Ο νεογνικός χειρισμός, ένα πειραματικό μοντέλο πρώιμων εμπειριών, είναι γνωστό οτι επηρεάζει τη λειτουργία του άξονα υποθαλάμου-υπόφυσης-επινεφριδίων, βελτιώνοντας έτσι την προσαρμοστικότητα, την αντιμετώπιση του στρες, τις διανοητικές ικανότητες και γενικά τις διεργασίες του εγκεφάλου που σχετίζονται με την πλαστικότητα. Προηγούμενη μελέτη έχει δείξει οτι ο νεογνικός χειρισμός παρουσιάζει εκλεκτικές επιδράσεις στους υποδοχείς του N-μεθυλο-D-ασπαρτικού οξέος (NMDA) του διεγερτικού νευροδιαβιβαστή γλουταμινικό στον εγκέφαλο των επίμυων. Οι υποδοχείς του γλουταμινικού που διαμεσολαβούν την ταχεία συναπτική νευροδιαβίβαση στις διεγερτικές συνάψεις του κεντρικού νευρικού συστήματος είναι οι υποδοχείς του α-αμινο-3-υδροξυ-5-μεθυλο-ισοξαζολο-προπιονικού οξέος (AMPA) και είναι κρίσιμοι κατά τη νευρωνική ανάπτυξη, τη συναπτική πλαστικότητα και τη δομική αναδιαμόρφωση του εγκεφάλου. Συντίθενται από τέσσερις υπομονάδες, τις GluRA, GluRB, GluRC και GluRD, οι οποίες συνδυάζονται για να σχηματίσουν τετραμερή. Οι περισσότεροι υποδοχείς AMPA είναι ετεροτετραμερή, αποτελούμενα από τουλάχιστον δύο από τις αναφερθείσες υπομονάδες. Οι υποδοχείς AMPA που είναι διαβατοί στο ασβέστιο δεν περιέχουν την υπομονάδα GluRB, ενώ τόσο η υπομονάδα GluRA, όσο και η GluRB, παίζουν σημαντικό ρόλο στη συναπτική διακίνηση των υποδοχέων AMPA. Ο σκοπός της παρούσας εργασίας ήταν να ερευνηθεί κατά πόσο ο νεογνικός χειρισμός μπορεί να επιδρά στους υποδοχείς AMPA, εφόσον έχει δειχθεί οτι η σύνθεση των υπομονάδων και κατά συνέπεια και οι ιδιότητες των υποδοχέων AMPA στη σύναψη μεταβάλλονται από την αισθητική εμπειρία και κατά πόσο αυτή η επίδραση είναι εξαρτώμενη από το φύλο. Σύμφωνα με το παρόν πρωτόκολλο νεογνικού χειρισμού, κάθε νεογνό απομακρυνόταν από τη φωλιά για 15 λεπτά καθημερινά από την πρώτη μεταγεννητική ημέρα μέχρι τον απογαλακτισμό του τρεις εβδομάδες μετά. Η τεχνική του in situ υβριδισμού χρησιμοποιήθηκε για την εντόπιση και ποσοτικοποίηση της έκφρασης του mRNA της κάθε υπομονάδας. Η έκφραση των υπομονάδων του υποδοχέα AMPA μελετήθηκε σε συγκεκριμένες εγκεφαλικές περιοχές που εμπλέκονται στο συναίσθημα, στη μάθηση, στη μνήμη και στην αισθητική αντίληψη, όπως ο ιππόκαμπος, η αμυγδαλή και ο εγκεφαλικός φλοιός ενήλικων αρσενικών και θηλυκών επίμυων. Τα αποτελέσματα της έρευνας έδειξαν οτι ο νεογνικός χειρισμός προκάλεσε διαφορετικές και σε κάποιες περιπτώσεις φυλετικά διμορφικές μεταβολές στην έκφραση των υπομονάδων του υποδοχέα AMPA, με τρόπο ειδικό ανά υπομονάδα και περιοχή. Πιο συγκεκριμένα, τα ζώα που είχαν υποστεί νεογνικό χειρισμό, αρσενικά και θηλυκά, είχαν μειωμένα επίπεδα mRNA της GluRB και της GluRC στο ραχιαίο ιππόκαμπο και στο σωματαισθητικό φλοιό, συγκριτικά με τα ζώα χωρίς χειρισμό. Επιπλέον, τα επίπεδα mRNA της GluRC ήταν μειωμένα στην αμυγδαλή και στον προμετωπιαίο φλοιό και τα επίπεδα mRNA της GluRD ήταν μειωμένα στον κοιλιακό ιππόκαμπο αρσενικών και θηλυκών ζώων που είχαν υποστεί χειρισμό. Επιπροσθέτως, ο νεογνικός χειρισμός είχε φυλετικά διμορφικές επιδράσεις, αυξάνοντας τα επίπεδα mRNA της GluRA στο ραχιαίο ιππόκαμπο των αρσενικών ζώων και μειώνοντάς τα στα θηλυκά, καθώς και μειώνοντας τα επίπεδα mRNA της GluRΒ στον κοιλιακό ιππόκαμπο και στην αμυγδαλή μόνο των θηλυκών επίμυων. Οι μεταβολές στο mRNA των υποδοχέων AMPA θεωρούμε οτι συντελούνται μέσω επιγενετικής ρύθμισης λόγω του νεογνικού χειρισμού. Τα αποτελέσματα μας στο ραχιαίο ιππόκαμπο υποδεικνύουν ότι ο νεογνικός χειρισμός είναι πιθανό να προκαλεί επαγόμενη από την εμπειρία συναπτική ενδυνάμωση και αποδυνάμωση αρσενικών και θηλυκών ζώων αντίστοιχα, δεδομένων των αντίθετων μεταβολών που παρατηρούνται στα επίπεδα του mRNA της υπομονάδας GluRA στα δύο φύλα. Επιπλέον, η παρατηρούμενη μείωση της έκφρασης του mRNA της GluRB πιθανόν να οδηγεί σε μειωμένο αριθμό αδιάβατων στο ασβέστιο υποδοχέων AMPA που περιέχουν την υπομονάδα GluRB, οπότε ο νεογνικός χειρισμός μπορεί να προκαλεί μεταβολή στο φαινότυπο του υποδοχέα AMPA από αδιάβατο σε διαβατό στο ασβέστιο και τελικά αύξηση της πλαστικότητας της σύναψης στις περιοχές όπου παρατηρούνται οι μεταβολές. Συμπερασματικά, στην παρούσα μελέτη δείξαμε οτι οι υπομονάδες του υποδοχέα AMPA μεταβάλλονται από το νεογνικό χειρισμό, ένα μοντέλο πρώιμης εμπειρίας, στον εγκέφαλο ενήλικων επίμυων, με τρόπο ειδικό ανά υπομονάδα και ανά περιοχή και σε κάποιες περιπτώσεις φυλετικά διμορφικό. Αυτό σημαίνει οτι η γλουταμινεργική νευροδιαβίβαση μεταβάλλεται από μια πρώιμη εμπειρία. Αυτή η επίδραση του νεογνικού χειρισμού μπορεί να είναι ένας από τους παράγοντες που υπόκεινται της αυξημένης πλαστικότητας του εγκεφάλου των ζώων που έχουν υποστεί νεογνικό χειρισμό, η οποία εκδηλώνεται τόσο σε κυτταρικό επίπεδο όσο και στο συμπεριφορικό επίπεδο. / Neonatal handling, an experimental model of early life experiences, is known to affect the hypo¬thalamic-pituitary-adrenal axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related pro¬cesses. Previous study has shown selective effects of neonatal handling on rat brain NMDA receptors. AMPA receptors (AMPARs) mediate fast synaptic trans¬mission at excitatory synapses in the CNS and are crucial during neuro¬nal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as GluRA, GluRB, GluRC and GluRD, which combine to form tetramers. Most AMPARs are heterotetramerics, made of at least two of the four pro¬per subunits GluRA-D. AMPA receptors that are permeable to Ca2+ lack the GluRB subunit, while both GluRA and GluRB subunits have an important role in AMPAR trafficking towards the synapse. The present study addressed the question of whether neonatal handling might have an effect on AMRARs, since it has been shown that the subunit composition and thus the synaptic properties of AMPARs, changes in response to sensory experience, and whether this effect is gender-specific. According to the current neonatal handling pro¬tocol, each pup of a litter was removed from the nest for 15 min daily from the first postnatal day 1 (PND1) until weaning (PND22). In situ hybridization was used in order to localize and quantify subunit mRNA expression, with specific cDNA oligo¬nucleotides. AMPAR subunit expression was studied in specific brain regions that are involved in emotions, learning, memory and sensory perception, such as the hippo¬campus, cerebral cortex and amydgala of adult male and female rats. We found that neonatal handling caused differential and in cases sexually dimorphic changes in AMPA receptor subunit expression, depending on the brain region and the subunit. More specifically, neonatally handled animals, both males and females, had lower GluRB and GluRC mRNA levels in the dorsal hippocampus and the somatosensory cortex, compared to the non-handled. Moreover, GluRC mRNA levels were decreased in the amygdala and the prefrontal cortex and GluRD mRNA levels were decreased in the ventral hippocampus of handled animals of both sexes. Furthermore, neonatal handling had sexually dimorphic effects, increasing GluRA mRNA levels in the dorsal hippocampus of males while decreasing them in females, as well as decreasing GluRB mRNA levels in the ventral hippocampus and the amygdala only of the females. The observed changes in AMPA receptor mRNA levels are thought to occur via epigenetic regulation in response to neonatal handling. Our results in dorsal hippocampus suggest that neonatal handling may induce experience-dependent synaptic strengthening in males and weakening in females, due to the opposite changes observed in GluRA mRNA levels of the two sexes. Moreover, the observed decrease in GluRB mRNA expression most likely leads to reduced GluRB-containing calcium-impermeable AMPA receptors, therefore neonatal handling may cause a switch in AMPA receptor phenotype from calcium-impermeable to calcium-permeable AMPA receptors, and thus induce a higher synaptic plasticity in the regions where the changes are observed. In the present study we show that neonatal handling, an experimental model of early life experiences, induces changes in the AMPA receptor subunits expression in the adult rat brain that are region- and subunit-specific and in cases sexually dimorphic. These results demonstrate that glutamatergic transmission changes in response to an early experience. This effect of handling could be one of the factors underlying the increased plasticity of the brain of neonatally handled animals, which is manifested both at the cellular and at the behavioural/systemic level.

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique. / Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity.

Le cerveau est formé d’un réseau complexe de neurones responsables de nos fonctions cognitives et de nos comportements. Les neurones reçoivent via des contacts spécialisés nommés « synapses », des signaux d’autres neurones.[...] Le mécanisme par lequel les neurones reçoivent, intègrent et transmettent ces informations est très complexe et n'est toujours pas parfaitement compris. Dans les synapses excitatrices, les récepteurs AMPA (AMPARs) sont responsables de la transmission synaptique rapide. Les récents développements en microscopie de super résolution ont permis à la communauté scientifique de changer la vision de la transmission synaptique. Une première avancée fait suite à l’observation que les AMPARs ne sont pas distribués de façon homogène dans les synapses, mais sont organisés en nanodomaines de ~ 80 nm de diamètre contenant ~ 20 récepteurs. Ce contenu est un facteur important pour déterminer l'amplitude de la réponse synaptique. En raison de la basse affinité des AMPARs pour le glutamate, un AMPAR ne peut être activé que lorsqu'il est situé dans une zone de ~ 150 nm en face du site de libération des neurotransmetteurs. Récemment, il a été montré que les nanodomaines d’AMPARs sont situés en face de ces sites de libération, formant des nano-colonnes trans-synaptiques à l'état basal. Cette organisation précise à l’échelle nanométrique semble être un facteur clé dans l'efficacité de la transmission synaptique. Une autre avancée a été l'observation que les AMPARs diffusent à la surface des neurones et sont immobilisés à la synapse pour participer à la transmission synaptique. L'échange dynamique entre le pool diffusif d’AMPARs et les récepteurs immobilisés dans les nanodomaines participe au maintien de l’efficacité de la réponse synaptique lors de stimulations à hautes fréquences. L'objectif de ma thèse a été de déterminer le rôle des paramètres indiqués ci-dessus sur les propriétés de la transmission synaptique, à l'état basal et au cours de phénomènes dits de plasticité synaptique. Tout d'abord, nous avons identifié le rôle crucial de la Neuroligine dans l'alignement des nanodomaines d’AMPARs avec les sites de libération du glutamate. En plus de cela, nous avons mis en évidence l’impact de cet alignement sur l’efficacité de la transmission synaptique en perturbant celui-ci. En parallèle, nous avons démontré que les AMPARs désensibilisés sont plus mobiles à la membrane plasmatique que les récepteurs ouverts ou fermés, et ce, en raison d'une diminution de leur affinité pour les sites d’immobilisation synaptiques. Nous avons montré que ce mécanisme permettait aux synapses de récupérer plus rapidement de la désensibilisation et d'assurer la fidélité de la transmission synaptique lors de stimulations à hautes fréquences. Enfin, les synapses peuvent moduler leurs intensités de réponse grâce à des mécanismes de plasticité synaptique à long terme, et plus particulièrement, la dépression à long terme (LTD) qui correspond à un affaiblissement durable de ce poids synaptique. [...] À la suite des découvertes précédentes concernant le rôle de la nano-organisation dynamique des AMPARs pour réguler le poids et la fiabilité de la transmission synaptique, j'ai décidé d'étudier leur rôle dans l'affaiblissement et la sélection des synapses. J'ai découvert que la quantité d’AMPAR par nanodomaine diminue rapidement et durablement. Cette première phase semble due à une augmentation de l’internalisation des AMPARs. Dans un deuxième temps, la mobilité des AMPARs augmente suite à une réorganisation moléculaire de la synapse. Ce changement de mobilité des AMPARs permet aux synapses déprimées de maintenir leur capacité à répondre aux signaux neuronaux à hautes fréquences. Ainsi, nous proposons que l'augmentation de la mobilité des AMPARs au cours de la LTD permet de transmettre une réponse fidèle dans les synapses stimulées à hautes fréquences et donc de sélectivement les maintenir tout en éliminant les synapses inactives. / The brain is a complex network of interconnected neurons responsible for all our cognitive functions and behaviors. Neurons receive inputs at specialized contact zones named synapses which convert an all or none electrical signal to a chemical one, through the release of neurotransmitters. This chemical signal is then turned back in a tunable electrical signal by receptors to neurotransmitters. However, a single neuron receives thousands of inputs coming from several neurons in a spatial- and temporal-dependent manner. The precise mechanism by which neurons receive, integrate and transmit this synaptic inputs is highly complex and is still not perfectly understood. At excitatory synapses, AMPA receptors (AMPARs) are responsible for the fast synaptic transmission. With the recent developments in super-resolution microscopy, the community has changed its vision of synaptic transmission. One breakthrough was the discovery that AMPARs are not randomly distributed at synapses but are organized in nanodomains of ~80 nm of diameter containing ~20 receptors. This content is an important factor since it will determine the intensity of the synaptic response. Due to their mM affinity for glutamate, AMPARs can only be activated when located in an area of ~150 nm in front of the neurotransmitter release site. Recently, AMPAR nanodomains have been shown to be located in front of glutamate release sites and to form trans-synaptic nanocolumns at basal state. Thus, the nanoscale organization of AMPARs regarding release sites seems to be a key parameter for the efficiency of synaptic transmission. Another breakthrough in the field was the observation that AMPARs diffuse at the cell surface and are immobilized at synapses to participate to synaptic transmission. The dynamic exchange between AMPAR diffusive pool and the receptors immobilized into the nanodomains participates to maintain the efficiency of synaptic response upon high-frequency stimulation.The overall aim of my PhD has been to determine the role of each above listed parameters on the intimate properties of synaptic transmission both at basal state and during synaptic plasticity. First, we identified the crucial role of Neuroligin in the alignment of AMPAR nanodomains with glutamate release sites. In addition, we managed to break this alignment to understand its impact on synaptic transmission properties. In parallel, we demonstrated that, due to a decrease in their affinity for synaptic traps, desensitized AMPARs diffuse more at the plasma membrane than opened or closed receptors. This mechanism allows synapses to recover faster from desensitization and ensure the fidelity of synaptic transmission upon high-frequency release of glutamate. Finally, synapses can modulate their strength through long-term synaptic plasticity, in particular, Long-Term Depression (LTD) corresponds to a long-lasting weakening of synaptic strength and is thought to be important in some cognitive processes and behavioral flexibility through synapse selective elimination. Following the previous discoveries about the impact of AMPAR dynamic nano-organization at synapses on the regulation of the synaptic transmission strength and reliability, I decided to investigate their role in the weakening of synapses. I found that AMPAR nanodomain content drops down rapidly and this depletion last several minutes to hours. The initial phase seems due to an increase of endocytosis events, but in a second phase, AMPAR mobility is increased following a reorganization of the post-synaptic density. This change in mobility allows depressed synapses to maintain their capacity to answer to high-frequency inputs. Thus, we propose that LTD-induced increase in AMPAR mobility allows to conduct a reliable response in synapses under high-frequency stimulation and thus to selectively maintain them while eliminating the inactive ones.

Lithium is a drug used to treat mood disorders and also has many side effects, including central nervous system (CNS) complications (such as cognitive dulling), associated with its use. The mechanism of its action still remains unknown. Over the years, many leads have started emerging. It has been shown to inhibit several enzymes in the cell and has been implicated in altering many neurotransmitter systems and signal transduction pathways (serotonin, dopamine and norepinephrine neurotransmissions). Effect of exposure to therapeutic levels of lithium on mature glutamatergic synapses is being studied and several changes in glutamate receptor subtypes have already been reported. Effects of lithium on developing glutamatergic synapses have not been studied. The thesis tries to document and understand the changes brought about by long term lithium treatment on developing glutamatergic synapses in vitro in hippocampal neuronal cultures. In the present work, patch clamp technique was used to monitor the changes in the postsynapse and fluorescence imaging to study the presynaptic changes. The hippocampal neuronal cultures were treated with 1 mM lithium for 6 days during the synaptogenesis stage (DIV 4-10) and termed as chronic Li treatment (CLi). Following CLi treatment the changes occurring in amplitude and rectification property of the AMPA receptor (AMPAR), a subtype of glutamate ionotropic receptor, mediated miniature excitatory postsynaptic currents (mEPSCs) have been reported (Chapter III). Lithium inhibits protein kinase A (PKA), glycogen synthase kinase–3β (GSK-3β) and glutamate reuptake. Effect of inhibiting PKA, GSK-3β and glutamate reuptake was also studied with a view to understand the molecular basis of lithium action on AMPAR mEPSCs (Chapter IV). It was found that chronic lithium treatment (CLi) caused a reduction in the mean amplitude of mEPSCs mediated by AMPARs and also changed the rectification property of these receptors from being more outwardly rectifying to being more inwardly rectifying, an indication probably of increase in contribution of Ca2+-permeable AMPARs to the synaptic events. AMPAR events in chronic lithium treated cultures were more sensitive to both N-acetyl spermine (NASPM) application and di-fluoro-methyl-ornithine (DFMO) treatment, both specific to Ca2+-permeable AMPARs, indicating that there was an increase in the contribution from Ca2+-permeable AMPARs to the synaptic events. PKA inhibition with H-89 treatment (starting from DIV 4 (for 6 days)) reduced the mean amplitude of AMPAR mEPSCs and increased the mean rectification index (RI). GSK-3β inhibition with SB415286 (starting from DIV 4 (for 6 days)) did not alter the mean mEPSC amplitude but reduced the mean RI. Transient (24 hrs) glutamate reuptake inhibition with threo-β-Hydroxy-Aspartic-Acid (THA) at DIV 4 followed by a period of recovery led to smaller amplitudes but no change in RI. The 24 hr glutamate reuptake block on DIV 4 had long term effects. It led to an increase in AMPAR mEPSC frequency while AMPAR mEPSC amplitudes were reduced. The mean RI decrease seen when glutamate reuptake was blocked for 24 hrs on DIV 10, was absent in DIV 4 THA treated neurons. However, when the neuronal cultures were maintained in the presence of PKA and GSK-3β inhibitors, the DIV 4 THA treated neurons showed AMPAR mEPSC characteristics similar to CLi neurons. Thus, it was seen that individual inhibition of PKA, GSK-3β and glutamate reuptake did not lead to changes in AMPAR mEPSCs similar to that seen in lithium treated neurons. The effect of lithium exposure during synapse development on AMPARs could be reproduced closely by co-inhibiting PKA, GSK-3β and glutamate reuptake. Using the styryl dye FM1-43, the changes induced in presynaptic release by a similar chronic lithium treatment was studied (Chapter V). It was found that lithium exposure (1 mM, DIV 4-10) brought down the extent of dye loading, destaining and also slowed down the rate of dye loss in response to high KCl stimulation (the τfast component of destaining was significantly slower). Minimum loading experiments did not reveal any difference in mode of exocytosis (kiss and run/full-collapse) in control and lithium treated cultures. Chlorpromazine treatment (that inhibits clathrin-mediated endocytosis) affected dye loading to a lesser extent in lithium treated cultures as compared to control. Surprisingly, exposure to hyperosmotic solution 10 minutes after dye wash out boosted the extent of dye loading and destaining in lithium treated cultures (a phenomenon not seen in control). This could happen if the FM1-43 is trapped away from the wash solution during the wash period. This would be possible if endocytosis in CLi takes place, differently from control, through a process involving membrane infoldings similar to bulk endocytosis albeit a slower/compromised one. Taken together, the data presented here indicates that lithium treatment during synaptogenesis affects vesicular recycling mostly at the endocytosis and docking/priming steps (mobilization of vesicles for release). Lithium treated cultures also did not show the high KCl associated presynaptic potentiation observed in control which is a significant finding. In conclusion, chronic lithium treatment affected both the presynaptic and postsynaptic compartments of the glutamatergic synapse. The effect of lithium on AMPAR mEPSC could not be reproduced by individual inhibitions of biochemical effectors but by multiple inhibitions. Thus, the study done here underscores the need to look at the manifold effect of lithium in an integrated way. The study also might have implications in understanding the CNS complications seen in patients taking lithium treatment and in babies perinatally exposed to lithium.

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The purpose of this research is to advance development of a photochemical tool designed to probe the role of ionotropic glutamate receptor signaling in neurodegenerative processes, and to delve more deeply into the biological processes underlying the role of these receptors in signaling and memory formation. This ligand-targeted nanoprobe was designed and developed in our lab to label endogenous calcium-permeable AMPARs (CP-AMPARs) in live cells with minimal disruption to native receptor activity. Nanoprobe is designed to use naphthyl acetyl spermine (NASPM) as a photocleavable ligand to target and covalently label native CP-AMPARs with a non-perturbing, fluorescent marker that then allows observation of these receptors using standard epifluorescence microscopy. My contribution to this work, outlined in the aims below, is the characterization of nanoprobe using electrophysiology and fluorescent imaging to evaluate its effectiveness as an endogenous CP-AMPAR label on live neurons. Aim 1: To use whole cell patch clamp electrophysiology to test the labeling of CP-AMPARs with nanoprobe by recording changes in glutamate-evoked current through heterologously expressed GluA1-L497Y homomultimers during, pre- and post- nanoprobe labeling. Aim 2: To use fluorescent imaging to evaluate nanoprobe labeling of glutamate receptors endogenously expressed in hippocampal neurons by co-labeling nanoprobe-treated neurons with traditional antibodies to AMPAR and synaptic targets. Aim 3: To use nanoprobe to detect endogenously expressed CP-AMPARs on live neurons during the course of neuron development. Live neuronal cultures will be imaged before and after labeling with nanoprobe in young dissociated cultures (DIV 1-2) and in maturing cultures (DIV 14-17). Conclusions: Whole cell patch clamp electrophysiology results provide evidence that nanoprobe will label CP-AMPARs in a minimally-perturbing fashion that allows the receptors to resume normal activity after photolytic-release of ligand as designed. Fixed cell imaging of CP-AMPAR nanoprobe labeling was largely ineffective, and live cell imaging was not conclusive, but provided supporting evidence that nanoprobe targets and labels NASPM-sensitive endogenous glutamate receptors on live dissociated hippocampal neurons

Depression is a common mood disorder. Although major ethical challenges make it nearly impossible to invasively and directly measure serotonin (5-hydroxytryptamine, 5-HT) levels in human brains, neuroimaging technologies have shown macroscopic structural and functional abnormalities in the prefrontal cortex (PFC) of depressed patients. The monoamine hypothesis of depression is based on the neurotransmitter imbalance, such as deceased serotonin brain levels are implicated in the cause of depression. Research has focused on the control mechanisms involved in the dorsal raphé nucleus (DRN) which is the serotonergic control center located in the midbrain. We hypothesized that activation 5-HT 2A receptor in PFC would increase serotonin levels by an AMPA-dependent mechanism in both DRN and PFC. Enhancement of the 5-HT in DRN may inhibit 5-HT level in PFC by 5-HT 1A receptor. This becomes the full feedback loop system. While 5-HT levels in the PFC have been well studied, pathway that modulate this DRN pool through upstream cascade interactions leading to a downstream feedback loop have been difficult to elucidate. Developing a mechanism-based pharmacokinetics (PK) and pharmacodynamics (PD) model to quantitatively describe the effect of 5-HT 2A receptors regulation to serotonin in the DRN and PFC would help us to better understand the complex brain. 5-HT 2A receptor agonist and AMPA receptor agonist and antagonist were used to activate or block the related receptor. Male Wistar rats underwent neurosurgery for implantation of microdialysis (MD) probes. Three to five rats were randomly assigned to experimental arms. Using the MD method, the drug combination was examined to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results, a mechanism-based PD model was developed. Phoenix WinNonlin ® and Berkeley Madonna™ were used for model estimation, external validation with secondary data set, and simulation. The result supports the possibility of a 5-HT 2A /AMPA feedback control circuit that originates in the PFC and modulates DRN and PFC 5-HT levels through feedback coupling of 5-HT. The time-course profiles of 5-HT in both DRN and PFC was well modeled and model parameters were estimated with good precision (CV% ranged from 1.37% to 35.03%). The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.