Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists.

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.

Converging synaptic and network dysfunctions in distinct autoimmune encephalitis.

Psychiatric and neurological symptoms, as well as cognitive deficits, represent a prominent phenotype associated with variable forms of autoimmune encephalitis, regardless of the neurotransmitter receptor targeted by autoantibodies. The mechanistic underpinnings of these shared major neuropsychiatric symptoms remain however unclear. Here, we investigate the impacts of patient-derived monoclonal autoantibodies against the glutamatergic NMDAR (NMDAR mAb) and inhibitory GABAaR (GABAaR mAb) signalling in the hippocampal network. Unexpectedly, both excitatory and inhibitory synaptic receptor membrane dynamics, content and transmissions are altered by NMDAR or GABAaR mAb, irrespective of the affinity or antagonistic effect of the autoantibodies. The effect of NMDAR mAb on inhibitory synapses and GABAaR mAb on excitatory synapses requires neuronal activity and involves protein kinase signalling. At the cell level, both autoantibodies increase the excitation/inhibition balance of principal cell inputs. Furthermore, NMDAR or GABAaR mAb leads to hyperactivation of hippocampal networks through distinct alterations of principal cell and interneuron properties. Thus, autoantibodies targeting excitatory NMDAR or inhibitory GABAaR trigger convergent network dysfunctions through a combination of shared and distinct mechanisms.

NMDA receptor autoantibodies primarily impair the extrasynaptic compartment.

Autoantibodies directed against the N-methyl-D-aspartate receptor (NMDAR-Ab) are pathogenic immunoglobulins detected in patients suffering from NMDAR encephalitis. NMDAR-Ab alter the receptor membrane trafficking, synaptic transmission and neuronal network properties, leading to neurological and psychiatric symptoms in patients. Patients often have very little neuronal damage but rapid and massive (treatment-responsive) brain dysfunctions related to an unknown early mechanism of NMDAR-Ab. Our understanding of this early molecular cascade remains surprisingly fragmented. Here, we used a combination of single molecule-based imaging of membrane proteins to unveil the spatiotemporal action of NMDAR-Ab on live hippocampal neurons. We first demonstrate that different clones of NMDAR-Ab primarily affect extrasynaptic (and not synaptic) NMDARs. In the first minutes, NMDAR-Ab increase extrasynaptic NMDAR membrane dynamics, declustering its surface interactome. NMDAR-Ab also rapidly reshuffle all membrane proteins located in the extrasynaptic compartment. Consistent with this alteration of multiple proteins, effects of NMDAR-Ab were not mediated through the sole interaction between the NMDAR and EphB2 receptor. In the long term, NMDAR-Ab reduce the NMDAR synaptic pool by slowing down receptor membrane dynamics in a cross-linking-independent manner. Remarkably, exposing only extrasynaptic NMDARs to NMDAR-Ab was sufficient to produce their full-blown effect on synaptic receptors. Collectively, we demonstrate that NMDAR-Ab initially impair extrasynaptic proteins, then the synaptic ones. These data thus shed new and unsuspected light on the mode of action of NMDAR-Ab and, probably, our understanding of (extra)synaptopathies.

Distance-dependent regulation of NMDAR nanoscale organization along hippocampal neuron dendrites.

Hippocampal pyramidal neurons are characterized by a unique arborization subdivided in segregated dendritic domains receiving distinct excitatory synaptic inputs with specific properties and plasticity rules that shape their respective contributions to synaptic integration and action potential firing. Although the basal regulation and plastic range of proximal and distal synapses are known to be different, the composition and nanoscale organization of key synaptic proteins at these inputs remains largely elusive. Here we used superresolution imaging and single nanoparticle tracking in rat hippocampal neurons to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDA receptors (NMDARs)-which play key roles in the use-dependent adaptation of glutamatergic synapses-along the dendritic arbor. We report significant changes in the nanoscale organization of GluN2B-NMDARs between proximal and distal dendritic segments, whereas the topography of GluN2A-NMDARs remains similar along the dendritic tree. Remarkably, the nanoscale organization of GluN2B-NMDARs at proximal segments depends on their interaction with calcium/calmodulin-dependent protein kinase II (CaMKII), which is not the case at distal segments. Collectively, our data reveal that the nanoscale organization of NMDARs changes along dendritic segments in a subtype-specific manner and is shaped by the interplay with CaMKII at proximal dendritic segments, shedding light on our understanding of the functional diversity of hippocampal glutamatergic synapses.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

Restoring glutamate receptosome dynamics at synapses rescues autism-like deficits in Shank3-deficient mice.

Shank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.

Consequences of NMDA receptor deficiency can be rescued in the adult brain.

N-methyl-D-aspartate receptors (NMDARs) are required to shape activity-dependent connections in the developing and adult brain. Impaired NMDAR signalling through genetic or environmental insults causes a constellation of neurodevelopmental disorders that manifest as intellectual disability, epilepsy, autism, or schizophrenia. It is not clear whether the developmental impacts of NMDAR dysfunction can be overcome by interventions in adulthood. This question is paramount for neurodevelopmental disorders arising from mutations that occur in the GRIN genes, which encode NMDAR subunits, and the broader set of mutations that disrupt NMDAR function. We developed a mouse model where a congenital loss-of-function allele of Grin1 can be restored to wild type by gene editing with Cre recombinase. Rescue of NMDARs in adult mice yields surprisingly robust improvements in cognitive functions, including those that are refractory to treatment with current medications. These results suggest that neurodevelopmental disorders arising from NMDAR deficiency can be effectively treated in adults.

NMDA receptor membrane dynamics tunes the firing pattern of midbrain dopaminergic neurons.

KEY POINTS: NMDA receptors (NMDARs) expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shapes synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics on cultured mesencephalic and VTA dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings, we demonstrate that NMDARs are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectedly, the NMDAR membrane dynamics per se regulates the firing pattern of VTA dopaminergic neurons, probably through a functional interplay between NMDARs receptors and small-conductance calcium-dependent potassium (SK) channels. ABSTRACT: Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated with neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small-conductance calcium-dependent potassium (SK) channels. However, the cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain unclear. Here, we investigated whether the surface dynamics and distribution of NMDARs tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDARs are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves the ionotropic function of the receptor intact, robustly altered the firing pattern of midbrain DA neurons without altering synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Together, these data show that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function.

Autoimmunity and NMDA receptor in brain disorders: Where do we stand?

Over the past decades, the identification of autoimmune encephalitis in which patients express autoantibodies directed against neurotransmitter receptors has generated great hope to shed new light on the molecular mechanisms underpinning neurological and psychiatric conditions. Among these autoimmune encephalitides, the discovery of autoantibodies directed against the glutamatergic NMDA receptor (NMDAR-Ab), in the anti-NMDAR encephalitis, has provided some key information on how complex neuropsychiatric symptoms can be caused by a deficit in NMDAR signalling. Yet, NMDAR-Abs have also been detected in several neurological and psychiatric conditions, as well as in healthy individuals. In addition, these various NMDAR-Abs appear to have different molecular properties and pathogenicities onto receptors and synaptic functions. Here, we discuss the current view on the variety of NMDAR-Abs and, in particular, how these autoantibodies can lead to receptor dysfunction in neuronal networks. Since our mechanistic understanding on patients' NMDAR-Abs is still in its infancy, several complementary processes can be proposed and further in-depth molecular and cellular investigations will surely reveal key insights. Autoantibodies represent a great opportunity to gain knowledge on the etiology of neuropsychiatric disorders and pave the way for innovative therapeutic strategies. ONE SENTENCE SUMMARY: Current view on patients' autoantibody against NMDAR.

Interplay between NMDA receptor dynamics and the synaptic proteasome.

Proteasome activity at the excitatory synapse plays an important role in neuronal communication. The proteasome translocation to synapses is mediated by neuronal activity, in particular the activation of N-methyl-d-aspartate receptors (NMDARs). These receptors are composed of different subunits with distinct trafficking properties that provide various signalling and plasticity features to the synapse. Yet whether the interplay between the proteasome and NMDAR relies on specific subunit properties remain unclear. Using a combination of single molecule and immunocytochemistry imaging approaches in rat hippocampal neurons, we unveil a specific interplay between GluN2B-containing NMDARs (GluN2B-NMDARs) and the synaptic proteasome. Sustained proteasome activation specifically increases GluN2B-NMDAR (not GluN2A-NMDAR) lateral diffusion. In addition, when GluN2B-NMDAR expression is downregulated, the proteasome localization decreases at glutamatergic synapses. Collectively, our data fuel a model in which the cellular dynamics and location of GluN2B-NMDARs and proteasome are intermingled, shedding new lights on the NMDAR-dependent regulation of synaptic adaptation.

Nanoscale exploration of the extracellular space in the live brain by combining single carbon nanotube tracking and super-resolution imaging analysis.

The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.

Characterization of the Functional Cross-Talk between Surface GABAA and Dopamine D5 Receptors.

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.

Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses.

NMDA-type glutamate receptors (NMDAR) are central actors in the plasticity of excitatory synapses. During adaptive processes, the number and composition of synaptic NMDAR can be rapidly modified, as in neonatal hippocampal synapses where a switch from predominant GluN2B- to GluN2A-containing receptors is observed after the induction of long-term potentiation (LTP). However, the cellular pathways by which surface NMDAR subtypes are dynamically regulated during activity-dependent synaptic adaptations remain poorly understood. Using a combination of high-resolution single nanoparticle imaging and electrophysiology, we show here that GluN2B-NMDAR are dynamically redistributed away from glutamate synapses through increased lateral diffusion during LTP in immature neurons. Strikingly, preventing this activity-dependent GluN2B-NMDAR surface redistribution through cross-linking, either with commercial or with autoimmune anti-NMDA antibodies from patient with neuropsychiatric symptoms, affects the dynamics and spine accumulation of CaMKII and impairs LTP. Interestingly, the same impairments are observed when expressing a mutant of GluN2B-NMDAR unable to bind CaMKII. We thus uncover a non-canonical mechanism by which GluN2B-NMDAR surface dynamics plays a critical role in the plasticity of maturing synapses through a direct interplay with CaMKII.

Astroglial glutamate transporters in the brain: Regulating neurotransmitter homeostasis and synaptic transmission.

Astrocytes, the major glial cell type in the central nervous system (CNS), are critical for brain function and have been implicated in various disorders of the central nervous system. These cells are involved in a wide range of cerebral processes including brain metabolism, control of central blood flow, ionic homeostasis, fine-tuning synaptic transmission, and neurotransmitter clearance. Such varied roles can be efficiently carried out due to the intimate interactions astrocytes maintain with neurons, the vasculature, as well as with other glial cells. Arguably, one of the most important functions of astrocytes in the brain is their control of neurotransmitter clearance. This is particularly true for glutamate whose timecourse in the synaptic cleft needs to be controlled tightly under physiological conditions to maintain point-to-point excitatory transmission, thereby limiting spillover and activation of more receptors. Most importantly, accumulation of glutamate in the extracellular space can trigger excessive activation of glutamatergic receptors and lead to excitotoxicity, a trademark of many neurodegenerative diseases. It is thus of utmost importance for both physiological and pathophysiological reasons to understand the processes that control glutamate time course within the synaptic cleft and regulate its concentrations in the extracellular space. © 2017 Wiley Periodicals, Inc.

Ultradian corticosterone pulses balance glutamatergic transmission and synaptic plasticity.

The rodent adrenal hormone corticosterone (CORT) reaches the brain in hourly ultradian pulses, with a steep rise in amplitude before awakening. The impact of a single CORT pulse on glutamatergic transmission is well documented, but it remains poorly understood how consecutive pulses impact on glutamate receptor trafficking and synaptic plasticity. By using high-resolution imaging and electrophysiological approaches, we report that a single pulse of CORT to hippocampal networks causes synaptic enrichment of glutamate receptors and increased responses to spontaneously released glutamatergic vesicles, collectively abrogating the ability to subsequently induce synaptic long-term potentiation. Strikingly, a second pulse of CORT one hour after the first--mimicking ultradian pulses--completely normalizes all aspects of glutamate transmission investigated, restoring the plastic range of the synapse. The effect of the second pulse is precisely timed and depends on a nongenomic glucocorticoid receptor-dependent pathway. This normalizing effect through a sequence of CORT pulses--as seen around awakening--may ensure that hippocampal glutamatergic synapses remain fully responsive and able to encode new stress-related information when daily activities start.

Surface trafficking of NMDA receptors: gathering from a partner to another.

Understanding the molecular and cellular pathways by which neurons integrate signals from different neurotransmitter systems has been among the major challenges of modern neuroscience. The ionotropic glutamate NMDA receptor plays a key role in the maturation and plasticity of glutamate synapses, both in physiology and pathology. It recently appeared that the surface distribution of NMDA receptors is dynamically regulated through lateral diffusion, providing for instance a powerful way to rapidly affect the content and composition of synaptic receptors. The ability of various neuromodulators to regulate NMDA receptor signaling revealed that this receptor can also serve as a molecular integrator of the ambient neuronal environment. Although still in its infancy, we here review our current understanding of the cellular regulation of NMDA receptor surface dynamics. We specifically discuss the roles of well-known modulators, such as dopamine, and membrane interactors in these regulatory processes, exemplifying the recent evidence that the direct interaction between NMDAR and dopamine receptors regulates their surface diffusion and distribution. In addition to the well-established modulation of NMDA receptor signaling by intracellular pathways, the surface dynamics of the receptor is now emerging as the first level of regulation, opening new pathophysiological perspectives for innovative therapeutical strategies.

Immuno-psychiatry: an agenda for clinical practice and innovative research.

BACKGROUND: The diagnostic scheme for psychiatric disorders is currently based purely on descriptive nomenclature given that biomarkers subtypes and clearly defined causal mechanisms are lacking for the vast majority of disorders. The emerging field of "immuno-psychiatry" has the potential to widen the exploration of a mechanism-based nosology, possibly leading to the discovery of more effective personalised treatment strategies. DISCUSSION: Disturbances in immuno-inflammatory and related systems have been implicated in the aetiology, pathophysiology, phenomenology and comorbidity of several psychiatric disorders, including major mood disorders and schizophrenia. A fundamental challenge in their clinical management is to identify bio-signatures that might indicate risk, state, trait, prognosis or theragnosis. Here, we provide the rationale for a clinical and research agenda to refine future clinical practice and conceptual views, and to delineate pathways toward innovative treatment discovery. CONCLUSION: The development of bio-signatures will allow clinicians to tailor interventions to the abovementioned biomarker subtypes - a major translational goal for research in this field.

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors.

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine-glutamate interplay.

Stress hormones and AMPA receptor trafficking in synaptic plasticity and memory.

The acquisition and consolidation of memories of stressful events is modulated by glucocorticoids, a type of corticosteroid hormone that is released in high levels from the adrenal glands after exposure to a stressful event. These effects occur through activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). The molecular mechanisms that underlie the effects of glucocorticoids on synaptic transmission, synaptic plasticity, learning and memory have recently begun to be identified. Glucocorticoids regulate AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor trafficking--which is crucially involved in synaptic transmission and plasticity--both rapidly and persistently. Stress hormones may, through modulation of AMPA receptor function, promote the consolidation of behaviourally relevant information.

[Anti-NR1 antibodies in anti-N-methyl-D-aspartate receptor encephalitis and schizophrenia]. / Anticorps anti-NR1 dans l'encéphalite anti-récepteur N-méthyl-D-aspartate et la schizophrénie.

The recent discovery of anti-NMDA receptor antibodies and proof of their pathogenic effects in limbic encephalitides raised many questions among neuroscientist and physicians working in the field of schizophrenia. Indeed, this two conditions share several major clinical, pathophysiological or etiological aspects and some authors tend to consider some forms of schizophrenia as mild-encephalitis cases. Some studies have reported the presence of these antibodies in schizophrenic patient's sera without neurological symptoms. These findings suggest new therapeutic perspectives in some schizophrenic patients, despite a low seroprevalence and pathogenic effects that remain to be demonstrated.