Synaptic NMDA Receptors Research Articles

Effects of GluN2A and GluN2B gain-of-function epilepsy mutations on synaptic currents mediated by diheteromeric and triheteromeric NMDA receptors

Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2AN615K, GluN2BN615I and GluN2BV618G gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying ‘artificial’ synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg2+ block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg2+ from 0 to 1 mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg2+ block. The GluN2AN615K mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2BN615I mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2BV618G mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2BN615I or GluN2BV618G mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes.

Chronic Stress Induces Persistent Hypertension and Increases NMDA Receptor Activity in the Paraventricular Nucleus in Borderline Hypertensive Rats

Stress activates sympathetic outflow and promotes hypertension development in humans with genetic predisposition. The enhanced N‐methyl‐D‐aspartate receptor (NMDAR) activity in the hypothalamus paraventricular nucleus (PVN) plays a critical role in controlling sympathetic output in hypertension. However, it is unclear how chronic stress affects NMDAR activity in the PVN in borderline hypertensive rats (BHR). BHR are the F1 male offspring from crossing female spontaneously hypertensive rats with male Wistars Kyoto rats (WKY). Arterial blood pressure (ABP) was monitored in free moving rats by using radiotelemetry. Using a 6‐week chronic unpredictable stress protocol, we found that stressed WKY rats had increased ABP during chronic stress but ABP returned to the normal level within a few days after stress. However, the stressed BHR showed a greater increase in ABP during stress and the elevated ABP was sustained for at least 2 weeks after discontinuing stress. Furthermore, whole‐cell voltage clamp recording in brain slices showed that the amplitude of evoked‐NMDAR currents in spinally projecting PVN neurons was significantly higher in stressed BHR than in non‐stressed BHR controls. Inhibiting α2δ‐1 with gabapentin normalized the increased synaptic NMDAR activity of spinally projecting PVN neurons in stressed BHR. In addition, the expression level of GluN1 and α2δ‐1 in the PVN was significantly greater in stressed BHR than in stressed WKY or non‐stressed BHR. These results suggest that chronic stress induces persistent hypertension in BHR, which is associated with α2δ‐1‐mediated NMDAR hyperactivity in PVN presympathetic neurons.Support or Funding InformationAcknowledgmentsSupported by NIH grants HL131161 and HL142133.

Neural Mechanisms Responsible for the Regulation of Caloric Intake Following Acute High Fat Diet are Developmentally Regulated

Previous studies have shown that, in adulthood, acute high fat diet (HFD) exposure is associated with an initial period of hyperphagia before caloric intake is restored within 3–5 days. This period of homeostasis is accompanied by activation of NMDA receptors (NMDA‐R) and increased excitability of identified vagal efferent motoneurons regulating gastric motility. The mechanisms responsible regulation of caloric intake, as well as how and why these mechanisms are lost, are critical to understanding the development of obesity. Alterations in vagal neurocircuit maturation following perinatal (p)HFD exposure, has profound effects on neural control of gastric motility in adulthood, including the failure to regulate caloric intake, which may increase the predisposition to develop obesity. Our previous studies have shown that, following pHFD, inhibitory synaptic inputs to vagal efferent motoneurons are increased, which may prevent the voltage‐dependent activation of synaptic NMDA‐Rs. The purpose of this study was to test the hypothesis that pHFD exposure attenuates the homeostatic activation of DMV NMDA‐Rs following acute HFD exposure in adulthood, leading to dysregulated gastric motility.Sprague Dawley rats were exposed to a control or HFD (14% or 60% kcal from fat, respectively) during postnatal (P) days 1–10. In adulthood, (>P28) rats were re‐exposed to a HFD for 3–5 days prior to experimentation. Whole‐cell patch clamp recordings were made from DMV neurons and the effects of the NMDA‐R antagonist, AP5 (25μM) was tested on miniature excitatory postsynaptic current (mEPSC) amplitude and action potential (AP) firing rates. The 13C octanoic acid breath test was used to determine the effects of pHFD (P1–10) exposure on basal gastric emptying rates. Food intake and body weight was measured twice daily for 10 days prior to, and throughout, adult exposure to HFD.pHFD (P1–10) exposure attenuated the actions of AP5 to inhibit DMV NMDA‐R activation (mEPSC amplitude: 103±5.7% vs. 35±9.4%, P<0.05, N=4–6), and to decrease neuronal excitability (AP firing rate: 102±7.7% vs. 21±13.7% of baseline, P<0.05; N=4–7) observed previously following acute HFD exposure in adulthood. Measurement of gastric emptying rates suggest that pHFD exposure itself significantly delayed baseline gastric emptying (73±1.4 vs. 63±4.3min, P<0.05 N=8–12), and, unlike control rats, gastric emptying was not delayed further by adult dietary challenge (102±13.0% vs. 144±15.8% of baseline, respectively, P>0.05, N=4–6). Additionally, rats that were exposed to a pHFD (P1–10) did not regulate their caloric intake within 3–5 days of adult HFD exposure, as observed control rats (AUC: 215±28.0 vs. 393±34.7, P<0.05, N=9–16).These data suggest that pHFD (P1–10) exposure disrupts central vagal neurocircuit development, increasing the inhibitory drive to DMV neurons, delaying gastric emptying, and preventing the homeostatic regulation of gastric emptying and caloric intake in response to acute adult HFD exposure. Understanding how diet affects the development of vagal neurocircuits is critical for identifying therapeutic targets for the development of obesity.Support or Funding InformationNIH DK111667 to KNB NIH F31 118833 to CC

Acute High‐Fat Diet Induced Regulation of Caloric Intake is Dependent on Activation of Extrasynaptic NMDA Receptors in Dorsal Motor Nucleus of the Vagus Neurons

During HFD exposure, both humans and experimental animal models undergo a 24hr hyperphagic period before caloric balance is restored within 3–5 days. We have demonstrated previously that regulation is associated with activation of synaptic NMDA receptors (NMDA‐Rsyn) on neurons of the dorsal motor nucleus of the vagus (DMV), which increases motoneuron excitability and efferent control of gastric functions (PMID: 29368945). Previous studies have suggested that activation of extrasynaptic NMDA‐R(ex) may cause a sufficient local depolarization to remove the Mg2+ block on NMDA‐Rsyn, allowing for their activation. The purpose of this study is to test the hypothesis that, following acute HFD exposure, increased activation of DMV NMDA‐Rsyn occurs subsequent to activation of NMDA‐Rex, and is responsible for the regulation of caloric intake.Whole‐cell patch clamp recordings were made from gastric‐projecting DMV neurons in thin (300μm) brainstem slices of Sprague‐Dawley rats, 4–6 weeks of age. Rats were fed a control or HFD (14%, 60% kcal from fat, respectively) for 3–5 days prior to experimentation. The effects of the NMDA‐Rex antagonist, memantine (30μM), and the glutamate uptake inhibitor, dihydrokainate (DHK; 30μM), on NMDAsyn‐mediated miniature excitatory postsynaptic currents (mEPSCs) and DMV neuronal excitability (action potential firing; AP) were assessed. The ability of 4th ventricular application of memantine or DHK (50 and 20pmol/2μL, respectively) to attenuate or uncover the inhibitory effects of the glutamate antagonist, kynurenic acid (Kyn A, 100pmol/60nL) on gastric tone and motility were recorded in anesthetized rats. Implantation of indwelling 4th ventricular cannulae allowed for chronic administration of memantine (50pmol/2mL); food intake was measured twice daily for 10 days prior to, and throughout, HFD exposure in freely moving rats, to assess the role of NMDA‐Rex on the homeostatic regulation of caloric intake.Following acute HFD, memantine prevented the inhibitory actions of the NMDA‐Rsyn antagonist AP5 (25mM) on mEPSC frequency and AP firing rate in DMV neurons. In control rats, however, increasing extracellular glutamate concentration with DHK uncovered this inhibitory effect pf AP5. Brainstem microinjection of memantine attenuated the glutamate‐mediated decrease in gastric tone and motility observed following HFD exposure, while DHK uncovered this effect in control rats. Chronic application of memantine attenuated the homeostatic regulation of caloric intake observed following acute HFD exposure (for all data and statistics, see Table 1).These data suggest that the acute HFD‐induced increase in NMDA‐Rsyn activation occurs following NMDA‐Rex activation, although the source of this glutamate remains to be elucidated. This mechanism is also responsible for the restoration of caloric balance. Understanding these mechanisms and, importantly, how they are lost following chronic HFD exposure, may provide a mechanistic understanding hyperphagia and weight gain as well as identify potential therapeutic targets for the treatment of obesity.Support or Funding InformationNIH DK111667 to KNB NIH F31 118833 to CC Memantine + AP5 in HFD DHK + AP5 in CTL In vitro mEPSC Frequency 96±6.2% of baseline, N=6 73±6.7% of baseline *, N=13 AP firing rate 111±11.0% of baseline, N=9 73±10.9% of baseline *, N=15 Memantine+ KynA in HFD DHK + KynA in CTL Antrum Corpus Antrum Corpus In vivo Gastric motility −4±8.2% of baseline, N=6 −1±7.5% of baseline, N=6 −53±4.6% of baseline *, N=6 −44±9.2% of baseline *, N=6 Gastric tone −6±10.8 mg, N=6 −15±18.0 mg, N=6 −86±17.3 mg *, N=6 −123±34.23mg *, N=6 4th Ventricular Memantine (AUC) 4th Ventricular PBS (AUC) Caloric Intake 251±22.9 *, N=6 178±16.2, N=6 *P<0.05 compared to controls or baseline from Students T‐Test

Targeting Synaptic NMDA Receptor Co-agonism as a Therapy for Alzheimer’s Disease?

Alzheimer's disease (AD) is associated with lower brain glucose metabolism. In this issue, Le Douce etal. (2020) show that this leads to insufficient astrocyte-dependent production of D-serine, co-agonist of synaptic NMDA receptors. Oral L-serine therapy rescues NMDA receptor hypofunction, plasticity, and cognition in an AD model, suggesting a new therapeutic strategy.

Impairment of Glycolysis-Derived l-Serine Production in Astrocytes Contributes to Cognitive Deficits in Alzheimer’s Disease

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.

A novel method using ambient glutamate for the electrophysiological quantification of extrasynaptic NMDA receptor function in acute brain slices.

We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83nM resulting in a block of more than 82% of ES-NMDARs during a 5min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.

Rapid exchange of synaptic and extrasynaptic NMDA receptors in hippocampal CA1 neurons.

N-methyl-d-aspartate receptors (NMDARs) are fundamental coincidence detectors of synaptic activity necessary for the induction of synaptic plasticity and synapse stability. Adjusting NMDAR synaptic content, whether by receptor insertion or lateral diffusion between extrasynaptic and synaptic compartments, could play a substantial role defining the characteristics of the NMDAR-mediated excitatory postsynaptic current (EPSC), which in turn would mediate the ability of the synapse to undergo plasticity. Lateral NMDAR movement has been observed in dissociated neurons; however, it is currently unclear whether NMDARs are capable of lateral surface diffusion in hippocampal slices, a more physiologically relevant environment. To test for lateral mobility in rat hippocampal slices, we rapidly blocked synaptic NMDARs using MK-801, a use-dependent and irreversible NMDAR blocker. Following a 5-min washout period, we observed a strong recovery of NMDAR-mediated responses. The degree of the observed recovery was proportional to the amount of induced blockade, independent of levels of intracellular calcium, and mediated primarily by GluN2B-containing NMDA receptors. These results indicate that lateral diffusion of NMDARs could be a mechanism by which synapses rapidly adjust parameters to fine-tune synaptic plasticity.NEW & NOTEWORTHYN-methyl-d-aspartate-type glutamate receptors (NMDARs) have always been considered stable components of synapses. We show that in rat hippocampal slices synaptic NMDARs are in constant exchange with extrasynaptic receptors. This exchange of receptors is mediated primarily by NMDA receptors containing GluN2B, a subunit necessary to undergo synaptic plasticity. Thus this lateral movement of synaptic receptors allows synapses to rapidly regulate the total number of synaptic NMDARs with potential consequences for synaptic plasticity.

LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses.

LAR-type receptor phosphotyrosine-phosphatases (LAR-RPTPs) are presynaptic adhesion molecules that interact trans-synaptically with multitudinous postsynaptic adhesion molecules, including SliTrks, SALMs, and TrkC. Via these interactions, LAR-RPTPs are thought to function as synaptogenic wiring molecules that promote neural circuit formation by mediating the establishment of synapses. To test the synaptogenic functions of LAR-RPTPs, we conditionally deleted the genes encoding all three LAR-RPTPs, singly or in combination, in mice before synapse formation. Strikingly, deletion of LAR-RPTPs had no effect on synaptic connectivity in cultured neurons or in vivo, but impaired NMDA-receptor-mediated responses. Deletion of LAR-RPTPs decreased NMDA-receptor-mediated responses by a trans-synaptic mechanism. In cultured neurons, deletion of all LAR-RPTPs led to a reduction in synaptic NMDA-receptor EPSCs, without changing the subunit composition or the protein levels of NMDA-receptors. In vivo, deletion of all LAR-RPTPs in the hippocampus at birth also did not alter synaptic connectivity as measured via AMPA-receptor-mediated synaptic responses at Schaffer-collateral synapses monitored in juvenile mice, but again decreased NMDA-receptor mediated synaptic transmission. Thus, LAR-RPTPs are not essential for synapse formation, but control synapse properties by regulating postsynaptic NMDA-receptors via a trans-synaptic mechanism that likely involves binding to one or multiple postsynaptic ligands.

A microRNA signature of toxic extrasynaptic N-methyl-D-aspartate (NMDA) receptor signaling

The cellular consequences of N-Methyl-D-Aspartate receptor (NMDAR) stimulation depend on the receptors’ subcellular localization. Synaptic NMDARs promote plasticity and survival whereas extrasynaptic NMDARs mediate excitotoxicity and contribute to cell death in neurodegenerative diseases. The mechanisms that couple activation of extrasynaptic NMDARs to cell death remain incompletely understood. We here show that activation of extrasynaptic NMDARs by bath application of NMDA or L-glutamate leads to the upregulation of a group of 19 microRNAs in cultured mouse hippocampal neurons. In contrast, none of these microRNAs is induced upon stimulation of synaptic activity. Increased microRNA expression depends on the pri-miRNA processing enzyme Drosha, but not on de novo gene transcription. These findings suggest that toxic NMDAR signaling involves changes in the expression levels of particular microRNAs.

The role of eEF2 kinase in the rapid antidepressant actions of ketamine.

Major depressive disorder is a prevalent and serious form of mental illness. While traditional antidepressants ameliorate some of the symptoms associated with depression, the onset of action typically takes several weeks leaving severely depressed individuals vulnerable to self-injurious behavior and possibly suicide. There has been a major unmet need for the development of pharmacological therapies that can quickly alleviate symptoms associated with depression. Clinical data shows that a single sub-psychomimetic dose of ketamine, a noncompetitive glutamatergic N-methyl-d-aspartate (NMDA) receptor antagonist, has rapid antidepressant responses in patients with treatment-resistant major depressive disorder. We have studied key signaling pathways and synaptic mechanisms underlying the rapid antidepressant action of ketamine. Our studies show ketamine blocks synaptic NMDA receptors involved in spontaneous synaptic transmission, which deactivates calcium/calmodulin-dependent kinase eukaryotic elongation factor 2 kinase (eEF2K), resulting in dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the subsequent desuppression of brain-derived neurotrophic factor (BDNF) protein synthesis in the hippocampus. This signaling pathway then potentiates synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor responses that results in a novel form of synaptic potentiation which corresponds with antidepressant efficacy. In this chapter, we focus on our studies examining ketamine's action and the instructive role of eEF2K in rapid antidepressant action. Our recent studies highlight eEF2K as a major molecular substrate mediating synaptic plasticity and the rapid antidepressant effects of ketamine.

GluN2A NMDA Receptor Enhancement Improves Brain Oscillations, Synchrony, and Cognitive Functions in Dravet Syndrome and Alzheimer’s Disease Models

SUMMARYNMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely un-known. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer’s disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12–20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments.

NYX-2925 induces metabotropic N-methyl-d-aspartate receptor (NMDAR) signaling that enhances synaptic NMDAR and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.

N‐methyl‐d‐aspartate receptors (NMDARs) mediate both physiological and pathophysiological processes, although selective ligands lack broad clinical utility. NMDARs are composed of multiple subunits, but N‐methyl‐d‐aspartate receptor subunit 2 (GluN2) is predominately responsible for functional heterogeneity. Specifically, the GluN2A‐ and GluN2B‐containing subtypes are enriched in adult hippocampus and cortex and impact neuronal communication via dynamic trafficking into and out of the synapse. We sought to understand if ((2S, 3R)‐3‐hydroxy‐2‐((R)‐5‐isobutyryl‐1‐oxo‐2,5‐diazaspiro[3,4]octan‐2‐yl) butanamide (NYX‐2925), a novel NMDAR modulator, alters synaptic levels of GluN2A‐ or GluN2B‐containing NMDARs. Low‐picomolar NYX‐2925 increased GluN2B colocalization with the excitatory post‐synaptic marker post‐synaptic density protein 95 (PSD‐95) in rat primary hippocampal neurons within 30 min. Twenty‐four hours following oral administration, 1 mg/kg NYX‐2925 increased GluN2B in PSD‐95‐associated complexes ex vivo, and low‐picomolar NYX‐2925 regulated numerous trafficking pathways in vitro. Because the NYX‐2925 concentration that increases synaptic GluN2B was markedly below that which enhances long‐term potentiation (mid‐nanomolar), we sought to elucidate the basis of this effect. Although NMDAR‐dependent, NYX‐2925‐mediated colocalization of GluN2B with PSD‐95 occurred independent of ion flux, as colocalization increased in the presence of either the NMDAR channel blocker (5R,10S)‐(–)‐5‐Methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate or glycine site antagonist 7‐chlorokynurenic acid. Moreover, while mid‐nanomolar NYX‐2925 concentrations, which do not increase synaptic GluN2B, enhanced calcium transients, functional plasticity was only enhanced by picomolar NYX‐2925. Thus, NYX‐2925 concentrations that increase synaptic GluN2B facilitated the chemical long‐term potentiation induced insertion of synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor GluA1 subunit levels. Basal (unstimulated by chemical long‐term potentiation) levels of synaptic GluA1 were only increased by mid‐nanomolar NYX‐2925. These data suggest that NYX‐2925 facilitates homeostatic plasticity by initially increasing synaptic GluN2B via metabotropic‐like NMDAR signaling. Cover Image for this issue: doi: 10.1111/jnc.14735.

Spatial Coupling Tunes NMDA Receptor Responses via Ca2+ Diffusion.

In the CNS, NMDA receptors generate large and highly regulated Ca2+ signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca2+-dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca2+ and calmodulin (CaM). We asked whether Ca2+ influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (κ). In the absence of extracellular Ca2+, we observed no cooperativity (κ < 0.1), whereas in 1.8 mm external Ca2+, both recombinant and native channels showed substantial negative cooperativity (κ = 0.27). Intracellular Ca2+ chelation or overexpression of a Ca2+-insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially (κ = 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents.SIGNIFICANCE STATEMENT At central synapses, NMDA receptors are a major class of excitatory glutamate-gated channels and a source of activity-dependent Ca2+ influx. In turn, fluxed Ca2+ ions bind to calmodulin-primed receptors and reduce further entry, through an autoinhibitory mechanism known as Ca2+ -dependent inactivation (CDI). Here, we show that the diffusion of fluxed Ca2+ between active channels situated within submicroscopic distances amplified receptor inactivation. Thus, calmodulin-mediated gating modulation, an evolutionarily conserved regulatory mechanism, endows synapses with sensitivity to both the temporal sequence and spatial distribution of Ca2+ signals. Perturbations in this mechanism, which coordinates the activity of NMDA receptors within a cluster, may cause signaling alterations that contribute to neuropsychiatric conditions.

Streptozotocin-Induced Diabetic Neuropathic Pain Is Associated with Potentiated Calcium-Permeable AMPA Receptor Activity in the Spinal Cord.

Neuronal hyperactivity in the spinal dorsal horn can amplify nociceptive input in diabetic neuropathic pain. The glutamate N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDA receptors and AMPA receptors, respectively) are involved in spinal nociceptive transmission. It is unclear, however, whether painful diabetic neuropathy is associated with changes in the activity of synaptic NMDA receptors and AMPA receptors in spinal dorsal horn neurons. AMPA receptors lacking GluA2 are Ca2+-permeable (CP-AMPA receptors), and their currents display characteristic inward rectification. In this study, we showed that evoked excitatory postsynaptic currents (EPSCs), induced by streptozotocin, exhibited inward rectification in spinal dorsal neurons in diabetic rats. Presynaptic and postsynaptic NMDA receptor activity in the spinal dorsal horn was similar in diabetic and control rats. In the dorsal spinal cord, the membrane GluA2 protein level was significantly lower in diabetic than in control rats, whereas the cytosolic GluA2 level was greater in diabetic than in control rats. In contrast, the GluA1 subunit levels in the plasma membrane and cytosol did not differ between the two groups. Blocking CP-AMPA receptors significantly reduced the amplitude of EPSCs of dorsal horn neurons in diabetic but not in control rats. Furthermore, blocking spinal CP-AMPA receptors reduced pain hypersensitivity in diabetic rats but had no effect on nociception in control rats. Our study suggests that diabetic neuropathy augments CP-AMPA receptor activity in the spinal dorsal horn by causing intracellular retention of GluA2 and impairing GluA2 membrane trafficking. Increased prevalence of spinal CP-AMPA receptors sustains diabetic neuropathic pain. SIGNIFICANCE STATEMENT: This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition.

NMDAR-Activated PP1 Dephosphorylates GluN2B to Modulate NMDAR Synaptic Content

SUMMARYIn mature neurons, postsynaptic N-methyl-D-aspartate receptors (NMDARs) are segregated into two populations, synaptic and extrasynaptic, which differ in localization, function, and associated intracellular cascades. These two pools are connected via lateral diffusion, and receptor exchange between them modulates synaptic NMDAR content. Here, we identify the phosphorylation of the PDZ-ligand of the GluN2B subunit of NMDARs (at S1480) as a critical determinant in dynamically controlling NMDAR synaptic content. We find that phosphorylation of GluN2B at S1480 maintains NMDARs at extrasynaptic membranes as part of a protein complex containing protein phosphatase 1 (PP1). Global activation of NMDARs leads to the activation of PP1, which mediates dephosphorylation of GluN2B at S1480 to promote an increase in synaptic NMDAR content. Thus, PP1-mediated dephosphorylation of the GluN2B PDZ-ligand modulates the synaptic expression of NMDARs in mature neurons in an activity-dependent manner, a process with profound consequences for synaptic and structural plasticity, metaplasticity, and synaptic neurotransmission.

BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons.

The effects of brain-derived neurotrophic factor (BDNF) in long-term synaptic potentiation (LTP) are thought to underlie learning and memory formation and are partly mediated by local protein synthesis. Here, we investigated the mechanisms that mediate BDNF-induced alterations in the synaptic proteome that are coupled to synaptic strengthening. BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors (NMDARs) and increased the amplitude of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) in cultured rat hippocampal neurons by a mechanism requiring activation of the protein tyrosine kinase Pyk2 and dependent on cellular protein synthesis. Single-particle tracking using quantum dot imaging revealed that the increase in the abundance of synaptic NMDAR currents correlated with their enhanced stability in the synaptic compartment. Furthermore, BDNF increased the local synthesis of Pyk2 at the synapse, and the observed increase in Pyk2 protein abundance along dendrites of cultured hippocampal neurons was mediated by a mechanism dependent on the ribonucleoprotein hnRNP K, which bound to Pyk2 mRNA and dissociated from it upon BDNF application. Knocking down hnRNP K reduced the BDNF-induced synaptic synthesis of Pyk2 protein, whereas its overexpression enhanced it. Together, these findings indicate that hnRNP K mediates the synaptic distribution of Pyk2 synthesis, and hence the synaptic incorporation of GluN2B-containing NMDARs, induced by BDNF, which may affect LTP and synaptic plasticity.

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.

The Synaptonuclear Messenger RNF10 Acts as an Architect of Neuronal Morphology.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

N-methyl-D-aspartate receptor dysfunction by unmutated human antibodies against the NR1 subunit.

Anti–N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody‐mediated synaptic dysfunction. Cerebrospinal fluid–derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline‐configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient. In primary hippocampal neurons, germline NR1 autoantibodies strongly and specifically reduced total and synaptic NMDAR currents in a dose‐ and time‐dependent manner. The findings suggest that functional NMDAR antibodies are part of the human naïve B cell repertoire. Given their effects on synaptic function, they might contribute to a broad spectrum of neuropsychiatric symptoms. Ann Neurol 2019;85:771–776