La cellule de Purkinje (CP) est la seule sortie anatomique du cortex cérébelleux. Des études récentes ont montré que les récepteurs NMDA (NMDA-R) jouaient un rôle essentiel dans le Depression à long terme (DLT) à la synapse entre les fibres parallèles (FP) et les CPs. Les NMDA-Rs pourraient jouer un rôle prépondérant dans l’intégration des informations somato-sensorielles des FPs et ainsi contribuer au rôle du cervelet dans l'apprentissage moteur. Nous montrons que les NMDA-Rs sont fonctionnels et recrutés uniquement lors de patrons de décharges des FPs haute fréquences. Ces résultats étant potentiellement liés aux propriétés biophysiques des NMDA-Rs, nous avons démontré que la PLT dépend des NMDA-Rs comportant les sous unité GluN2A et que l'expression post synaptique de la plasticité s'effectuait à travers une diffusion anterograde du monoxyde d'azote (MA). De plus, nous avons confirmé et disséqué les propriétés de filtre passe haut des NMDA-Rs in vivo et in vitro.Nous avons montré que la PLT nécessitait des trains d'activité des FPs plus long que dans le cadre de la DLT, nous postulons que la quantité de MA produite est plus importante lors de l'induction de PLT. Utilisant nos données, nous avons implémenté un model mathématique de plasticité à la synapses FP-CP pouvant prédire le signe de plasticité synaptique selon les patrons d'activité rencontrés par cette synapse. / Synaptic plasticity is thought to be the cellular mechanism underlying learning and memory and has been the subject of intense experimental and theoretical research. The experimental work has led to detailed knowledge of the receptors and signalling pathways involved in the induction of different types of synaptic plasticity. In parallel, theoretical studies have built ’plasticity rules’, formal descriptions linking spike timings to changes in synaptic efficacy, such as the spike-timing-dependent plasticity (STDP) rule [Gerstner et al., 1996, Song et al., 2000]. However, these plasticity rules are generally quite abstract and their link to the underlying biophysical mechanisms is often unclear. The best known mechanisms in synaptic plasticity are linked to N-methyl-D-aspartate receptor (NMDA-R) function. NMDA-Rs are biophysical coincidence detectors of glutamate and membrane depolarization [Mayer et al., 1984, Nowak et al., 1984]. The activation of postsynaptic NMDA-Rs defines learning rules where the relative timing of pre- and post-synaptic activity is a key parameter [Debanne et al., 1994, Nevian and Sakmann, 2006, Sjostrom et al., 2003]. In the few cases where the participation of presynaptic NMDA-Rs has been proposed, these have invariably been involved in presynaptically-expressed LTD [Rodríguez-Moreno and Paulsen, 2008b, Sjostrom et al., 2003]. Cerebellar parallel fibre-Purkinje cell (PF–PC) synaptic plasticity follows non-Hebbian plasticity rules. We have previously reported that PF-PC LTD induction needs PF bursting activity (at least pairs of spikes) [Bidoret et al., 2009] and is linked to the presence of presynaptic NMDA-Rs [Casado et al., 2002b]. In this thesis, we set out to characterise the activity requirements for bidirectional synaptic plasticity in young and adult animals, and to investigate the signalling pathways involved. Surprisingly, we found that LTP induction shares many properties with LTD induction, including a similar frequency-dependence for presynaptic activity and an absolute requirement for NMDA-R activation and NO production. However, LTP requires a different source of post-synaptic calcium increase [Ly et al., 2013a]. In contrast with other synapses [Bender et al., 2006, Fino, 2010], our data indicate that both LTP and LTD share signalling mechanisms. These involve presynaptically produced NO and postsynaptic Ca rises. Supporting the notion that the frequency dependence of plasticity arises from the involvement of presynaptic NMDA-Rs, we provide the first direct evidence for Ca influx through presynaptic NMDA-Rs in PFs in young and adult animals, settling a long-lasting controversy [Bidoret et al., 2009, Casado et al., 2002a, Shin and Linden, 2005a, Wang et al., 2014a]. Based on our data, we propose a novel mechanistic plasticity rule. This deliberately parsimonious rule can be used to interpret and predict the plasticity arising from arbitrary patterns of PF and climbing fibre (CF) activity. Our results support the notion that bidirectional synaptic plasticity depends on multi-spike activity patterns in an intricate fashion [Bidoret et al., 2009, Froemke and Dan, 2002, Pfister and Gerstner, 2006, Sjöström et al., 2001].

The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.

Acid-sensing ion channels (ASIC) are a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and contribute to the demise of neurons. Pharmacological block of ASIC1a provides neuroprotection at delayed time points. However, no endogenous receptors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC1a function and ASIC1a-induced [Ca²?]i elevations during acidosis and ischemia, which may be a mechanism by which sigma ligands provide neuroprotection following stroke. This hypothesis is based on the following observations: First, sigma receptors regulate multiple ion channels that become activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and sigma receptors have been shown to be neuroprotective at delayed time points following stroke. Ratiometric Ca²+ fluorometry and whole-cell patch clamp experiments showed that sigma-1 receptor activation depresses elevations in [Ca²+]i and membrane currents mediated by ASIC1a channels in cortical neurons. Furthermore, most of the elevations in [Ca²+]i triggered by acidosis are the result of Ca²+ channels opening downstream of ASIC1a activation. Stimulation of sigma-1 receptors effectively suppressed these secondary Ca²+ fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking sigma-1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca²+]i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that sigma-1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendritic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of [Ca²+]i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated [Ca²+]i increases after ischemia onset. Moreover, presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission and thus enhance postsynaptic [Ca²+]i elevations. Sigma-1 receptor activation decreased ischemia-mediated Ca²+ dysregulation at pH values of 7.4 - 6.0 and prevented the synergistic interaction between ischemia and acidosis.

Migrän är en av de vanligaste sjukdomarna i världen och förekommer hos både barn och vuxna. Den uppskattade prevalensen i Sverige är 15 % och prevalensen världen över är drygt 10 %. Det är idag känt att både gener och miljöfaktorer är inblandade i uppkomsten av migrän men det är dock oklart exakt hur migrän uppstår. Migrän är en kostnadskrävande sjukdom dels i form av behandlingskostnader men även på grund av inkomstbortfall då sjukdomen utgör ett stort funktionshinder. Behandlingsmetoder vid migrän indelas i tre kategorier: icke-farmakologisk, anfallskuperande farmakologisk, respektive profylaktisk farmakologisk behandling. Det finns ett behov av att hitta substanser som ger mildare biverkningar vid migrän, jämfört med de behandlingar som används idag, framförallt eftersom många barn är drabbade. Syftet med examensarbetet var att undersöka om magnesiumtillskott är ett effektivt behandlingsalternativ vid migrän, dels för behandling vid migränanfall och dels som förebyggande behandling av migrän. Då magnesium har en rad viktiga funktioner i kroppen leder låga magnesiumnivåer till ökade risker för sjukdomar och komplikationer. Studier visar att det finns samband mellan brist på magnesium och en ökad sjuklighet med kopplingar till bland annat typ 2 diabetes, högt blodtryck, kardiovaskulär sjukdom, migränhuvudvärk och spänningshuvudvärk. För att undersöka om magnesium kan användas för att behandla migränanfall analyserades fyra olika studier där magnesium har använts som anfallskuperande behandling. För att undersöka om magnesium kan användas i profylaktisk behandling av migrän analyserades även fyra olika studier där magnesium har getts i förebyggande syfte. För sökning efter studier användes databasen PubMed. Magnesiums smärtlindrande verkan i anfallskuperande behandling av migrän var inte övertygande vid jämförelse mot placebo. Av de studier där magnesium användes som förebyggande behandling visade dock samtliga av dessa fyra studier fördelaktiga resultat gällande bland annat migränanfallens intensitet vid jämförelse mot placebo. I två studier där man dagligen använde magnesiumcitrat som förebyggande behandling sågs även signifikanta sänkningar i migränanfallens frekvens jämfört med placebo. De analyserade studierna i detta examensarbete visar att magnesium verkar fungera som förebyggande behandling av migrän. Fler studier behövs för att undersöka om magnesium kan användas som smärtlindrande behandling vid migränanfall. Då det finns ett behov av alternativa metoder för behandling av migrän som ger milda biverkningar motiverar detta till att göra fortsatta studier inom området.

N-methyl-D-aspartate (NMDA) receptors are glutamatergic ionotropic receptors involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They are heteromeric complexes of GluN1 combined with GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Many allosteric modulators can influence the activity of these receptors including neurosteroids. Pregnanolone sulfáte (3α5βS) is an endogenous neurosteroid that inhibits NMDA receptors in a use-dependent manner and has neuroprotective effect. Binding site for 3α5βS on the NMDA receptor molecule is still not indentified. The aim of my work was to contribute to the identification of the biding site by kinetic analysis of rate of response return from 3α5βS inhibition. Using the point mutation we also attempted to identify the amino acids residues that could be involved in the neurosteroid binding. In order to study the effect of 3α5βS on NMDA receptors the electropfysiological recordings on human embryonic kidney 293T cells expressing recombinant GluN1/GluN2B receptors was performed. We confirm that the effect of 3α5βS on GluN1/GluN2B receptors is voltage-independent. The results of my work indicate that steroids can reach the binding site on the NMDA receptors through the membrane rather than directly from the aqueous...

NMDA receptor is necessary for excitatory transmission in the central nervous system. Altered funtion of the NMDA receptors is associated with many neurodegenerative and neuropsychiatric diseases. All available crystal structures of the NMDAR meant great shift towards our understanding of details of the receptor and its function. Unfortunately, these up- to-date available structures present only certain functional states of receptors and also a few structural data are still missing. For complete comprehension of the process of activation and deactivation of NMDA receptors, we need to supplement the current information with more data. The aim of this thesis was to employ a combination of different approaches (computational modelling, cloning, biochemistry, protein expression and purification and mass spectrometry) to obtain new structural data, by which we would be able to fill in the gaps in current receptor models, especially at various functional states of the receptor. Key words: NMDA receptor, glutamate receptor, computational modelling, structure, cloning, protein expression

Ketamine and phencyclidine (PCP) are noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) type of ligand-gated glutamate receptors. Both agents have high abuse liability, and may produce dependence. Tolerance to the reinforcing effects of drugs of abuse is widely regarded as a key component of the dependence process. Therefore, the present study was conducted to examine whether tolerance develops to the reinforcing effects of ketamine, and whether PCP and dizocilpine, a noncompetitive NMDA antagonist with negligible abuse liability, produce cross-tolerance to the reinforcing effects of ketamine. Further, identification of the neural mechanisms that underlie tolerance to the reinforcing effects of drugs may yield information regarding drug dependence.

Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 1314 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.

It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

Schizophrenia is a troubling and severe mental illness that is only incompletely treated by currently available drugs. New drug development is hindered by a scarcity of functionally relevant pharmacodynamic biomarkers that are translatable across preclinical and human subjects. Although psychosis is a major feature of schizophrenia, cognitive and negative symptoms determine the long-term functional outcomes for patients. Stimulus-evoked neural synchrony at gamma (~ 40 Hz) frequency plays an important role in the processing and integration of sensory information. Not surprisingly, schizophrenia patients show deficits in gamma oscillations. NMDA receptor (NMDAR) activation on fast-spiking parvalbumin-positive interneurons is deemed important for the generation of gamma oscillations. NMDA hypofunction has been proposed as an alternative hypothesis to the well-known dopamine dysregulation to explain the neurochemical abnormalities associated with schizophrenia. For this dissertation, we validated a preclinical model to pharmacologically probe NMDA-mediated gamma oscillations by further characterizing the auditory-steady state response (ASSR) in female Sprague Dawley rats. The ASSR is a measure of cortical neural synchrony evoked in response to periodic auditory stimuli. ASSR at 40 Hz is consistently disrupted in patients. First, we established the reliability of click train-evoked 40 Hz ASSR and tone-evoked gamma oscillations in 6 separate sessions, spread over a 3-week period. Then we established the sensitivity of these neural synchrony measures to acute NMDAR blockade using the high affinity NMDA channel blocker MK-801, using a repeated measures design. Next, we compared the reliability and sensitivity of the 40 Hz ASSR from two distinct recording sites. Results from this study showed that as compared to vertex, temporal recording showed a greater gamma synchrony. However, the temporal recording had poor test-retest reliability and lower sensitivity to MK-801-induced disruption. Lastly, we characterized the dose-response profiles of an NMDA co-agonist D-serine, an atypical (clozapine) and a typical (haloperidol) antipsychotic, on the 40 Hz ASSR. Results from these studies showed that only clozapine was effective in robustly augmenting 40 Hz ASSR. Furthermore, only clozapine pretreatment had partial protective effect against MK-801 induced ASSR disruption. Overall, this work establishes that vertex recorded 40 Hz ASSR is a reliable neural synchrony biomarker in female SD rats that is amenable for bidirectional pharmacodynamic modulation.