Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.

Major depressive disorder (MDD) is a growing health problem. Current treatment options are not always effective and take several weeks of regular administration before an improvement can be seen in symptoms. Sub-anaesthetic doses of ketamine have been found to have antidepressant effects in previously treatment-resistant MDD after just one dose. However, ketamine also produces short term psychosis-like side effects which are undesirable for MDD patients. Ketamine is known to be an NMDA receptor antagonist, binding within the channel pore to block ion flow, however the molecular mechanism(s) underlying its antidepressant and psychosis-like effects are still unclear. In this thesis several genetically modified mouse lines were used to probe the molecular events involved in ketamine's actions. Firstly, a mouse line in which the c-terminal domain (CTD) of the NMDAR subtype GluN2B had been replaced with that of GluN2A, and a second line in which the opposite replacement had taken place, were used to investigate the role of the CTD in the NMDAR response to ketamine. It was found that the GluN2B CTD is required for the short-term psychosis-like response to a sub-anaesthetic dose of ketamine. This is interesting as the channel pore region, containing the binding site for ketamine, is unaltered in these mutants. Therefore, this finding implicates GluN2B CTD specific intracellular signalling molecules in this action of ketamine and raises the question of whether the CTD itself is able to respond to ketamine binding within the pore to induce signalling changes, perhaps via a conformational change. Secondly, a mouse line, in which the activity-regulated synaptic protein Arc has been tagged with a fluorescent marker, was used to investigate the response of synapses to both anaesthetic and sub-anaesthetic doses of ketamine. In this experiment tagged Arc protein was visible as punctate accumulations at synapses. A novel method termed 'synaptome mapping' was used to image these accumulations across entire coronal sections and to quantify their number, size and intensity. Using this method alterations to the Arc synaptome map were detected 1h, 6h and 24h following ketamine administration. The two doses used produced different changes to this map, with the sub-anaesthetic antidepressant dose inducing increases in Arc puncta number across many brain regions, whereas the anaesthetic dose induced short term (1h) increases followed by longer term decreases in Arc puncta number. This finding links long-term increases in Arc at the synapse with an antidepressant response to ketamine.

Our memories are fundamental components of who we are as individuals. They influence almost every aspect of our lives such as our decisions, our personalities, our emotions, and our purpose in life. Diseases that affect memory have devastating impacts on the individuals who bear them. Imagine not being able to recall pleasant memories or even the faces of close family members. It's important to understand the biology of memory formation not only because it's an intriguing scientific question, but because of its consequences when these processes are lost. N-methyl-D-aspartate-type glutamate receptors (NMDARs) and calcium/calmodulin-dependent kinase II (CaMKII) are essential molecules involved in learning and its physiological correlate, synaptic plasticity. Calcium influx through NMDARs activates CaMKII, which translocates to the postsynaptic signaling sites through its interactions with the NMDAR subunits NR1 and NR2B. The significance of CaMKII's translocation is not fully known, however we hypothesize that it is an early molecular event that is necessary for the expression of synaptic plasticity and learning. Our laboratory has developed two strains of mice with targeted mutations to NR1 and NR2B (NR1KI and NR2BKI) that are deficient in their ability to bind to CaMKII to test the role of CaMKII binding to NMDARs in synaptic plasticity and learning. We found that CaMKII binding to NR2B is necessary for long-term potentiation (LTP), the most commonly studied form of synaptic plasticity. NR2BKI mice are able to learn spatial and cued tasks normally, however they are unable to consolidate spatial tasks for long-term memory storage. On the other hand, we found that CaMKII binding to NR1 is not necessary for LTP. Furthermore NR1KI mice do not show impairments in contextual or cued learning. We found that NR1 mutations resulted in an age-dependent truncation of the intracellular domains of NR1 that reduced its activity leading to severe impairments in synaptic transmission, LTP, and learning. Our results suggest that CaMKII binding to NR2B is the more important for synaptic plasticity and memory formation than NR1. However, we found that the intracellular domains of NR1 are critical for NMDAR and synapse function.

<p>At excitatory synapses, NMDAR-mediated synaptic plasticity occurs in response to activity inputs by modifying synaptic strength. While comprehensive studies have been focused on the induction and expression mechanisms underlying synaptic plasticity, it is less clear whether and how synaptic plasticity itself can be subjected to regulations. The presence of "plasticity of plasticity", or meta-plasticity, has been proposed as an essential mechanism to ensure a proper working range of plasticity, which may also offer an additional layer of information storage capacity. However, it remains elusive whether and how meta-plasticity occurs at single synapses and what molecular substrates are locally utilized. Here, I develop systems allowing for sustained alterations of individual synaptic inputs. By implementing a history of inactivity at single synapses, I demonstrate that individual synaptic inputs control synaptic molecular composition homosynaptically, while allowing heterosynaptic integration along dendrites. Furthermore, I report that subunit-specific regulation of NMDARs at single synapses mediates a novel form of input-specific metaplasticity. Prolonged suppression of synaptic releases at single synapses enhances synaptic NMDAR-mediated currents and increases the number of functional NMDARs containing NR2B. Interestingly, synaptic NMDAR composition is adjusted by spontaneous glutamate release rather than evoked activity. I also demonstrate that inactivated synapses with more NMDARs containing NR2B acquire a lower induction threshold for long-term synaptic potentiation. Together, these results suggest that at single synapses, spontaneous release primes the synapse by modifying its synaptic state with specific molecular compositions, which in turn determine the synaptic gain in an input-specific manner.</p> / Dissertation

Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.

No description available.

Calcium-influx through ionotropic glutamate receptors expressed on non-excitable cells, such as CNS glia, may regulate important cell events via intracellular signaling mechanisms. Oligodendrocytes and oligodendrocyte progenitors (OPCs), two glial populations supporting CNS myelination and myelin repair, express AMPA and NMDA receptors. Although calcium-influx through these receptors is thought to cause glutamate excitotoxicity to oligodendrocytes in CNS injuries, more recent studies suggest that AMPA or NMDA receptor-mediated synaptic transmission between neurons and OPCs plays a positive role in neuronal activity-dependent oligodendrocyte development and regeneration. Given the opposing roles of glutamate receptors in oligodendrocyte death and repair, the clinical relevance of these receptors in white matter injuries remain unclear. Another major challenge for exploring the role of these receptors in white matter injuries is that OPCs and neurons express a similar complement of AMPA and NMDA receptor subunits, which has complicated the interpretation of pharmacological manipulations and global genetic deletion approaches. To define the cell autonomous role of AMPA and NMDA receptor-mediated calcium signaling in oligodendroglia, I abolished the calcium influx through glutamate receptors using two different genetic approaches, and examined their impacts on oligodendrocyte development, injury-induced cell death, and regeneration. First, I employed a new mouse line which allows overexpression of GluA2, the calcium-impermeable AMPA receptor subunit, in a Cre activity-dependent manner. After crossing these mice with OPC- or oligodendrocyte-lineage-specific Cre mice, I applied hypoxic-ischemic injury to these multiple transgenic mice. Surprisingly, even though AMPA receptor-mediated calcium influx was blocked in OPCs, oligodendrogenesis or myelin integrity was not affected. However, GluA2 overexpression significantly promoted oligodendrocyte regeneration and OPC proliferation after injury, while the same manipulation in oligodendrocytes did not protect them from the initial cell loss. Moreover, GluA2 overexpression also stimulated transcriptional activities linked to myelinogenesis, even without injury. Second, I used conditional knockout mice for Grin1, the gene encoding an essential subunit of NMDA receptor complexes. As with GluA2 overexpressing mice, the removal of NMDA receptors from OPCs or all oligodendroglia did not significantly change normal oligodendrocyte development. However, the ablation of NMDA receptor in OPCs exacerbated oligodendrocyte loss by impairing new oligodendrogenesis in hypoxic-ischemic injury. These results suggest that neither AMPA receptors nor NMDA receptors mediate glutamate excitotoxicity in oligodendrocytes in neonatal hypoxic-ischemic injury. Instead, these receptors play distinct roles in post-injury oligodendrocyte development: AMPA receptor-mediated calcium suppresses oligodendrocyte regeneration, and NMDA receptor signaling supports oligodendrocyte regeneration after injury. / Biomedical Sciences

Long-term depression (LTD) plays an important role in the refinement of neocortical maps during early postnatal development. Synapse formation and refinement in the cortex during development rely on synaptic plasticity, the cellular mechanisms of which are poorly understood. The aim of this thesis was to investigate timing- and pattern-dependent LTD at excitatory synapses in the mouse barrel cortex during development. This thesis first describes the developmental prole and N-methyl-D-aspartate (NMDA) receptor GluN2 subtype-dependence of timing-dependent plasticity at layer 4-to-layer 2/3 synapses. A developmental dissociation of timing-dependent plasticity was observed where timing-dependent LTD (t-LTD) was found during early development (postnatal day, P6-8) but disappeared after P25. In contrast, timing-dependent LTP (t-LTP) only appeared in the second postnatal week of development (P11-15) and persisted in the adult cortex. This bidirectional plasticity also showed a GluN2 subtype-dependent dissociation. Whereas t-LTP was dependent on GluN2A subunit-containing NMDA receptors, t-LTD was dependent on GluN2C/D subunit-containing NMDA receptors. This thesis also reports a novel anti-Hebbian form of NMDA receptor-dependent plasticity, in which presynaptic layer 4 neurons drive their presynaptic long-term self-depression without the involvement of postsynaptic action potentials or calcium. This mechanism suggests that, during development, presynaptic self-depression occurs when specific spike patterns (presynaptic burst-spike) in the presynaptic neuron are unsuccessful in driving postsynaptic activity. Finally, this thesis addresses how t-LTD induction rules differ in vertical intra-columnar layer 4-to-layer 2/3 and horizontal cross-columnar layer 2/3-to-layer 2/3 synapses in the barrel cortex. Distinct GluN2 subunit expression in vertical and horizontal synapses regulated the time-window of t-LTD induction. It is also shown that different excitatory intra- and cross-columnar synapses onto the same postsynaptic layer 2/3 neurons can have different molecular requirements for the induction of t-LTD, and that they also interact to induce heterosynaptic associative LTD. These findings may have important implications for understanding the cellular mechanisms of experience-dependent plasticity and its relevance to the computational principles of cortical circuit operation.

Résumé: Les récepteurs du glutamate de type NMDA (NMDAR) sont des canaux ioniques impliqués dans les phénomènes de plasticité de la transmission synaptique dans le système nerveux central, des mécanismes supposés être à la base du développement neuronal, de l’apprentissage et de la formation de la mémoire. Les NMDAR forment des tétramères à la membrane plasmique, constitués de deux sous-unités obligatoires GluN1 et deux sous-unités variables GluN2 (GluN2A-D) ou GluN3. Dans le prosencéphale, les récepteurs comportant les sous-unités GluN2A (GluN2A-NMDAR) et GluN2B (GluN2B-NMDAR) sont les plus abondants et présentent des profils d’expression différents au cours du développement, les GluN2B-NMDAR étant fortement exprimés aux stades précoces tandis que l’expression des GluN2A-NMDAR augmente progressivement au cours du développement postnatal. Des contributions relatives de ces deux sous-types majoritaires de NMDAR aux propriétés de signalisation distinctes dépendent directement les phénomènes de plasticité neuronale, tels que l’adaptation des synapses glutamatergiques et des circuits neuronaux excitateurs. Bien que la régulation moléculaire des NMDAR ait fait l’objet d’intenses recherches ces dernières décennies, la localisation précise de ces deux sous-types de récepteurs dans la membrane postsynaptique demeurait méconnue. Pour répondre à cette question, nous avons étudié la distribution des NMDAR à la surface de neurones d’hippocampe de rats en combinant deux techniques de microscopie de super-résolution - la microscopie de reconstruction optique stochastique directe (dSTORM) et la déplétion d’émission stimulée (STED) - permettant de dépasser la limite de résolution inhérente à la diffraction de la lumière. Ces techniques nous ont permis de mettre en évidence que les sous-types de récepteurs GluN2A- et GluN2B-NMDAR présentent une nano-organisation différente à la surface neuronale. En effet, ils sont organisés en structures nanoscopiques (nanodomaines) qui diffèrent en nombre, en surface et en morphologie, notamment au niveau des synapses. Au cours du développement, l’organisation membranaire des deux sous-types de NMDAR évolue, avec en particulier de profonds changements de distribution des GluN2A-NMDAR. De plus, cette organisation nanoscopique est impactée différemment par des modulations de l’interaction avec les protéines d’échafaudage à domaine PDZ ou de l’activité de la kinase CaMKII suivant le sous-type de NMDAR considéré. En effet, la réorganisation des GluN2A-NMDAR implique principalement des changements de nombre de récepteurs dans les nanodomaines sans modification de leur localisation, tandis que la réorganisation des GluN2B-NMDAR passe essentiellement par des modifications de localisation des nanodomaines sans changements du nombre de récepteurs qu’ils contiennent. Ainsi, les GluN2A- et GluN2B-NMDAR présentent des nano-organisations différentes dans la membrane postsynaptique, reposant vraisemblablement sur des voies de régulation et des complexes de signalisation distincts. / NMDA-type glutamate receptors (NMDARs) are a type of ion permeable channels playing critical roles in excitatory neurotransmission in the central nervous system by mediating different forms of synaptic plasticity, a mechanism thought to be the molecular basis of neuronal development, learning and memory formation. NMDARs form tetramers in the postsynaptic membrane, most generally associating two obligatory GluN1 subunits and two modulatory GluN2 (GluN2A-D) or GluN3 (GluN3A-B) subunits. In the hippocampus, the dominant GluN2 subunits are GluN2A and GluN2B, displaying different expression patterns, with GluN2B being highly expressed in early development while GluN2A levels increase gradually during postnatal development. In the forebrain, the plastic processes mediated by NMDARs, such as the adaptation of glutamate synapses and excitatory neuronal networks, mostly rely on the relative implication of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular regulation of synaptic NMDARs has been under intense investigation over the last decades, the exact topology of these two subtypes within the postsynaptic membrane has remained elusive. Here we used a combination of super-resolution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) and stimulated emission depletion (STED) microscopy to characterize the surface distribution of GluN2A- or GluN2B-containing NMDARs. Both dSTORM and STED microscopy, based on different principles, enable to overcome the resolution barrier due to the diffraction limit of light. Using these techniques, we here unveil a differential nanoscale organization of native GluN2A- and GluN2B-NMDARs in rat hippocampal neurons. Both NMDAR subtypes are organized in nanoscale structures (termed nanodomains) that differ in their number, area, and shape. These observed differences are also maintained in synaptic structures. During development of hippocampal cultures, the membrane organization of both NMDAR subtypes evolves, with marked changes for the topology of GluN2A-NMDARs. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially affected by alterations of either interactions with PDZ scaffold proteins or CaMKII activity. The regulation of GluN2A-NMDARs mostly implicates changes in the number of receptors in fixed nanodomains, whereas the regulation of GluN2B-NMDARs mostly implicates changes in the nanodomain topography with fixed numbers of receptors. Thus, GluN2A- and GluN2B-NMDARs have distinct organizations in the postsynaptic membrane, likely implicating different regulatory pathways and signaling complexes.

It is considered that coincident inputs from multiple presynaptic axons are required to achieve both the suprathreshold level of excitation for the central neurons and the activation of NMDA receptors. The aim of the present study was to determine, whether a discharge of single retinal ganglion cell, which axon terminates in the tectum layer F, can evoke a suprathreshold excitation of frog tectum neurons and activate the NMDA receptors. Extracellular recordings of the neuronal activity elicited by the electrical stimulation of single optic fiber were made in frog tectum. We conclude, that: (1) a train of action potentials of single optic fiber, which terminates in the frog tectum layer F, surely elicits a suprathreshold excitation of tectal neurons at physiological conditions. The suprathreshold level is achieved due to the frequency facilitation of the fast non-NMDA retinotectal synaptic potentials. (2) The train of action potentials of higher frequency activates the NMDA receptors of the tectal neurons at physiological conditions. The activation of NMDA receptors is achieved due to the temporal summation and frequency facilitation of the fast synaptic potentials. (3) Two different kinds of optic fibers (likely darkness and moving edge detectors) exhibit different efficiency to achieve both the suprathreshold excitation of the tectal neurons and the activation of NMDA receptors. Findings of the present study improve the understanding of how the local neural network operates... [to full text]