Afin d’identifier de nouveaux régulateurs impliqués dans le contrôle du nombre des RGABAs à la synapse, nous avons utilisé la jonction neuromusculaire GABAergique du nématode Caenorhabditis elegans comme système modèle. Après mutagénèse aléatoire d’une souche knock-in exprimant les RGABAs tagués avec une protéine fluorescente (TagRFP), nous avons isolé plusieurs mutants présentant des défauts de localisation des récepteurs. Nous avons mis au point une nouvelle stratégie, basée sur l’analyse bio-informatique de données issues du séquençage du génome entier (WGS), en combinant identification et cartographie des mutations causales sans étape préalable de cartographie génétique. Sur 36 mutants analysés, nous avons retrouvé plusieurs gènes connus pour leur rôle dans la synaptogénèse GABAergique, validant ainsi notre approche. Nous avons initié la caractérisation fonctionnelle d’un nouveau gène candidat, provisoirement appelé nsp-3, qui code pour une protéine transmembranaire hautement conservée au cours de l’évolution. L’absence de nsp-3 induit la localisation ectopique de RGABAs au sein du muscle. Les récepteurs ectopiques colocalisent partiellement avec des marqueurs endosomaux. Des données d’électrophysiologie combinées à des analyses quantitatives du nombre de récepteurs synaptiques, montrent que NSP-3 régule la formation d’un pool de réserve de récepteurs sous-synaptiques. Des données pharmacologiques montrent que le recrutement de ce pool est essentiel dans la plasticité synaptique de la JNM GABAergique après un traitement aigu à l’aldicarbe, un inhibiteur de l’acétylcholine estérase (AChE). L’observation d’un reporteur transcriptionnel montre que nsp-3 est exprimé dans la plupart des tissus du vers. Des expériences de sauvetage phénotypique tissu-spécifiques et des données de colocalisation in vivo suggèrent que NSP-3 agit dans le muscle, à l’interface RE-Golgi, où elle régule le trafic des RGABAAs vers la surface. Cette étude décrit un rôle des nonaspanines dans un nouveau processus cellulaire où elles régulent le trafic des RGABAAs à la jonction neuromusculaire de C. elegans / To identify novel genes and mechanisms involved in the formation and regulation of inhibitory synapses, we used the inhibitory GABAergic neuromuscular junction of the nematode C. elegans as a genetically tractable model. After random mutagenesis of a knock-in strain expressing fluorescently tagged GABAA receptors (GABAAR), we screened for mutants with abnormal fluorescence pattern in vivo. We analyzed 36 mutant strains using a novel whole-genome sequencing strategy to simultaneously map and identify causative mutation without any prior time-consuming genetic mapping. We undertook the functional characterization of a non-characterized gene, tentatively named nsp-3, which encodes an evolutionarily conserved transmembrane protein. nsp-3 deletion using CRISPR technology causes ectopic localization of GABAAR in intracellular compartments of the muscle cell. We found partial colocalization of these ectopic receptors with endosomal markers. Interestingly, we observed a 50 % decrease of GABAAR at synapses while we saw no change in GABA neurotransmission by electrophysiology. These and additional data predict the presence of a subsynaptic pool of GABAARs, which is depleted in the absence of NSP-3. Additional pharmacological data set suggests that this pool of receptors is recruited for GABAergic synaptic plasticity upon acute aldicarb (acetylcholine esterase inhibitor) treatment. A transcriptional reporter of endogenous nsp-3 expression detected expression in most tissues of the worm. Tissue-specific rescue experiments and colocalization data show that NSP-3 functions in muscles at ER-Golgi interface to regulate GABAARs trafficking to cell surface. Our data identified a novel function of the nonaspanins in the traffic of neurotransmitter receptors in the nervous system

GABA is the main inhibitory neurotransmitter of the central nervous system. GABA[subscript A]Rs are multimeric transmembrane receptors, which are composed of 5 subunits. It is known that there are 19 subunits that can make up the GABA[subscript A]Rs, allowing for a vast array of receptor subtypes. In addition to the GABA binding site GABA[subscript A]Rs have distinct allosteric binding sites for benzodiazepines, barbiturates, ethanol, certain general anaesthetics and neuroactive steroids. The molecular heterogeneity of the GABA[subscript A]R is accompanied by distinct pharmacological profiles of the different receptor subtypes. The advance of transgenic mouse models has allowed the functional significance of this heterogeneity to be studied in vivo. Therefore, this thesis utilises a variety of transgenic mouse models carrying either mutations or deletions of certain subunits to study the functional significance of the receptor heterogeneity. Mice lacking the α1 subunit (α1[superscript(-/-)]), carrying a point mutation of the α1 subunit (α1H101R), and mice lacking the δ subunit (δ[superscript(-/-)]) have been utilised to investigate the role of these subunits in the sedative actions of benzodiazepines and the GABA[subscript A]R agonist THIP. Although there are limitations to the interpretation of these results due genetic background of the α1[superscript(-/-)] and α1H101R, experiments suggest that the α1H101R mutation is not behaviourally silent as previously suggested and provide further evidence that the α1 subunit mediates the sedative properties of benzodiazepines. These experiments also reveal that the extrasynaptic δ containing receptors are responsible for mediating the sedative effects of THIP, and these findings combined with evidence from collaborators, implicates the thalamus as an anatomical mediator of these effects. An investigation of the putative cognitive enhancing effects of THIP using an attentional set-shifting task for mice suggested that pre-treatment with THIP reduces the number of errors to reach criterion. δ[superscript(-/-)] mice could not be trained to perform the task, therefore further behavioural investigation of these mice was performed, which suggested a heightened level of anxiety and reduced motivation for a food reward. This thesis has furthered our understanding of the functional role of GABA[subscript A]R subtypes. With the advance in genetic manipulations that allow for regionally selective mutations of the receptor the anatomical structures involved in these functions can be identified.

Une réduction de l'inhibition segmentaire contribue vraisemblablement à l'hypersensibilité douloureuse persistante – qui se manifeste par l'hyperalgie, l'allodynie, et la douleur spontanée – au cours d'états douloureux chroniques. L'association fréquente d'une allodynie avec la migraine – une céphalée épisodique – suggère qu'une perte de l'inhibition synaptique contribue aussi à la manifestation de la douleur migraineuse. Cependant, la grande prévalence de la migraine – plus de 10% de la population générale – soulève la question de savoir si le traitement des informations méningées par le réseau neuronal – associant interneurones excitateurs et inhibiteurs – dans le complexe trigéminal, premier relais sur les voies nociceptives de la face et des méninges, est le même que celui des autres informations, par exemple cutanées. Nous avons caractérisé l'effet du blocage pharmacologique des récepteurs à la glycine (GlyR) et des récepteurs GABAA (GABAAR) sur la transmission synaptique entre fibres afférentes primaires, cutanées ou méningées, et neurones de second ordre en enregistrant des potentiels de champ dans le sous-noyau caudal superficiel (Sp5C). Une stimulation électrique transcutanée évoque trois potentiels de champ négatifs dus à l'activation, du plus précoce au plus tardif, de fibres afférentes primaires de type Aβ, Aδ et C. Bloquer les GlyRs et/ou GABAARs segmentaires facilite les potentiels de champ polysynaptiques excitateurs évoqués par l'activation des fibres afférentes primaires de type A et, au contraire, inhibe, ou même abolit, les potentiels de champ C. Bloquer les récepteurs GABAB (GABABR) segmentaires prévient cette suppression. Il est intéressant de noter que bloquer les GABABRs, potentialise aussi les potentiels de champ C en condition controle. Une stimulation électrique méningée évoque deux potentiels de champ négatifs dus à l'activation, du plus précoce au plus tardif, des fibres afférentes primaires de type Aδ et C. Au contraire du potentiel de champ C cutané, le potentiel de champ C méningé est potentialisé après blocage des GlyRs et/ou GABAARs segmentaires. Ces résultats démontrent que le traitement des informations cutanées et méningées par le Sp5C est différent. Seule l'activation des fibres afférentes primaires cutanées de type A inhibe les inputs cutanés de type C vers le Sp5C par l'intermédiaire d'un circuit polysynaptique excitateur, d'interneurones GABAergiques de dernier ordre et de GABABRs présynaptiques. La théorie du "gate control" postule que l'activité des afférences non-nociceptives ferme la porte à la transmission des inputs nociceptifs vers les centres supérieurs. Nos résultats suggèrent que l'état de la porte dépend de l'activité non seulement dans les fibres afférentes primaires de type A mais aussi dans les circuits polysynaptiques excitateurs de la corne dorsale. / Pathological disruption of segmental inhibition is thought to contribute to persistent pain hypersensitivity – including hyperalgesia, allodynia and spontaneous pain – that occurs during chronic pain states. That allodynia is also often associated with migraine – an episodic headache – suggests that a loss of synaptic inhibition is also involved in the manifestation of headache pain. However, the very high prevalence of migraine – more than 10% of the general population – raises the question as to whether processing of meningeous inputs by local neuronal network – consisting of excitatory and inhibitory interneurons – within the trigeminal nucleus, the first relay station for incoming nociceptive signals of the face and meninges, is the same as that of others, for instance cutaneous. We sought to characterize how pharmacological blockade of glycine and GABAA receptors modifies synaptic transmission between either cutaneous or meningeous primary afferent fibers and second order neurons by recording field potentials in the rat superficial medullary dorsal horn (MDH). Transcutaneous electrical stimulation evokes three negative field potentials elicited by, from the earliest to the latest, Aβ-, Aδ- and C-fiber primary afferents. Blocking segmental glycine and/or GABAA receptors strongly facilitates A-fiber-activated polysynaptic excitatory field potentials but, conversely, inhibits, or even abolishes, C-fiber field potentials. Blocking segmental GABAB receptors reverses such suppression. Interestingly, it also potentiates C-fiber field potentials under control conditions. Meningeous electrical stimulation evokes two negative field potentials elicited by, from the earliest to the latest, Aδ- and C-fiber primary afferents. Unlike cutaneous C-fiber field potentials, meningeous ones are facilitated by blocking segmental glycine and/or GABAA receptors. These results demonstrate that MDH processing of cutaneous and meningeous inputs are different. Only activation of cutaneous A-fiber primary afferents inhibits cutaneous C-fiber inputs to the MDH by the way of polysynaptic excitatory pathways, last-order GABAergic interneurons and presynaptic GABAB receptors. In view of the gate control theory postulating that afferent volleys in non-nociceptive afferents close the gate to central transmission of nociceptive inputs, our results suggest that the state of the gate depends on firing activities of both A-fiber primary afferents and polysynaptic excitatory circuits, i.e. the inhibitory tone, within the dorsal horn.

Abstract not available

Η παρούσα διατριβή ασχολήθηκε με τη μελέτη της έκφρασης των υπομονάδων των υποδοχέων του γλουταμινικού οξέος και του γ-αμινοβουτυρικού οξέος (GABA) στα βασικά γάγγλια και τον φλοιό των εγκεφαλικών ημισφαιρίων του μυός weaver. Παράλληλα, μελετήθηκε η έκφραση των νευροπεπτιδίων εγκεφαλίνης και δυνορφίνης στα βασικά γάγγλια του μυός weaver. O μυς weaver χαρακτηρίζεται από προοδευτική, γενετικά επαγόμενη εκφύλιση των ντοπαμινεργικών κυττάρων του μεσεγκεφάλου, κυρίως αυτών οι οποίοι καταλήγουν στο ραβδωτό σώμα. Για αυτόν τον λόγο, θεωρείται ένα καλό μοντέλο της νόσου Parkinson και η μελέτη των νευροχημικών μεταβολών που συμβαίνουν στον εγκέφαλο του παραπάνω μυός, αποτελεί πολύτιμο εργαλείο για τη διερεύνηση των παθογενετικών μηχανισμών της νόσου. Mε την τεχνική του υβριδισμού in situ, προσδιορίστηκαν τα επίπεδα mRNA των υπομονάδων z1, ε1 και ε2 του υποδοχέα NMDA, των υπομονάδων KA2 και GluR6 του υποδοχέα καϊνικού οξέος, των υπομονάδων α1, α2, α4, β2 και β3 του υποδοχέα GABAA, καθώς και των πρόδρομων πολυπεπτιδίων προ-προεγκεφαλίνη και προδυνορφίνη. Η μελέτη πραγματοποιήθηκε σε φυσιολογικούς μύες (+/+) και μύες weaver (wv/wv), στις ηλικίες των 26 ημερών, 3 μηνών και 6 μηνών μετά τη γέννηση. Όσον αφορά στους υποδοχείς του γλουταμινικού οξέος, τα αποτελέσματά μας υπέδειξαν αύξηση στην έκφραση των υπομονάδων z1, ε2, ΚΑ2 και GluR6 στο ραβδωτό σώμα των μυών weaver, σε σχέση με τους φυσιολογικούς. Η αύξηση στο mRNA των υπομονάδων z1, ε2 και GluR6 παρατηρήθηκε μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της υπομονάδας KA2, παρουσίασε αύξηση και στις τρεις ηλικίες που μελετήθηκαν. Οι αυξήσεις της έκφρασης των υπομονάδων z1, ε2, ΚΑ2 και GluR6 συμφωνούν και πιθανόν εξηγούν τις αυξήσεις στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς NMDA και μη-NMDA, οι οποίες έχουν βρεθεί από παλαιότερες μελέτες του εργαστηρίου μας στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Με βάση βιβλιογραφικά δεδομένα, υποστηρίζουμε ότι η καθυστερημένη αύξηση στην έκφραση των υπομονάδων z1, ε2 και GluR6 κατά πάσα πιθανότητα συντελείται μέσω επαγωγής του μεταγραφικού παράγοντα ΔfosB, σε απόκριση προς τη μείωση της ντοπαμίνης. Στον σωματοαισθητικό φλοιό των μυών weaver ηλικίας 26 ημερών, παρατηρήθηκε αύξηση στην έκφραση των υπομονάδων z1, ε1, ε2 και KA2, η οποία θα μπορούσε να οφείλεται στη μειωμένη θαλαμοφλοιϊκή γλουταμινεργική είσοδο. Όσον αφορά στους υποδοχείς GABAA, παρατηρήθηκε αύξηση στα επίπεδα mRNA των υπομονάδων α4 και β3, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών, η οποία συμφωνεί και μπορεί να εξηγήσει την αύξηση στα επίπεδα των θέσεων δέσμευσης για τους υποδοχείς GABAA, η οποία έχει βρεθεί σε προηγούμενη μελέτη του εργαστηρίου μας, στο ραβδωτό σώμα των μυών weaver ηλικίας 6 μηνών. Σκοπεύουμε να ελέγξουμε την πιθανότητα, η αύξηση της έκφρασης της υπομονάδας α4, να υποδεικνύει μία αύξηση του αριθμού των εξωσυναπτικών υποδοχέων GABAA στους νευρώνες προβολής του ραβδωτού σώματος. Στην ωχρά σφαίρα των μυών weaver ηλικίας 6 μηνών, παρατηρήθηκε μείωση των επιπέδων mRNA των υπομονάδων α1 και β2, υποδεικνύοντας μία μείωση του αριθμού των υποδοχέων GABAA, η οποία ήταν αναμενόμενη, λόγω της αυξημένης GABAεργικής εισόδου στην εν λόγω εγκεφαλική περιοχή του μυός weaver. Στον σωματοαισθητικό φλοιό, παρατηρήθηκε μείωση στην έκφραση των υπομονάδων α2 και β2 και ταυτόχρονα αύξηση στην έκφραση των υπομονάδων α4 και β3. Με βάση βιβλιογραφικά δεδομένα, προτείνουμε ότι οι μεταβολές αυτές μπορεί να αντανακλούν μείωση στον αριθμό των συναπτικών και αύξηση στον αριθμό των εξωσυναπτικών υποδοχέων GABAA, σε απόκριση προς τη μειωμένη GABAεργική είσοδο προς τους νευρώνες του σωματοαισθητικού φλοιού του μυός weaver. Όσον αφορά στην έκφραση των πολυπεπτιδίων, το mRNA της προ-προεγκεφαλίνης, παρουσίασε αύξηση στο ραβδωτό σώμα των μυών weaver, μόνο στην ηλικία των 6 μηνών, ενώ το mRNA της προδυνορφίνης, παρουσίασε μείωση στην παραπάνω περιοχή, στην ηλικία των 26 ημερών και αύξηση στις μεγαλύτερες ηλικίες. Σύμφωνα με τα βιβλιογραφικά δεδομένα υποστηρίζουμε ότι: α) η καθυστερημένη αύξηση της έκφρασης της προ-προεγκεφαλίνης στο ραβδωτό σώμα του μυός weaver, οφείλεται στη μείωση της τονικής ανασταλτικής ρυθμιστικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου και πιθανώς συντελείται μέσω του μεταγραφικού παράγοντα ΔfosB, β) ο παραπάνω μεταγραφικός παράγοντας είναι κατά πάσα πιθανότητα υπεύθυνος και για την καθυστερημένη επαγωγή της έκφρασης της προδυνορφίνης στο ραβδωτό σώμα των μυών weaver και γ) η μείωση του παραπάνω mRNA στην ηλικία των 26 ημερών οφείλεται στη μείωση της τονικής διεγερτικής δράσης της ντοπαμίνης στην έκφραση του εν λόγω γονιδίου. Τέλος, το γεγονός ότι οι μεταβολές των mRNA των διαφόρων υπομονάδων και νευροπεπτιδίων δεν ήταν οι ίδιες στις διάφορες ηλικίες που μελετήθηκαν υποδεικνύει ότι κατά την πρόοδο της ντοπαμινεργικής εκφύλισης των ντοπαμινεργικών νευρώνων του μεσεγκεφάλου διαφορετικοί μηχανισμοί ευθύνονται για την πρόκληση των αλλαγών στην έκφραση των υπό μελέτη γονιδίων. / In the present study we investigated the expression of the subunits of glutamate and γ-aminobutyric acid (GABA) receptors in basal ganglia and cerebral cortex of the weaver mouse. We also studied the expression of striatal neuropeptides, which are important neuromodulators of the synaptic transmission in the basal ganglia circuitry. The weaver mouse is characterized by a progressive, genetically induced degeneration of the mesencephalic dopaminergic neurons, especially those that project to the striatum. For this reason, the weaver mouse is a useful model for clarifying the pathogenetic mechanisms that underly Parkinson’s disease. Using the in situ hybridization method, the mRNA levels of the ΝΜDA subunits z1, ε1 and ε2, the kainate subunits KΑ2 and GluR6, the GABAA subunits α1, α2, α4, β2 and β3, as well as the mRNA levels of the precursor polypeptides pre-proenkephalin and prodynorphin, were estimated. The study was performed using wild-type (+/+) and weaver mice (wv/wv) of the following ages: 26 days, 3 months and 6 months. Concerning the glutamate receptors, an increase in the mRNA levels of z1, ε2, KA2 and GluR6 subunits was indicated in the weaver striatum, compared to the wild type. The z1, ε2 and GluR6 mRNA increases were observed only at the age of 6 months, whereas the KA2 mRNA increase was observed at all three ages studied. The increases in z1, ε2, ΚΑ2 and GluR6 mRNA expression are in agreement and probably explain the increased levels of ΝΜDA- and non-NMDA-sensitive binding sites that we had previously found in the 6 months old weaver striatum. Based on bibliographic data, we suggest that the delayed increases in z1, ε2 and GluR6 mRNA levels, are probably mediated by the delayed induction of the ΔfosB transcription factor, in response to the reduction of striatal dopamine levels. In the somatosensory cortex of 26 day old weaver mice, an increase in the levels of z1, ε1, ε2 and ΚΑ2 mRNAs was observed. The above increases can be attributed to the decreased thalamocortical glutamatergic imput. Concerning the GABAA receptors, the observed increases of the α4 and β3 mRNA levels in the 6 months old weaver striatum are in agreement and probably explain the increased levels of GABAA binding sites that we had previously found in the 6 months old weaver striatum. We are going to test the hypothesis, that the α4 mRNA increase might indicate an increase in the number of extrasynaptic GABAA receptors in striatal projection neurons. In the 6 months old weaver globus pallidus, the observed decrease in α1 and β2 mRNA levels was expected, since the GABAergic transmission is increased in the above region of the weaver brain. In the weaver somatosensory cortex, a decrease in the α2 and β2 mRNA levels and an increase in the α4 and β3 mRNA levels were observed. Based on bibliographic data, we suggest that the above alterations probably indicate a differential regulation of the synaptic versus extrasynaptic cortical GABAA receptors, in response to the decreased GABAergic presynaptic input to the weaver cortical neurons. Concerning the expression of the striatal neuropeptides, the pre-proenkephalin mRNA was increased in the weaver striatum, only at the age of 6 months. In contrast, prodynorphin mRNA was decreased in the 26 day old weaver striatum, whereas it was increased in the 3 and 6 months old weaver striatum. Based on bibliographic data, we suggest that: a) the delayed increase in the expression of pre-proenkephalin could be caused by the reduction of the tonic dopaminergic inhibitory control on the expression of the above gene in the dopamine-depleted weaver striatum and is probably mediated by the ΔfosB transcription factor; b) the above transcription factor could be responsible for the delayed induction of the prodynorphin expression in the weaver striatum as well, and c) the decrease of prodynorphin mRNA in the 26 day old weaver striatum could be attributed to the reduction of the dopaminergic stimulatory control on the expression of the above gene. Finally, the different pattern of expression alterations among the three ages studied indicates that distinct mechanisms are responsible for the observed changes, during the progress of the dopaminergic degeneration of the weaver brain.

GABA is the major inhibitory neurotransmitter in the central nervous system. It binds to two types of receptors –ionotropic GABAA and metabotropic GABAB. The GABAA receptor directly gates a Clionophore that causes hyperpolarization in mature excitatory neurons while GABAB receptor mediates a slower hyperpolarizing response via G-protein coupled receptor (GPCR) activated potassium channels. This signaling mechanism gets further complicated by the heterogeneous GABA receptor subunit composition that influences the response kinetics in the postsynaptic membrane. In this thesis, the focus has been to decipher the role of GABAA receptors in relation to cellular excitability in the subiculum under physiological and pathophysiological conditions. The subiculum, considered as the output structure of hippocampus, modulates information flow from hippocampus to various cortical and sub-cortical areas and has been implicated in learning and memory, rhythm generation and various neurological disorders. It gates hippocampal activity with its well orchestrated and fine tuned intrinsic and local network properties. Over the years many studies have shown the involvement of subiculum in temporal lobe epilepsy where it forms the focal point of epileptiform activities with altered cellular and network properties. The subiculum is characterized by the presence of a significant population of burst firing neurons that lead local epileptiform activity. By virtue of its bursting nature and recurrent connections, it is a potential site for seizure generation and maintenance. Epileptiform activities are dynamic in nature and change temporally and spatially according to the alterations in electrophysiological properties of neurons. Transitions to different electrical activities in neurons following a prolonged challenge with epileptogenic stimulus have been shown in other brain structures, but not in the subiculum. Considering the importance of the subicular burst firing neurons in the propagation of epileptiform activity to the entorhinal cortex, we have explored the phenomenon of electrophysiological phase transitions in the burst firing neurons of the subiculum in an in vitro brain slice model of epileptogenesis. Whole-cell patch clamp and extracellular field recordings revealed a distinct phenomenon in the subiculum wherein an early hyperexcitable phase was followed by a late suppressed phase upon continuous perfusion with epileptogenic 4-amino pyridine and magnesium-free medium. The late suppressed phase was characterized by inhibitory post-synaptic potentials (IPSPs) in pyramidal excitatory neurons and bursting activity in local fast spiking interneurons at a frequency of 0.1-0.8 Hz. The IPSPs were mediated by GABAA receptors that coincided with excitatory synaptic inputs to attenuate action potential discharge. These IPSPs ceased following a cut between the CA1 and subiculum. Our results suggest the importance of feedforward inhibition in the suppression of epileptiform activity in subiculum to mediate a homeostatic response towards the induced hyper-excitability. GABA release from presynaptic nerve endings activates postsynaptic GABAA receptors, which evoke faster phasic inhibitory postsynaptic currents (IPSCs) and non-inactivating inhibitory tonic current, mediated through extrasynaptic GABAA receptors. These receptors are heteropentameric GABA-gated channels assembled from 19 possible subunits (α1-6, β1-3, γ1-3, δ, π, ρ1-3, θ, and ε). The 2 major subunits involved in tonic GABAA currents in the hippocampus are α5 and δ subunits. Tonic GABAA receptor mediated inhibitory current plays an important role in neuronal physiology as well as pathophysiology such as mood disorders, insomnia, epilepsy, autism spectrum disorders and schizophrenia. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons having hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. In the present study, we show the involvement of α5βγ GABAA receptors in mediating picrotoxin sensitive tonic current in subicular pyramidal neurons using known pharmacological agents that target specific GABAA receptor subunits. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. Our experiments suggest that tonic GABAergic inhibition can actively modulate subthreshold properties of subicular pyramidal neurons including resonance due to HCNchannels that may potentially alter the response dynamics in an oscillating neuronal network.

No description available.

Yes / Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. / BBSRC, MRC and the European Research Council

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / Extrasynaptic GABAA receptors, often identified as those containing both α4 and δ subunits, appear to be a target for the actions of alcohol (ethanol) at relatively low concentrations, perhaps suppressing the activity of GABAergic interneurons which regulate activity in the mesolimbocortical circuit. Pharmacological studies in rodents using the δ-subunit selective agonist Gaboxadol (THIP) have found both promotional and inhibitory effects on alcohol consumption. The goal of this project was to determine the role of extrasynaptic GABAA receptors located in the infralimbic cortex (ILC) in the binge-like alcohol intake of male C57BL/6J (B6) mice. The ILC is of interest due to its demonstrated involvement in stress reactivity and alcohol exposure has been shown to interfere with extinction learning; impairments of which may be related to inflexible behavior (i.e. problematic alcohol consumption). Adult male B6 mice were bilaterally implanted with stainless steel guide cannulae aimed at the ILC and were offered limited access to 20% ethanol or 5% sucrose for 6 days. On day 7, mice were bilaterally injected with 50 or 100 ng THIP (25 or 50 ng per side respectively) or saline vehicle into the ILC. It was found that the highest dose of THIP (100 ng/mouse) increased alcohol intake relative to vehicle controls, although control animals consumed relatively little ethanol following infusion. Furthermore, THIP had no effect on sucrose consumption (p > 0.05), suggesting that the effect of THIP was selective for ethanol consumption. Together, these findings suggest that the mice that consumed ethanol may have been particularly reactive to the microinfusion process relative to animals that consumed sucrose, perhaps because ethanol consumption was not as reinforcing as sucrose consumption. In addition, the observation that THIP effectively prevented the decrease in ethanol intake on day 7 induced by the microinjection process may be related to a role for the ILC in adaptive learning processes, which in turn, promote behavioral flexibility.