Post-traumatic stress disorder (PTSD) is a psychological disorder that can cause great social/economic hardship. Progress towards treating PTSD has been slow due to a lack of understanding of its pathogenesis. This dissertation aimed to address this issue by investigating the involvement of the astrocytic glutamate reuptake transporter, GLT-1, and regional differences in expression of NMDA receptor subtypes in the development of a rat model of PTSD. We hypothesized that impaired astrocytic glutamate reuptake inhibits long-term memory processes, and that concurrent presence of glucocorticoids (GCs) during situational trauma selectively inhibits fear extinction memory processes in the prefrontal cortex, but not of conditioned fear memory processes in the amygdala, due to differences between these brain regions in expression of NMDA receptor subtypes. The effect of GLT-1 manipulation was studied in vivo. Utilizing the Single Prolonged Stress (SPS) model of PTSD, rats were either exposed to SPS or not. Within these groups, rats were administered a saline sham, a GLT-1 facilitator (ceftriaxone (CEF)), or a GLT-1 inhibitor (dihydrokainic acid (DHK)). Using Classical Fear Conditioning (CFC) and Fear Extinction (EXT) paradigms, retention of fear extinction memories was measured to determine the effect of GLT-1 manipulation on SPS-induced behavior (i.e., impaired fear extinction retention). From the brain of each rat, the amygdala, hippocampus, and prefrontal cortex (PFC) were collected and expression of GLT-1, p-CREB (a molecular indicator of long-term memory), and glucocorticoid receptor (GR, a molecular indicator of a PTSD-like state) were quantified. Analysis of the behavioral data showed that SPS exposure alone reduced the retention of extinction memories, but CEF and DHK both eliminated this effect. Analysis of the brain tissues revealed that SPS induced an increase in GR expression in the hippocampus. SPS also increased GLT-1 expression, but not p-CREB, in the PFC and amygdala. To evaluate the involvement of regional differences in NMDA receptor subtype expression ex vivo, tissue sections of amygdala, hippocampus, and PFC were taken from SPS and non-SPS exposed rats. Synaptic transmission was stimulated in these tissues using bicuculline in the presence of glucocorticoids, NVP-AA077 (a NR2A NMDA receptor subtype inhibitor), or Ro-25 (a NR2B NMDA receptor subtype inhibitor). P-CREB was measured in the tissues treated with GCs to determine if GCs exert greater inhibition of long-term memory in the PFC (a region reported to express high NR2A) than in the amygdala (a region reported to express high NR2B). P-CREB was also measured in the tissues treated with NVP or Ro-25 to determine if these reported receptor profile differences could be demonstrated, and if they changed following SPS exposure. Contrary to the stated hypothesis, analysis of non-SPS exposed rats revealed that GCs, NVP, and Ro-25 decreased p-CREB in all three regions with no differences between regions. However, in the SPS exposed group, p-CREB was not decreased in PFC and hippocampal tissues treated with GCs, amygdalar and PFC tissues treated with NVP, and PFC tissue treated with Ro-25. Overall, the results of the in vivo experiment did not convincingly demonstrate a role of glutamate spill-over in the pathogenesis of PTSD, but did show that modulation of glutamate reuptake can mitigate some of the behavioral consequences of exposure to situational trauma. The results of the ex vivo experiment did not reveal evidence that regional differences in NMDA receptor profiles exist across the three regions analyzed, nor did they show that GCs exert a region specific inhibition of long-term memory formation. However, it was demonstrated that SPS may affect long-term memory by altering expression of synaptic NMDA receptors. This study provides evidence that glial cells may play a role in the pathogenesis of PTSD, and thus may serve as targets for future therapy. / Ph. D.

There is evidence that while polyamines facilitate seizures by positively modulating N-methyl-D-aspartate receptors (NMDAr), selective antagonists of the NR2B-subunit decrease seizures. However, it remains undetermined whether traxoprodil (CP-101,606), an ifenprodil analog that acts as a selective antagonist of the NR2B subunit of the NMDAr, decreases seizure activity. In the current study we investigated whether traxoprodil alters PTZ-induced seizures in adult male Wistar rats by behavioral and electroencephalographical methods. Spermidine (SPD) (2 nmol/site; i.c.v.) facilitated behavioral and electroencephalographical seizures induced by a normally subeffective dose of PTZ (35 mg/kg; i.p.), but did not alter seizure activity induced by convulsant dose of PTZ (70 mg/kg; i.p). Traxoprodil (20 nmol i.c.v.) increased the latency to generalized tonic clonic seizures induced by PTZ (70 mg/kg; i.p). The oral administration of traxoprodil (60 mg/kg) increased the latency to clonic and tonic-clonic seizures, and decreased total time spent in seizures. These data constitute pharmacological evidence supporting a role for NR2B subunit in PTZ-induced seizures. While more studies are necessary to determine whether traxoprodil is a useful anticonvulsant in clinical settings, NR2B subunits may represent new targets of drug development for convulsive disorders. / Há evidências de que as poliaminas facilitam convulsões por modular positivamente os receptores N-metil-D-aspartato (NMDAr), e que os antagonistas seletivos a subunidade NR2B do NMDAr têm atividade anticonvulsivante. Entretanto, permanece indeterminado se o traxoprodil (CP-101,606), um análogo do ifenprodil que age como antagonista seletivo na subunidade NR2B do NMDAr, tem efeito anticonvulsivante. Neste estudo investigamos se o traxoprodil altera as convulsões induzidas por pentilenotetrazol (PTZ) em ratos Wistar machos por meio de métodos comportamentais e eletroencéfalográficos (EEG). Espermidina (SPD) (2 nmol/sítio; i.c.v.) facilita as convulsões comportamentais e eletroencéfalográficas induzidas por doses subconvulsivantes de PTZ (35 mg/kg; i.p.), mas não altera a atividade convulsiva induzida por dose plenamente convulsivante de PTZ (70 mg/kg; i.p.). Traxoprodil (20 nmol i.c.v.) aumenta a latência para convulsão tônico-clônica generalizada induzida por PTZ (70 mg/kg; i.p.). A administração oral de traxoprodil (60 mg/kg) aumenta as latências para convulsão clônica e tônico-clônica generalizada e diminui a duração total das convulsões induzidas por PTZ (70 mg/kg; i.p.). Esses dados mostram que o traxoprodil diminui as convulsões induzidas por PTZ, um modelo animal com bom poder de predição de atividade convulsivante em humanos, e sugerem um papel para a subunidade NR2B nas convulsões induzidas por PTZ. Enquanto mais estudos são necessários para determinar se o traxoprodil tem, de fato, atividade anticonvulsivante na clínica, as subunidades NR2B podem representar um novo alvo para o desenvolvimento de drogas anticonvulsivantes.

La stimulation à haute fréquence (SHF) du noyau subthalamique (NST) joue un rôle essentiel chez les patients Parkinsoniens dans l'amélioration des troubles moteurs pour lesquels la dopa-thérapie n'est plus satisfaisante. Tout comme l'administration à long terme de L-DOPA, la SHF du NST, peut aussi, selon l'intensité de stimulation, évoquer des mouvements dyskinétiques. Ces dyskinésies sont considérées comme un phénomène d'apprentissage moteur pathologique, secondaire à une altération de la transmission glutamatergique et sont sous-tendues par des modifications durables d'expression génique, notamment dans le striatum. L'objectif de ce travail de thèse est d'étudier et de comparer les mécanismes moléculaires des dyskinésies induites par la L-DOPA à celles induites par la SHF, en se focalisant plus particulièrement sur la sous unité NR2B des récepteurs NMDA. Dans un premier temps, nous avons montré par immunohistochimie que la sous unité NR2B est hyperphosphorylée dans le NST et l'EP suite à l'induction de dyskinésie par la SHF du NST chez l'animal sain. Ces résultats ont été confirmés par la suite dans un modèle animal de la maladie de Parkinson, le rat 6-OHDA. La comparaison de ces modifications avec celles observées chez le rat 6-OHDA rendus dyskinétique par un traitement chronique à la L-DOPA nous permet de suggérer que l'induction des dyskinésies est associée à une hyperphosphorylation de NR2B au sein d'une voie subthalamo-entopédonculaire alors qu'une activation de NR2B dans le striatum semble être impliquée dans l'expression des dyskinésies. Enfin, nos résultats mettent également en évidence une implication différentielle des deux structures de sorties des ganglions de la base dans les processus akinétiques et dyskinésiogènes. / High frequency stimulation of the subthalamic nucleus (STN-HFS) alleviates parkinsonian motor symptoms and indirectly improves dyskinesia by decreasing L-DOPA requirement. However, inappropriate stimulation can also trigger dyskinetic movements Dyskinesia are thought to be a pathological learning process due to an overactive glutamate transmission within the basal ganglia. Moreover, several molecular changes seem to be involved in this process. The aim of the present study is to compare the molecular mechanisms of dyskinesia induced by L-DOPA and by STN-HFS, by focusing more particularly on the NR2B-containing NMDA receptor. We show by immunohistochemistry that NR2B subunit is hyperphosphorylated within the STN and the EP during a dyskinesiogenic STN-HFS in normal rats. Similar results are obtained from 6-OHDA rats, a model of Parkinson disease. Comparison of these results with those observed in 6-OHDA dyskinetic rats chronically treated with L-DOPA suggest that dyskinesia induction is associated with an hyperphosphorylation of NR2B within a subthalamo-entopeduncular network while activation of NR2B within the striatum seem to be involved in the expression of dyskinesia. A different implication of the two output of the basal ganglia in akinetic and dyskinesiogenic process is also demonstrated. STAR Date de soutenance : 8 juillet 2011 Thèse sur travaux: non

Anesthesia-induced spatial learning impairments in aged rats model postoperative cognitive dysfunction (POCD) in the elderly surgical population. Mechanisms underlying both normal age-related cognitive decline and anesthesia-induced spatial learning deficits in aged rats were investigated. With respect to the involvement of inflammasome activation and age-related cognitive decline, I hypothesized that the aged hippocampus exhibits an elevated activation of inflammasome components contributing to elevated levels of IL-1β in the aged brain. Age-related cognitive decline was identified in a subpopulation of male Fischer 344 rats. Activation of the NLRP1 inflammasome was elevated in the aged brain, contributing to spatial learning deficits in aged rats. With respect to anesthesia-induced spatial learning impairment in aged rats, I hypothesized that an increase in NR2B subunit in the hippocampus and cortex during and following isoflurane anesthesia exposure resulting in spatial learning impairment in aged rats via disruption of downstream signaling molecule, extracellular-signal regulated protein kinase (ERK). Anesthesia exposure resulted in chronic spatial learning impairment in aged rats that were previously unimpaired in spatial learning tasks. Additionally, anesthesia induced elevated levels of N-methyl-D-aspartate (NMDA) receptor NR2B subunit protein expression in aged. It was concluded that various factors contribute to age-related spatial impairment including: NLRP1 inflammasome activation and NMDA receptor NR2B protein expression elevation. It was also concluded that anesthesia exposure exacerbates the elevation in NR2B protein expression in the aged brain, with subsequent disruption of ERK activation leading to chronic spatial learning deficits in aged rats. In the final chapter, a relationship for the interplay between inflammation and NMDA receptor function in the aged brain is discussed. In addition, a novel mechanism for anesthesia-induced cognitive deficits is presented. Therapeutic treatments for cognitive decline and anesthesia-induced cognitive deficits are explored. Finally, future lines of research are proposed.

Οι περισσότερες πληροφορίες που αφορούν μοριακούς μηχανισμούς πλαστικότητας στο κεντρικό νευρικό σύστημα, πηγάζουν από μελέτες συγκεκριμένων τύπων μακρόχρονης ενδυνάμωσης (LTP) και μακρόχρονης αναστολής (LTD), οι οποίοι εξαρτώνται από την ενεργότητα των υποδοχέων N-methyl-D-aspartate (NMDA) του γλουταμινικού οξέος. Αλλαγές στην φωσφορυλίωση των υποδοχέων NMDA από διάφορες κινάσες και φωσφατάσες διαμεσολαβούν ή και ρυθμίζουν την έκφραση του LTP και του LTD. Η οπτική αποστέρηση, όπως η εκτροφή πειραματόζωων στο σκοτάδι, επιμηκύνει την κρίσιμη περίοδο πλαστικότητας του οπτικού φλοιού. Οι υποδοχείς NMDA του γλουταμινικού οξέος, έχει αποδειχθεί ότι εμπλέκονται σημαντικά στην πλαστικότητα του οπτικού φλοιού. Η φωσφορυλίωση τους εμπλέκεται στη συναπτική πλαστικότητα μέσω της ρύθμισης της διακίνησης τους στην σύναψη και της έκφρασης τους στη επιφάνεια του κυττάρου, καθώς και μέσω της επίδρασης στη λειτουργία του ιοντικού διαύλου του υποδοχέα. Η σερίνη 1303 της υπομονάδας NR2B των υποδοχέων NMDA αποτελεί την κύρια περιοχή φωσφορυλίωσης από την CaM κινάση II και την PKC. Σκοπός της παρούσας μελέτης είναι η συσχέτιση των μεταβολών του υποδοχέα NMDA, ο οποίος συμμετέχει σε φαινόμενα πλαστικότητας του εγκεφάλου, με την ευαίσθητη περίοδο καθορισμού της αρχιτεκτονικής του οπτικού φλοιού. Για το σκοπό αυτό εξετάσαμε τις μεταβολές της φωσφορυλίωσης της υπομονάδας NR2B των υποδοχέων NMDA σε επίμυες μετά από οπτική αποστέρηση για την περίοδο Ρ0-Ρ21 και Ρ0-Ρ30 (Ρ: postnatal day). Τα αποτελέσματα της παρούσας διπλωματικής εργασίας δείχνουν ότι στον οπτικό φλοιό τα επίπεδα της φωσφορυλίωσης του καταλοίπου σερίνης 1303 της NR2B υπομονάδας των υποδοχέων NMDA αυξήθηκαν στις μεταγεννητικές ημέρες 21 και 30 (κατά 27% και 47% αντίστοιχα) στα πειραματόζωα που υπέστησαν οπτική αποστέρηση συγκριτικά με πειραματόζωα ίδιας ηλικίας των οποίων η εκτροφή πραγματοποιήθηκε σε ημερήσιο κύκλο 12 ώρες φως/ 12 ώρες σκοτάδι. Τα αποτελέσματά μας υποδεικνύουν μια εξαρτώμενη από την εμπειρία ρύθμιση της φωσφορυλίωσης του καταλοίπου σερίνης 1303 της NR2B υπομονάδας των υποδοχέων NMDA . Υπάρχουν ενδείξεις ότι η φωσφωρυλίωση του καταλοίπου ser1303 της υπομονάδας NR2B του υποδοχέα NMDA μέσω της PKC ενισχύει τα NMDA-επαγώμενα ρεύματα (Liao et al., 2001). Επιπλέον μελέτες κατά την ανάπτυξη του οπτικού φλοιού επίμυων και μυών που έχουν υποστεί οπτική αποστέρηση (Carmignoto and Vicini, 1992; Yashiro et al. 2005) έχουν δείξει λειτουργικές μεταβολές του υποδοχέα NMDA και συγκεκριμένα ενίσχυση της διάρκειας του NMDA-επαγώμενου ρεύματος Τα αποτελέσματα της παρούσας μελέτης προτείνουν ότι η φωσφορυλίωση της υπομονάδας NR2B του υποδοχέα NMDA μπορεί να είναι ένας από τους μοριακούς μηχανισμούς οι οποίοι ενέχονται στην λειτουργική μεταβολή του υποδοχέα NMDA, η οποία παρατηρείται κατά την ανάπτυξη του οπτικού φλοιού που έχουν υποστεί οπτική αποστέρηση. Τέλος, τα αποτελέσματα αυτά συμφωνούν και συμπληρώνουν την υπάρχουσα βιβλιογραφία που υποστηρίζει ότι η φωσφορυλίωση αποτελεί έναν από τους παράγοντες που συμβάλλουν στην πλαστικότητα. / Phosphorylation of ligand-gated ion channels is recognized as a potentially important mechanism for short and long-term modulation of ion-channel function. Ionotropic glutamate receptors mediate most excitatory neuronal transmission in the brain and play essential roles in the regulation of synaptic activity. Depending on their specific response to different pharmacological agents, ionotropic glutamate receptors are subdivided into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors. NMDA receptors are heteromultimers mainly consisting of the obligatory NR1 subunit in combination with NR2A-NR2D and NR3A-B subunits. Many serine/threonine phosphorylation sites have been identified in NMDA receptor subunits, which are substrates for several kinases. Phosphorylation of NMDA receptors mediate synaptic plasticity through regulation of synaptic trafficking and surface expression of these receptors, in addition to the effects on channel function. Phosphorylation on NR2B-Ser1303 is mediated by CaM kinase II and PKC. Visual deprivation has been a powerful tool for investigating the anatomical and physiological correlates of experience-dependent plasticity. Rearing in the dark during a developmentally sensitive period blocks the development of retinal circuitry. In visual cortex, dark-rearing prolongs the critical period for ocular dominance plasticity, reduces LTD and increases LTP, attenuates the maturation of NMDA receptors and attenuates the developmental increase in inhibition relative to excitation. To test the role of neurotransmitter receptor phosphorylation on visual system plasticity, we examined the effects of dark rearing on phosphorylation of NMDA receptor subunit NR2B in rat visual cortex. In visual cortex, we found that dark rearing rats from birth leads to an increase of the percentage of NR2B subunits of the NMDA receptor that are phosphorylated on Serine 1303. Increases in NR2B subunit phosphorylation in dark-reared rats were observed at both P21 (27%) and P30 (47%) which implies that modulation of NMDA subunit phosphorylation appears at the onset and continues during the critical period for ocular dominance plasticity in rats. Our results suggest that NR2B phosphorylation at Ser1303 is regulated by activity in rat visual cortex. There is evidence for PKC-mediated enhancement of NMDA receptor currents by direct phosphorylation of NR2B at Ser1303. Several studies have shown that visual deprivation increases the current duration of synaptic NMDA receptors. A putative molecular basis for this current enhancement could be the increased phosphorylation of NR2B subunit at ser1303.

Master of Science / Department of Psychological Sciences / Mary Cain / Research has demonstrated that an enriched rearing environment improves learning in many tasks. However, growing evidence suggests that an enriched environment may not provide the same benefits during a fear conditioning paradigm. In fact, it appears that an isolated rearing environment may facilitate acquisition of fear to an aversive stimulus. The neural mechanisms responsible for this disparity in fear learning among differentially reared animals are currently unknown. The NR2B subunit of the NMDA receptor has been shown to be involved in the acquisition of fear and influenced by differential rearing, making it a prime candidate to begin investigating these underlying neural mechanisms. Therefore, this study assessed the expression of the NR2B subunit in brain regions important for the acquisition of fear (amygdala and hippocampus) among differentially reared rats. Rats were reared in an enriched, an isolated, or a standard condition for 30 days. They received four tone-footshock pairings, after which their brains were removed and expression of the NR2B subunit was quantified in the basolateral amygdala (BLA), central nucleus of the amygdala (ACe), and the CA3 region of the hippocampus. Analyses found that the isolated rats began to acquire fear to the aversive stimulus faster than the enriched and standard housed rats. However, the isolated rats showed the least amount of NR2B expression in the BLA while there were no rearing differences in expression within the ACe or the CA3. The results from this study provide further insight to the importance of the rearing environment in learning and memory, especially the learning of fear, and its central neural basis.

Le récepteur du glutamate de type N-méthyl-D-aspartate (NMDAR) est un transducteur clef dans la physiologie du système nerveux et dans nombre de ses pathologies, selon qu’il est localisé à la synapse ou en position extra-synaptique respectivement. Son activité est sous le contrôle étroit du ‘site-glycine’, dont l’activation est gouvernée par la disponibilité en coagoniste. Pourtant, on ignore encore largement les règles qui régissent cette étape limitante de l’activation des NMDARs in situ. Par ailleurs, l’ensemble des onnaissances actuelles suggère que les astrocytes pourraient contrôler les NMDARs dans le contexte des interactions entre cellules gliales et neurones, en particulier via la libération du gliotransmetteur D-sérine. Le principal objectif de ce travail de thèse a été de comprendre les modalités du contrôle endogène des NMDARs par leur site co-agoniste, dans la région CA1 de l’hippocampe. Nous avons porté notre attention, avant tout, sur les acteurs de ce contrôle : la glycine et la D-sérine, qui sont les ligands endogènes du site-co-agoniste. Nous nous sommes intéressés à leur contribution respective dans le contrôle des NMDARs, aux dynamiques de ce contrôle en fonction de l’activité neuronale, à ses variations en fonction de la localisation des NMDARs, ainsi qu’à ses modifications développementales. Nous montrons par des approches d’électrophysiologie que la D-sérine, et non la glycine, est le co-agoniste endogène des NMDARs à la synapse CA3-CA1 chez l’adulte. Elle est délivrée par les prolongements astrocytaires environnants, d’une manière qui est influencée par l’activité synaptique. Sa libération répond à un mécanisme vésiculaire et est dépendante de la signalisation calcique intra-astrocytaire. De cette manière, les astrocytes exercent un contrôle étroit et dynamique des NMDARs à l’état basal et au cours de phénomènes de plasticité synaptique. En contre partie, à l’inverse de leurs homologues localisés à la synapse, les NMDARs extrasynaptiques sont contrôlés par la glycine à l’âge adulte. Cette compartimentation spatiale est dictée par une disponibilité différentielle des deux co-agonistes aux différents sites. Elle est également favorisée par une composition en sous-unités des NMDARs synaptiques et extra-synaptiques différente qui leur confère une affinité distincte pour la glycine et la D-sérine. Enfin, le contrôle des NMDARs par la D-sérine astrocytaire observé à l’âge adulte n’est pas opérationnel à la naissance. En effet, il ne se met en place qu’au cours du premier mois post-natal, de façon concomitante au changement de composition en sous-unités des NMDARs. / N-methyl D-aspartate receptors (NMDARs) are central to many aspects of brain physiology and pathology, which they impact differently depending on their synaptic or extrasynaptic location, respectively. In addition to glutamate, they are gated by the necessary binding of a co-agonist on the so-called ‘glycine-binding site’. However, very little is known about the rules that govern the control of NMDARs through this site, in situ. Evidence now suggests that astrocytes could play a critical role in controlling NMDARs activity, in particular through the release of the gliotransmitter D-serine. In the present work, we aimed at understanding how NMDARs are endogenously controlled through their co-agonist binding site, in the CA1 region of rat hippocampus. We primarily focused on the role of two endogenous ligands of this site: glycine and D-serine. We investigated their relative contribution in the control of NMDARs at the different subcellular locations, the dynamics of such control according to synaptic activity, as well as possible changes during post-natal development. Using elecrophysiological approaches, we demonstrate that NMDARs are gated by Dserine, but not glycine, at CA3-CA1 synapses in adults. D-serine is supplied at least in part by surrounding astrocytes in an activity-dependant manner. Its release occurs in response to calcium signalling within the astrocyte and in a vesicular way. Correspondingly, we found astrocytic supply of D-serine to be essential for NMDARs-dependant functions such as synaptic plasticity. In contrast with their synaptic counterparts, extrasynaptic NMDARs are gated by endogenous glycine and not by D-serine. We provide evidence that this compartmentation relies on the differential availability of the two co-agonists at synaptic and extrasynaptic sites. Besides, due to differences in their subunit composition, synaptic and extrasynaptic NMDARs may have preferential affinity for D-serine and glycine respectively. Finally, we show that the control of the NMDAR co-agonist site is developmentally regulated. Early after birth, glycine is the endogenous co-agonist of synaptic NMDARs. The control exerted by D-serine only progressively appears during the first post-natal month, as the switch in NMDARs subunit composition occurs, suggesting a maturation of cellular interactions at the tripartite synapse.

<p>The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions. </p><p>The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor. </p><p>The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells. </p><p>Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace ³H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for ³H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse.</p><p>The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.</p>

The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions. The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor. The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells. Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace 3H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for 3H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse. The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.

Nonspecific NMDA receptor antagonists induce hyperlocomotion in rats. The aim of this work is to determine whether the NMDA receptor antagonist specific for NR2 subunit exhibit similar negative effect as nonspecific antagonists. This subunit is predominant in the brain in the early postnatal period. The introduction summarizes the data on NMDA receptors and the development of rat. The experimental part deals with the action of a specific NMDA receptor antagonist Ro 25-6981 on motor performance of developing rats. Substance was repeatedly administered to rats at postnatal days 7 to 11. Spontaneous locomotion and motor performance of the animals were repeatedly tested up to adulthood by battery of tests appropriate for individual ages. Our research demonstrated that this substance does not have significant effect on motor system of laboratory rat and that it might be further tested as a possible age-bound antiepileptic drug.