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Neurosteroids and benzodiazepines are modulators of the GABAA receptors, thereby causing anxiolysis. Furthermore, benzodiazepines such as midazolam are known to cause adverse side-effects on cognition upon administration. We previously found that midazolam at nanomolar concentrations (10 nM) blocked long-term potentiation (LTP). Here, we aim to study the effect of neurosteroids and their synthesis using XBD173, which is a synthetic compound that promotes neurosteroidogenesis by binding to the translocator protein 18 kDa (TSPO), since they might provide anxiolytic activity with a favourable side-effect profile. By means of electrophysiological measurements and the use of mice with targeted genetic mutations, we revealed that XBD173, a selective ligand of the translocator protein 18 kDa (TSPO), induced neurosteroidogenesis. In addition, the exogenous application of potentially synthesised neurosteroids (THDOC and allopregnanolone) did not depress hippocampal CA1-LTP, the cellular correlate of learning and memory. This phenomenon was observed at the same concentrations that neurosteroids conferred neuroprotection in a model of ischaemia-induced hippocampal excitotoxicity. In conclusion, our results indicate that TSPO ligands are promising candidates for post-ischaemic recovery exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity., International Journal of Molecular Sciences, 24 (10), ISSN:1422-0067

Slobodan M Janković,1 Miralem Dješević,2 Snežana V Janković1 1Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; 2Cardiology Department, Private Policlinic Center Eurofarm, Sarajevo, Bosnia and HercegovinaCorrespondence: Slobodan M JankovićUniversity of Kragujevac, Faculty of Medical Sciences, Svetozara Markovića 69 Street, Kragujevac, 34000, SerbiaEmail slobnera@gmail.comAbstract: GABA A receptors are ubiquitous in the central nervous system and there is a huge diversity of receptor subtypes in almost all regions of the brain. However, the expression of GABA A receptor subtypes is altered in both the gray and white matter of patients with focal epilepsy. Although there is a number of anticonvulsants with marketing authorization for the treatment of focal epilepsy which act through GABA A receptors, potentiating the inhibitory effects of GABA, it is necessary to develop more potent and more specific GABAergic anticonvulsants that are effective in drug-resistant patients with focal epilepsy. There are three orthosteric and at least seven allosteric agonist binding sites at the GABA A receptor. In experimental and clinical studies, full agonists of GABA A receptors showed a tendency to cause desensitization of the receptors, tolerance, and physical dependence; therefore, partial orthosteric agonists and positive allosteric modulators of GABA A receptors were further developed. Preclinical studies demonstrated the anticonvulsant efficacy of positive allosteric modulators with selective action on GABA A receptors with α2/α3 subunits, but only a handful of them were further tested in clinical trials. The best results were obtained for clobazam (already marketed), ganaxolone (in phase III trials), CVL-865 (in phase II trials), and padsevonil (in phase III trials). Several compounds with more selective action on GABA A receptors, perhaps only in certain brain regions, have the potential to become effective drugs against specific subtypes of focal-onset epilepsy. However, their development needs time, and in the near future we can expect only one or two new GABA A agonists to obtain marketing authorization for focal epilepsy, an advance that would be of use for just a fraction of patients with drug-resistant epilepsy.Keywords: GABA A receptors, positive allosteric modulators, focal-onset epilepsy

GABA A receptors are ubiquitous in the central nervous system and there is a huge diversity of receptor subtypes in almost all regions of the brain. However, the expression of GABA A receptor subtypes is altered in both the gray and white matter of patients with focal epilepsy. Although there is a number of anticonvulsants with marketing authorization for the treatment of focal epilepsy which act through GABA A receptors, potentiating the inhibitory effects of GABA, it is necessary to develop more potent and more specific GABAergic anticonvulsants that are effective in drug-resistant patients with focal epilepsy. There are three orthosteric and at least seven allosteric agonist binding sites at the GABA A receptor. In experimental and clinical studies, full agonists of GABA A receptors showed a tendency to cause desensitization of the receptors, tolerance, and physical dependence; therefore, partial orthosteric agonists and positive allosteric modulators of GABA A receptors were further developed. Preclinical studies demonstrated the anticonvulsant efficacy of positive allosteric modulators with selective action on GABA A receptors with α2/α3 subunits, but only a handful of them were further tested in clinical trials. The best results were obtained for clobazam (already marketed), ganaxolone (in phase III trials), CVL-865 (in phase II trials), and padsevonil (in phase III trials). Several compounds with more selective action on GABA A receptors, perhaps only in certain brain regions, have the potential to become effective drugs against specific subtypes of focal-onset epilepsy. However, their development needs time, and in the near future we can expect only one or two new GABA A agonists to obtain marketing authorization for focal epilepsy, an advance that would be of use for just a fraction of patients with drug-resistant epilepsy.

International audience; Muscle dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane β-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells.

A procedure is described for the preparation of 6-thiapregnanes in five steps from pregnenolone via a 5-oxo-7-iodo-secopregnane intermediate. The 6-thiasteroid obtained was converted into 6-thia-allopregnanolone and its sulfoxide and sulfone derivatives. The trans stereochemistry at the A/B ring junction was accomplished by stereoselective reduction of an intermediate hemithioketal with triethylsilane/BF3·Et2O. The compounds synthesized are analogs of natural neurosteroids, and exhibited GABAA receptor activity comparable to allopregnanolone. Fil: Duran, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Ghini, Alberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Coirini, Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Burton, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina

Progesterone is a well-known steroid hormone, synthesized by ovaries and placenta in females, and by adrenal glands in both males and females. Several tissues are targets of progesterone and the nervous system is a major one. Progesterone is also locally synthesized by the nervous system and qualifies, therefore, as a neurosteroid. In addition, the nervous system has the capacity to bio-convert progesterone into its active metabolite allopregnanolone. The enzymes required for progesterone and allopregnanolone synthesis are widely distributed in brain and spinal cord. Increased local biosynthesis of pregnenolone, progesterone and 5α-dihydroprogesterone may be a part of an endogenous neuroprotective mechanism in response to nervous system injuries. Progesterone and allopregnanolone neuroprotective effects have been widely recognized. Multiple receptors or associated proteins may contribute to the progesterone effects: classical nuclear receptors (PR), membrane progesterone receptor component 1 (PGRMC1), membrane progesterone receptors (mPR), and γ-aminobutyric acid type A (GABAA) receptors after conversion to allopregnanolone. In this review, we will succinctly describe progesterone and allopregnanolone biosynthetic pathways and enzyme distribution in brain and spinal cord. Then, we will summarize our work on progesterone receptor distribution and cellular expression in brain and spinal cord; neurosteroid stimulation after nervous system injuries (spinal cord injury, traumatic brain injury, and stroke); and on progesterone and allopregnanolone neuroprotective effects in different experimental models including stroke and spinal cord injury. We will discuss in detail the neuroprotective effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABAA receptors. Fil: Gennoun, Rachida. Inserm; Francia. Universite Paris Sud; Francia Fil: Labombarda, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Gonzalez Deniselle, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Liere, P.. Inserm; Francia Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Schumacher, M.. Inserm; Francia. Universite Paris Sud; Francia

Hypnotic zolpidem produces its effects via the benzodiazepine binding site in α1-containing GABAA receptors. The aim of the study was to assess the influence of duration of zolpidem treatment and its withdrawal, as well as the role of alpha1-containing GABAA receptors in the development of physical dependence and tolerance. Namely, recombinant receptors can be used to characterize mechanisms involved in different processes in the brain and to delineate the contribution of specific receptor subtypes. To address the influence of chronic zolpidem treatment we exposed HEK293 cells stably expressing alpha1beta2gamma2S recombinant GABAA receptors for seven consecutive days, while withdrawal periods lasted for 24, 48, 72 and 96 hours. Using radioligand binding studies we determined that chronic zolpidem treatment did not induce changes in either GABAA receptor number or in the expression of subunit mRNAs. We observed the enhancement of binding sites and upregulated expression of subunit mRNAs only following 96-hour withdrawal. Moreover, zolpidem treatment and its withdrawal (All time points) induced functional uncoupling between GABA and benzodiazepine binding sites in the GABAA receptor complex. Accordingly, it might be assumed that zolpidem withdrawal-induced uncoupling of GABAA receptors is associated with altered GABAA receptor subunit mRNA expression. The results presented here provide an insight into molecular and cellular mechanisms probably underlying adaptive changes of GABAA receptor function in response to chronic usage and withdrawal of zolpidem and perhaps the observed molecular changes could be linked to the tolerance and dependence produced upon prolonged treatment with other GABAergic drugs.

Cys-loop neurotransmitter-gated ion channels are vital for communication throughout the nervous system. Following activation, these receptors enter into a desensitized state in which the ion channel shuts even though the neurotransmitter molecules remain bound. To date, the molecular determinants underlying this most fundamental property of Cys-loop receptors have remained elusive. Here we present a generic mechanism for the desensitization of Cys-loop GABAA (GABAARs) and glycine receptors (GlyRs), which both mediate fast inhibitory synaptic transmission. Desensitization is regulated by interactions between the second and third transmembrane segments, which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker picrotoxin prevented desensitization, consistent with its deep channel-binding site overlapping a physical desensitization gate., Following activation, Cys-loop neurotransmitter-gated ion channels gradually desensitize and conductance shuts down. Here Gielen et al. present a mechanism for desensitization in which the interface between transmembrane domains is remodelled to constrict the channel pore at the intracellular end.

La activación del receptor GABAA durante un breve período de tiempo (5-10minutos) por GABA, en neuronas de corteza cerebral de rata, induce horas más tarde unareducción en la interacción entre los sitios de unión de GABA y benzodiacepinas, llamadadesacoplamiento, en ausencia de cambios en el número de receptores. El objetivo delpresente trabajo fue estudiar el mecanismo molecular del desacoplamiento inducido por GABA. Nuestros resultados sugieren que el desacoplamiento inducido por GABA estáasociado a una reducción en el porcentaje de receptores GABAA conteniendo la subunidadα3. Por otro lado, los resultados de nuestros experimentos indicarían que eldesacoplamiento está también mediado por un aumento en el grado de fosforilación de lasubunidad γ2 del receptor. Por lo tanto, el desacoplamiento sería el resultado de al menosdos mecanismos regulatorios que actuando en forma conjunta producen una alteraciónadaptativa en la función del receptor GABAA. Si bien no se conocen las vías deseñalización intracelulares activadas, es posible que un aumento en la internalización delreceptor GABAA inducido por GABA represente la señal que desencadenaría los procesosresponsables del desacoplamiento. GABAA receptor activation during a brief period of time (5-10 minutes) by GABA, inrat cerebral cortical neurons, induces hours later a reduction in the interaction between GABA and benzodiazepine binding sites, named uncoupling, in the absence of changes inreceptor number. The aim of the present work was to study the molecular mechanism of GABA-induced uncoupling. Our results suggest that GABA-induced uncoupling is associated with a reduction inthe percentage of α3-containing GABAA receptors. On the other hand, results from ourexperiments might indicate that uncoupling is also mediated by an enhancement in thephosphorylation degree of γ2 receptor subunit. In conclusion, uncoupling would be theresult of at least two regulatory mechanisms that act together to produce an adaptivealteration of the GABAA receptor function. The intracellular signaling pathways areunknown, however, a GABA-induced increase in GABAA receptor internalization couldprovide a signal that triggers the processes responsible for uncoupling. Fil: Gutiérrez, María Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Several biological, psychological, and cultural differences can explain the difference in pain perception between men and women. It is known that gonadal hormones influence the nociceptive response in animals and humans. The brain also has the ability to synthesize its own "sex hormones", also named neurosteroids. The aims of this thesis were: 1) to assess the physiological and psychological factors influencing the difference in pain perception between men and women, 2) to relate the levels of androgens and cortisol with clinical symptoms and pain perception in healthy volunteers and patients with fibromyalgia, and 3) to evaluate the involvement of gonadal hormones and of their 3α5α-reduced metabolites in the transmission of pain and the effectiveness of descending pain modulation systems (DPMS) in males and females using behavioral pain model in rats and mice., Plusieurs variables biologiques, psychologiques, ainsi que des différences culturelles, ont été mises en cause afin d'expliquer la différence de perception de la douleur existante entre les hommes et les femmes. Il est connu que les hormones gonadiques influencent la réponse nociceptive chez l’animal et chez l’humain. Le cerveau a aussi la capacité de synthétiser ses propres "hormones sexuelles", les neurostéroïdes. L'objectif de cette thèse était: 1) évaluer les facteurs physiologiques et psychologiques influençant de perception de la douleur chez les hommes et les femmes, 2) évaluer l'implication des androgènes et du cortisol sur les symptômes cliniques et la perception de la douleur chez des sujets atteints de fibromyalgie et sains, et 3) évaluer l'implication des hormones gonadiques et de leurs métabolites 3α5α-réduits dans la transmission et la modulation de la douleur chez animaux les mâles et les femelles par l'utilisation de modèles de douleur comportementaux.

Background: In Nepali and Indian system of traditional medicine, Withania somnifera (WS) is considered as a rejuvenative medicine to maintain physical and mental health and has also been shown to improve memory consolidation. Objective: In this study, a methanolic extract of WS (mWS) was applied on mice hippocampal CA1 neurons to identify the receptors activated by the WS. Materials and Methods: The whole cell patch clamp recordings were performed on CA1 pyramidal neurons from immature mice (7-20 postnatal days). The cells were voltage clamped at -60 mV. Extract of WS root were applied to identify the effect of mWS. Results: The application of mWS (400 ng/μl) induced remarkable inward currents (-158.1 ± 28.08 pA, n = 26) on the CA1 pyramidal neurons. These inward currents were not only reproducible but also concentration dependent. mWS-induced inward currents remained persistent in the presence of amino acid receptor blocking cocktail (AARBC) containing blockers for the ionotropic glutamate receptors, glycine receptors and voltage-gated Na + channel (Control: -200.3 ± 55.42 pA, AARBC: -151.5 ± 40.58 pA, P > 0.05) suggesting that most of the responses by mWS are postsynaptic events. Interestingly, these inward currents were almost completely blocked by broad GABA A receptor antagonist, bicuculline- 20 μM (BIC) (BIC: -1.46 ± 1.4 pA, P < 0.001), but only partially by synaptic GABA A receptor blocker gabazine (1 μM) (GBZ: -18.26 ± 4.70 pA, P < 0.01). Conclusion: These results suggest that WS acts on synaptic/extrasynaptic GABA A receptors and may play an important role in the process of memory and neuroprotection via activation of synaptic and extrasynaptic GABA A receptors.