of Ph.D. thesis Molecular mechanisms of regulation of trafficking and function of different subtypes of NMDA receptors in hippocampal neurons Mgr. Kristýna Skřenková N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that play a key role in the mammalian central nervous system. Under physiological conditions, these receptors are important for excitatory synaptic transmission and memory formation. However, under pathological conditions, their abnormal regulation or activation may lead to many neurological and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy or schizophrenia. Previous studies have shown that the number and type of NMDA receptors on the cell surface are regulated at multiple levels, including their synthesis, folding, internalization or degradation. During the trafficking of NMDA receptors to the cell surface membrane, both the agonist binding and receptor activation are examined. Moreover, NMDA receptors undergo many posttranslational modifications such as palmitoylation, phosphorylation or N-glycosylation. In this thesis, we studied the molecular mechanisms that may affect the trafficking and functional properties of NMDA receptors in mammalian cells and rat hippocampal neurons. Specifically, we studied i)...

Several trials have reported rapid antidepressant response from the anesthetic drug ketamine although the mechanism behind this effect is not fully understood. Research has focused mainly on ketamine’s action in the brain, including its effects on chemical balance, connections between brain cells and networks, and cognition. Trials with psychedelic drugs have had similar antidepressant results as ketamine, and the quality of the subjective psychedelic experience seems to mediate antidepressant action. Ketamine causes similar alterations of consciousness, which have been viewed as side effects. This thesis examines whether ketamine works in a similar way as psychedelics, where the ketamine-induced dissociative-like experience has a relationship to antidepressant response. Leading theories of depression and ketamine’s action in the brain are presented, and eight studies examining the relationship between ketamine-induced subjective experience and antidepressant response are reviewed. Three included studies found a relationship between psychedelic- and dissociative-like symptoms and reduction in depression, while five did not. The supposed relationship between psychedelic- and dissociative-like symptoms and antidepressant action has not been adequately explored and needs further examination in clinical trials.

Alzheimer¿s disease is associated with aging and is characterised by a progressive cognitive decline. Its onset in women coincides with the abrupt depletion of ovarian steroids prompting the investigation of utilising oestrogen replacement therapy as restoration or a preventative measure. Gonadal steroids have also recently been implicated in other disease states, particularly schizophrenia. In addition to the cognitive decline, sufferers of Alzheimer¿s disease and schizophrenia display anxiety related behaviour which gonadal steroids have also been shown to ameliorate. In this thesis several paradigms were used to investigate the effects of oestradiol benzoate (EB) on cognition and anxiety, utilising the NMDA receptor antagonist PCP, the muscarinic receptor antagonist scopolamine and the dopamine releasing agent amphetamine to induce a cognitive deficit in rats by different pharmacological mechanisms. The thesis also investigated the effects of EB on a delay dependent cognitive deficit model of forgetfulness in natural aging. Results showed that subchronic PCP dosing failed to induce a significant deficit in the novel object recognition task. Locomotor activity tests demonstrated that the PCP treated rats were sensitised to the treatment suggesting that the PCP dosing regimen was successful. There was no significant effect of oestrogen in the reversal learning model or in the plus maze task designed to explore EB¿s effects on anxiety. However, in the latter task there was a trend towards an anxiogenic effect of EB. Results from the delay dependent model of forgetfulness in natural aging demonstrated that EB could enhance recognition memory, but not spatial memory. The results are discussed in the context of the role of gonadal steroids especially oestrogen in combating the cognitive decline seen in schizophrenia, neurodegenerative disease and natural aging.

<p>The superior olivary complex (SOC) is comprised of nuclei involved in sound localization. To compute interaural sound level differences, lateral superior olive (LSO) neurons integrate converging glutamatergic inputs from the cochlear nucleus with glycinergic inputs from the medial nucleus of the trapezoid body (MNTB). To compute interaural timing differences, the medial superior olive (MSO) integrates converging glutamatergic inputs from the ipsilateral and contralateral cochlear nucleus. The MSO also receives a major inhibitory input from the MNTB. N-methyl-D-aspartate receptors (NMDARs) are thought to play a role in the developmental refinement of these auditory brainstem pathways. The GluN2A and GluN2B NMDAR subunits confer widely different properties on NMDARs, substantially affecting plasticity. We assessed postnatal developmental messenger RNA (mRNA) expression of GluN1, GluN2A and GluN2B subunits in the LSO, MSO and MNTB using quantitative <em>in-situ</em> hybridization in tissue from 10 litters, ages postnatal day 1 to 36 (P1-36).</p> <p>GluN1 mRNA expression in the LSO, MSO and MNTB decreased with age. In all three nuclei, GluN2B mRNA expression was highest during the first postnatal week, dropping to low levels thereafter. In the LSO, GluN2A levels increased, then decreased to moderate levels. In the MNTB, GluN2A levels decreased from initially high levels. In the MSO, GluN2A levels increased to intermediate levels. The GluN2A/2B ratio increased 2-fold between P1 and P8 in the MNTB, whereas the ratio increased 3-fold between P8 and P15 in the LSO and MSO. The changes in GluN2A:GluN2B ratio are consistent with a developmental switch from GluN2B-containing NMDARs to GluN2A-containing NMDARs. These results are consistent with prior electrophysiological experiments that show NMDAR-mediated currents declining with age in the aVCN-MNTB, aVCN-LSO and MNTB-LSO pathways. The GluN2A subunit exhibited different developmental expression patterns in MNTB, LSO and MNTB, which suggests that GluN2A mRNA expression is locally regulated between nuclei, whereas GluN2B may be globally regulated.</p> / Master of Science (MSc)

Schizophrenia is a debilitating neuropsychiatric disorder that affects approximately one percent of the global population. Aberrant N-methyl-D-aspartate receptors and endoplasmic reticulum stress have been implicated in the pathogenesis of schizophrenia. Despite a century of extensive research, outcomes from best-practice treatments remain dismal. Lurasidone hydrochloride is a novel atypical antipsychotic drug with a unique receptor binding profile that can potentially treat the heterogeneous symptomology of schizophrenia. However, discrepancies in experimental design (i.e. animal models used, symptoms assessed etc.) have yielded conflicting results surrounding the procognitive and antidepressant properties of lurasidone. Furthermore, the limited aqueous solubility of lurasidone poses a considerable challenge for improving antipsychotic drug delivery to the brain and limiting the prevalence of adverse side effects. These obstacles coupled with the elusive pathophysiology of schizophrenia and its incurable nature, highlight the importance of investigating novel therapeutic targets and their underlying mechanisms to improve treatment and enhance the quality of life of patients with schizophrenia. This thesis sought to accomplish three primary objectives: (1) validate the behavioural efficacy of lurasidone hydrochloride; (2) investigate the role of mesencephalic astrocyte-derived neurotrophic factor as a potential therapeutic target of lurasidone; and (3) evaluate the therapeutic potential of intranasal lurasidone administration as a novel method for antipsychotic drug delivery. The data presented within this thesis suggest that repeated lurasidone treatment may be effective at treating the positive, negative, and cognitive symptoms of schizophrenia, but not sensorimotor gating deficits. Furthermore, sub-chronic lurasidone treatment in rats significantly increased the relative expression of mesencephalic astrocyte-derived neurotrophic factor in the rat prefrontal cortex, a primary site of impairment observed in schizophrenia. Lastly, we conclude that lurasidone administered via the nasal route using a novel poly(oligo ethylene glycol methacrylate)-based nanogel formulation required four times less drug to achieve a therapeutic response comparable to traditional intraperitoneal routes. The findings presented within this thesis suggest that lurasidone might be a favourable atypical antipsychotic drug that exerts its therapeutic effects through the modulation of neurotrophic factor expression in the brain regions affected by schizophrenia. This thesis offers new insight that can help guide future studies toward improving the prognosis of patients suffering from schizophrenia. / Thesis / Master of Science (MSc)

<p>Obsessive-compulsive disorder (OCD) is a devastating illness that afflicts around 2% of the world's population with recurrent distressing thoughts (obsessions) and repetitive ritualistic behaviors (compulsions). While dysfunction at excitatory glutaminergic excitatory synapses leading to hyperactivity of the orbitofrontal cortex and head of the caudate - brain regions involved in reinforcement learning - are implicated in the pathology of OCD, clinical studies involving patients are unable to dissect the molecular mechanisms underlying this cortico-striatal circuitry defect. Since OCD is highly heritable, recent studies using mutant mouse models have shed light on the cellular pathology mediating OCD symptoms. These studies point toward a crucial role for deltaFosB, a persistent transcription factor that accumulates with chronic neuronal activity and is involved in various diseases of the striatum. Furthermore, elevated deltaFosB levels results in the transcriptional upregulation of Grin2b, which codes GluN2B, an N-methyl-D-aspartate glutamate receptor (NMDAR) subunit required for the formation and maintenance of silent synapses. Taken together, the current evidence indicates that deltaFosB-mediated expression of aberrant silent synapses in caudate medium spiny neurons (MSNs), in particular D1 dopamine-receptor expressing MSNs (D1 MSNs), mediates the defective cortico-striatal synaptic transmission that underlies compulsive behavior in OCD.</p> / Dissertation

La synapse glutamatergique assure la majeure partie de la transmission excitatrice du cerveau et des changements de sa force constituent un corrélat cellulaire des processus d’apprentissage et de mémoire. Ces processus adaptatifs nécessitent souvent l’activation des récepteurs ionotropiques au glutamate de type NMDA (NMDAR) et l’influx calcique dans le compartiment postsynaptique qui suit leur ouverture. Jusqu’alors, l’activation des voix de signalisations sous-jacentes était considérée comme le seul mécanisme essentiel à la plasticité synaptique. Il est apparu récemment que les NMDAR diffusent à la surface des neurones, assurant un remodelage dynamique de leur distribution. La possibilité que la dynamique de surface des NMDAR joue un rôle déterminant dans les propriétés plastiques des synapses a donc émergé. Au cours de ma thèse, je me suis intéressé à cette problématique à l’aide d’approches d’imagerie dynamique à haute-résolution (ex. suivi de nanoparticules uniques, FRAP) et d’outils moléculaires de haute spécificité (ex. ligand biomimétique, x-link de récepteurs via les anticorps). J’ai dans un premier temps étudié la dynamique de surface des NMDAR endogènes au cours de la plasticité synaptique au sein de réseaux neuronaux hippocampiques in vitro. Mes résultats révèlent que l’induction de la potentialisation à long terme (LTP) des synapses glutamatergiques s’accompagne d’une redistribution latérale des NMDAR de surface dans la région postsynaptique. De façon remarquable, la réduction de la diffusion de surface des NMDAR via des anticorps commerciaux, mais aussi des anticorps purifiés de patients atteints d’encéphalite auto-immune, ciblant des épitopes extracellulaires des NMDAR, bloque la LTP. Dans un second temps, je me suis intéressé à la régulation de cette dynamique des NMDAR. En collaboration avec le groupe de Stéphane Oliet (CRI, INSERM), nous avons découvert qu’une redistribution rapide de surface des NMDAR s’opère différemment sous l’effet des co-agonistes du récepteur, la glycine et la D-sérine, et cela de façon dépendante des sous-unités GluN2A/GluN2B des NMDAR. De plus, j’ai démontré que l’interaction directe entre les NMDAR et les récepteurs dopaminergiques D1 membranaires contrôle la distribution des deux types de récepteurs aux abords de la synapse et module la plasticité synaptique. L’ensemble de ces données indique que la dynamique de surface des NMDAR est régulée par la présence d’un neuromodulateur, la dopamine, et de co-agonistes, contrôlant de façon dynamique la fenêtre plastique des synapses. / Glutamate synapse mediates most synaptic excitation in the brain and changes in its strength constitute a cellular basis for learning and memory processes. These adaptive properties often require ionotropic glutamate NMDA receptor (NMDAR) and the calcium influx in the postsynaptic compartment following their opening. So far, the activation of the subsequent signaling pathways was considered as the only mechanism essential for synaptic plasticity. It recently appeared that NMDAR diffuse at the neuronal surface, dynamically shaping their distribution. Whether the NMDAR surface dynamics and its potential regulators play an instrumental role in the plastic properties of synapses emerged thus as a possibility. During my PhD, I tackled this question using a combination of high resolution imaging techniques (e.g. single nanoparticle tracking, FRAP) and high specificity molecular approaches (e.g. biomimetic ligand, antibody based receptor cross-link). First, I studied surface dynamics of endogenous NMDAR during synaptic plasticity on hippocampal neurons in vitro. My results reveal that the induction of glutamate synapse long-term potentiation (LTP) is accompanied by a lateral redistribution of surface NMDAR within the postsynaptic area. Strikingly, reducing the surface diffusion of NMDAR using both commercial and purified antibodies from autoimmune encephalitis patients targeting extracellular epitopes of the NMDAR prevents LTP. Second I investigated whether NMDAR dynamics were regulated. In collaboration with Stephane Oliet’s group (CRI, INSERM), we uncovered that rapid surface redistribution can also be achieved differentially using the NMDAR co-agonists, glycine and D-serine, in a GluN2A/GluN2B NMDAR subunit dependent manner. In addition, I demonstrated that the direct interaction between NMDAR and dopamine D1 receptor at the membrane controls both receptors distribution in the synaptic area and modulates synaptic plasticity. Altogether, these data indicate that the NMDAR surface dynamics is regulated by ambient neuromodulators such as dopamine and co-agonists, dynamically controlling then the plastic range of synapses.

Gliotransmitters dérivés de l'astrocyte glutamate et l'ATP modulent l'activité neuronale. Cependant, il reste à savoir comment les astrocytes contrôlent la libération et coordonnent les actions de ces gliotransmetteurs. Dans la première partie de ma thèse, en utilisant l'expression transgénique de la canalrhodopsine 2 (ChR2) sensible à la lumière dans les astrocytes, nous avons observé que la photostimulation augmentait de manière fiable le potentiel d'action des neurones pyramidaux de l'hippocampe. Cette excitation repose principalement sur une libération de glutamate dépendant du Ca2+ par les astrocytes qui active les NMDR neuronaux extrasynaptiques. Remarquablement, nos résultats montrent que l'augmentation de Ca2+ induite par ChR2 et la libération ultérieure de glutamate sont amplifiées par l'activation autocrine induite par l'ATP/ADP des récepteurs P2Y1 sur les astrocytes. Ainsi, l'excitation neuronale est favorisée par une action synergique de la signalisation glutamatergique et purinergique autocrine dans les astrocytes. Ce nouveau mécanisme peut être particulièrement pertinent pour les conditions pathologiques dans lesquelles la concentration extracellulaire d'ATP est augmentée et agit comme un signal de danger majeur. Dans la seconde partie de ma thèse, nous rapportons que la photostimulation sélective des astrocytes ChR2 dans le gyrus denté facilite la transmission synaptique excitatrice sur les cellules granulaires via l'activation des NMDR pré-synaptiques contenant GluN2B. De plus, nous avons découvert que l'élévation intracellulaire du Ca2+ induite par l'ATP et dérivée de l'ATP contrôlait étroitement la libération du glutamate par les astrocytes au cours de la photostimulation des astrocytes. Nos résultats fournissent des preuves d'une relation étroite entre l'ATP dérivé d'astrocyte et le glutamate. / Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. In the first part of my thesis, using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a Ca2+-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDRs. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal. In the second part of my thesis, we report that selective photostimulation ChR2 positive astrocytes in dentate gyrus facilitates excitatory synaptic transmission onto granule cells via the activation of pre-synaptic GluN2B-containing NMDRs. Moreover, we discovered that astrocyte-derived ATP-mediated intracellular Ca2+ elevation tightly controls glutamate release from astrocytes during astrocyte photostimulation. Our results provide evidence for a close relationship between astrocytic-derived ATP and glutamate.