 - Caratterizzazione e modulazione dei canali operati dai recettori per il GABA e per il Glutammato." [Capitolo/Saggio]
Vicini, S; Puja, Giulia; Mereu, G.
abstract

L'eterogeneita' strutturale dei recettori inibitori ed eccitatori apre un capitolo importante nella neurobiologia modernae nel campo delle neuroscienze.La comprensione dei correlati funzionali alla molteplicità delle forme molecolari dei canali ionici ed il loro ruolo nella funzione e nella patologia del sistema nervoso centrale e' importante per il futuro della farmacologia.

1992 - Different sites of action of neurosteroids and benzodiazepines on natural and recombinant GABAA receptors [Articolo su rivista]
Puia, G.; Vicini, S.; Seeburg, P. H.; Costa, E.
abstract

1992 - "Expression pattern of GABAA receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella." [Articolo su rivista]
Bovolin, P; SANTI M., R; Puja, Giulia; Costa, E; AND GRAYSON, D.
abstract

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.

1992 - "Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at GABAA receptor." [Articolo su rivista]
Puja, Giulia; Ducic, I; Vicini, S; AND COSTA, E.
abstract

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.

1992 - Positive and negative modulation of GABA gated Cl- currents and structural microheterogeneity of GABA(A) receptors [Relazione in Atti di Convegno]
Costa, E.; Mereu, G.; Puia, G.; Vicini, S.
abstract

1992 - "Purification and characterization of naturally occurring benzodiazepine receptor ligands in rat and human brain." [Articolo su rivista]
ROTHSTEIN J., D; Garland, W; Puja, Giulia; Guidotti, A; WEBER R., J; AND COSTA, E.
abstract

Chemicals that are active at the benzodiazepine receptor (endozepines) are naturally present in the CNS. These substances are present in tissue from humans and animals and in plants and fungi. Using selective extraction protocols, HPLC purification, receptor binding displacement studies, and selective anti-benzodiazepine antibodies, we have identified six or seven peaks of endozepines in rat and human brain. All material could competitively displace [3H]flunitrazepam binding to cerebellar benzodiazepine binding sites. Two peaks also competitively displaced Ro 5-4864 binding to the mitochondrial benzodiazepine binding site. Total amounts of brain endozepines were estimated to be present in potentially physiological concentrations, based on their ability to displace [3H]flunitrazepam binding. Although endozepine peaks 1 and 2 had HPLC retention profiles similar to those of nordiazepam and diazepam, respectively, gas chromatography-mass spectrometry as well as high-performance TLC revealed biologically insignificant amounts of diazepam (less than 0.02 pg/g) and nordiazepam (less than 0.02 pg/g) in the purified material. Electrophysiologically, some purified endozepines positively modulated gamma-aminobutyric acid (GABA) action on Cl- conductance, monitored in patch-clamped cultured cortical neurons or in mammalian cells transfected with cDNA encoding various GABAA receptor subunits. These studies demonstrate that mammalian brains contain endozepines that could serve as potent endogenous positive allosteric modulators of GABAA receptors.

1992 - Synthesis of (2‐Arylindol‐3‐yl)acetamides as Probes of Mitochondrial Steroidogenesis—A New Mechanism for GABAA Receptor Modulation [Articolo su rivista]
Kozikowski, A. P.; Ma, D.; Romeo, E.; Auta, J.; Papadopoulos, V.; Puia, G.; Costa, E.; Guidotti, A.
abstract

High affinity and selectivity for mitochondrial DBI receptors characterize indolylacetamides 1. These compounds stimulate the production of neurosteroids in glial cells allowing possible indirect modulation of the γ‐aminobutyric acid receptor complex. In behavioral experiments with rats, 1a was found to have antineophobic action, that is, it reduces the fear of novelty. (Figure Presented.) Copyright © 1992 by VCH Verlagsgesellschaft mbH, Germany

1992 - 2-ARYL-3-INDOLEACETAMIDES (FGIN-1) - A NEW CLASS OF POTENT AND SPECIFIC LIGANDS FOR THE MITOCHONDRIAL DBI RECEPTOR (MDR) [Articolo su rivista]
Romeo, E; Auta, J; Kozikowski, Ap; Ma, D; Papadopoulos, V; Puia, G; Costa, E; Guidotti, A
abstract

The 2 aryl-3-indoleacetamides (FGIN-1) are a new class of compounds that potently (nM) and selectively bind to glial mitochondrial diazepam binding inhibitor (DBI) receptors (MDR), previously called peripheral benzodiazepine receptors, and increase mitochondrial steroidogenesis. The high-affinity binding of FGIN-1 to MDR derivatives depends on the following chemical characteristics: 1) the dialkylation of the amide; 2) the chain length of this alkyl substitution; and 3) the halogenation of aryl groups appended to the indole nucleus. FGIN-1 derivatives do not bind to gamma-aminobutyric acid (GABAA), GABAB, glycine, glutamate, dopamine, serotonin, opiate, cholecystokinin, beta adrenergic, cannabinoid or sigma receptors. FGIN-1-27 [N, N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide] enters the brain, and for this reason, this FGIN-1 compound is potent and efficacious behaviorally. Like the neurosteroid 3 alpha-5 alpha tetrahydrodeoxycorticosterone (THDOC), FGIN-1-27 delays the onset of isoniazid-induced convulsions, but fails to delay the onset of bicuculline-induced convulsions. However, differently from THDOC, the FGIN-1-27 anticonvulsant action is blocked by the isoquinoline carboxamide PK 11195. In the elevated plus maze test, FGIN-1-27 inhibits neophobia manner that is antagonized by PK 11195 but not by flumazenil. Because FGIN-1-27 binds to MDR and does not bind to the GABAA receptors, it is inferred that FGIN-1-27 may act on GABAA receptors indirectly, presumably via a stimulation of neurosteroid synthesis and release from glial cells.

1991 - Development of voltage-dependent ionic currents in rat cerebellar granule cells grown in primaryculture [Articolo su rivista]
Galdziki, Z; Fan, L; Moran, O; Novelli, A; Puja, Giulia; AND SCIANCALEPORE, M.
abstract

In this work we studied the excitable properties of cerebellar granule cells grown in primary culture in the presence of "high" KCl (25 mM). Whole cell patch-clamp records of currents were obtained from cells at 1-33 days in culture (DIC). Sodium currents, blocked by TTX, were present from the first DIC and did show slow developmental changes. Two types of potassium currents were detected at all DIC: a delayed rectifier current (IK) and an inactivating current (IA). Both IK and IA increased until 7 to 9 DIC (four and two times respectively). Most of the IA increase, however, correlated with an increase in cell size, monitored by measurements of cell membrane capacitance (Cm) and the current density thus did not change. Conversely, delayed rectifier potassium current density did increase in the initial DIC (3-6) and did not change significantly after this time. Calcium currents were not detectable at any DIC under our experimental conditions.

1991 - "Influence of recombinant GABAA receptor subunit composition on the allosteric modulators of GABA-gated Cl- currents." [Articolo su rivista]
Puja, Giulia; Vicini, S; Seeburg, P. H. AND COSTA E.
abstract

gamma-Aminobutyric acid (GABA)-activated Cl- currents in neonatal rat cortical neurons and in cultured cells engineered for the expression of specific molecular forms of the GABAA receptor alpha, beta, and gamma subunits, were recorded with the patch-clamp technique in the whole-cell configuration. The effects of various allosteric modulators of GABAA receptors were determined. Diazepam and clonazepam showed greater efficacy as positive modulators of GABA-elicited currents in alpha 2 beta 1 gamma 2 or alpha 3 beta 1 gamma 2 receptors than in alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Alpidem was more efficacious at alpha 1 beta 1 gamma 2 or alpha 2 beta 1 gamma 2 receptors than at alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Conversely, zolpidem was equally efficacious for all these receptors except for alpha 5 beta 1 gamma 2. Both imidazopyridines (alpidem and zolpidem) were virtually ineffective at modulating the GABA response of alpha 5 beta 1 gamma 2 receptors and in almost all the receptors assembled from alpha 1, alpha 2, alpha 3 or alpha 5 subunits together with beta 1 and gamma 1 subunits. The beta-carboline derivatives methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and methyl-beta-carboline-3-carboxylate (beta-CCM) elicited a positive allosteric modulation of alpha 1 beta 1 gamma 1 or alpha 2 beta 1 gamma 1 receptors, whereas they acted as negative allosteric modulators at nearly all other receptors tested, as they do in cortical neurons. Although the positive allosteric modulation by beta-carbolines never exceeded a doubling of the GABA response, DMCM was more efficacious at alpha 1 beta 1 gamma 1 receptors and beta-CCM was more efficacious at alpha 2 beta 1 gamma 1 receptors. DMCM was inactive at alpha 3 beta 1 gamma 1 receptors, whereas beta-CCM was virtually inactive at alpha 5 beta 1 gamma 1 receptors. The benzodiazepine 4'-chlorodiazepam, which is a negative modulator resistent to flumazenil inhibition, acted at all the various GABAA receptors that contained a gamma subunit.

1990 - "Neurosteroids act on recombinant human GABAA receptors" [Articolo su rivista]
Puja, Giulia; SANTI M., R; Vicini, S; PRITCHETT D., B; PURDY R., H; PAUL S., M; SEEBURG P., H; AND COSTA, E.
abstract

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

1990 - "Voltage-dependent calcium currents in trigeminal chick neurons." [Articolo su rivista]
Puja, Giulia
abstract

The presence of action potentials, when sodium and potassium currents were blocked, has been investigated in trigeminal ganglion neurons, using the patch-clamp technique. In this conditions, inward currents, sensitive to the external application of cadmium, were detected. Activation and inactivation properties were investigated, as well as the behaviour of the current in the presence of extracellular Barium. The properties of these inward currents in trigeminal neurons are correlated to high threshold voltage-dependent calcium channels.

1989 - Differences in the negative allosteric modulation of gamma-aminobutyric acid (GABA) receptors elicited by 4'- chlorodiazepam and by beta-carboline-3-carboxilate ester: a study with natural and reconstituted receptors." [Articolo su rivista]
Puja, Giulia; SANTI M., R; Vicini, S; PRITCHETT D., B; SEEBURG P., H; AND COSTA, E.
abstract

Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.

Università degli studi di Modena e Reggio Emilia

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