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Rita BARDONI

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


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Pubblicazioni

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 - Experimental Protocols and Analytical Procedures for Studying Synaptic Transmission in Rodent Spinal Cord Dorsal Horn [Articolo su rivista]
Bardoni, R.
abstract

Synaptic modulation and plasticity are key mechanisms underlying pain transmission in the spinal cord and supra-spinal centers. The study and understanding of these phenomena are fundamental to investigating both acute nociception and maladaptive changes occurring in chronic pain. This article describes experimental protocols and analytical methods utilized in electrophysiological studies to investigate synaptic modulation and plasticity at the first station of somatosensory processing, the spinal cord dorsal horn. Protocols useful for characterizing the nature of synaptic inputs, the site of modulation (pre- versus postsynaptic), and the presence of short-term synaptic plasticity are presented. These methods can be employed to study the physiology of acute nociception, the pathological mechanisms of persistent inflammatory and neuropathic pain, and the pharmacology of receptors and channels involved in pain transmission. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Spinal cord dissection and acute slice preparation Basic Protocol 2: Stimulation of the dorsal root and extracellular recording (compound action potentials and field potentials) Basic Protocol 3: Patch-clamp recording from dorsal horn neurons: action potential firing patterns and evoked synaptic inputs Basic Protocol 4: Analysis of parameters responsible for changes in synaptic efficacy Basic Protocol 5: Recording and analysis of currents mediated by astrocytic glutamate.


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.


2022 - Osteoporosis in Light of a New Mechanism Theory of Delayed Onset Muscle Soreness and Non-Contact Anterior Cruciate Ligament Injury [Articolo su rivista]
Sonkodi, Balázs; Bardoni, Rita; Poór, Gyula
abstract

Osteoporosis is a disorder, with a largely unknown pathomechanism, that is often marked as a "silent thief", because it usually only becomes undisguised when fractures occur. This implies that the pathological damage occurs earlier than the sensation of pain. The current authors put forward a non-contact injury model in which the chronic overloading of an earlier autologously microinjured Piezo2 ion channel of the spinal proprioceptor terminals could lead the way to re-injury and earlier aging in a dose-limiting and threshold-driven way. As a result, the aging process could eventually lead the way to the metabolic imbalance of primary osteoporosis in a quad-phasic non-contact injury pathway. Furthermore, it is emphasised that delayed onset muscle soreness, non-contact anterior cruciate injury and osteoporosis could have the same initiating proprioceptive non-contact Piezo2 channelopathy, at different locations, however, with different environmental risk factors and a different genetic predisposition, therefore producing different outcomes longitudinally. The current injury model does not intend to challenge any running pathogenic theories or findings, but rather to highlight a principal injury mechanism.


2022 - 5-HT7 Receptors Regulate Excitatory-Inhibitory Balance in Mouse Spinal Cord Dorsal Horn [Articolo su rivista]
Comitato, Antonella; Lacivita, Enza; Leopoldo, Marcello; Bardoni, Rita
abstract

Serotonergic receptors of the 5-HT7 type (5-HT7Rs) are widely expressed in the central nervous system (CNS), where they modulate several functions, such as pain. Behavioral experiments in vivo have shown both anti- and pro-nociceptive actions of 5-HT7Rs, although an analgesic effect seems to be prevalent. In the spinal cord dorsal horn, the mechanisms involved in 5-HT7R-mediated synaptic modulation are still poorly understood, especially those regarding the control of synaptic inhibition. The present study investigated the modulation exerted by 5-HT7Rs on dorsal horn excitatory and inhibitory synaptic circuits, by performing patch-clamp recordings from lamina II neurons in mouse spinal cord slices. Our results show that applying the selective 5-HT7 agonist LP-211 facilitates glutamatergic release by enhancing the frequency of spontaneous postsynaptic currents (sEPSCs) and increasing the peak amplitude of excitatory postsynaptic currents (EPSCs) evoked by dorsal root stimulation. The effects on sEPSCs were still observed in the presence of the 5-HT1A antagonist WAY-100635, while the 5-HT7 antagonist SB-269970 blocked them. LP-211 was also able to increase the release of gamma-aminobutyric acid (GABA) and glycine, as shown by the increase of spontaneous inhibitory currents (sIPSC) frequency and evoked inhibitory postsynaptic currents (IPSC) amplitude. LP-211 was proved to be more effective in potentiating synaptic inhibition as compared to excitation: consistently, 5-HT7R activation significantly enhanced the excitability of tonic firing neurons, mainly corresponding to inhibitory interneurons. Our data bring new insights into the mechanisms of synaptic modulation mediated by 5-HT7Rs in the dorsal horn. Stronger impact on synaptic inhibition supports the hypothesis that these receptors may play an anti-nociceptive role in the spinal cord of naïve animals.


2021 - Does compression sensory axonopathy in the proximal tibia contribute to noncontact anterior cruciate ligament injury in a causative way?—a new theory for the injury mechanism [Articolo su rivista]
Sonkodi, B.; Bardoni, R.; Hangody, L.; Radak, Z.; Berkes, I.
abstract

Anterior cruciate ligament injury occurs when the ligament fibers are stretched, partially torn, or completely torn. The authors propose a new injury mechanism for non-contact anterior cruciate ligament injury of the knee. Accordingly, non-contact anterior cruciate ligament injury could not happen without the acute compression microinjury of the entrapped peripheral proprioceptive sensory axons of the proximal tibia. This would occur under an acute stress response when con-comitant microcracks-fractures in the proximal tibia evolve due to the same excessive and repetitive compression forces. The primary damage may occur during eccentric contractions of the acceleration and deceleration moments of strenuous or unaccustomed fatiguing exercise bouts. This primary damage is suggested to be an acute compression/crush axonopathy of the proprioceptive sensory neurons in the proximal tibia. As a result, impaired proprioception could lead to injury of the anterior cruciate ligament as a secondary damage, which is suggested to occur during the deceleration phase. Elevated prostaglandin E2, nitric oxide and glutamate may have a critical neuro-modulatory role in the damage signaling in this dichotomous neuronal injury hypothesis that could lead to mechano-energetic failure, lesion and a cascade of inflammatory events. The presynaptic modulation of the primary sensory axons by the fatigued and microdamaged proprioceptive sensory fibers in the proximal tibia induces the activation of N-methyl-D-aspartate receptors in the dorsal horn of the spinal cord, through a process that could have long term relevance due to its contribution to synaptic plasticity. Luteinizing hormone, through interleukin-1β, stimulates the nerve growth factor-tropomyosin receptor kinase A axis in the ovarian cells and promotes tropomyosin receptor kinase A and nerve growth factor gene expression and prostaglandin E2 release. This luteinizing hormone induced mechanism could further elevate prostaglandin E2 in excess of the levels generated by osteocytes, due to mechanical stress during strenuous athletic moments in the pre-ovulatory phase. This may explain why non-contact anterior cruciate ligament injury is at least three-times more prevalent among female athletes.


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.


2021 - Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity [Articolo su rivista]
Ciarpella, F.; Zamfir, R. G.; Campanelli, A.; Ren, E.; Pedrotti, G.; Bottani, E.; Borioli, A.; Caron, D.; Di Chio, M.; Dolci, S.; Ahtiainen, A.; Malpeli, G.; Malerba, G.; Bardoni, R.; Fumagalli, G.; Hyttinen, J.; Bifari, F.; Palazzolo, G.; Panuccio, G.; Curia, G.; Decimo, I.
abstract

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10–20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling.


2021 - Presynaptic Inhibition of Pain and Touch in the Spinal Cord: From Receptors to Circuits [Articolo su rivista]
Comitato, Antonella; Bardoni, Rita
abstract

Sensory primary afferent fibers, conveying touch, pain, itch, and proprioception, synapse onto spinal cord dorsal horn neurons. Primary afferent central terminals express a wide variety of receptors that modulate glutamate and peptide release. Regulation of the amount and timing of neurotransmitter release critically affects the integration of postsynaptic responses and the coding of sensory information. The role of GABA (γ-aminobutyric acid) receptors expressed on afferent central terminals is particularly important in sensory processing, both in physiological conditions and in sensitized states induced by chronic pain. During the last decade, techniques of opto- and chemogenetic stimulation and neuronal selective labeling have provided interesting insights on this topic. This review focused on the recent advances about the modulatory effects of presynaptic GABAergic receptors in spinal cord dorsal horn and the neural circuits involved in these mechanisms.


2021 - Serotonergic modulation by 5-HT7 receptors in mouse spinal cord dorsal horn [Relazione in Atti di Convegno]
Comitato, A.; Lacivita, E.; Leopoldo, M.; Bardoni, R.
abstract

Background Serotonergic receptors of the 5-HT7 type (5-HT7Rs) are widely expressed in the central nervous system, where they modulate seve ral functions, such as sleep induction, learning, mood, and vegetative behaviours. Along the pain axis, 5-HT7Rs are expressed on nociceptive primary afferent fibers and in the dorsal horn, both on neurons and astrocytes. [1]. Behavioural experiments have produ ced controversial results about anti- and pro-nociceptive actions of 5-HT7Rs. The low agonist selectivity and the different pain animal models used have likely contributed to the heterogeneity of the results [2]. To investigate the effects of 5-HT7Rs on spinal pain, we have performed an electrophysiological study on mouse spinal cord slices, using the selective agonist LP-211 [3]. The recorded neurons have been functionally characterized, in order to identify the neural circuits involved in the serotonergic modulation. Methods Patch-clamp recording was performed on lamina II neurons in spinal cord slices obtained from postnatal CD1 mice (P15-P25) [4]. Excitatory postsynaptic currents (EPSCs) were recorded in voltage clamp; evoked EPSCs were elicited by stimulating the dorsal root with a suction electrode in the Aδ and C fibers range. Results Application of 1 μM LP-211 to the spinal cord slice induced a facilitation of glutamatergic transmission: the frequency of spontaneous EPSCs was significantly increased in a subpopulation of neurons (control: 0.9±0.2 Hz; LP-211: 1.8±0.6 Hz; 5 responsive neurons out of 8). The recorded neurons were characterized from their firing pattern: significant effects of LP-211 were observed in both tonic and de layed firing neurons, corresponding to inhibitory and excitatory interneurons, respectively. Application of 1 μM LP-211 in the presence of 10 μM SB269970 (a 5-HT7R antagonist) did not alter spontaneous EPSC frequency in 11 lamina II neurons, confirming the involvement of 5-HT7Rs in glutamate release facilitation. EPSCs evoked by dorsal root stimulation were also tested with LP-211. The currents, evoked by paired pulse protocol, were significantly potentiated by the compound (mean potentiation: 19±4.2%; 4 responsive neurons out 7). The second EPSC was less potentiated than the first and the paired pulse ratio decreased in 3 neurons. Conclusion The compound LP-211 is able to selectively activate 5-HT7Rs in the dorsal horn, causing a facilitatory effect of both spontaneous and evoked EPSCs. The decrease of paired-pulse ratio suggests that LP-211 activates presynaptic 5-HT7Rs, increasing glutamate release. The study of specific effects of these receptors on the different neuron populations will be critical to determine whether 5-HT7Rs exert anti- or pro-nociceptive effects at the spinal level. References 1. Cortes-Altamirano JL et al., Curr. Neuropharm, 2018, 16:210-221 2. Bardoni R, Curr. Neuropharm, 2019, 17:1133-1145 3. Hedlund PB et al, Neurosci Lett. 2010 481:12-6. 4. Betelli C et al., Mol. Pain, 2015, 11:6


2020 - A spinal neural circuitry for converting touch to itch sensation [Articolo su rivista]
Chen, S.; Gao, X. -F.; Zhou, Y.; Liu, B. -L.; Liu, X. -Y.; Zhang, Y.; Barry, D. M.; Liu, K.; Jiao, Y.; Bardoni, R.; Yu, W.; Chen, Z. -F.
abstract

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2Cre) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.


2020 - Development of mouse hippocampal organoids [Abstract in Atti di Convegno]
Ciarpella, F; Zamfir, R; Campanelli, A; Dolci, S; Dichio, M; Patuzzo, C; Ren, E; Bardoni, R; Pedrotti, G; Mannino, L; Fumagalli, G; Ahtiainen, A; Malerba, G; Hyttinen, J; Panuccio, G; Palazzolo, G; Curia, G; Decimo, I
abstract


2020 - Identification of a Potent and Selective 5-HT1AReceptor Agonist with In Vitro and In Vivo Antinociceptive Activity [Articolo su rivista]
Linciano, P.; Sorbi, C.; Comitato, A.; Lesniak, A.; Bujalska-Zadrozny, M.; Pawlowska, A.; Bielenica, A.; Orzelska-Gorka, J.; Kedzierska, E.; Biala, G.; Ronsisvalle, S.; Limoncella, S.; Casarini, L.; Cichero, E.; Fossa, P.; Satala, G.; Bojarski, A. J.; Brasili, L.; Bardoni, R.; Franchini, S.
abstract

Opioids are the gold standard drugs for the treatment of acute and chronic severe pain, although their serious side effects constitute a big limitation. In the search for new and safer drugs, 5-HT1AR agonists are emerging as potential candidates in pain relief therapy. In this work, we evaluated the affinity and activity of enantiomers of the two newly synthesized, potent 5-HT1AR agonists N-[(2,2-diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyridin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate (rac-1) and N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-methyl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydrogenoxalate (rac-2) in vitro and in vivo. The role of chirality in the interaction with 5-HT1AR was evaluated by molecular docking. The activity of the rac-1 was tested in mouse models of acute pain (hot plate) and severe tonic nociceptive stimulation (intraplantar formalin test). Rac-1 was active in the formalin test with a reduction in paw licking in both phases at 10 mg/kg, and its effect was abolished by the selective 5-HT1AR antagonist, WAY-100635. The eutomer (S)-1, but not the racemate, was active during the hot plate test at 10 and 20 mg/kg, and this effect was abolished by 30 min treatment with WAY-100635 at 30 min. Similarly to 8-OH-DPAT, (S)-1 evoked a slow outward current and depressed spontaneous glutamatergic transmission in superficial dorsal horn neurons, more effectively than rac-1. The eutomer (S)-1 showed promising developability properties, such as high selectivity over 5-HT subtypes, no interaction with the μ receptors, and low hepato- and cardiotoxicity. Therefore, (S)-1 may represent a potential candidate for the treatment of acute and chronic pain without having the adverse effects that are commonly associated with the classic opioid drugs.


2019 - IDENTIFICATION OF POTENT 1,3-DIOXOLANE-BASED 5-HT1A RECEPTOR AGONISTS FOR THE TREATMENTS OF CNS DISORDERS AND PAIN [Poster]
Linciano, Pasquale; Sorbi, Claudia; Franchini, Silvia; Tait, Annalisa; Bardoni, Rita; Ronsisvalle, Simone; Denora, Nunzio; Lesniak, Anna; Bujalska-Zadrożny, Magdalena; Pawłowska, Agata; Chichero, Elena; Fossa, Paola; Brasili, Livio
abstract


2019 - Pain Inhibits GRPR Neurons via GABAergic Signaling in the Spinal Cord [Articolo su rivista]
Bardoni, Rita; Shen, Kai-Feng; Li, Hui; Jeffry, Joseph; Barry, Devin M.; Comitato, Antonella; Li, Yun-Qing; Chen, Zhou-Feng
abstract

It has been known that algogens and cooling could inhibit itch sensation; however, the underlying molecular and neural mechanisms remain poorly understood. Here, we show that the spinal neurons expressing gastrin releasing peptide receptor (GRPR) primarily comprise excitatory interneurons that receive direct and indirect inputs from C and Aδ fibers and form contacts with projection neurons expressing the neurokinin 1 receptor (NK1R). Importantly, we show that noxious or cooling agents inhibit the activity of GRPR neurons via GABAergic signaling. By contrast, capsaicin, which evokes a mix of itch and pain sensations, enhances both excitatory and inhibitory spontaneous synaptic transmission onto GRPR neurons. These data strengthen the role of GRPR neurons as a key circuit for itch transmission and illustrate a spinal mechanism whereby pain inhibits itch by suppressing the function of GRPR neurons.


2019 - Serotonergic modulation of nociceptive circuits in spinal cord dorsal horn [Articolo su rivista]
Bardoni, R.
abstract

Background: Despite the extensive number of studies performed in the last 50 years, aimed at describing the role of serotonin and its receptors in pain modulation at the spinal cord level, several aspects are still not entirely understood. The interpretation of these results is often complicated by the use of different pain models and animal species, together with the lack of highly selective agonists and antagonists binding to serotonin receptors. Method: In this review, a search has been conducted on studies investigating the modulatory action exerted by serotonin on specific neurons and circuits in the spinal cord dorsal horn. Particular attention has been paid to studies employing electrophysiological techniques, both in vivo and in vitro. Conclusion: The effects of serotonin on pain transmission in dorsal horn depend on several factors, including the type of receptors activated and the populations of neurons involved. Recently, studies performed by activating and/or recording from identified neurons have importantly contributed to the understanding of serotonergic modulation on dorsal horn circuits.


2018 - Non-canonical Opioid Signaling Inhibits Itch Transmission in the Spinal Cord of Mice [Articolo su rivista]
Munanairi, A; Liu, Xy; Barry, Dm; Yang, Q; Yin, Jb; Jin, H; Li, H; Meng, Qt; Peng, Jh; Wu, Zy; Yin, J; Zhou, Xy; Wan, L; Mo, P; Kim, S; Huo, Fq; Jeffry, J; Li, Yq; Bardoni, R; Bruchas, Mr; Chen, Zf
abstract

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies


2017 - Distinct roles of NMB and GRP in itch transmission [Articolo su rivista]
Wan, Li; Jin, Hua; Liu, Xian-Yu; Jeffry, Joseph; Barry, Devin M.; Shen, Kai-Feng; Peng, Jia-Hang; Liu, Xue-Ting; Jin, Jin-Hua; Sun, Yu; Kim, Ray; Meng, Qing-Tao; Mo, Ping; Yin, Jun; Tao, Ailin; Bardoni, Rita; Chen, Zhou-Feng
abstract

A key question in our understanding of itch coding mechanisms is whether itch is relayed by dedicated molecular and neuronal pathways. Previous studies suggested that gastrin-releasing peptide (GRP) is an itch-specific neurotransmitter. Neuromedin B (NMB) is a mammalian member of the bombesin family of peptides closely related to GRP, but its role in itch is unclear. Here, we show that itch deficits in mice lacking NMB or GRP are non-redundant and Nmb/Grp double KO (DKO) mice displayed additive deficits. Furthermore, both Nmb/Grp and Nmbr/Grpr DKO mice responded normally to a wide array of noxious stimuli. Ablation of NMBR neurons partially attenuated peripherally induced itch without compromising nociceptive processing. Importantly, electrophysiological studies suggested that GRPR neurons receive glutamatergic input from NMBR neurons. Thus, we propose that NMB and GRP may transmit discrete itch information and NMBR neurons are an integral part of neural circuits for itch in the spinal cord.


2017 - GABAB receptors-mediated tonic inhibition of glutamate release from Aβ fibers in rat laminae III/IV of the spinal cord dorsal horn. [Articolo su rivista]
Salio, C; Merighi, A; Bardoni, Rita
abstract

Presynaptic GABAB receptors (GABABRs) are highly expressed in dorsal root ganglion neurons and spinal cord dorsal horn. GABABRs located in superficial dorsal horn play an important antinociceptive role, by acting at both pre- and postsynaptic sites. GABABRs expressed in deep dorsal horn could be involved in the processing of touch sensation and possibly in the generation of tactile allodynia in chronic pain. The objective of this study was to characterize the morphological and functional properties of GABABRs expressed on Aβ fibers projecting to lamina III/IV and to understand their role in modulating excitatory synaptic transmission. We performed high-resolution electron microscopic analysis, showing that GABAB2 subunit is expressed on 71.9% of terminals in rat lamina III-IV. These terminals were engaged in axodendritic synapses and, for the 46%, also expressed glutamate immunoreactivity. Monosynaptic excitatory postsynaptic currents, evoked by Aβ fiber stimulation and recorded from lamina III/IV neurons in spinal cord slices, were strongly depressed by application of baclofen (0.1-2.5 µM), acting as a presynaptic modulator. Application of the GABABR antagonist CGP 55845 caused, in a subpopulation of neurons, the potentiation of the first of two excitatory postsynaptic currents recorded with the paired-pulse protocol, showing that GABABRs are endogenously activated. A decrease in the paired-pulse ratio accompanied the effect of CGP 55845, implying the involvement of presynaptic GABABRs. CGP 55845 facilitated only the first excitatory postsynaptic current also during a train of four consecutive stimuli applied to Aβ fibers. These results suggest that GABABRs tonically inhibit glutamate release from Aβ fibers at a subset of synapses in deep dorsal horn. This modulation specifically affects only the early phase of synaptic excitation in lamina III-IV neurons


2015 - Gabab receptor mediated presynaptic inhibition in laminae III-IV of rat dorsal horn [Abstract in Atti di Convegno]
Bardoni, Rita
abstract

The neurotransmitter GABA exerts a strong inhibitory control on spinal cord dorsal horn, by acting both at ionotropic (GABAA) and metabotropic (GABAB) receptors. Several electrophysiological studies have shown that GABAB receptors expressed on Adelta and C primary afferents inhibit glutamate release in superficial laminae (I and II). Although GABAB receptors have been localized also on large diameter fibers, projecting to deeper dorsal horn laminae, their function in presynaptic inhibition has not been fully characterized. I performed patch-clamp recordings from lamina III-IV neurons, in spinal cord slices from postnatal rats (P17-P23), to determine GABAB receptor role in modulating glutamate release from low threshold afferent fibers (mainly Abeta). Glutamatergic monosynaptic EPSCs were evoked by stimulating the dorsal root at low intensities and applying the paired pulse protocol. Application of the GABAB receptor agonist baclofen (1 microMol) reversibly inhibited both EPSC peaks (1st peak decrease: 76.3±3.1%; 2nd peak: 42.6±8.7%, n=9) and increased the paired pulse ratio (PPR) (PPR increase: 147.7±20.6%). Baclofen 2.5 microMol produced a stronger inhibition of both peaks (1st peak decrease: 86.2±3.1%; 2nd peak: 57.6±5.1%, n=7 ) and a larger increase of PPR (288.4±60.9%). The effects of baclofen at both concentrations were blocked by the GABAB antagonist CGP 55845 (5 microMol). The intracellular perfusion of the recorded neuron with the G protein inhibitor GDP-beta-S did not alter the inhibitory effects of baclofen, confirming a presynaptic site of action (n=10). In order to assess whether presynaptic GABAB receptors are endogenously activated, I tested the effect of CGP 55845 alone on evoked EPSCs (paired pulse protocol). The GABAB antagonist caused a significant increase of the first EPSC peak in 4 neurons out of 15 tested, accompanied by a smaller increase of the second peak (1st peak increase: 42.5±15.9%; 2nd peak: 10.3±6.7%). PPR was decreased by CGP 55845 in this group of cells (PPR ctl=0.60±0.04; PPR CGP=0.47±0.05). These findings indicate that presynaptic GABAB receptors are effective in modulating glutamate release from low threshold A fibers in dorsal horn laminae III-IV and suggest that they could be endogenously activated by GABA.


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 - Transient, activity dependent inhibition of transmitter release from low threshold afferents mediated by GABA A receptors in spinal cord lamina III/IV [Articolo su rivista]
Betelli, Chiara; Macdermott, Amy; Bardoni, Rita
abstract

Background Presynaptic GABA A receptors (GABA A Rs) located on central terminals of low threshold afferent fibers are thought to be involved in the processing of touch and possibly in the generation of tactile allodynia in chronic pain. These GABA A Rs mediate primary afferent depolarization (PAD) and modulate transmitter release. The objective of this study was to expand our understanding of the presynaptic inhibitory action of GABA released onto primary afferent central terminals following afferent stimulation. Results We recorded evoked postsynaptic excitatory responses (eEPSCs and eEPSPs) from lamina III/IV neurons in spinal cord slices from juvenile rats (P17–P23, either sex), while stimulating dorsal roots. We investigated time and activity dependent changes in glutamate release from low threshold A fibers and the impact of these changes on excitatory drive. Blockade of GABA A Rs by gabazine potentiated the second eEPSC during a train of four afferent stimuli in a large subset of synapses. This resulted in a corresponding increase of action potential firing after the second stimulus. The potentiating effect of gabazine was due to inhibition of endogenously activated presynaptic GABA A Rs, because it was not prevented by the blockade of postsynaptic GABA A Rs through intracellular perfusion of CsF. Exogenous activation of presynaptic GABA A Rs by muscimol depressed evoked glutamate release at all synapses and increased paired pulse ratio (PPR). Conclusions These observations suggest that afferent driven release of GABA onto low threshold afferent terminals is most effective following the first action potential in a train and serves to suppress the initial strong excitatory drive onto dorsal horn circuitry.


2014 - Delta Opioid Receptors Presynaptically Regulate Cutaneous Mechanosensory Neuron Input to the Spinal Cord Dorsal Horn [Articolo su rivista]
Bardoni, R.; Tawfik, V. L.; Wang, D.; Francois, A.; Solorzano, C.; Shuster, S. A.; Choudhury, P.; Betelli, C.; Cassidy, C.; Smith, K.; deNooij, J. C.; Mennicken, F.; O'Donnell, D.; Kieffer, B. L.; Woodbury, C. J.; Basbaum, A. I.; MacDermott, A. B.; Scherrer, G.
abstract


2014 - Delta opioid receptors presynaptically regulate cutaneous mechanosensory neuron input to the spinal cord dorsal horn [Articolo su rivista]
Bardoni, Rita; TAWFIK V., L; Wang, D; François, A; Solorzano, C; Shuster, Sa; Choudhury, P; Betelli, C; Cassidy, C; Smith, K; DE NOOIJ, Jc; Mennicken, F; O’Donnell, D; Kieffer, Bl; Woodbury, Cj; Basbaum, Ai; Macdermott, Ab; Scherrer, G.
abstract

Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.


2013 - Pre- and postsynaptic inhibitory control in the spinal cord dorsal horn. [Articolo su rivista]
Bardoni, Rita; Takazawa, T; Tong, Ck; Choudhury, P; Scherrer, G; Macdermott, A. B.
abstract

Sensory information transmitted to the spinal cord dorsal horn is modulated by a complex network of excitatory and inhibitory interneurons. The two main inhibitory transmitters, GABA and glycine, control the flow of sensory information mainly by regulating the excitability of dorsal horn neurons. A presynaptic action of GABA has also been proposed as an important modulatory mechanism of transmitter release from sensory primary afferent terminals. By inhibiting the release of glutamate from primary afferent terminals, activation of presynaptic GABA receptors could play an important role in nociceptive and tactile sensory coding, while changes in their expression or function could be involved in pathological pain conditions, such as allodynia.


2013 - Role of presynaptic glutamate receptors in pain transmission at the spinal cord level. [Articolo su rivista]
Bardoni, Rita
abstract

Nociceptive primary afferents release glutamate, activating postsynaptic glutamate receptors on spinal cord dorsal horn neurons. Glutamate receptors, both ionotropic and metabotropic, are also expressed on presynaptic terminals, where they regulate neurotransmitter release. During the last two decades, a wide number of studies have characterized the properties of presynaptic glutamatergic receptors, particularly those expressed on primary afferent fibers. This review describes the subunit composition, distribution and function of presynaptic glutamate ionotropic (AMPA, NMDA, kainate) and metabotropic receptors expressed in rodent spinal cord dorsal horn. The role of presynaptic receptors in modulating nociceptive information in experimental models of acute and chronic pain will be also discussed.


2012 - CONTROL OF Abeta LOW-THRESHOLD MECHANORECEPTOR INPUTS TO SPINAL CORD NEURONS BY DELTA OPIOID RECEPTORS [Abstract in Atti di Convegno]
G., Scherrer; Bardoni, Rita; P., Choudhury; B. L., Kieffer; A., Basbaum; A. B., Macdermott
abstract

Aim of Investigation: Sensory neurons of the dorsal root ganglia (DRG) are heterogeneous in order to respond to a variety of environmental stimuli (e.g. noxious heat or light touch) and transmit somatosensory information to spinal neurons. Opioids relieve pain in part by acting on DRG neurons, but the precise identity of the cells in which activity is regulated by opioids remains unclear. Here we determined the molecular identity of DRG neurons that express opioid receptors and tested the hypothesis that the delta opioid receptor (DOR) controls Abeta low-threshold mechanoreceptor inputs to spinal cord neurons.Methods: We used a combination of mouse genetics, neuroanatomical and electrophysiological approaches. We first used knockin mice expressing a DOReGFP receptor and a panoply of markers for distinct populations of DRG neurons to localize DOR in DRG and skin by immunohistochemistry. We next performed whole cell patch clamp recording in spinal cord slices to investigate the effect of opioid agonists on synaptic transmission between DRG and spinal dorsal horn neurons.Results: We found that DOR is expressed by several populations of mechanonociceptors, as well as by Ret and TrkC coexpressing Abeta low-threshold mechanoreceptors that innervate touch-sensing organs in the skin. By contrast, the mu opioid receptor (MOR) is predominantly expressed by thermonociceptors, as well as by a small population of putative AM mechanonociceptors in which MOR and DOR are colocalized at the plasma membrane. In spinal cord slices from wild-type mice, we found that the DOR agonist deltorphin depresses Abeta synaptic input to laminae III-V spinal neurons, consistent with our anatomical findings.Conclusions: Collectively our results indicate that the function of the opioid system in somatosensation is not restricted to the regulation of nociceptive inputs to the spinal cord but also includes control of touch information, thereby providing a rationale for targeting DOR in clinical situations where pain results from a dysfunction of Abeta low-threshold mechanoreceptors.


2012 - ROLE OF PERIPHERALLY EXPRESSED CALCIUM-PERMEABLE AMPA RECEPTORS IN CHRONIC INFLAMMATORY PAIN [Abstract in Atti di Convegno]
Vijayan, Gangadharan; Rui, Wang; Bettina, Ulzhöfer; Ceng, Luo; Bardoni, Rita; Kiran Kumar, Bali; Nitin, Agarwal; Irmgard, Tegeder; Ullrich, Hildebrandt; Gergely G., Nagy; Andrew J., Todd; Alessia, Ghirri; Annette, Häussler; Rolf, Sprengel; Peter H., Seeburg; Amy B., Macdermott; Gary R., Lewin; Rohini, Kuner
abstract

Aim of Investigation: Mechanisms underlying chronic, pathological pain are not well-understood. Plasticity in peripheral nociceptors and their synapses with spinal neurons can serve as a cellular basis for the development and maintenance of chronic pain following inflammation or nerve injury. Although there are anatomical evidence for the expression of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type (AMPA-type) glutamate receptors (AMPARs) in the peripheral nervous system, either their role in chronic pain or their relative contribution of peripheral versus central in chronic pain is unknown. To elucidate the function of AMPARs expressed in peripheral nociceptive neurons in pain modulation, we generated a conditional knock-out mouse lacking GluA1 or GluA2 subunit of AMPA receptors specifically in nociceptors, while preserving their expression in the spinal cord and brain using cre-lox recombinase system under the control of Nav1.8 promoter. Methods: All animal experiments were approved by the local ethical committee (Regierungspräsidium Karlsruhe). Mice lacking GluA1 or GluA2 specifically in peripheral nociceptors were generated using Cre-loxP mediated recombination system without affecting its expression in spinal cord and brain, being referred to as SNS-GluA1-/-or SNS-GluA2-/-. Chronic pain hypersensitivity and underlying mechanisms in these mice were tested using a combination of molecular, biochemical, electrophysiological and behavioral analyses. Pain models used in this study includes; capsaicin test, Formalin test, Complete Freund's adjuvant and knee arthritis inflammatory pain models. Results: In this study, we established the role of AMPA receptors located at the peripheral side of the pain pathway in physiological and inflammatory pain states. To this end, we deleted individual subunits and this approach affects the activation, gating, signaling properties of the AMPA receptors without deleting the function of AMPA receptors completely. Interestingly, we observed that the deletion of GluRA1 but not GluRA2 affected the role of AMPA receptor in pain processing. Deletion of GluRA1 reduced the calcium influx triggered by its native agonist, glutamate, by 50%. It also decreases the excitability of nerve fibers in response to exposure of peripheral terminals to different algogens. In contrast, GluRA1 is also contributing to depression of neurotransmitter release in central terminals of nociceptors. Nonetheless, the overall in vivo contributions of GluRA1 at peripheral and central terminals of nociceptors clearly emerged as a pronociceptive, sensitizing role for GluA1-containing AMPARs in inflammatory hyperalgesia. Conclusions: In summary, the results of this study demonstrate an important contribution of GluA1-containing calcium-permeable AMPARs expressed in the peripheral nervous system in modulating the activation properties of nociceptive neurons in response to algogens or in an inflammatory milieu. Furthermore, these findings clearly elucidated and delineated the central versus peripheral components of actions of calcium permeable AMPA receptor in chronic pain. This distinction is clinically very important because if chronic pain is completely centralized, therapeutic approaches targeting peripheral mechanisms are unlikely to be therapeutically beneficial. Conversely, if the peripheral contribution is strong, targeting peripheral mechanisms are offers the unique advantage of bypassing deleterious side effects.


2011 - Gabaa receptors modulate synaptic transmission mediated by Abeta fibers in lamina III of rat dorsal horn [Abstract in Atti di Convegno]
Bardoni, Rita; C., Betelli; A. B., Macdermott
abstract

GABAA receptors contribute to depolarization of primary afferent fibers in the spinal cord following the stimulation of primary afferent fibers. This suggests that GABAA receptors are expressed at primary afferent terminals, where they could modulate glutamate release either by causing membrane depolarization or by shunting. We have investigated the effect of presynaptic GABAA receptor activation on synaptic transmission in the dorsal horn by recording Abeta fiber mediated EPSCs from lamina III neurons in rat spinal cord slices. The monosynaptic nature of the EPSCs was assessed by stimulating the attached dorsal root at 20 Hz and low intensity. Exogenous activation of GABAA receptors by 2 microMol muscimol caused depression of the EPSCS amplitudes and changed paired pulse ratio (PPR) in all cells tested (PPR increase:77.9±17%, n=12). Similar results were obtained from both unidentified and NK1 receptor expressing neurons. Application of muscimol at different concentrations (ranging from 0.5 to 5 microMol showed a dose-dependent increase of PPR. Muscimol also caused an increase of miniature EPSC frequency in lamina III neurons (mean frequency: 2.9±0.7 Hz in control, 6.5±1.5 Hz in muscimol, n=7), while mEPSC amplitude was not affected. Application of 2 microMol muscimol in the presence of 10 microMol bumetanide, a blocker of the chloride transporter NKCC1 in primary afferent fibers, still caused an increase of the evoked EPSC PPR, although the effect was significantly reduced compared to control (PPR increase in bumetanide: 24.3±7%, n=12). In order to assess whether presynaptic GABAA receptors are endogenously activated , we tested the effect of the GABAA antagonist gabazine (10 µM) on Abeta fiber mediated EPSCs in lamina III neurons, evoked by repetitive stimulation (4 pulses at 10-50 Hz). Gabazine application increased the second peak (138.4±38%, n=17), while the other peaks were unaffected or slightly decreased. Our results suggest that functional GABAA receptors are expressed on Abeta fiber terminals, where they depress synaptic transmission during repetitive stimulation. This mechanism of presynaptic inhibition could be impaired during chronic pain, leading to the facilitation of Abeta mediated synaptic transmission and to the generation of allodynia.


2011 - Peripheral calcium-permeable AMPA receptors regulate chronic inflammatory pain in mice. [Articolo su rivista]
V., Gangadharan; R., Wang; B., Ulzhöfer; C., Luo; Bardoni, Rita; K. K., Bali; N., Agarwal; I., Tegeder; U., Hildebrandt; G. G., Nagy; A. J., Todd; Ghirri, Alessia; A., Häussler; R., Sprengel; P. H., Seeburg; A. B., Macdermott; G. R., Lewin; R., Kuner
abstract

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type (AMPA-type) glutamate receptors (AMPARs) play an important role in plasticity at central synapses. Although there is anatomical evidence for AMPAR expression in the peripheral nervous system, the functional role of such receptors in vivo is not clear. To address this issue, we generated mice specifically lacking either of the key AMPAR subunits, GluA1 or GluA2, in peripheral, pain-sensing neurons (nociceptors), while preserving expression of these subunits in the central nervous system. Nociceptor-specific deletion of GluA1 led to disruption of calcium permeability and reduced capsaicin-evoked activation of nociceptors. Deletion of GluA1, but not GluA2, led to reduced mechanical hypersensitivity and sensitization in models of chronic inflammatory pain and arthritis. Further analysis revealed that GluA1-containing AMPARs regulated the responses of nociceptors to painful stimuli in inflamed tissues and controlled the excitatory drive from the periphery into the spinal cord. Consequently, peripherally applied AMPAR antagonists alleviated inflammatory pain by specifically blocking calcium-permeable AMPARs, without affecting physiological pain or eliciting central side effects. These findings indicate an important pathophysiological role for calcium-permeable AMPARs in nociceptors and may have therapeutic implications for the treatment chronic inflammatory pain states.


2011 - Presynaptic AMPA receptors modulate glutamate release in mouse spinal cord dorsal horn [Abstract in Rivista]
Bardoni, Rita; A. B., Macdermott; R., Kuner
abstract

Purpose of the study was the investigation of the role of AMPA receptors, expressed on nociceptive fiber terminals, in modulating glutamate release. We also intended to characterize the contribution of different AMPA receptor subunits to presynaptic modulation. We used the patch-clamp technique on dorsal horn neurons in mouse spinal cord slices. Excitatory postsynaptic currents (EPSCs), recorded from lamina II neurons, were evoked by stimulation of the attached dorsal root. Recordings were performed from both wild-type animals and transgenic mice carrying a nociceptor-specific deletion of GluA1 or GluA2 subunit. We observed that application of AMPA receptor agonists (AMPA or kainate) caused a significative depression of evoked glutamatergic EPSCs in wild type mice. The effect was reversible in wash. The inhibition of EPSCs was accompanied, in most neurons, by an increase of synaptic failures and a change in the coefficient of variation (CV) of EPSC peak amplitudes. Both effects are considered as indicators of presynaptic modulation. We observed similar effects, in the presence of AMPA, also in mice with nociceptor-specific deletion of the GluA2 subunit. However, in mice with deletion of the GluA1 subunit, presynaptic modulation exerted by AMPA (expressed as a change of CV) was significantly reduced. We showed that functional AMPA receptors are expressed on nociceptive primary afferents in mouse spinal cord. Their exogenous activation causes the depression of glutamate release. The decrease of presynaptic modulation observed in mice carrying a deletion of the GluA1 subunit, but not of GluA2, suggests that calcium-permeable AMPA receptors are particularly important in regulating glutamate release from primary afferents


2010 - Glutamate-mediated astrocyte-to-neuron signaling in the rat dorsal horn [Articolo su rivista]
Bardoni, Rita; Ghirri, Alessia; Micaela, Zonta; Betelli, Chiara; Vitale, Giovanni; Ruggieri, Valentina; Sandrini, Maurizio; Giorgio, Carmignoto
abstract

By releasing neuroactive agents, including proinflammatory cytokines, prostaglandinsand neurotrophins, microglia and astrocytes are proposed to be involved in nociceptivetransmission, especially in conditions of persistent, pathological pain. The specificaction on dorsal horn neurons of agents released from astrocytes, such as glutamate, hasbeen, however, poorly investigated. By using patch-clamp and confocal microscopecalcium imaging techniques in rat spinal cord slices, we monitored the activity of dorsalhorn lamina II neurons following astrocyte activation. Results obtained revealed thatstimuli that triggered Ca2+ elevations in astrocytes, such as the purinergic receptoragonist BzATP and low extracellular Ca2+, induce in lamina II neurons slow inwardcurrents (SICs). Similarly to SICs triggered by astrocytic glutamate in neurons fromother central nervous system regions, these currents: i) are insensitive to TTX, ii) areblocked by the NMDA receptor antagonist D-AP5, iii) lack an AMPA component, andiv) have slow rise and decay times. Ca2+ imaging also revealed that astrocytic glutamateevokes NMDAR-mediated episodes of synchronous activity in groups of substantiagelatinosa neurons. Importantly, in a model of peripheral inflammation, thedevelopment of thermal hyperalgesia and mechanical allodynia was accompanied by a significant increase of spontaneous SICs in dorsal horn neurons. The NMDARmediatedastrocyte-to-neuron signaling thus represents a novel pathway that maycontribute to the control of central sensitization in pathological pai


2009 - BDNF and TrkB mediated mechanisms in the spinal cord [Capitolo/Saggio]
Bardoni, R.; Merighi, A.
abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an essential role during development, promoting the survival of specific populations of central and peripheral neurons. During adulthood, BDNF also acts as a synaptic modulator in several areas of the central nervous system (CNS), including the spinal cord, and is involved in short and long term changes of synaptic efficacy. In spinal cord dorsal horn BDNF is expressed in the peptidergic terminals originating from primary afferent fibres, while its high affinity receptor trkB has been detected on both primary afferent terminals and dorsal horn neurons. In superficial dorsal horn, exogenous BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotransmission. Conditions of inflammatory and neuropathic pain alter the expression of BDNF and trkB receptors in dorsal horn. In experimental pain models, modulation of synaptic transmission by BDNF plays an important role in the induction and maintenance of central sensitization.


2009 - BDNF and TrkB mediated mechanisms in the spinal cord. In "Synaptic plasticity in pain", Editor Marzia Malcangio [Capitolo/Saggio]
Bardoni, Rita; A., Merighi
abstract

BDNF acts as a synaptic modulator in spinal cord, inducing short and long term changes in pain transmission


2009 - Postnatal development shifts the balance of pain descending control [Articolo su rivista]
Bardoni, Rita
abstract

Non presente


2008 - BDNF as a pain modulator [Articolo su rivista]
A., Merighi; C., Salio; Ghirri, Alessia; L., Lossi; F., Ferrini; Betelli, Chiara; Bardoni, Rita
abstract

At least some neurotrophins may be powerful modulators of synapses, thereby influencing short- and long-term synaptic efficiency. BDNF acts at central synapses in pain pathways both at spinal and supraspinal levels. Neuronal synthesis, subcellular storage/co-storage and release of BDNF at these synapses have been characterized on anatomical and physiological grounds, in parallel with trkB (the high affinity BDNF receptor) distribution. Histological and functional evidence has been provided, mainly from studies on acute slices and intact animals, that BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotrasmission in spinal cord. Recent studies have unraveled some of the neuronal circuitries and mechanisms involved, highlighting the key role of synaptic glomeruli in lamina II as the main sites for such a modulation.


2008 - Pre-synaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of post-natal rat spinal cord [Articolo su rivista]
A., Merighi; Bardoni, Rita; C., Salio; L., Lossi; F., Ferrini; Prandini, Massimiliano; M., Zonta; S., Gustincich; G., Carmignoto
abstract

A subset of primary sensory neuronsproduces BDNF, which is implicated in control of nociceptiveneurotransmission. We previously localized full-lengthtrkB receptors on their terminals within lamina II. To functionallystudy these receptors, we here employed patchclamprecordings, calcium imaging and immunocytochemistryon slices from 8–12 days post-natal rats. In this preparation,BDNF (100–500 ng/mL) enhances the release of sensoryneurotransmitters (glutamate, substance P, CGRP) in laminaII by acting on trkB receptors expressed by primaryafferent fibers of the peptidergic nociceptive type (PNPAFs).Effect was blocked by trk antagonist K252a or antitrkBantibody clone 47.Apre-synaptic mechanism was demonstratedafter (i) patch-clamp recordings where the neurotrophininduced a significant increase in frequency, but notamplitude, of AMPA-mediated mEPSCs, (ii) real time calciumimaging, where sustained application of BDNF evokedan intense response in up to 57% lamina II neurons with asignificant frequency rise. Antagonists of ionotropic glutamatereceptors and NK1 receptors completely inhibited thecalcium response to BDNF. Reduction of CGRP (a specificmarker of PN-PAFs) and substance P content in dorsal hornfollowing BDNF preincubation, and analysis of the calciumresponse after depletion with capsaicin, confirmed that theneurotrophin presynaptically enhanced neurotransmitterrelease from PN-PAFs. This is the first demonstration thattrkB receptors expressed by PN-PAF terminals in lamina IIare functional during postnatal development. Implicationsof this finding are discussed considering that BDNF can bereleased by these same terminals and microglia, a fraction ofwhich (as shown here) contains BDNF also in unactivatedstate


2007 - BDNF-mediated modulation of GABA and glycine release in dorsal horn lamina II from postnatal rats [Articolo su rivista]
Bardoni, Rita; Ghirri, Alessia; C., Salio; Prandini, Massimiliano; A., Merighi
abstract

Recent studies show that excitatoryglutamatergic transmission is potentiated by BDNF insuperficial dorsal horn, both at the pre- and the postsynapticsite. The role of BDNF in modulating GABA andglycine-mediated inhibitory transmission has not beenfully investigated. To determine whether the neurotrophinis effective in regulating the spontaneous release of thetwo neurotransmitters, we have recorded miniature inhibitorypostsynaptic currents (mIPSCs) in lamina II of postnatalrats. We show that application of BDNF enhancedthe spontaneous release of GABA and glycine, in presenceof tetrodotoxin. The effect was blocked by the trk-receptorinhibitor k-252a. Amplitude and kinetics of mIPSCswere not altered. Evoked GABA and glycine IPSCs(eIPSCs) were depressed by BDNF and the coefficient ofvariation of eIPSC amplitude was significantly increased.By recording glycine eIPSCs with the paired-pulse protocol,an increase of paired-pulse ratio during BDNF applicationwas observed. We performed parallel ultrastructuralstudies to unveil the circuitry involved in the effectsof BDNF. These studies show that synaptic interactionsbetween full length functional trkB receptors and GABAcontainingprofiles only occur at non peptidergic synapticglomeruli of types I and II. Expression of trkB in presynapticvesicle-containing dendrites originating fromGABAergic islet cells, indicates these profiles as key structuresin the modulation of inhibitory neurotransmissionby the neurotrophin. Our results thus describe a yetuncharacterized effect of BDNF in lamina II, giving furtherstrength to the notion that the neurotrophin plays animportant role in pain neurotransmission.


2007 - Controllo Motorio. In "Fisiologia. Molecole, cellule e sistemi" Editors: E.D'Angelo e A. Peres. [Capitolo/Saggio]
Bardoni, Rita
abstract

non disponibile


2007 - Glutamate released from astrocytes mediates slow inward currents in rat spinal cord dorsal horn. [Abstract in Atti di Convegno]
Bardoni, Rita; Ghirri, A; Betelli, C; Carmignoto, C.
abstract

Activation of glial cells (microglia and astrocytes) in spinal cord dorsal horn is involved in the maintenance and possibly the generation of inflammatory and neuropathic pain (Wieseler-Frank et al., 2004). In other brain regions, activated astrocytes release glutamate that acts on NMDA receptors, preferentially at the extrasynaptic membrane, to generate in nearby neurons slow inward currents (SICs). We used here patch-clamp recording and calcium imaging techniques to investigate whether astrocytes exert a similar action on dorsal horn neurons. In spinal cord slices from young postnatal rats, in the absence of extracellular Mg2+ and in the presence of tetrodotoxin, we observed spontaneous SICs in 5 of 42 lamina II neurons (12%), while occasional recordings revealed SICs also in laminae III-IV neurons. The percentage of cells exhibiting SICs increased by application of stimuli that induced Ca2+ oscillations in astrocytes, such as the purinerigic receptor agonist 2-& 3-O-(4-benzoylbenzoyl)ATP (100 µM) and low extracellular Ca2+. These stimuli triggered SICs in 33% and 61 % of neurons, respectively. These events had the typical slow kinetics of SICs: a rise time of 70 ± 14 ms, a decay time of 372 ± 102 ms and a mean amplitude of 175 ± 37 pA. By using pharmacological tools, we confirmed the identity of these events as SICs. Indeed, they resulted to be mediated by NMDA receptors since they were blocked by 100 µM D-APV (only one SIC was observed in 1 out of 9 cells in low extracellular Ca2+), and the AMPA receptor blocker NBQX (10 µM) had no effects (under these conditions, 55% of neurons displayed SICs upon low Ca2+ stimulation). Furthermore, in 1 mM Mg2+, astrocytic glutamate was still capable of activating neuronal NMDA receptors since low Ca2+ induced SICs in 36% of lamina II neurons tested. Confocal microscope Ca2+ imaging in spinal cord slices loaded with the Ca2+ indicator Oregon Green BAPTA-1 revealed that astrocytic glutamate, as previously observed in CA3 and CA1 hippocampal regions, evoked synchronous Ca2+ elevations in small groups of dorsal horn neurons.Our observations reveal that NMDA receptor-mediated signals characterize the action of astrocytes also in the dorsal horn and raise questions about their possible pathophysiological role.


2007 - Modulazione della trasmissione sinaptica inibitoria da parte della neurotrofina BDNF nelle corna dorsali del midollo spinale di ratto [Abstract in Atti di Convegno]
Bardoni, Rita
abstract

Studi compiuti in diverse aree del sistema nervoso centrale hanno mostrato che la neurotrofina BDNF è in grado di controllare la trasmissione sinaptica, modulando sia il rilascio del neurotrasmettitore che la funzionalità dei recettori postsinaptici. E’ noto che nelle corna dorsali del midollo spinale il BDNF, rilasciato dalle fibre afferenti primarie, modula la trasmissione sinaptica glutammatergica. Studi comportamentali hanno suggerito che il BDNF abbia un effetto pro-nocicettivo, anche se sono state osservate azioni analgesiche della neurotrofina in determinate condizioni. Effetti del BDNF sul rilascio di GABA e glicina nelle corna dorsali non erano ancora stati studiati in dettaglio. Il nostro studio si è avvalso sia di tecniche elettrofisiologiche che immunocitochimiche applicate alla microscopia elettronica. Tramite registrazioni in patch-clamp da fettine di midollo spinale di ratti neonati abbiamo osservato che il BDNF provoca un aumento della frequenza delle correnti postsinaptiche in miniatura, mediate sia da GABA che da glicina, senza alterarne l’ampiezza. La neurotrofina provoca anche una depressione delle correnti sinaptiche inibitorie evocate, causando una variazione significativa della variabilità delle risposte sinaptiche e del “paired pulse ratio”. Queste osservazioni suggeriscono che il BDNF possa modulare le sinapsi inibitorie delle corna dorsali, agendo a livello presinaptico. A supporto di ciò, gli studi ultrastrutturali mostrano la co-localizzazione di vescicole di GABA con i recettori per la neurotrofina (recettori trkB) a livello dei glomeruli sinaptici nella lamina II. I nostri dati suggeriscono quindi un ruolo del BDNF nel controllare la trasmissione nocicettiva a livello spinale, tramite la modulazione del rilascio dei due neurotrasmettitori inibitori.


2006 - COMUNICAZIONE CELLULARE:RECETTORI E SISTEMI DI TRASDUZIONE. In: Fisiologia.Molecole, cellule, sistemi. Editors: E. D'Angelo e A. Peres [Capitolo/Saggio]
Bardoni, Rita
abstract

non disponibile


2006 - Fisiologia: molecole, cellule e sistemi [Monografia/Trattato scientifico]
A. M., Angioy; Bardoni, Rita; Bigiani, Albertino; M., Brunelli; A., Contestabile; M., Crispino; E., D'Angelo; F., Franciolini; E., Fugassa; S., Fulle; A., Giuditta; R., Levi; M. G., Lionetto; V., Lombardi; E., Macchi; D., Negrini; P., Paggi; S., Palmero; A., Peres; M., Piccolino; C., Poggesi; G., Rispoli; P., Rossi; V. F., Sacchi; T., Schettino; R., Serio; G., Valenti; Vellani, Vittorio; A., Zaza
abstract

Questo libro nasce con l’intento di offrire agli studenti dei Corsi universitari di carattere biomedico uno strumento di riferimento per lo studio dei meccanismi alla base del funzionamento degli esseri viventi, in particolare nel regno animale: l’attenzione è principalmente rivolta ai processi fondamentali e unificanti dei fenomeni fisiologici, quelli che costituiscono le basi, di solito molecolari e cellulari, di funzioni fisiologiche anche apparentemente lontane tra loro. Poiché questo libro è destinato agli studenti di Corsi di Laurea differenti, dalle Scienze biologiche alle Biotecnologie, ma anche di Scienze naturali e Farmacia, si è ritenuto utile includere anche capitoli di fisiologia dei sistemi.La sequenza degli argomenti si articola dal livello molecolare e cellulare, per considerare successivamente le interazioni fra le cellule e concludersi con la trattazione dei più importanti organi e sistemi d’organo. Questa progressione dal microscopico al macroscopico segue una logica inversa rispetto alla cronologia della storia dell’indagine dei processi fisiologici, che è naturalmente partita molti secoli fa dallo studio del funzionamento degli apparati e degli organi più evidenti e considerati maggiormente importanti. La progressiva comprensione dei meccanismi di funzionamento e il parallelo sviluppo tecnologico degli strumenti d’indagine hanno facilitato nel corso del tempo dei processi a livelli via via più nascosti e microscopici per trovare una spiegazione soddisfacente dell’evento macroscopico. Laddove questo approccio è riuscito ad addentrarsi fino al livello molecolare si è constatato che molte funzioni macroscopiche, a prima vista significativamente diverse tra loro, si basano in realtà su processi microscopici molto simili. Per questo motivo ci è sembrato preferibile trattare inizialmente una serie di processi elementari che vengono successivamente ritrovati in azione per contribuire alla realizzazione di funzioni fisiologiche complesse.A loro volta i processi fisiologici elementari, a livello molecolare e cellulare, si basano sulle leggi della fisica, della chimica e della biologia molecolare e cellulare. Queste nozioni costituiscono un’indispensabile base propedeutica per lo studio della fisiologia e non sono normalmente contenute nei testi di fisiologia. Per comodità del lettore, tuttavia, sono stati inclusi alcuni capitoli che sintetizzano alcuni punti fondamentali delle discipline fisiche, chimiche e biologiche di particolare interesse in fisiologia.La grande variabilità dei programmi degli insegnamenti che fanno riferimento alla fisiologia – e la diversa estensione e importanza che nelle diverse Università viene a essi data – ha consigliato di impostare il libro in modo da facilitarne un utilizzo in una certa misura “personalizzato”. Così, accanto a un percorso base, contenente tutti gli argomenti più classici trattati in modo rigoroso, ma senza eccessivi approfondimenti, sono presenti parti differenziate, che potranno essere utilizzate secondo le necessità degli indirizzi di studio. Il testo in corpo ridotto riguarda approfondimenti non strettamente necessari alla preparazione di base. I riquadri trovano spazio nei diversi capitoli, per consentire approfondimenti specifici in aree diverse: biofisica molecolare (per le Classi di Biotecnologie e di Scienze Biologiche con indirizzi cellulari o di Neuroscienze), fisiologia comparata (per le Classi di Scienze naturali e di Scienze biologiche con indirizzi naturalistici), ricerca applicata (Biotecnologie e Farmacia), area storica (destinata a tutti coloro che sono interessati a conoscere la storia delle idee sul funzionamento degli esseri viventi e del corpo umano in particolare, una storia di grandissimo interesse scientifico, filosofico e umano, che può essere tracciata fin dai primordi delle civiltà conosciute).


2006 - Modulation of glutamate release in nociceptive transmission at the spinal cord level [Abstract in Atti di Convegno]
Ghirri, Alessia; Magherini, Pier Cosimo; Carmignoto, G; Bardoni, Rita
abstract

Glutamatergic ionotropic receptors expressed on primary afferent central terminals in spinal cord dorsal horn are functional and can modulate glutamate release, presynaptic glutamate receptors could be activated by glutamate released during synaptic activity or by astrocytes.


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 - Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn [Articolo su rivista]
Bardoni, Rita; C., Torsney; Ck, Tong; Prandini, Massimiliano; Ab, Macdermott
abstract

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.


2003 - Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol [Articolo su rivista]
Ml, Baccei; Bardoni, Rita; M., Fitzgerald
abstract

To study the postnatal development of nociceptive synaptic inputs in the superficial dorsal horn of the neonatal rat spinal cord, we examined the effect of capsaicin and menthol on glutamatergic mEPSCs in postnatal day (P) 0-1, P5-6 and P9-11 slices of spinal cord. Capsaicin (100 nM to 2 muM) increased the mEPSC frequency in a concentration-dependent manner at all ages tested, with a significant enhancement of the effect between P5 and P10. This effect was sensitive to vanilloid receptor (VR) antagonists. The elevation in mEPSC frequency occurred at concentrations of capsaicin (100 nM) that did not alter the distribution of mEPSC amplitudes and was abolished by a dorsal rhizotomy, demonstrating that capsaicin acts via presynaptic VR1 receptors localized on primary afferents. Menthol significantly increased the mEPSC frequency with a similar developmental pattern to capsaicin without consistently affecting mEPSC amplitude. The increase in mEPSC frequency following capsaicin did not depend on transmembrane calcium influx since it persisted in zero [Ca2+](o). The facilitation of spontaneous glutamate release by capsaicin was sufficient to evoke action potentials in neonatal dorsal horn neurons but was accompanied by a block of EPSCs evoked by electrical stimulation of the dorsal root. These results indicate that VR1-expressing nociceptive primary afferents; form functional synaptic connections in the superficial dorsal horn from birth and that activation of the VR1 receptor increases spontaneous glutamate release via an undetermined mechanism. In addition, the data suggest that immature primary afferents express functional menthol receptors that are capable of modulating transmitter release. These results have important functional implications for infant pain processing.


2002 - Functional expression of AMPA receptors on central terminals of rat dorsal root ganglion neurons and presynaptic inhibition of glutamate release [Articolo su rivista]
Cj, Lee; Bardoni, Rita; Ck, Tong; Hs, Engelman; Dj, Joseph; Magherini, Pier Cosimo; Ab, Macdermott
abstract

No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.


2001 - Excitatory synaptic transmission in neonatal dorsal horn: NMDA and ATP receptors [Articolo su rivista]
Bardoni, Rita
abstract

Postnatal development sees a strong synaptogenesis in rat superficial dorsal born. My studies show that synapses mediated by two excitatory neurotransmitters, glutamate and ATP, are functional since the very first postnatal days. Using an electrophysiological approach, the functional properties of two receptors activated by these neurotransmitters, glutamatergic NMDA and ATP ionotropic receptors, are described.


2000 - Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats [Articolo su rivista]
Bardoni, Rita; Magherini, Pier Cosimo; Ab, Macdermott
abstract

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina Ii over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors. NeuroReport 11:1721-1727


1998 - Effects of the P-2-purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2 ',4 '-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord [Articolo su rivista]
Jgg, Gu; Bardoni, Rita; Magherini, Pier Cosimo; Ab, Mcdermott
abstract

The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 mu M significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 mu M had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P-2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2(x) receptor function at excitatory synapses mediated by AMPA receptors. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.


1998 - NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat [Articolo su rivista]
Bardoni, Rita; Magherini, Pier Cosimo; Ab, Macdermott
abstract

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau 1, 24 VS 42 msec; tau 2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).


1997 - ATP mediates synaptic transmission in rat spinal cord lamina II (Substantia gelatinosa) [Abstract in Atti di Convegno]
Bardoni, Rita; Goldstein, Pa; Lee, Cj; Gu, J; Macdermott, Ab
abstract

ATP has been proposed to mediate synaptic transmission in the spinal cord dorsal horn, particularly in the pathway carrying nociceptive information. Using transverse spinal cord slices from postnatal rats, we show that EPSCs mediated by P2X receptors, and presumably activated by synaptically released ATP, are evoked in a subpopulation of spinal cord lamina II neurons, a region known to receive strong input from nociceptive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurons are nondesensitizing, insensitive to alphabeta methylene ATP, and show strong but variable sensitivity to the antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These characteristics are consistent with a heterogeneous population of P2X receptors, the composition of which includes P2X2, P2X4, and P2X6 receptor subtypes. Our results suggest that ATP-activated P2X receptors in lamina II of the rat spinal cord may play a role in transmitting or modulating nociceptive information.


1997 - ATP P-2X receptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord [Articolo su rivista]
Bardoni, Rita; Goldstein, Pa; Lee, Cj; Gu, Jg; Macdermott, Ab
abstract

ATP has been proposed to mediate synaptic transmission in the spinal cord dorsal horn, particularly in the pathway carrying nociceptive information. Using transverse spinal cord slices from postnatal rats, we show that EPSCs mediated by P2X receptors, and presumably activated by synaptically released ATP, are evoked in a subpopulation of spinal cord lamina II neurons, a region known to receive strong input from nociceptive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurons are nondesensitizing, insensitive to alphabeta methylene ATP, and show strong but variable sensitivity to the antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These characteristics are consistent with a heterogeneous population of P2X receptors, the composition of which includes P2X2, P2X4, and P2X6 receptor subtypes. Our results suggest that ATP-activated P2X receptors in lamina II of the rat spinal cord may play a role in transmitting or modulating nociceptive information.


1997 - Electrical properties of periglomerular cells in the frog olfactory bulb. [Articolo su rivista]
Bardoni, Rita; O., Belluzzi; Magherini, Pier Cosimo
abstract

Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) preparation to study the basic electrical properties of these inhibitory interneurons. The cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous excitatory synaptic activity at rest, usually leading to overshooting, TTX-sensitive action potentials. The passive cable properties of the cell membrane have been carefully characterised. Depolarisation of this neurone under voltage-clamp conditions activated a complex pattern of current flow, that has been dissected into its main components. The currents have been isolated resorting to their different kinetic and pharmacological properties. Four main voltage dependent ionic currents have been isolated, two inward currents, I(Na) and I(Ca), and two outward currents carried by potassium ions, one fast transient, I(A)-type and another similar to the delayed rectifier type. These currents have been characterised kinetically and pharmacologically. The functional implications of their properties are discussed.


1997 - NMDA receptor mediated synaptic transmission in rat spinal cord lamina I and II during postnatal development [Abstract in Atti di Convegno]
Bardoni, Rita; Magherini, Pier Cosimo; Macdermott, Ab
abstract

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV.


1996 - Excitatory synapses in the glomerular triad of frog olfactory bulb in vitro [Articolo su rivista]
Bardoni, Rita; O., Belluzzi; Magherini, Pier Cosimo
abstract

WHOLE-CELL patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stymulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component.


1996 - Potassium currents in periglomerular cells of frog olfactory bulb in vitro [Articolo su rivista]
Bardoni, Rita; M., Puopolo; P. C., Magherini; O., Belluzzi
abstract

Voltage-activated currents have been recorded from periglomerular cells in thin slices of frog olfactory bulb. Cells were examined with whole-cell patch clamp methods. The voltage-dependent potassium currents were studied after pharmacological block of inward currents. Depolarising steps from -130 mV gave an early transient, A-type, outward current and a delayed rectifier K+ current (IKV). The two currents could be isolated on the basis of the differences in their kinetic properties. The A-current developed following a third-order kinetics when the membrane was depolarised to potentials more positive than -40 mV after preconditioning to potentials more negative than -60 mV. Once activated (tau a 2.5 ms at 0 mV), IA inactivated following a single exponential (tau ha about 60 ms). IKV activated with a second-order kinetics above -30 mV with a time constant of 4 ms at 0 mV. IA and IKV were sensitive, respectively, to 4-aminopyridine (4-AP) and tetraethylammonium (TEA).


1995 - Sodium current in periglomerular cells of frog olfactory bulb in vitro [Articolo su rivista]
Bardoni, Rita; P. C., Magherini; O., Belluzzi
abstract

Kinetic properties of the sodium current in periglomerular (PG) cells were investigated by applying whole-cell patch-clamp techniques to thin slices of the frog olfactory bulb. Eight of the cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous activity at rest. Na current was isolated from other current contributions by equimolar substitution of K+ with Cs+ in the intracellular solution to prevent K-currents, and 100 microM Cd2+ in the external solution to block Ca-current. Depolarisations beyond -40 mV activated a fast transient TTX-sensitive inward current. Once activated, INa declined exponentially to zero following a single exponential. The underlying conductance showed a sigmoidal activation between -40 and +30 mV, with half activation at -17.4 mV and a maximal value of 9.7 nS per neurone. The steady-state inactivation was complete at -30 mV and completely removed at -90 mV, with a midpoint at -56 mV. The activation process could be adequately described by third order kinetics, with time constants ranging from 260 microseconds at -20 mV to 70 microseconds at +50 mV.


1994 - MODIFICATIONS OF A-CURRENT KINETICS IN MAMMALIAN CENTRAL NEURONS INDUCED BY EXTRACELLULAR ZINC [Articolo su rivista]
Bardoni, Rita; Belluzzi, O.
abstract

1. Whole-cell voltage clamp recordings were used to study the action of the transition ion zinc on the A-current kinetics in granule cells from rat cerebellar slices. 2. The effects of zinc have been tested in the concentration range from 1 mu M to 1 mM, and fully characterized on all kinetic parameters at 100 and 300 mu M. All the effects observed were rapid, concentration dependent and fully reversible. 3. Steady-state inactivation curves are strongly shifted towards depolarized potentials, with activation curves much less so. These shifts lead to an increase of the peak current amplitude around physiological resting membrane potentials and to a decrease at hyperpolarized potentials. 4. The forward 'on' rate constants are slowed by Zn2+ at a concentration of 100-300 mu M by a factor from 1.5 to 4. The backward 'off' rate constants are unaffected by Zn2+. 5. The development of I-A inactivation, as measured from the current decay, is not affected by Zn2+ up to 1 mM. Removal of inactivation is, on the contrary, significantly slowed. 6. The results are neither compatible with the theory of the surface charge screening effect nor with a mechanism involving channel block. It seems more likely that Zn2+ interferes with the channel gating by binding to a specific domain of the channel protein. 7. After treatment with Hg2+, which is irreversible, Zn2+ still maintains its effects, which suggest that the two divalents act at different sites. 8. In view of the widespread distribution of zinc throughout the brain, its actions on the A-current could play an important role in physiological function.


1993 - Azione modulatrice dello zinco extracellulare sulla corrente potassio transiente in cellule dei granuli di cervelletto di ratto [Articolo su rivista]
Bardoni, Rita; O., Belluzzi
abstract

Caratterizzazione dell'azione modulatoria dello ione zinco sulle proprietà di voltaggio-dipendenza della corrente potassio nelle cellule dei granuli del cervelletto


1993 - Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices [Articolo su rivista]
Bardoni, Rita; O., Belluzzi
abstract

1. Whole-cell voltage-clamp techniques were used to study voltage-activated transient potassium currents in a large sample (n = 143) of granule cells (GrC) from rat cerebellar slices. Tetrodotoxin (TTX; 0.1 microM) was used to block sodium currents, while calcium current was too small to be seen under ordinary conditions. 2. Depolarizing pulses from -50 mV evoked a slow, sustained outward current, developing with a time constant of 10 ms, inactivating over a time scale of seconds and which could be suppressed by 20 mM tetraethylammonium (TEA). By preventing the Ca2+ inflow, this slow outward current could be further separated into a Ca(2+)-dependent and a Ca(2+)-independent component. 3. After conditioning hyperpolarizations to potentials negative to -60 mV, depolarizations elicited transient outward current, peaking in 1-2 ms and inactivating rapidly (approximately 10 ms at 20 degrees C), showing the overall kinetic characteristics of the A-current (IA). The current activated following third-order kinetics and showed a maximal conductance of 12 nS per cell, corresponding to a normalized conductance of 3.8 nS/pF. 4. IA was insensitive to TEA and to the Ca(2+)-channel blockers. 4-Aminopyridine (4-AP) reduced the A-current amplitude by approximately 20%, and the delayed outward currents by > 80%. 5. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzmann function with a slope factor of 8.4 mV and half-inactivation occurring at -78.8 mV. Activation of IA was characterized by a Boltzmann curve with the midpoint at -46.7 mV and with a slope factor of 19.8 mV. 6. IA activation and inactivation was best fitted by the Hodgkin-Huxley m3h formalism. The rate of activation, tau a, was voltage-dependent, and had values ranging from 0.55 ms at -40 mV to 0.2 ms at +50 mV. Double-pulse experiment showed that development and removal of inactivation followed a single-exponential time course; the inactivation time constant, tau ha, was markedly voltage-dependent and ranged from approximately 10 ms at -40 and -100 mV and 70 ms at -70 mV. 7. A set of continuous equations has been developed describing the voltage-dependence of both the steady-state and time constant of activation and inactivation processes, allowing a satisfactory numerical reconstruction of the A-current over the physiologically significant membrane voltage range


1992 - ELECTROPHYSIOLOGICAL EFFECTS OF A NEUROTOXIN EXTRACTED FROM THE SKIN OF THE AUSTRALIAN FROG PSEUDOPHRYNE-CORIACEA [Articolo su rivista]
Sacchi, O; Bardoni, Rita; Magherini, Pier Cosimo; Belluzzi, O.
abstract

1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryine coriacea (PsC) have been studied in rat superior cervical ganglia at 37-degrees-C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10-20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.


1991 - Analisi cinetica di una corrente potassio transiente nelle cellule dei granuli di cervelletto di ratto "in vitro" [Articolo su rivista]
Bardoni, Rita; O., Belluzzi
abstract

Caratterizzazione delle correnti voltaggio-dipendenti mediate dal potassio nelle cellule dei granuli del cervelletto