NMDA Receptor-mediated EPSCs Research Articles

Early expression of GluN2A-containing NMDA receptors in a model of fragile X syndrome.

NMDA-type glutamate receptors (NMDARs) play a crucial role in synaptogenesis, circuit development, and synaptic plasticity, serving as fundamental components in cellular models of learning and memory. Their dysregulation has been implicated in several neurological disorders and synaptopathies. NMDARs are heterotetrameric complexes composed of two GluN1 and two GluN2 subunits. The composition of GluN2 subunits determines the main biophysical properties of the channel, such as calcium permeability and gating kinetics, and influences the ability of the receptor to interact with postsynaptic proteins involved in normal synaptic physiology and plasticity, including scaffolding proteins and signaling molecules. During early development, NMDARs in the forebrain contain solely the GluN2B subunit, a necessary subunit for proper synaptogenesis and synaptic plasticity. As the animal matures, the expression of the GluN2A subunit increases, leading to a partial replacement of GluN2B-containing synaptic NMDARs with GluN2A-containing receptors. The switch in the synaptic GluN2A-to-GluN2B ratio has a significant impact on the kinetics of excitatory postsynaptic currents and diminishes the synaptic plasticity capacity. In this study, we present findings indicating that GluN2A expression occurs earlier in a mouse model of fragile X syndrome (FXS). This altered timing of GluN2A expression affects various important parameters of NMDAR-mediated excitatory postsynaptic currents, including maximal current amplitude, decay time, and response to consecutive stimuli delivered in close temporal proximity. These observations suggest that the early expression of GluN2A during a critical period when synapses and circuits are developing could be an underlying factor contributing to the formation of pathological circuits in the FXS mouse model.NEW & NOTEWORTHY NMDA receptors (NMDARs) play important roles in synaptic transmission and are involved in multiple neurological disorders. During development, GluN2A in the forebrain becomes incorporated into previously GluN2B-dominated NMDARs, leading to the "GluN2A/GluN2B ratio switch." This is a crucial step for normal brain development. Here we present findings indicating that GluN2A expression occurs earlier in the fragile X mouse and this could be an underlying factor contributing to the pathology found in the fragile X model.

Activation of CRF/CRFR1 Signaling in the Central Nucleus of the Amygdala Contributes to Chronic Stress-Induced Exacerbation of Neuropathic Pain by Enhancing GluN2B-NMDA Receptor-Mediated Synaptic Plasticity in Adult Male Rats

Exacerbation of pain by chronic stress and comorbidity of pain with stress-related disorders such as depression and post-traumatic stress disorder, represent significant clinical challenges. Previously we have documented that chronic forced swim (FS) stress exacerbates neuropathic pain in spared nerve injury (SNI) rats, associated with an up-regulation of GluN2B-containing N-methyl-D-aspartate receptors (GluN2B-NMDARs) in the central nucleus of the amygdala (CeA). However, the molecular mechanisms underlying chronic FS stress (CFSS)-mediated exacerbation of pain sensitivity in SNI rats still remain unclear. In this study, we demonstrated that exposure of CFSS to rats activated the corticotropin-releasing factor (CRF)/CRF receptor type 1 (CRFR1) signaling in the CeA, which was shown to be necessary for CFSS-induced depressive-like symptoms in stressed rats, and as well, for CFSS-induced exacerbation of pain hypersensitivity in SNI rats exposed to chronic FS stress. Furthermore, we discovered that activation of CRF/CRFR1 signaling in the CeA upregulated the phosphorylation of GluN2B-NMDARs at tyrosine 1472 (pGluN2BY1472) in the synaptosomal fraction of CeA, which is highly correlated to the enhancement of synaptic GluN2B-NMDARs expression that has been observed in the CeA in CFSS-treated SNI rats. In addition, we revealed that activation of CRF/CRFR1 signaling in the CeA facilitated the CFSS-induced reinforcement of long-term potentiation as well as the enhancement of NMDAR-mediated excitatory postsynaptic currents in the basolateral amygdala (BLA)-CeA pathway in SNI rats. These findings suggest that activation of CRF/CRFR1 signaling in the CeA contributes to chronic stress-induced exacerbation of neuropathic pain by enhancing GluN2B-NMDAR-mediated synaptic plasticity in rats subjected to nerve injury. PerspectiveOur present study provides a novel mechanism for elucidating stress-induced hyperalgesia and highlights that the CRF/CRFR1 signaling and the GluN2B-NMDAR-mediated synaptic plasticity in the CeA may be important as potential therapeutic targets for chronic stress-induced pain exacerbation in human neuropathic pain. Data AvailabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

P2X7 purinergic receptor modulates dentate gyrus excitatory neurotransmission and alleviates schizophrenia-like symptoms in mouse

ATP-gated P2X7 receptors (P2X7Rs) play a crucial role in brain disorders. However, how they affect normal and pathological synaptic transmission is still largely unclear. Here, by using whole-cell patch-clamp technique to record AMPA- and NMDA receptor-mediated excitatory postsynaptic currents (s/mEPSCs) in dentate gyrus granule cells (DG GCs), we revealed a modulation by P2X7Rs of presynaptic sites, especially originated from entorhinal cortex (EC)-GC path but not the mossy cell (MC)-GC path. The involvement of P2X7Rs was confirmed using a pharmacological approach. Additionally, the acute activation of P2X7Rs directly elevated calcium influx from EC-GC terminals. In postnatal phencyclidine (PCP)-induced mouse model of schizophrenia, we observed that P2X7R deficiency restored the EC-GC synapse alteration and alleviated PCP-induced symptoms. To summarize, P2X7Rs participate in the modulation of GC excitatory neurotransmission in the DG via EC-GC pathway, contributing to pathological alterations of neuronal functions leading to neurodevelopmental disorders.

Synaptic Dysfunction by Mutations in GRIN2B: Influence of Triheteromeric NMDA Receptors on Gain-of-Function and Loss-of-Function Mutant Classification.

GRIN2B mutations are rare but often associated with patients having severe neurodevelopmental disorders with varying range of symptoms such as intellectual disability, developmental delay and epilepsy. Patient symptoms likely arise from mutations disturbing the role that the encoded NMDA receptor subunit, GluN2B, plays at neuronal connections in the developing nervous system. In this study, we investigated the cell-autonomous effects of putative gain- (GoF) and loss-of-function (LoF) missense GRIN2B mutations on excitatory synapses onto CA1 pyramidal neurons in organotypic hippocampal slices. In the absence of both native GluN2A and GluN2B subunits, functional incorporation into synaptic NMDA receptors was attenuated for GoF mutants, or almost eliminated for LoF GluN2B mutants. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) from synaptic GoF GluN1/2B receptors had prolonged decays consistent with their functional classification. Nonetheless, in the presence of native GluN2A, molecular replacement of native GluN2B with GoF and LoF GluN2B mutants all led to similar functional incorporation into synaptic receptors, more rapidly decaying NMDA-EPSCs and greater inhibition by TCN-201, a selective antagonist for GluN2A-containing NMDA receptors. Mechanistic insight was gained from experiments in HEK293T cells, which revealed that GluN2B GoF mutants slowed deactivation in diheteromeric GluN1/2B, but not triheteromeric GluN1/2A/2B receptors. We also show that a disease-associated missense mutation, which severely affects surface expression, causes opposing effects on NMDA-EPSC decay and charge transfer when introduced into GluN2A or GluN2B. Finally, we show that having a single null Grin2b allele has only a modest effect on NMDA-EPSC decay kinetics. Our results demonstrate that functional incorporation of GoF and LoF GluN2B mutants into synaptic receptors and the effects on EPSC decay times are highly dependent on the presence of triheteromeric GluN1/2A/2B NMDA receptors, thereby influencing the functional classification of NMDA receptor variants as GoF or LoF mutations. These findings highlight the complexity of interpreting effects of disease-causing NMDA receptor missense mutations in the context of neuronal function.

Enhanced function of NR2C/2D-containing NMDA receptor in the nucleus accumbens contributes to peripheral nerve injury-induced neuropathic pain and depression in mice.

N-methyl-d-aspartate receptors (NMDARs) dysfunction in the nucleus accumbens (NAc) participates in regulating many neurological and psychiatric disorders such as drug addiction, chronic pain, and depression. NMDARs are heterotetrameric complexes generally composed of two NR1 and two NR2 subunits (NR2A, NR2B, NR2C and NR2D). Much attention has been focused on the role of NR2A and NR2B-containing NMDARs in a variety of neurological disorders; however, the function of NR2C/2D subunits at NAc in chronic pain remains unknown. In this study, spinal nerve ligation (SNL) induced a persistent sensory abnormity and depressive-like behavior. The whole-cell patch clamp recording on medium spiny neurons (MSNs) in the NAc showed that the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) was significantly increased when membrane potential held at −40 to 0 mV in mice after 14 days of SNL operation. In addition, selective inhibition of NR2C/2D-containing NMDARs with PPDA caused a larger decrease on peak amplitude of NMDAR-EPSCs in SNL than that in sham-operated mice. Appling of selective potentiator of NR2C/2D, CIQ, markedly enhanced the evoked NMDAR-EPSCs in SNL-operated mice, but no change in sham-operated mice. Finally, intra-NAc injection of PPDA significantly attenuated SNL-induced mechanical allodynia and depressive-like behavior. These results for the first time showed that the functional change of NR2C/2D subunits-containing NMDARs in the NAc might contribute to the sensory and affective components in neuropathic pain.

β3-adrenoceptor activation exhibits a dual effect on behaviors and glutamate receptor function in the prefrontal cortex

β-adrenoceptor (β-AR), especially the β1- and β2-AR subtypes, is known to participate in stress-related behavioral changes. Recently, SR58611A, a brain-penetrant β3-AR agonist, exhibits anxiolytic- and antidepressant-like effects. In this study, we sought to study the role of SR58611A in behavioral changes and its potential cellular and molecular mechanism in the prefrontal cortex (PFC). We found that rats with SR58611A (1 mg/kg) enhanced PFC-mediated recognition memory, whereas administration of higher dosage of SR58611A (20 mg/kg) caused hyperlocomotion, and exhibited an impairment effect on recognition memory. Electrophysiological data also indicated that SR58611A (1 mg/kg) selectively enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSC) through interacting with norepinephrine (NE) system and activating β3-AR, whereas higher dosage of SR58611A (20 mg/kg) reduced both AMPA receptor- and NMDA receptor-mediated EPSC. SR58611A-induced different effects on EPSC linked with the change of the surface expression quantity of NMDA receptor and/or AMPA receptor subunits. Synaptosomal-associated protein 25 (SNAP-25), which is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein involved in incorporation of NMDA receptor to postsynaptic membrane, contributed to SR58611A (1 mg/kg)-induced enhancement of recognition memory and NMDA receptor function. Moreover, SR58611A (1 mg/kg) could rescue repeated stress-induced defect of both recognition memory and NMDA receptor function through a SNAP-25-dependent mechanism. These results provide a potential mechanism underlying the cognitive-enhancing effects of SR58611A (1 mg/kg).

A potential of methoxpropamine to be a widespread recreational drug: it blocks NMDA receptors and inhibits NMDA receptor-mediated synaptic transmission in a brain preparation of mice

The activity of N-methyl-d-aspartate (NMDA) receptors in the central nervous system is affected by many psychoactive drugs, such as 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine) and its analog 2-(3-methoxyphenyl)-2-(amino)cyclohexan-1-one (methoxetamine, MXE). Recreational use of MXE can cause acute toxicity, such as dissociative state and tachycardia. After MXE use, confusion, agitation, ataxia, and nystagmus are induced. Moreover, MXE-related deaths have been reported, and this compound is banned in many countries. Recently, MXE’s derivative, 2-(3-methoxyphenyl)-2-(propylamino)cyclohexan-1-one (methoxpropamine, MXPr), was reported as a new psychoactive substance and sold online as a designer drug. The aims were to determine how MXPr affects NMDA receptors in neurons and to compare the potency with MXE. Cartwheel cells in the dorsal cochlear nucleus of mice were used as a model of NMDA receptor-expressing neurons, and patch-clamp method was performed for the recordings. NMDA-induced inward current was initially evoked by microiontophoresis application of NMDA onto the cartwheel cells, and the effects of MXPr, MXE, and (5R,10S)-( +)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801, as a positive control substance) on NMDA-induced response were examined. Moreover, the effects of MXPr on NMDA receptor-mediated excitatory postsynaptic currents were also investigated. The IC50 of MXPr, MXE, and MK-801 on NMDA-induced response decreased in the following order: MXPr (1.647 μM) > MXE (0.841 μM) > MK-801 (0.060 μM), indicating that MXPr and MXE act as potent antagonists of NMDA receptors. MXPr suppressed NMDA receptor-mediated excitatory synaptic transmission in a dose-dependent manner. MXPr, which may cause health and social damages to humans by blocking NMDA receptors, is a serious concern like MXE.

Caffeine has a dual influence on NMDA receptor-mediated glutamatergic transmission at the hippocampus.

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A1R and A2R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca2+ homeostasis. We found that caffeine (30-200μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50nM), thus likely mediated by blockade of inhibitory A1Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A2AR antagonist, SCH58261 (50nM). Furthermore, caffeine increased the frequency of Ca2+ transients in neuronal cell culture, an action mimicked by the A1R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca2+ homeostasis.

Comparisons of Nociceptive Behavior and NMDA receptor‐Mediated Synaptic Transmission between Mice Lacking D‐Serine Production and Degradation Enzymes

D‐serine is the endogenous co‐agonist for the glycine‐binding site of the N‐methyl‐D‐aspartate (NMDA) glutamate receptor. D‐serine is produced by the enzyme serine racemase (SR) and is degraded by the enzyme D‐amino acid oxidase (DAO). Co‐activation of spinal NMDA receptors by D‐serine is known to play a significant role in the etiological mechanism of neuropathic and inflammatory pain. The present study aims to obtain further information on how D‐serine and its related enzymes (SR and DAO) are involved in the modulation of persistent pain.The experiments were performed on both SR knock‐out (SR‐KO) mice and the DAO‐mutant mice that lack the enzymatic activity of DAO. We have previously shown that SR‐KO mice have less than half of the D‐serine content in the spinal cord and that DAO‐mutant mice have a significantly elevated level of D‐serine.The DAO‐mutant and SR‐KO mice used in this study are kindly provided by R. Konno (International University of Health and Welfare, Tochigi, Japan).First, the nociceptive behavioral responses to formalin that is subcutaneously injected into a hind paw were analyzed. Results showed that the responses in the second phase, which are known be associated with NMDA receptor activation, were augmented both in SR‐KO mice and in DAO‐mutant mice.Second, tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn (SDH) in the lumbar spinal slices that were prepared from SR‐KO mice or from DAO‐mutant mice. Non‐NMDA and NMDA receptor‐mediated excitatory postsynaptic currents (non‐NMDAEPSCs and NMDA‐EPSCs, respectively) were pharmacologically isolated. Results show that the ratio of the amplitudes of NMDA‐EPSCs to that of non‐NMDA‐EPSCs was significantly smaller in SR‐KO mice and larger in DAO‐mutant mice compared with each corresponding control group and that the time constant of decay time course of NMDA‐EPSCs was significantly longer in SR‐KO mice.Finally, quantitative real‐time RT‐PCR experiments were performed on RNA extracted from the SDH tissue. The results showed that the subunit composition of NMDA receptors altered in SR‐KO mice and DAO‐mutant mice. The most significant observation is that the expression level of the NR2B subunit of NMDA receptors increased in the SR‐KO mice.These observations might indicate that D‐serine and its related enzymes (SR and DAO) influence the activation level of NMDA receptors and their subunit composition and thus affect nociceptive synaptic transmission in the SDH of the spinal cord and influence nociceptive behavior.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number JP18K16462 (EK).

PTC-174, a positive allosteric modulator of NMDA receptors containing GluN2C or GluN2D subunits

NMDA receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. The diverse functions of these receptors are tuned by deploying different combinations of GluN1 and GluN2 subunits (GluN2A-D) to form either diheteromeric NMDA receptors, which contain two GluN1 and two identical GluN2 subunits, or triheteromeric NMDA receptors, which contain two GluN1 and two distinct GluN2 subunits. Here, we characterize PTC-174, a novel positive allosteric modulator (PAM) of receptors containing GluN2C or GluN2D subunits. PTC-174 potentiates maximal current amplitudes by 1.8-fold for diheteromeric GluN1/2B receptors and by > 10-fold for GluN1/2C and GluN1/2D receptors. PTC-174 also potentiates responses from triheteromeric GluN1/2B/2D and GluN1/2A/2C receptors by 4.5-fold and 1.7-fold, respectively. By contrast, PTC-174 produces partial inhibition of responses from diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors. PTC-174 increases potencies of co-agonists glutamate and glycine by 2- to 5-fold at GluN1/2C and GluN1/2D receptors, and NMDA receptor activation facilitates allosteric modulation by PTC-174. At native NMDA receptors in GluN2D-expressing subthalamic nucleus neurons, PTC-174 increases the amplitude of responses to NMDA application and slows the decay of excitatory postsynaptic currents (EPSCs) evoked by internal capsule stimulation. Furthermore, PTC-174 increases the amplitude and slows the decay of EPSCs in hippocampal interneurons, but has not effect on the amplitudes of NMDA receptor-mediated EPSCs in hippocampal CA1 pyramidal neurons. Thus, PTC-174 provides a useful new pharmacological tool to investigate the molecular pharmacology and physiology of GluN2C- and GluN2D-containing NMDA receptors.

N-terminal alternative splicing of GluN1 regulates the maturation of excitatory synapses and seizure susceptibility

The majority of NMDA receptors (NMDARs) in the brain are composed of 2 GluN1 and 2 GluN2 subunits. The inclusion or exclusion of 1 N-terminal and 2 C-terminal domains of GluN1 results in 8 splicing variants that exhibit distinct temporal and spatial patterns of expression and functional properties. However, previous functional analyses of Grin1 variants have been done using heterologous expression and the in vivo function of Grin1 splicing is unknown. Here we show that N-terminal splicing of GluN1 has important functions in the maturation of excitatory synapses. The inclusion of exon 5 of Grin1 is up-regulated in several brain regions such as the thalamus and neocortex. We find that deletion of Grin1 exon 5 disrupts the developmental remodeling of NMDARs in thalamic neurons and the effect is distinct from that of Grin2a (GluN2A) deletion. Deletion of Grin2a or exon 5 of Grin1 alone partially attenuates the shortening of NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) during early life, whereas deletion of both Grin2a and exon 5 of Grin1 completely abolishes the developmental change in NMDAR-EPSC decay time. Deletion of exon 5 of Grin1 leads to an overproduction of excitatory synapses in layer 5 pyramidal neurons in the cortex and increases seizure susceptibility in adult mice. Our findings demonstrate that N-terminal splicing of GluN1 has important functions in synaptic maturation and neuronal network excitability.

Neuronal NMDAR Currents of the Hippocampus and Learning Performance in Autoimmune Anti-NMDAR Encephalitis and Involvement of TNF-α and IL-6.

Among autoimmune encephalitis, patients with anti-N-methyl D- aspartate receptor (NMDAR) encephalitis typically present epileptic seizures, memory deficits and psychiatric symptoms. However, the signal mechanisms leading to the functional disorders of autoantibodies are largely unclear. In this study, anti-NMDAR antibody was administered into dentate gyri against the NR1 subunit of the NMDAR. The purpose of the study examined the effects of pro-inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on neuronal NMDAR currents of the hippocampus in rats with anti-NMDAR encephalitis and we further determined the role played by TNF-α and IL-6 in modulating learning performance. In results, we observed a decrease in amplitude of the NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) in the hippocampal neurons of animals treated with anti-NMDAR. In those rats with anti-NMDAR, we also observed impaired learning performance in the Morris water maze and spatial working memory test. Of note, cerebral infusion of TNF-α and IL-6 worsened NMDAR-EPSCs and this was accompanied with exaggeration of impaired learning performance. In conclusion, our findings suggest that the role played by neuroinflammation in exacerbating the memory impairment found in animals treated with anti-NMDAR. Anti-inflammation is a potential target in improving the memory impairment induced by anti-NMDA encephalitis.

Persistent activation of histamine H1 receptors in the hippocampal CA1 region enhances NMDA receptor-mediated synaptic excitation and long-term potentiation in astrocyte- and D-serine-dependent manner

Behavioral studies using pharmacological tools have implicated histamine H1 receptors in cognitive function via their interactions with N-methyl-D-aspartate receptors (NMDARs) in the hippocampus. However, little is known about the neurophysiological mechanism that underlies the interaction between H1 receptors and NMDARs. To explore how H1 receptor activation affects hippocampal excitatory neurotransmission and synaptic plasticity, this study aimed to examine the effect of H1 receptor ligands on both NMDAR-mediated synaptic currents and long-term potentiation (LTP) at synapses between Schaffer collaterals and CA1 pyramidal neurons using acute mouse hippocampal slices. We found that the H1 receptor antagonist/inverse agonists, pyrilamine (0.1 μM) and cetirizine (10 μM), decreased the NMDAR-mediated component of stimulation-induced excitatory postsynaptic currents (EPSCs) recorded from CA1 pyramidal neurons without affecting the AMPA receptor-mediated component of EPSCs and its paired pulse ratio. Pretreatment of slices with either the glial metabolism inhibitor, fluoroacetate (5 mM), or D-serine (100 μM) diminished the pyrilamine- or cetirizine-induced attenuation of the NMDAR-mediated EPSCs. Furthermore, the LTP of field excitatory postsynaptic potentials induced following high frequency stimulation of Schaffer collaterals was attenuated with application of pyrilamine or cetirizine. Pretreatment with D-serine again attenuated the pyrilamine-induced suppression of LTP. Our data suggest that H1 receptors in the CA1 can undergo persistent activation induced by their constitutive receptor activity and/or tonic release of endogenous histamine, resulting in facilitation of the NMDAR activity in a manner dependent of astrocytes and the release of D-serine. This led to the enhancement of NMDA-component EPSC and LTP at the Schaffer collateral-CA1 pyramidal neuron synapses.

Effects of matrix metalloproteinase inhibitors on N-methyl-D-aspartate receptor and contribute to long-term potentiation in the anterior cingulate cortex of adult mice

Matrix metalloproteinases (MMPs) have been suggested to contribute to long-term potentiation, behavioral learning, and memory. In the dorsal horn of spinal cord, MMPs were reported to contribute to injury-related changes, and inhibitors of MMPs have been proposed as potential analgesics. However, it is unclear whether MMP inhibitors produce these effects by inhibiting the function of N-methyl-D-aspartate receptor (NMDAR), a key receptor for the induction of long-term potentiation. In this study, we wanted to examine if MMP inhibitors affect NMDAR-mediated excitatory postsynaptic currents in the anterior cingulate cortex of adult mice. Among different subtype inhibitors we used, we found that MMP-9 and MMP-2/9 inhibitors did not change NMDAR-mediated excitatory postsynaptic currents. However, MMP-3 and broad-spectrum MMP inhibitors reduced the NMDAR-mediated excitatory postsynaptic currents. Consistently, MMP-9 and MMP-2/9 inhibitors had no effect on NMDAR-dependent long-term potentiation, but MMP-3 and broad-spectrum MMP inhibitors inhibited the induction of long-term potentiation. Our results suggest that MMP inhibitors may produce their effects by inhibiting NMDAR functions in central synapses.

Calcitonin gene-related peptide potentiated the excitatory transmission and network propagation in the anterior cingulate cortex of adult mice.

The neuropeptide of calcitonin gene-related peptide (CGRP) plays critical roles in chronic pain, especially in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas including pain perception-related prefrontal anterior cingulate cortex. However, less information is available for the functional roles of CGRP in cortical regions such as the anterior cingulate cortex (ACC). Recent studies have consistently demonstrated that long-term potentiation is a key cellular mechanism for chronic pain in the ACC. In the present study, we used 64-electrode array field recording system to investigate the effect of CGRP on excitatory transmission in the ACC. We found that CGRP induced potentiation of synaptic transmission in a dose-dependently manner (1, 10, 50, and 100 nM). CGRP also recruited inactive circuit in the ACC. An application of the calcitonin receptor-like receptor antagonist CGRP8-37 blocked CGRP-induced chemical long-term potentiation and the recruitment of inactive channels. CGRP-induced long-term potentiation was also blocked by N-methyl-D-aspartate (NMDA) receptor antagonist AP-5. Consistently, the application of CGRP increased NMDA receptor-mediated excitatory postsynaptic currents. Finally, we found that CGRP-induced long-term potentiation required the activation of calcium-stimulated adenylyl cyclase subtype 1 (AC1) and protein kinase A. Genetic deletion of AC1 using AC1−/− mice, an AC1 inhibitor NB001 or a protein kinase A inhibitor KT5720, all reduced or blocked CGRP-induced potentiation. Our results provide direct evidence that CGRP may contribute to synaptic potentiation in important physiological and pathological conditions in the ACC, an AC1 inhibitor NB001 may be beneficial for the treatment of chronic headache.

Colocalization of distinct NMDA receptor subtypes at excitatory synapses in the entorhinal cortex.

The subunit composition of N-methyl-d-aspartate receptors (NMDARs) at synaptic inputs onto a neuron can either vary or be uniform depending on the type of neuron and/or brain region. Excitatory pyramidal neurons in the frontal and somatosensory cortices (L5), for example, show pathway-specific differences in NMDAR subunit composition in contrast with the entorhinal cortex (L3), where we now show colocalization of NMDARs with distinct subunit compositions at individual synaptic inputs onto these neurons. Subunit composition was deduced electrophysiologically based on alterations of current-voltage relationship ( I-V) profiles, amplitudes, and decay kinetics of minimally evoked, pharmacologically isolated, NMDAR-mediated excitatory postsynaptic currents by known subunit-preferring antagonists. The I-Vs were outwardly rectifying in a majority of neurons assayed (~80%), indicating expression of GluN1/GluN2/GluN3-containing triheteromeric NMDARs ( t-NMDARs) and of the conventional type, reversing close to 0 mV with prominent regions of negative slope, in the rest of the neurons sampled (~20%), indicating expression of GluN1/GluN2-containing diheteromeric NMDARs ( d-NMDARs). Blocking t-NMDARs in neurons with outwardly rectifying I-Vs pharmacologically unmasked d-NMDARs, with all responses antagonized using D-AP5. Coimmunoprecipitation assays of membrane-bound protein complexes isolated from the medial entorhinal area using subunit-selective antibodies corroborated stoichiometry and together suggested the coexpression of t- and d-NMDARs at these synapses. Colocalization of functionally distinct NMDAR subtypes at individual synaptic inputs likely enhances the repertoire of pyramidal neurons for information processing and plasticity within the entorhinal cortex. NEW & NOTEWORTHY The subunit composition of a N-methyl-d-aspartate (NMDA) receptor, which dictates most aspects of its function, can vary between neurons in different brain regions and/or between synaptic inputs onto single neurons. Here we demonstrate colocalization of tri- and diheteromeric-NMDA receptors at the same/single synaptic input onto excitatory neurons in the entorhinal cortex. Synaptic colocalization of distinct NMDAR subtypes might endow entorhinal cortical neurons with the ability to encode distinct patterns of neuronal activity through single synapses.

Agmatine preferentially antagonizes GluN2B-containing N-methyl-d-aspartate receptors in spinal cord.

The role of the N-methyl-d-aspartate receptor (NMDAr) as a contributor to maladaptive neuroplasticity underlying the maintenance of chronic pain is well established. Agmatine, an NMDAr antagonist, has been shown to reverse tactile hypersensitivity in rodent models of neuropathic pain while lacking the side effects characteristic of global NMDAr antagonism, including sedation and motor impairment, indicating a likely subunit specificity of agmatine's NMDAr inhibition. The present study assessed whether agmatine inhibits subunit-specific NMDAr-mediated current in the dorsal horn of mouse spinal cord slices. We isolated NMDAr-mediated excitatory postsynaptic currents (EPSCs) in small lamina II dorsal horn neurons evoked by optogenetic stimulation of Nav1.8-containing nociceptive afferents. We determined that agmatine abbreviated the amplitude, duration, and decay constant of NMDAr-mediated EPSCs similarly to the application of the GluN2B antagonist ifenprodil. In addition, we developed a site-specific knockdown of the GluN2B subunit of the NMDAr. We assessed whether agmatine and ifenprodil were able to inhibit NMDAr-mediated current in the spinal cord dorsal horn of mice lacking the GluN2B subunit of the NMDAr by analysis of electrically evoked EPSCs. In control mouse spinal cord, agmatine and ifenprodil both inhibited amplitude and accelerated the decay kinetics. However, agmatine and ifenprodil failed to attenuate the decay kinetics of NMDAr-mediated EPSCs in the GluN2B-knockdown mouse spinal cord. The present study indicates that agmatine preferentially antagonizes GluN2B-containing NMDArs in mouse dorsal horn neurons. NEW & NOTEWORTHY Our study is the first to report that agmatine preferentially antagonizes the GluN2B receptor subunit of the N-methyl-d-aspartate (NMDA) receptor in spinal cord. The preferential targeting of GluN2B receptor is consistent with the pharmacological profile of agmatine in that it reduces chronic pain without the motor side effects commonly seen with non-subunit-selective NMDA receptor antagonists.

Effect of Ketamine on LTP and NMDAR EPSC in Hippocampus of the Chronic Social Defeat Stress Mice Model of Depression.

Depression is a common mental disorder that is associated with memory dysfunction. Ketamine has recently been demonstrated to be a rapid antidepressant. The mechanisms underlying how depression induces memory dysfunction and how ketamine relieves depressive symptoms remain poorly understood. This work compared three groups of male C57BL/6J mice: mice exposed to chronic social defeat stress (CSDS) to induce a depression-like phenotype, depression-like mice treated with ketamine, and control mice that were not exposed to CSDS or treated with ketamine. Spatial working memory and long term memory were assessed by spontaneous alternation Y-maze and fear conditioning tests, respectively. We used western blot to analyze the density of N-methyl-D-aspartate receptor (NMDAR) subunits in the hippocampus. We recorded long term potentiation (LTP) and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal slices. We observed that compared with control mice, depression-like mice had significant reductions in spatial working memory and contextual fear memory. The level of NR2B, LTP and NMDA receptor-mediated EPSCs of depression-like mice were decreased. Ketamine treatment attenuated the memory impairment, and increased the density of NR2B and the amplitude of LTP and NMDA receptor-mediated EPSCs in the hippocampus of depression-like mice. In conclusion, depression-like mice have deficits in working memory and contextual fear memory. The decrease of NR2B, LTP induction and NMDA receptor-mediated EPSCs in the hippocampus may be involved in this process. Ketamine can improve expression of NR2B, LTP induction and NMDA receptor-mediated EPSCs in the hippocampus of depression-like mice, which might be part of the reason why ketamine can alleviate the memory dysfunction induced by depression.

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices.

Manipulation of metabolic pathways (e.g., ketogenic diet (KD), glycolytic inhibition) alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a “branching” metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP) in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP). Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa) in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5–5 mM) blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min), suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531), nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA). In contrast, F1,6BP caused moderate reduction (~50%) in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 μM), suggesting that these bursts are calcium dependent. Altogether, these data demonstrate that the glycolytic metabolite, F1,6BP, blocks epileptiform activity via a previously unrecognized extracellular effect on ICa, which provides new insight into the metabolic control of neural excitability.

HttQ111/+ Huntington's Disease Knock-in Mice Exhibit Brain Region-Specific Morphological Changes and Synaptic Dysfunction.

Background:Successful disease-modifying therapy for Huntington’s disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion.Objective:To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events.Methods:Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses.Results:B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures.Conclusions:These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life.