Ο σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη της έκφρασης των υπομονάδων του GABAΑ υποδοχέα σε τομές από τον διαφραγματικό και τον κροταφικό ιππόκαμπο αμέσως μετά την θανάτωση του ζώου και κατά την διάρκεια της ανάπτυξης των "in vitro" οξέων κυμάτων που έχουν παρατηρηθεί μόνο σε τομές του κροταφικού ιππόκαμπου. Επιπλέον, μελετήθηκε η ποσοτική και τοπογραφική κατανομή των Α1 υποδοχέων αδενοσίνης με την χρήση του ραδιενεργού ιχνηθέτη [3H]-CHA (αγωνιστής των Α1 υποδοχέων) στον κροταφικό και τον διαφραγματικό ιππόκαμπο αμέσως μετά την θανάτωση του επίμυος. Η μελέτη της κατανομής των Α1 υποδοχέων αδενοσίνης έδειξε ότι η δέσμευση της [3H]-CHA ήταν μικρότερη στον κροταφικό σε σύγκριση με τον διαφραγματικό ιππόκαμπο με την μεγαλύτερη διαφορά να εντοπίζεται στην CA1 περιοχή. Το παραπάνω αποτέλεσμα έρχεται σε συμφωνία με την υπόθεση, ότι οι συνάψεις του κροταφικού ιππόκαμπου εμφανίζουν μεγαλύτερη πιθανότητα απελευθέρωσης γλουταμινικού οξέος σε σύγκριση με αυτές του διαφραγματικού ιππόκαμπου, καθώς οι Α1 υποδοχέων αδενοσίνης εντοπίζονται στην CA1 περιοχή κυρίως προσυναπτικά όπου ελέγχουν την απελευθέρωση γλουταμινικού οξέος. Στη συνέχεια της παρούσας μελέτης δείξαμε ότι η έκφραση του mRNA και των πρωτεϊνών για τις κυριότερες υπομονάδες του GABAA υποδοχέα είναι διαφορετική μεταξύ του διαφραγματικού και του κροταφικού ιπποκάμπου. Ειδικά, στην CA1 περιοχή του ιπποκάμπου η έκφραση των α1, β2 και γ2 υπομονάδων ήταν μικρότερη, ενώ αντίθετα η έκφραση των α2 και β1 υπομονάδων ήταν μεγαλύτερη στον κροταφικό ιπποκάμπο σε σύγκριση με τον διαφραγματικό ιππόκαμπο. Σύμφωνα με προηγούμενες μελέτες που αφορούν την συνέκφραση των υπομονάδων στο σύμπλοκο του GABAA υποδοχέα τα αποτελέσματα μας υποδηλώνουν ότι ο α1β2 υποτύπος του GABAA υποδοχέα επικρατεί στον διαφραγματικό ιππόκαμπο, ενώ ο α2β1 υπότυπος κυριαρχεί στον κροταφικό ιππόκαμπο. Η διαφορετική κατανομή των υποτύπων στους δυο πόλους του ιπποκάμπου που είναι εντονότερη στην CA1 περιοχή, μπορεί να επηρεάζει τις ιδιότητες του διαύλου (αγωγιμότητα, πλάτος και διάρκεια των IPSCs), δείχνοντας ότι οι υπότυποι του GABAA υποδοχέα που εντοπίζονται στον κροταφικό ιππόκαμπο έχουν μικρότερη ανασταλτική αποτελεσματικότητα, η οποία συμφωνεί με την μικρότερη GABAA-προερχόμενη αναστολή που έχει δειχθεί στην CA1 περιοχή του κροταφικού ιππόκαμπου. Επιπλέον θα μπορούσε να εξηγήσει την μεγαλύτερη επιρρέπεια του κροταφικού ιππόκαμπου στην επιληψία. Η χαμηλότερη έκφραση του mRNA για τις α4, β3 και δ υπομονάδες του GABAA υποδοχέα στην περιοχή της οδοντωτής έλικας του κροταφικού ιπποκάμπο υποδεικνύει ότι η έκφραση του α4β3δ υποτύπου είναι μικρότερη στον κροταφικό σε σύγκριση με τον διαφραγματικό ιππόκαμπο. Καθώς έχει δειχθεί ότι ο α4β3δ υπότυπος παίζει σημαντικό ρόλο στην τονική αναστολή στα κοκκιώδη κύτταρα της οδοντωτής έλικας, τα παραπάνω αποτέλεσμα μας πιθανώς σημαίνει ότι η τονική αναστολή είναι διαφορετική στους δυο πόλους του ιππόκαμπου. Η αύξηση της έκφρασης του mRNA της α5 υπομονάδας στην CA1 περιοχή του κροταφικού ιπποκάμπου μπορεί να επηρεάζει την ικανότητα για συναπτική βραχυ- και μακρο-χρόνια πλαστικότητα η οποία έχει βρεθεί να είναι διαφορετική μεταξύ του κροταφικού και του διαφραγματικού ιπποκάμπου καθώς έχει δειχθεί ότι οι α5-υπότυποι παίζουν ρόλο σε διαδικασίες μνήμης και μάθησης. Επίσης, οι α5-υπότυποι του GABAA υποδοχέα στην CA1 περιοχή του ιππόκαμπου συμμετέχουν στην τονική αναστολή. Τα υψηλότερα επίπεδα στην έκφραση του mRNA για την α5 υπομονάδα στον κροταφικό ιππόκαμπο σε σύγκριση με τον διαφραγματικό ιππόκαμπο πιθανώς υποδεικνύουν ότι η τονική αναστολή είναι διαφορετική στην CA1 περιοχή των δυο πόλων του ιππόκαμπου. Στο δεύτερο μέρος της παρούσας διατριβής μελετήσαμε την πιθανή συσχέτιση του GABAεργικού συστήματος με την οργάνωση των "in vitro" οξέων κυμάτων η οποία έχει παρατηρηθεί, σε κανονικές "in vitro" συνθήκες, μόνο σε τομές του κροταφικού ιππόκαμπου. Για το λόγο αυτό πραγματοποιήσαμε αναλυτική μελέτη της έκφρασης του mRNA των υπομονάδων (α1, α2, α5, β1, β2, β3, γ2) του GABAΑ υποδοχέα σε διάφορα χρονικά διαστήματα κατά την κανονική "in vitro" διατήρηση των τομών (15min, 1, 3, 5 και 8h). Αρχικά μελετήσαμε την έκφραση των πρώιμων γονιδίων (c-fos, zif-268), που είναι δείκτες της νευρωνικής ενεργότητας, μετά από 5 ώρες κανονικής "in vitro" διατήρησης των τομών με σκοπό τη πιθανή συχέτιση της έκφρασης τους με την οργάνωση των "in vitro" οξέων κυμάτων. Τα αποτελέσματα μας έδειξαν και στους δυο πόλους του ιπποκάμπου παρόμοια αύξηση της έκφρασης του mRNA τόσο για το c-fos όσο και για zif-268 γεγονός που υποδηλώνει ότι γονιδιακή ενεργότητα είναι παρόμοια και όσο αφορά τα συγκεκριμένα πρώιμα γονίδια ανεξάρτητη της ανάπτυξης των "in vitro" οξέων κυμάτων. Στην CA1 περιοχή του κροταφικού ιππόκαμπου παρατηρήθηκε σημαντική αύξηση της έκφρασης του mRNA των α1, β2 και γ2 υπομονάδων του GABAΑ υποδοχέα η οποία ξεκινάει την 1η ώρα, δηλαδή πριν την οργανωμένη εμφάνιση της αυθόρμητης δραστηριότητας, γίνεται μέγιστη στις 4 ώρες παραμονής των τομών σε Τεχνητό Εγκεφαλονωτιαίο Υγρό (ΤΕΝΥ) και συμβαδίζει χρονικά με την οργάνωση των "in vitro" οξέων κυμάτων. Δεν παρατηρήθηκαν σημαντικές αλλαγές σε τομές που προέρχονται από τον διαφραγματικό ιππόκαμπο. Έχει δειχθεί ότι οι α1-υπότυποι παίζουν σημαντικό ρόλο στην ανασταλτική ικανότητα του υποδοχέα. Επίσης η παρουσία της β2 υπομονάδας στον δίαυλο χαρακτηρίζει μεγαλύτερα σε πλάτος και διάρκεια ανασταλτικά ρεύματα συγκρινόμενη με τις β1 υπομονάδες. Φαίνεται λοιπόν ότι ο υπότυπος α1β2γ2, του οποίου η έκφραση αυξάνει πριν την έναρξη της οργανωμένης ρυθμικής δραστηριότητας, λόγω της συγκρότησης του από τις συγκεκριμένες υπομονάδες, έχει μεγάλη ανασταλτική αποτελεσματικότητα η οποία μπορεί να συμμετέχει στην ανάπτυξη των "in vitro" οξέων κυμάτων. Η δέσμευση της [3H]–muscimol αυξάνει σε τομές που προέρχονται μόνο από τον κροταφικό ιππόκαμπο και έχουν παραμείνει σε κανονικές "in vitro" συνθήκες για 8 ώρες σε σύγκριση με αντίστοιχες τομές που προέρχονται αμέσως μετά την θανάτωση του ζώου. Καμία αλλαγή δεν παρατηρήθηκε στην δέσμευση της [3H]–muscimol σε τομές που προέρχονται από τον διαφραγματικό ιππόκαμπο. Η αύξηση της δέσμευσης της [3H]–muscimol μόνο στις τομές που προέρχονται από τον κροταφικό ιππόκαμπο είναι σε συμφωνία με την αύξηση της έκφρασης του α1β2γ2-υποτύπου καθώς έχει δειχθεί ότι η θέση δέσμευσης της muscimol στο δίαυλο του GABAA υποδοχέα είναι μεταξύ των α1 και β2 υπομονάδων. Συμπερασματικά, η εκλεκτική αύξηση της έκφρασης του α1β2γ2-υπότυπου μόνο στην CA1 περιοχή του κροταφικού δηλώνει μεγαλύτερη ανασταλτική αποτελεσματικότητα των GABAA υποδοχέων, δεδομένου ότι ο α1β2γ2-υπότυπος προκαλεί μεγαλύτερα ανασταλτικά μετασυναπτικά ρεύματα. Το παραπάνω μπορεί να σχετίζεται με την "in vitro" εμφάνιση των οξέων κυμάτων καθώς η αυθόρμητη δραστηριότητα προέρχεται από GABAA-επαγόμενες υπερπολώσεις των πυραμιδικών κυττάρων, ενώ και η αύξηση στην έκφραση του α1β2γ2-υποτύπου συμπίπτει χρονικά με την εμφάνιση των "in vitro" οξέων κυμάτων. / The hippocampus in the rat appears grossly as an elongated structure with its long axis bending in a C-shaped manner from the septal nuclei rostrodorsally to the incipient temporal lobe caudoventrally. The long axis of the hippocampal formation is referred as the dorsoventral axis. Although hippocampus has been traditionally thought as a homogeneous structure, several studies have been demonstrated differences at several organization levels (from the behavioural to the cellular) between its dorsal (DH) and ventral (VH) pole. In the present study, we examined whether the recently reported differences in the GABA-mediated somatic inhibition between the DH and VH could be related to variations in the GABAA receptors. We therefore studied the quantitative distribution, the kinetic parameters and the subunit composition of the GABAA receptors in the two parts of hippocampus. We also studied the A1 adenosine receptors in order to examine the involvement of the adenosinergic system in the glutamate release between the two hippocampal poles. The study of [3H]-CHA binding on A1 adenosine receptors by using "in vitro" quantitative autoradiography, revealed a weaker A1 receptor binding in VH compared to DH in all regions we examined. Taken into consideration that the A1 adenosine receptors are localized in the CA1 glutamatergic terminals, these results may to some extend explain our hypothesis that synapses in the VH have greater probability of glutamate release compared to those in the DH counterpart. Recent data have demonstrated a weaker somatic GABAergic inhibition in CA1 region of VH compared to DH. We therefore examined possible differences in the GABAA receptor subunit composition and receptor binding parameters between DH and VH by using "in situ" hybridization, western blotting and the specific binding of the GABAA receptor agonist [3H]-muscimol using quantitative autoradiography and saturation experiments. The experiments demonstrated that the VH compared to DH displayed: 1) lower levels of mRNA expression for α1, β2, γ2 but higher levels for α2 and β1 subunits in CA1, CA2 and CA3, with the differences being more pronounced in CA1 region. Western blot analysis confirmed the mRNA expression data, showing lower levels for α1, β2 and higher levels for α2 subunits’ protein. Only in the CA1 region the mRNA levels of α5 were higher, while those of α4 subunit were slightly lower; in dentate gyrus, the mRNA levels of α4, β3 and δ subunits were significantly lower in VH compared to DH presumably suggesting a lower expression of the α4/β3/δ receptor subtype; 2) lower levels of [3H]-muscimol binding in the VH, with the lowest value observed in CA1, apparently resulting from weaker affinity for GABA and not from a decreased receptor density, since the KD values were higher in VH, while the Bmax values were similar between DH and VH. In conclusion, the differences in the subunit mRNA and protein expression and the lower affinity of GABAA receptor observed predominantly in CA1 region of VH, suggest that the α1 subunit-containing GABAA receptors dominate in the DH, while the α2 subunit-containing receptors prevail in VH. This could underlie the lower GABAA mediated somatic inhibition observed in VH and, to some extent, explain: a) the higher liability of VH for epileptic activity and b) the differential involvement of DH and VH in cognitive and emotional processes. Recent electrophysiological experiments have been shown that slices from the VH of adult rats generate rhythmical activity during their maintenance in the recording chamber. This activity is fully organized during the first 3 hours of in vitro maintenance and resembles the in vivo recorded hippocampal "sharp waves", therefore called "in vitro sharp waves". The field manifestation of this spontaneous activity results from GABAA receptor-mediated hyperpolarizations in pyramidal cells. The aim of the second part of the present thesis focused on the possible relationship between the characteristics of GABAA receptors and the development of "in vitro sharp waves". Using the "in situ hybridisation" technique, we examined the mRNA expression of the alpha1/2/5,beta1/2/3 and gamma2 subunits of GABAA receptor and the binding of GABAA receptor agonist [3H]-muscimol in a time course including periods before and during the development of the "in vitro sharp waves". Six sets of transversely cut DH and VH slices were prepared: slices frozen immediately after killing the animal (naive slices), and slices maintained in vitro and frozen at different time points (15min, 1, 3, 5 and 8h) during the electrophysiological experiment. The results showed: A) Upregulation of alpha1, beta2 and gamma2 subunits mRNA in VH but not in DH slices at 1h of their maintenance, which became significant at 3h as compared to the respective naive slices; B) Increase in [3H]-muscimol binding only in VH slices, obtained at 8h compared to the respective naive ones. The upregulation of the α1β2γ2 GABAA receptor subtype (starting at 1h) in VH but not in DH presumably suggests an increase in GABAergic activity, which could be related with the appearance of "in vitro sharp waves" observed only in VH; C) Τhe similar mRNA expression of the early genes c-fos and zif-268 in the two hippocampal poles showing a comparable general gene activity in DH and VH. In conclusion, the α1β2γ2 subtype dominates in DH while the α2β2-subtype prevails in VH and this could be related to the weaker somatic inhibition observed in the CA1 region of VH, and also to the distinct involvement of DH and VH in cognitive and emotional processes. Moreover, the higher expression of the GABAA receptor subtype α4β4δ in the DG of DH compared to VH may imply a higher tonic inhibition in the former hippocampal pole. The upregulation of the α1β2γ2- subtype only in VH slices during their in vitro maintenance may reflect an increase in the impact of GABAA receptor-mediated transmission, which is required for the organization of "in vitro" sharp waves.

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Sensory information is processed and encoded by neural networks. In order to understand how the nervous system is able to rapidly integrate and store sensory information, knowledge of the connections and properties of the neurons in these circuits is required. The fruit fly Drosophila melanogaster provides a particularly powerful species to investigate the neural circuits of the olfactory system because in addition to possessing a simple olfactory system amenable to circuit analysis, a host of genetic reagents are available, including the GAL4-UAS system for targeted gene expression. The mushroom bodies, paired structures historically implicated in olfactory learning and memory, receive olfactory information at the mushroom body calyx from second-order olfactory projection neurons (PNs). Within the calyx, individual PN axonal boutons are surrounded by dendritic arborizations from intrinsic Kenyon cells (KCs) and each tiny cluster constitutes a single microglomerulus. Cells that connect the calyx with other areas of the brain, extrinsic neurons (ENs), also contribute to microglomeruli. Most of these contain the neurotransmitter, GABA, and are presumed to be inhibitory. In this study, the synaptic characteristics, neural circuits, and plasticity of calycal cells have been investigated using a combination of serial section electron and confocal microscopy. The findings reveal several new features of the circuits in the calyx: 1) The calyx contains three ultrastructurally distinct types of PN boutons that are heterogeneous in shape and exhibit subtle differences in synaptic densities. 2) All PN boutons form both ribbon and non-ribbon synapses, and from their smaller size and fewer postsynaptic partners, non-ribbon synapses may possibly become converted to ribbon synapses after activity; the olfactory signal may then be transmitted more strongly and efficiently at ribbon synapses. 3) PN boutons with an electron-dense cytoplasm have the most ribbon synapses per unit area of membrane as well as the highest ratio of ribbon to non-ribbon synapses, and thus may be more active and efficient than other boutons. 4) KC neurites are not exclusively postsynaptic in the calyx and can form occasional ribbon synapses, the functional interpretation of which awaits identification of their postsynaptic partners and vesicle contents. 5) Each PN bouton may contribute input to a single dendritic KC claw at about three presynaptic sites. For the postsynaptic side, a single claw receives input from individual presynaptic sites that must be highly redundant. 6) There may be important processing of the olfactory signal by local circuits formed by ENs in the calyx; ENs form synaptic connections with PNs, KCs, and other ENs. 7) Extensive serial synapses link EN terminals into a network, presumed to be GABAergic and inhibitory, that extends between microglomeruli and may be autaptic. 8) The structure and synaptic connectivity of microglomeruli may undergo changes after adult emergence. 9) vGAT and GAD1-GAL4 lines drive ectopic expression of marker genes in KCs and are not reliable reporters of GABA-positive cells. 10) Previously identified calycal ENs (MB-C1, MB-C2/C3, MB-CP1) are not immunopositive for GAD1, a marker of GABA-containing cells. 11) A network of ENs expressing a GABA phenotype differently innervates anatomically and functionally discrete areas of the honeybee calyx, and in addition the density of innervation may change with alterations in age and/or experience.

Les interneurones GABAergiques constituent une population mineure de cellules par rapport aux neurones glutamatergiques dans le néocortex. Cependant ils contrôlent fortement l'excitabilité neuronale, la dynamique des réseaux neuronaux et la plasticité synaptique. L'importance des circuits GABAergiques dans le processus fonctionnel et la plasticité des réseaux corticaux est soulignée par des résultats récents qui montrent que des modifications très précises et fiables des circuits GABAergiques sont associées à divers troubles du développement neurologique et à des défauts dans les fonctions cérébrales. De ce fait, la compréhension des mécanismes cellulaires et moléculaires impliquant le développement des circuits GABAergiques est la première étape vers une meilleure compréhension de la façon dont les anomalies de ces processus peuvent se produire. La molécule d’adhésion cellulaire neurale (NCAM) appartient à la super-famille des immunoglobulines de reconnaissance cellulaire et est impliquée dans des interactions homophiliques et hétérophiliques avec d’autres molécules. Même si plusieurs rôles de NCAM ont été démontrés dans la croissance neuronale, la fasciculation axonale, la formation et la maturation de synapses, de même que dans la plasticité cellulaire de plusieurs systèmes, le rôle de NCAM dans la formation des synapses GABAergiques reste inconnu. Ce projet visait donc à déterminer le rôle précis de NCAM dans le processus de maturation des synapses GABAergiques dans le néocortex, en modulant son expression à différentes étapes du développement. L’approche choisie a été de supprimer NCAM dans des cellules GABAergiques à paniers avant la maturation des synapses (EP12-18), pendant la maturation (EP16-24), ou durant le maintien de celles-ci (EP24-32). Les méthodes utilisées ont été le clonage moléculaire, l’imagerie confocale, la culture de coupes organotypiques et des techniques morphométriques de quantification de l’innervation GABAergique. Nos résultats montrent que l’inactivation de NCAM durant la phase de maturation des synapses périsomatiques (EP16-24) cause une réduction du nombre de synapses GABAergiques périsomatiques et du branchement de ces axones. En revanche, durant la phase de maintien (EP26-32), l’inactivation de NCAM n’a pas affecté ces paramètres des synapses GABAergiques. Or, il existe trois isoformes de NCAM (NCAM120, 140 et 180) qui pourraient jouer des rôles différents dans les divers types cellulaires ou à des stades développementaux différents. Nos données montrent que NCAM120 et 140 sont nécessaires à la maturation des synapses périsomatiques GABAergiques. Cependant, NCAM180, qui est l’isoforme la plus étudiée et caractérisée, ne semble pas être impliquée dans ce processus. De plus, l’inactivation de NCAM n’a pas affecté la densité des épines dendritiques ou leur longueur. Elle est donc spécifique aux synapses périsomatiques GABAeriques. Finalement, nos résultats suggèrent que le domaine conservé C-terminal KENESKA est essentiel à la maturation des synapses périsomatiques GABAergiques. Des expériences futures nous aiderons à mieux comprendre la mécanistique et les différentes voies de signalisation impliquées. / GABAergic interneurons, though a minor population in the neocortex, play an important role in cortical function and plasticity. Alterations in GABAergic circuits are implicated in various neurodevelopmental disorders. The GABAergic network comprises diverse interneuron subtypes that have different morphological and physiological characteristics, and localize their synapses onto distinct subcellular locations on the postsynaptic targets. Precisely how activity and molecularly driven mechanisms conspire to achieve the remarkable specificity of GABAergic synapse localization and formation is unknown. Therefore, unravelling the cellular and molecular mechanisms involved in this process is crucial for a better understanding of both cortical function and the basis of various neurological disorders. Here we focus our study on a subtype of GABAergic neurons - the basket interneurons which localize synapses, called perisomatic synapses, onto the soma and proximal dendrites of the postsynaptic targets, and tightly regulate their firing patterns. Although recent studies have shown the activity dependence of basket synapse formation, the molecular mechanisms implicated in the perisomatic synapse formation process are poorly understood. NCAM, the neural cell adhesion molecule, is a prime molecular player implicated both in early synaptogenesis events, and during maturation of glutamatergic synapses in the hippocampus. Recent studies have implicated the polysialylated form of NCAM (PSA-NCAM) in basket synapse formation. However, whether and how NCAM per se plays a role in the formation of GABAergic synapses is unknown. Using single cell genetics to knock down NCAM in individual basket interneurons at specific developmental time periods, we characterized the role of NCAM during perisomatic synapse formation and maintenance. Here we show that loss of NCAM during perisomatic synapse formation from equivalent postnatal day (EP) 16 to EP24, in organotypic slices from mouse visual cortex, significantly retards the process of basket cell axonal branching and bouton formation. However, loss of NCAM at a later stage (EP26 to EP32), when the synapses are already formed, did not affect the number or intricacy of perisomatic synapses. NCAM is therefore implicated in perisomatic synapse formation but not in its maintenance. Further studies also show that isoforms of NCAM, such as NCAM140 and NCAM120 are involved in perisomatic GABAergic synapse maturation. However, NCAM180 is not implicated in this process. Also, NCAM does not affect dendritic spine density and length during maturation and maintenance phases, therefore its action is specific only to GABAergic perisomatic synapses. Finally, the highly conserved C-terminal domain KENESKA is essential for GABAergic perisomatic synapse maturation. Future experiments will help us clarify this mechanism and the involved signalling pathways related to NCAM.

Background Obesity is currently one of the major causes of ill health and it is clear that overeatingis the cause of obesity. However, the actions of many endogenous factors that contribute to overeating are still not well understood. Gamma-aminobutyric acid (GABA)-ergic transmission has been shown to be of great importance for food intake regulation. The progesterone metabolite allopregnanolone is a potent positive GABAA receptor modulating steroid (GAMS) and in humans, elevated allopregnanolone levels have been suggested to be involved in increased food intake, and also with overweight and obesity. GABAA receptors that express the α2 and α3 subunits are proposed to be the main subtypes involved in food intake regulation. Therefore, the aims of the work in this thesis were to further investigate the effect of allopregnanolone on food intake, feeding behaviour, possible effects on weight gain and also to characterize a possible antagonist at α2β3γ2and α3β3γ2 GABAA receptors. Methods Allopregnanolone effects on food intake of different food items were recorded in male Wistar rats. Feeding patterns were analyzed. Food preference tests were also conducted and rats were repeatedly exposed to allopregnanolone under different feeding conditions to elucidate possible effects on body weight gain. To deeper investigate GABAA receptor subtypes suggested to be involved in food intake regulation, electrophysiological whole-cell patch-clamp recordings were performed to identify the specificity of the GAMS antagonist UC1020, at human α2β3γ2 and α3β3γ2 GABAA receptors expressed in HEK293-cells. Results Allopregnanolone increased the intake of standard chow, cookies and a high fat diet in male Wistar rats. Preferentially, allopregnanolone increased the rats´intake of the more calorie dense food type. Allopregnanolone reduced feeding latency and prolonged feeding duration. The increased chow intake induced by allopregnanolone was more pronounced at the beginning of the rats´ active period compared to the inactive. Repeated allopregnanolone administration during 5 consecutive days led to an increased body weight gain, more evident in schedule fed rats on a high fat diet. Both obesity prone and obesity resistant rats gained significantly more weight with repeated allopregnanolone exposure and the increased body weight gain correlated with increased food intake. The compound UC1020 was a potent antagonist of GAMS-enhanced GABA evoked currents at human α3β3γ2 GABAA receptors, whereas it had no effect at α2β3γ2 GABAA receptors. Conclusions Our findings indicate that allopregnanolone induced hyperphagia may be one of the endogenous factors involved in weight gain, especially when the diet is energy-rich. The compound UC1020 may prove useful for investigating the involvement of the α2 and α3 GABAA receptor subtypes in GAMS-induced hyperphagia.

The brain is a major target site in acute cyanide intoxication, as indicated by several symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and inhibited production of energy. In this thesis, some neurotoxic effects of cyanide, in particular, the effects on dopaminergic pathways were studied. In a previous study, decreased levels of striatal dopamine and HVA were found after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine were found in rats showing convulsions after infusion of low doses of cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose). Increased striatal dopamine synthesis was observed in rats after cyanide treatment and in vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose- dependently increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but not that of PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of dopamine. In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine Dj- and D2-receptor binding 1 hour after injection. Increased extracellular levels of striatal dopamine and homovanillic acid were also shown after cyanide (2.0 mg/kg; i.p.). DOPAC and 5-HIAA were slightly decreased. This indicates an increased release or an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Thus the effects of cyanide on dopamine Dj- and D2~receptors could in part be due to cyanide-induced release of dopamine. Because of reported changes in intracellular calcium in cyanide-treated animals, the effects of cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not to affect the inositol phospholipid breakdown in vitro. The effects of cyanide on the synthesis and metabolism of brain GAB A were also examined. A decreased activity of both GAD and GAB A-T were found in the rat brain tissue. The reduced activity of GAB A-T, but not that of GAD returned to the control value after adding PLP in the incubation media. The cyanide-produced reduction of GABA levels will increase the susceptibility to convulsions, and could partly be due to GAD inhibition. In conclusion, cyanide affects the central nervous system in a complex manner. Some effects are probably direct. The main part, however, appears to be secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 7 uppsatser</p> / digitalisering@umu

Les ganglions de la base, un ensemble de noyaux sous-corticaux interconnectés, sont impliqués dans l'élaboration, le contrôle et la mémorisation de comportements cognitivo-moteurs. L'une des principales structures de sortie de ce réseau, la substance noire réticulée (SNr), intègre les différentes informations neuronales puis les transmet au cortex via un relais thalamique. Cependant, cette transmission nécessite une régulation fine de l'activité neuronale de la SNr car celle-ci exerce une inhibition constante de ces structures cibles en raison de son activité GABAergique spontanée. Parmi les acteurs de cette régulation, le glutamate et le GABA sont à l'origine d'un équilibre fin entre excitation et inhibition des neurones nigraux. De nombreuses études se sont intéressées aux mécanismes de régulation de l'activité neuronale de la SNr mais, paradoxalement, aucune ne s'est intéressée au rôle des astrocytes. L'objet de ce travail de thèse a donc été d'étudier les relations entre neurones et astrocytes au sein de la SNr, afin de définir une potentielle implication des astrocytes dans la régulation de l'activité neuronale de cette structure. Nous avons étudié les excitabilités calciques des astrocytes et électriques des neurones grâce aux techniques d'imagerie calcique et de patch-clamp, dans un modèle de tranche parasagittale de cerveau de rat préservant les connexions subthalamo-nigrales et pallido-nigrales. Nous avons ainsi montré que les astrocytes nigraux possèdent une activité calcique spontanée, à la fois autonome et dépendante des libérations toniques de glutamate et de GABA. D'autre part, nous avons mis en évidence que l'activité de ces cellules est modulée par la stimulation à haute fréquence du noyau sous-thalamique. Nous avons montré qu'en retour, ces activités calciques spontanées astrocytaires sont impliquées dans la régulation de la fréquence de décharge des neurones de la SNr. Enfin, nous avons mis en évidence que la recapture astrocytaire du glutamate, et probablement du GABA, intervient également dans la régulation de l'activité de décharge neuronale nigrale. En conclusion, ce travail met en évidence une communication bidirectionnelle entre les neurones et les astrocytes de la SNr. Cette communication semble jouer un rôle important dans la régulation de l'activité de cette structure.

Gamma-aminobutyric acid (GABA) and short neuropeptide F (sNPF) are widespread signalling molecules in the brain of insects. In order to understand more about the signalling and to some extent start to unravel the functional roles of these two substances, this study has examined the locations of the transmitters and their receptors in the brain of the fruit fly Drosophila melanogaster using immunocytochemistry in combination with Gal4/UAS technique. The main focus is GABA and sNPF in feeding circuits and in the olfactory system. We found both GABA receptor types in neurons in many important areas of the Drosophila brain including the antennal lobe, mushroom body and the central body complex. The metabotropic GABAB receptor (GABABR) is expressed in a pattern similar to the ionotropic GABAAR, but some distribution differences can be distinguished (paper I). The insulin producing cells contain only GABABR, whereas the GABAAR is localized on neighbouring neurons. We found that GABA regulates the production and release of insulin-like peptides via GABABRs (paper II). The roles of sNPFs in feeding and growth have previously been established, but the mechanisms behind this are unclear. We mapped the distribution of sNPF with antisera to the sNPF precursor and found the peptide in a large variety of interneurons, including the Kenyon cells of the mushroom bodies, as well as in olfactory sensory neurons that send axons to the antennal lobe (paper III). We also mapped the distribution of the sNPF receptor in larval tissues and found localization in six median neurosecretory cells that are not insulin-producing cells, in neuronal branches in the larval antennal lobe and in processes innervating the mushroom bodies (paper IV). In summary, we have studied two different signal substances in the Drosophila brain (GABA and sNPF) in some detail. We found that these substances and their receptors are widespread, that both sNPF and GABA act in very diverse systems and that they presumably play roles in feeding, metabolism and olfaction. / At the time of doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

The first part of my thesis involves an investigation into mechanisms underlying the presynaptic regulation of transmitter release from myelinated hindlimb sensory afferents in rodents. The central hypothesis is that in addition to chemical transmission in spinal neuronal networks, electrical synapses formed by connexins are critically involved in presynaptic inhibition of large diameter sensory afferents. Subsequent sections of the thesis present a detailed examination of the distribution of connexins in the rodent spinal cord with a particular emphasis on the neuronal connexin, Cx36. Connexin36 (Cx36) is widely believed to be the protein forming the neuronal gap junctions that create electrical synapses between mammalian neurons in many areas of the central nervous system (Condorelli et al 1998). The first part of thesis concerns a previously unknown role of neuronal connexins in interneurone pathways involved in presynaptic control of synaptic transmission in the lumbar spinal cord of rodents. As far as we are aware, the idea that electrical contacts between spinal neurons contribute to spinal presynaptic inhibition is a novel hypothesis. Evidence will be presented: 1) that Cx36 is present in regions of the spinal cord containing interneurons involved in presynaptic inhibition, 2) that the lack of Cx36 in Cx36-/- knockouts mice results in a severe impairment of presynaptic inhibition, and 3) that blocking gap junctions pharmacologically in wild type mice impairs presynaptic inhibition. The exploration of this hypothesis will involve a combination of electrophysiological and immunohistochemical approaches in juvenile wild-type and knockout mice lacking Cx36, as well as immunohistochemical observations in adult rodents. This first section of the thesis begins with the development of a preparation in which several measures of presynaptic inhibition described in the in vivo adult cat preparation can be examined in vitro in young mice. The following sections of the thesis describe the distribution and features of Cx36 on neurons in mice and rats of different ages in four parts. The first will show that Cx36 is the only connexin associated with spinal neurons and refutes claims in the literature about the existence of a variety of connexions on spinal neurons. The second part will show that while gap junctions between some spinal neurons are only a transient developmental phenomenon, they persist in abundance in adult animals. The third part will present evidence of a previously unsuspected III association of Cx36 gap junctions at the chemical synapse between muscle afferent fibres and motoneurons. Specifically, an association between Cx36 and the glutamate transporter used in primary afferents, Vglut1 will be described. To our knowledge these results are the first to suggest the existence of mixed (electrical and chemical) synapses between primary afferents and motoneurons in the mature mammalian spinal cord. The final part of the thesis will describe the presence of Cx36 gap junctions on adult sacral motoneurons involved in control of sexual, urinary and defecation functions in the rodent.

The first part of my thesis involves an investigation into mechanisms underlying the presynaptic regulation of transmitter release from myelinated hindlimb sensory afferents in rodents. The central hypothesis is that in addition to chemical transmission in spinal neuronal networks, electrical synapses formed by connexins are critically involved in presynaptic inhibition of large diameter sensory afferents. Subsequent sections of the thesis present a detailed examination of the distribution of connexins in the rodent spinal cord with a particular emphasis on the neuronal connexin, Cx36. Connexin36 (Cx36) is widely believed to be the protein forming the neuronal gap junctions that create electrical synapses between mammalian neurons in many areas of the central nervous system (Condorelli et al 1998). The first part of thesis concerns a previously unknown role of neuronal connexins in interneurone pathways involved in presynaptic control of synaptic transmission in the lumbar spinal cord of rodents. As far as we are aware, the idea that electrical contacts between spinal neurones contribute to spinal presynaptic inhibition is a novel hypothesis. Evidence will be presented: 1) that Cx36 is present in regions of the spinal cord containing interneurones involved in presynaptic inhibition, 2) that the lack of Cx36 in Cx36-/- knockouts mice results in a severe impairment of presynaptic inhibition, and 3) that blocking gap junctions pharmacologically in wild type mice impairs presynaptic inhibition. The exploration of this hypothesis will involve a combination of electrophysiological and immunohistochemical approaches in juvenile wild-type and knockout mice lacking Cx36, as well as immunohistochemical observations in adult rodents. This first section of the thesis begins with the development of a preparation in which several measures of presynaptic inhibition described in the in vivo adult cat preparation can be examined in vitro in young mice. The following sections of the thesis describe the distribution and features of Cx36 on neurones in mice and rats of different ages in four parts. The first will show that Cx36 is the only connexin associated with spinal neurons and refutes claims in the literature about the existence of a variety of connexions on spinal neurons. The second part will show that while gap junctions between some spinal neurons are only a transient developmental phenomenon, they persist in abundance in adult animals. The third part will present evidence of a previously unsuspected III association of Cx36 gap junctions at the chemical synapse between muscle afferent fibres and motoneurones. Specifically, an association between Cx36 and the glutamate transporter used in primary afferents, Vglut1 will be described. To our knowledge these results are the first to suggest the existence of mixed (electrical and chemical) synapses between primary afferents and motoneurones in the mature mammalian spinal cord. The final part of the thesis will describe the presence of Cx36 gap junctions on adult sacral motoneurones involved in control of sexual, urinary and defecation functions in the rodent.