NMDA Receptor NR2B Research Articles

Expression Changes of NMDA and AMPA Receptor Subunits in the Hippocampus in rats with Diabetes Induced by Streptozotocin Coupled with Memory Impairment.

Cognitive impairment in diabetes (CID) is a severe chronic complication of diabetes mellitus (DM). It has been hypothesized that diabetes can lead to cognitive dysfunction due to expression changes of excitatory neurotransmission mediated by N-methyl-D-aspartate receptors (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR); however, the pathogenesis involved in this has not been fully understood, especially at early phase of DM. Here, we sought to determine the cognitive changes and aim to correlate this with the expression changes of NMDAR and AMPAR of glutamate signaling pathways in the rat hippocampus from early phase of DM and in the course of the disease progression. By Western blot analysis and immunofluorescence labeling, the hippocampus in diabetic rats showed a significant increase in protein expression NMDAR subunits NR1, NR2A and NR2B and AMPAR subunit GluR1. Along with this, behavioral test by Morris water maze showed a significant decline in their performance when compared with the control rats. It is suggested that NR1, NR2A, NR2B and GluR1are involved in learning and memory and that their expression alterations maybe correlated with the occurrence and development of CID in diabetic rats induced by streptozotocin.

S-nitrosylation of Src by NR2B-nNOS signal causes Src activation and NR2B tyrosine phosphorylation in levodopa-induced dyskinetic rat model.

Abnormality in Src PSD-95 NR2B signaling complex assemble occurs in levodopa-induced dyskinesia (LID). N-methyl-D-aspartate receptor (NMDAR) subunit NR2B tyrosine phosphorylation mediated by Src family protein tyrosine kinases is closely associated with dyskinesia. Src autophosphorylation (p-Src) is an important part of Src-catalyzed phosphorylation of NR2B. In addition, the neuronal nitric oxide synthase (nNOS)-derived NO (nNOS/NO) signal which was also involved in dyskinesia recently was proved to participate in the regulation of Src function. Yet, the detailed signal mechanism about the interactions of NR2B, nNOS, and Src is still unknown. In the present study, we investigated the influences of nNOS on Src activation and NR2B tyrosine phosphorylation in dyskinetic rat model by immunoblotting and immunoprecipitation. The results demonstrated that chronic levodopa treatment resulted in downregulation of p-nNOS-S847, one marker of nNOS overactivation. Coinstantaneously, the S-nitrosylation (SNO-Src) and autophosphorylation (p-Src) of Src and NR2B tyrosine phosphorylation were upregulated in dyskinetic rat model. Conversely, administration of 7-NI, one nNOS inhibitor, reversed all these effects of levodopa treatment. Besides, NR2B-containing NMDAR (NR2B/NMDAR) antagonist CP-101,606 could upregulate p-nNOS-S847 and thus attenuate nNOS activation and simultaneously reduce the SNO-Src, p-Src, and NR2B tyrosine phosphorylation. Taken together, the S-nitrosylation of Src is caused by nNOS/NO signal, which is overactivated via Ca2+ influx dependent on NR2B/NMDAR, and subsequently facilitates Src auto-tyrosine phosphorylation and further phosphorylates NR2B. The "NR2B/NMDAR-nNOS/NO-SNO-Src-p-Src-NR2B/NMDAR" signaling cycle may be the molecular basis of NR2B tyrosine phosphorylation upward positive feedback, which demonstrates the possibility as one latent target for dyskinesia therapy.

A Rho-kinase inhibitor reverses learning and memory deficits in a Rat model of chronic cerebral ischemia by altering Bcl-2/Bax-NMDAR signaling in the cerebral cortex

The current study investigated whether a Rho-kinase inhibitor alleviated impairments in a rat model of chronic cerebral ischemia and examined the specific pathological mechanisms by which Rho-kinase impacts neuronal damage and cognitive dysfunction. Adult Sprague–Dawley rats underwent permanent bilateral carotid artery occlusion (BCAO) to establish our chronic cerebral ischemia model. Chronic Y27632 administration reversed the abnormal behaviors of BCAO-treated rats in the Morris water maze. We performed Western blot analyses of the apoptosis-related proteins Bcl-2 and Bax to examine the potential mechanism underlying the beneficial effects of Y27632 on cerebral ischemia and showed for the first time that Y27632 reversed the decrease in the Bcl-2/Bax ratio in BCAO model rats. Y27632 restored the depression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors (NMDARs) in the cerebral cortex of BCAO model rats. We also investigated these effects on middle cerebral artery occlusion (MCAO) model rats and observed some differences between the two models. In summary, our data provide evidence supporting the hypothesis that Rho-kinase inhibitors exert neuroprotective effects on cerebral ischemia. The Bcl-2/Bax-NMDAR signaling pathway in the cerebral cortex may be responsible for the protective effects of the Rho-kinase inhibitor, and this pathway may represent a pharmacological target for curative clinical strategies.

Dual action of L-Lactate on the activity of NR2B-containing NMDA receptors: from potentiation to neuroprotection

L-Lactate is a positive modulator of NMDAR-mediated signaling resulting in plasticity gene induction and memory consolidation. However, L-Lactate is also able to protect neurons against excito-toxic NMDAR activity, an indication of a mitigating action of L-Lactate on NMDA signaling. In this study, we provide experimental evidence that resolves this apparent paradox. Transient co-application of glutamate/glycine (1 μM/100 μM; 2 min) in primary cultures of mouse cortical neurons triggers a NMDA-dependent Ca2+ signal positively modulated by L-Lactate (10 mM) or DTT (1 mM) but decreased by Pyruvate (10 mM). This L-Lactate and DTT-induced potentiation is blocked by Ifenprodil (2 μM), a specific blocker of NMDARs containing NR2B sub-units. In contrast, co-application of glutamate/glycine (1 mM/100 μM; 2 min) elicits a NMDAR-dependent excitotoxic death in 49% of neurons. L-Lactate and Pyruvate significantly reduce this rate of cell death processes (respectively to 23% and 9%) while DTT has no effect (54% of neuronal death). This L-Lactate-induced neuroprotection is blocked by carbenoxolone and glibenclamide, respectively blockers of pannexins and KATP. In conclusion, our results show that L-Lactate is involved in two distinct and independent pathways defined as NMDAR-mediated potentiation pathway (or NADH pathway) and a neuroprotective pathway (or Pyruvate/ATP pathway), the prevalence of each one depending on the strength of the glutamatergic stimulus.

Annexin 1 inhibits remifentanil-induced hyperalgesia and NMDA receptor phosphorylation via regulating spinal CXCL12/CXCR4 in rats

Chemokines related neuroinflammation and N-methyl-d-aspartate receptor (NMDAR) mediated nociceptive transmission are pivotal determinants in the pathogenesis of opioid-induced hyperalgesia (OIH), but little is known about specific mechanism and treatment. Chemokine CXCL12 with its receptor CXCR4 is implicated in different pathological pain, moreover, neurotoxicity of CXCL12 is associated with NMDAR activation. Recent studies recapitulate the anti-nociception of Annexin 1 (ANXA1) in inflammatory pain. This study examined whether ANXA1 prevented remifentanil-caused OIH through modulating CXCL12 and NMDAR pathway in rats. Acute exposure to remifentanil induced mechanical allodynia and thermal hyperalgesia, which was accompanied by the increase of spinal ANXA1 and CXCL12/CXCR4 expression. Central injection of Anxa12-26 attenuated behavioral OIH in a dose-dependent manner, facilitated ANXA1 production, and inhibited up-regulation of CXCL12/CXCR4 level and NR2B-containing NMDAR phosphorylation. Moreover, pretreatment with AMD3100 reduced hyperalgesia and NR2B-containing NMDAR phosphorylation. Also, exogenous CXCL12 elicited pain hypersensitivity and NMDAR activation in naïve rats, which was reversed by the supplemental delivery of Anxa12-26. These current findings indicate the participation of spinal CXCL12/CXCR4 and NR2B-containing NMDAR pathway in anti-hyperalgesic action of ANXA1 in OIH.

The spinal NR2BR/ERK2 pathway as a target for the central sensitization of collagen-induced arthritis pain.

ObjectivePain management is a huge challenge in the treatment of rheumatoid arthritis (RA), and central sensitization is reportedly involved in the development of pain. The current study was undertaken to explore the possible role of N-methyl-D-aspartate receptors (NMDARs) in the spinal mechanism of central sensitization in RA using a collagen-induced arthritis (CIA) model.MethodsMechanical hypersensitivity was assessed in C57BL/6 mice, before and after the induction of CIA via administration of chick type II collagen. Analgesic drugs, receptor antagonist, and kinase inhibitor were administrated intrathecally in the spinal cord. Protein expression and phosphorylation changes were detected via immunoblotting.ResultsCIA mice developed significant mechanical hypersensitivity, and spinal administration of the NMDAR antagonist D-2-amino-5-phosphonovaleric acid (D-APV) effectively attenuated peripheral pain hypersensitivity. There was specific enhancement of synaptic NR2B-containing NMDAR (NR2BR) expression in the spinal dorsal horns of the mice. Both the increased total protein expression of NR2B subunit and the enhanced total phosphorylation level of NR2B subunit at 1472 tyrosine promoted the synaptic expression of NMDAR in the mice. Intrathecal injection of tramadol suppressed synaptic NMDAR expression mainly by changing the synaptic phosphorylation state of NR2B subunit at Tyr1472. Extracellular signal-regulated protein kinases 2 (ERK2) activity synchronized with the synaptic expression of NR2BR, which was downregulated by the action of tramadol.ConclusionSpecific enhancement of NR2BR in the spinal dorsal horn may be vital for central sensitization in the CIA model of RA. The NR2BR/ERK2 pathway may be a promising target for pain management in RA patients.

The Neurogenesis Actuator and NR2B/NMDA Receptor Antagonist Ro25-6981 Consistently Improves Spatial Memory Retraining Via Brain Region-Specific Gene Expression

NR2B-containing NMDA (NR2B/NMDA) receptors are important in controlling neurogenesis and are involved in generating spatial memory. Ro25-6981 is a selective antagonist at these receptors and actuates neurogenesis and spatial memory. Inter-structural neuroanatomical profiles of gene expression regulating adult neurogenesis and neuroapoptosis require examination in the context of memory retrieval and reversal learning. The aim was to investigate spatial memory retrieval and reversal learning in relation to gene expression-linked neurogenetic processes following blockade of NR2B/NMDA receptors by Ro25-6981. Rats were trained in Morris water maze (MWM) platform location for 5 days. Ro25-6981 was administered (protocol days 6–7) followed by retraining (days 15–18 or 29–32). Platform location was tested (on days 19 or 33) then post-mortem brain tissue sampling (on days 20 or 34). The expression of three genes known to regulate cell proliferation (S100a6), differentiation (Ascl1), and apoptosis (Casp-3) were concomitantly evaluated in the hippocampus, prefrontal cortex, and cerebellum in relation to the MWM performance protocol. Following initial training, Ro25-6981 enhanced visuospatial memory retrieval performance during further retraining (protocol days 29–32) but did not influence visuospatial reversal learning (day 33). Hippocampal Ascl1 and Casp-3 expressions were correspondingly increased and decreased while cerebellar S100a6 and Casp-3 activities were decreased and increased respectively 27 days after Ro25-6981 treatment. Chronological analysis indicated a possible involvement of new mature neurons in the reconfiguration of memory processes. This was attended by behavioral/gene correlations which revealed direct links between spatial memory retrieval enhancement and modified gene activity induced by NR2B/NMDA receptor blockade and upregulation.

Rhynchophylline suppresses soluble Aβ1-42-induced impairment of spatial cognition function via inhibiting excessive activation of extrasynaptic NR2B-containing NMDA receptors

Rhynchophylline (RIN) is a significant active component isolated from the Chinese herbal medicine Uncaria rhynchophylla. The overproduction of soluble amyloid β protein (Aβ) oligomers in the hippocampus is closely involved in impairments in cognitive function at the early stage of Alzheimer's disease (AD). Growing evidences show that RIN possesses neuroprotective effects against Aβ-induced neurotoxicity. However, whether RIN can prevent soluble Aβ1-42-induced impairments in spatial cognitive function and synaptic plasticity is still unclear. Using the combined methods of behavioral tests, immunofluorescence and electrophysiological recordings, we characterized the key neuroprotective properties of RIN and its possible cellular and molecular mechanisms against soluble Aβ1-42-related impairments in rats. Our findings are as follows: (1) RIN efficiently rescued the soluble Aβ1-42-induced spatial learning and memory deficits in the Morris water maze test and prevented soluble Aβ1-42-induced suppression in long term potentiation (LTP) in the entorhinal cortex (EC)-dentate gyrus (DG) circuit. (2) Excessive activation of extrasynaptic GluN2B-NMDAR and subsequent Ca2+ overload contributed to the soluble Aβ1-42-induced impairments in spatial cognitive function and synaptic plasticity. (3) RIN prevented Aβ1-42-induced excessive activation of extrasynaptic NMDARs by reducing extrasynaptic NMDARs -mediated excitatory postsynaptic currents and down regulating GluN2B-NMDAR expression in the DG region, which inhibited Aβ1-42-induced Ca2+ overload mediated by extrasynanptic NMDARs. The results suggest that RIN could be an effective therapeutic candidate for cognitive impairment in AD.

Activation of spinal dorsal horn P2Y13 receptors can promote the expression of IL-1β and IL-6 in rats with diabetic neuropathic pain.

ObjectiveThe dorsal horn P2Y13 receptor is involved in the development of pain behavior induced by peripheral nerve injury. It is unclear whether the expression of proinflammatory cytokines interleukin (IL)-1β and IL-6 at the spinal dorsal horn are influenced after the activation of P2Y13 receptor in rats with diabetic neuropathic pain (DNP).MethodsA rat model of type 1 DNP was induced by intraperitoneal injection of streptozotocin (STZ). We examined the expression of P2Y13 receptor, Iba-1, IL-1β, IL-6, JAK2, STAT3, pTyr1336, and pTyr1472 NR2B in rat spinal dorsal horn.ResultsCompared with normal rats, STZ-diabetic rats displayed obvious mechanical allodynia and the increased expression of P2Y13 receptor, Iba-1, IL-1β, and IL-6 in the dorsal spinal cord that was continued for 6 weeks in DNP rats. The data obtained indicated that, in DNP rats, administration of MRS2211 significantly attenuated mechanical allodynia. Compared with DNP rats, after MRS2211 treatment, expression of the P2Y13 receptor, Iba-1, IL-1β, and IL-6 were reduced 4 weeks after the STZ injection. However, MRS2211 treatment did not attenuate the expression of the P2Y13 receptor, Iba-1, IL-1β, and IL-6 at 6 weeks after the STZ injection. MRS2211 suppressed JAK2 and STAT3 expression in the early stage, but not in the later stage. Moreover, pTyr1336 NR2B was significantly decreased, whereas pTyr1472 NR2B was unaffected in the dorsal spinal cord of MRS2211-treated DNP rats.ConclusionIntrathecal MRS2211 produces an anti-nociceptive effect in early-stage DNP. A possible mechanism involved in MRS2211-induced analgesia is that blocking the P2Y13 receptor downregulates levels of IL-1β and IL-6, which subsequently inhibit the activation of the JAK2/STAT3 signaling pathway. Furthermore, blocking the activation of the P2Y13 receptor can decrease NR2B-containing NMDAR phosphorylation in dorsal spinal cord neurons, thereby attenuating central sensitization in STZ-induced DNP rats.

Critical role of sigma-1 receptors in central neuropathic pain-related behaviours after mild spinal cord injury in mice

Sigma-1 receptor (σ1R) knockout (KO) CD1 mice, generated by homologous recombination, and separate pharmacological studies in wild type (WT) mice were done to investigate the role of this receptor in the development of pain-related behaviours (thermal hyperalgesia and mechanical allodynia) in mice after spinal cord contusion injury (SCI) – a model of central neuropathic pain. The modulatory effect of σ1R KO on extracellular mediators and signalling pathways in the spinal cord was also investigated. In particular, changes in the expression of inflammatory cytokines (tumour necrosis factor TNF-α, interleukin IL-1β) and both the expression and activation (phosphorylation) of the N-methyl-D-aspartate receptor subunit 2B (NR2B-NMDA) and extracellular signal-regulated kinases (ERK1/2) were analysed. Compared with WT mice, both mechanical and thermal hypersensitivity were attenuated in σ1R KO mice following SCI. Accordingly, treatment of WT mice with the σ1R antagonist MR309 (previously developed as E-52862; S1RA) after SCI exerted antinociceptive effects (i.e. reduced mechanical allodynia and thermal hyperalgesia). Attenuated nociceptive responses in σ1R KO were accompanied by reduced expression of TNF- α and IL-1β as well as decreased activation/phosphorylation of NR2B-NMDA receptors and ERK1/2. These findings suggest that σ1R may modulate central neuropathic pain and point to regulation of sensitization-related phenomena as a possible mechanism.

Role of α7nAChR-NMDAR in sevoflurane-induced memory deficits in the developing rat hippocampus.

Detrimental effects of volatile anaesthetics, including sevoflurane, on the structure and function of the developing brain have been reported. The internalization of N-methyl-D-aspartate receptors (NMDARs) contributes to anaesthetic neurotoxicity. Both nicotinic acetylcholine receptors (nAChRs) and NMDAR play a critical role in the development of the nervous system. Moreover, nAChR can interact with NMDAR, and previous studies have demonstrated modulation of NMDAR by nAChR. In our study, we used an α7 nicotinic acetylcholine receptor (α7nAChR) agonist and α7nAChR antagonist to explore the role of α7nAChR and NMDAR in sevoflurane-induced long-term effects on memory and dendritic spine both in vivo and in vitro. The results revealed that the activation of α7nAChR attenuated the development of sevoflurane-induced memory deficit and dendritic spine changes, which might be by regulating NR2B-containing NMDAR trafficking from the intracellular pool to the cell surface pool in the hippocampus. Moreover, we demonstrated that α7nAChR could regulate NR2B-containing NMDAR via Src-family tyrosine kinase (SFK). Thus, our current study indicates that the trafficking of NR2B-containing NMDAR is regulated by α7nAChR via SFK in neonatal rat hippocampus, which may be secondary to sevoflurane-induced cognitive deficits in the developing hippocampus.

Novel rare variations in genes that regulate developmental change in N-methyl-d-aspartate receptor in patients with schizophrenia

The mechanism underlying the vulnerability to developing schizophrenia (SCZ) during adolescence remains elusive. Hypofunction of N-methyl-d-aspartate receptors (NMDARs) has been implicated in the pathophysiology of SCZ. During development, the composition of synaptic NMDARs dramatically changes from NR2B-containing NMDARs to NR2A-containing NMDARs through the phosphorylation of NR2B S1480 or Y1472 by CDK5, CSNK2A1, and EphB2, which plays a pivotal role in the maturation of neural circuits. We hypothesized that the dysregulation of developmental change in NMDARs could be involved in the onset of SCZ. Using next-generation sequencing, we re-sequenced all the coding regions and splice sites of CDK5, CSNK2A1, and EphB2 in 474 patients with SCZ and 475 healthy controls. Variants on the database for human control subjects of Japanese origin were removed and all the nonsynonymous and nonsense variants were validated using Sanger sequencing. Four novel variants in CDK5 were observed in patients with SCZ but were not observed in controls. The total number of variants, however, was not significantly different between the SCZ and control groups (P=0.062). In silico analyses predicted P271T to be damaging. Further genetic research using a larger sample is required to examine whether CDK5 is involved in the pathophysiology of SCZ.

NR2B-containing NMDA receptors contribute to diarrhea-predominant irritable bowel syndrome

// Wenxue Zhang 1, 2, 3, 4 , Dongyan Zhao 5 , Qingqing Qi 1, 2 , Xin Long 1, 2 , Yueyue Li 1, 2 , Peng Wang 1, 2 , Yanbo Yu 1, 2 , Lixiang Li 1, 2 , Yiyuan Sun 1, 2 , Zhen Li 1, 2 , Yanqing Li 1, 2 and Xiuli Zuo 1, 2 1 Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, China 2 Laboratory of Translational Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, China 3 Department of Gastroenterology, China-Japan Friendship Hospital, Beijing 100029, China 4 Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China 5 Department of Gastroenterology, General Hospital of Puyang Oilfield, Puyang 457000, China Correspondence to: Xiuli Zuo, email: zuoxiuli@sina.com Keywords: NMDARs; NR2B; proBDNF; visceral hypersensitivity; diarrhea-predominant irritable bowel syndrome Received: December 01, 2016 Accepted: January 02, 2018 Published: January 02, 2018 ABSTRACT Colonic mucosal N-methyl-D-aspartate receptors (NMDARs) contribute to visceral hypersensitivity in diarrhea-predominant irritable bowel syndrome (IBS-D) by increasing the production of brain-derived neurotrophic factor (BDNF). We investigated which of the multiple NMDAR subtypes and BDNF isoforms are responsible for this effect. Immunohistochemistry and Western blotting were used to detect levels of colonic mucosal NR2A-D subunits and the precursor (proBDNF) and mature (mBDNF) forms of BDNF in 67 participants. Only NR2B subunit expression was elevated in the colonic mucosa of IBS-D patients, in parallel with increased total BDNF and proBDNF expression. Expression of 15 kDa mBDNF was not detected in the colonic mucosa. NR2B, total BDNF and proBDNF levels correlated with abdominal pain scores. Quantitative real-time PCR and Western blotting showed that NMDAR activation substantially induced total BDNF/proBDNF expression in HT-29 cells, while the NMDAR inhibitor MK-801 and the NR2B subunit antagonist Ro25-6981 each completely blocked these effects. Thus, colonic mucosal NR2B-containing NMDARs may contribute to visceral hypersensitivity in IBS-D patients by upregulating BDNF, especially proBDNF.

Differential Modulation of GABAA and NMDA Receptors by an α7-nicotinic Acetylcholine Receptor Agonist in Chronic Glaucoma.

Presynaptic modulation of γ-aminobutyric acid (GABA) release by an alpha7 nicotinic acetylcholine receptor (α7-nAChR) agonist promotes retinal ganglion cell (RGC) survival and function, as suggested by a previous study on a chronic glaucomatous model from our laboratory. However, the role of excitatory and inhibitory amino acid receptors and their interaction with α7-nAChR in physiological and glaucomatous events remains unknown. In this study, we investigated GABAA and N-methyl-D-aspartate (NMDA) receptor activity in control and glaucomatous retinal slices and the regulation of amino acid receptor expression and function by α7-nAChR. Whole-cell patch-clamp recordings from RGCs revealed that the α7-nAChR specific agonist PNU-282987 enhanced the amplitude of currents elicited by GABA and reduced the amplitude of currents elicited by NMDA. The positive modulation of GABAA receptor and the negative modulation of NMDA receptor (NMDAR) by PNU-282987-evoked were prevented by pre-administration of the α7-nAChR antagonist methyllycaconitine (MLA). The frequency and the amplitude of glutamate receptor-mediated miniature glutamatergic excitatory postsynaptic currents (mEPSCs) were not significantly different between the control and glaucomatous RGCs. Additionally, PNU-282987-treated slices showed no alteration in the frequency or amplitude of mEPSCs relative to control RGCs. Moreover, we showed that expression of the α1 subunit of the GABAA receptor was downregulated and the expression of the NMDAR NR2B subunit was upregulated by intraocular pressure (IOP) elevation, and the changes of high IOP were blocked by PNU-282987. In conclusion, retina GABAA and NMDARs are modulated positively and negatively, respectively, by activation of α7-nAChR in in vivo chronic glaucomatous models.

Xanthoceraside modulates NR2B-containing NMDA receptors at synapses and rescues learning-memory deficits in APP/PS1 transgenic mice.

Alzheimer's disease (AD) is characterized by memory loss and synaptic damage. Previous studies suggested that xanthoceraside decreases glutamate-induced PC12 cell death, ameliorates memory deficits, and increases the number of dendritic spines in AD mice. These results indicated that xanthoceraside might have activities that protect synaptic plasticity. Herein, we detected the effect of xanthoceraside on synaptic function. Three-month-old APP/PS1 transgenic mice were orally treated with xanthoceraside (0.02, 0.08, or 0.32mg/kg) once daily for 4months and then behavioral tests were performed. LTP and Fluo-4/AM were carried out in vivo and in vitro, respectively. CaMKII-GluR1 and NR2B-associated proteins on synapses were measured. Xanthoceraside administration alleviated learning-memory deficits and increased the LTP in APP/PS1 transgenic mice. Meanwhile, xanthoceraside increased the expression of pT286-CaMKII in synaptic and extrasynaptic pools and CaMKII, pS831-GluR1, and GluR1 in synaptic pools. In addition, xanthoceraside increased the total pY1472-NR2B and NR2B expression and increased the levels of pY1472-NR2B in synaptic and extrasynaptic pools and NR2B in synaptic pools. However, NR2B was decreased in extrasynaptic pools, which might be associated with decreased expression of STEP61 and pY531-Fyn. In vitro studies showed that xanthoceraside inhibited intracellular calcium overload and increased the number of and extended the length of dendrites in primary hippocampal neurons compared with the Aβ25-35 group. The mechanism of xanthoceraside on ameliorating learning-memory deficits might be related to decrease intracellular calcium overload, increase CaMKII-GluR1 proteins, and up-regulate trafficking of pY1472-NR2B at synapse, thereby improving LTP in APP/PS1 transgenic mice.

Inhibition of Cdk5 rejuvenates inhibitory circuits and restores experience-dependent plasticity in adult visual cortex

Cyclin-dependent kinase 5 (Cdk5) acts as an essential modulator for neural development and neurological disorders. Here we show that Cdk5 plays a pivotal role in modulating GABAergic signaling and the maturation of visual system. In adult mouse primary visual cortex, Cdk5 formed complex with the GABA synthetic enzyme glutamate decarboxylase GAD67, but not with GAD65. In addition to enhancement in the surface level of NR2B-containing NMDA receptors, inhibition of Cdk5 reduced the protein levels of GADs and Otx2, while leaving intact the expression of vesicular GABA transporter and subunits of GABAA or AMPA receptors. Whole-cell patch-clamp recording in layer II/III pyramidal neurons revealed a decrease in the frequency of miniature inhibitory postsynaptic current (mIPSC). Consequently, pharmacological inhibition and genetic knockdown of Cdk5 in adult mice led to a restoration of juvenile-like ocular dominance plasticity in vivo and long-term synaptic potential in layer II/III induced by white matter stimulation in vitro. Interestingly, we did not observe an alteration of perineuronal nets of extracellular matrix, but a reinstatement of the capability to evoke long-term depression at inhibitory synapses (iLTD), which depended on presynaptic endocannabinoid receptors and was a sign of the rejuvenated GABAergic synapses. Enhancement of GABA signaling by diazepam impeded ocular dominance plasticity rescued by Cdk5 inhibition. These results thus suggest that a physiological role of Cdk5 in visual cortex is to consolidate and stabilize neural circuits through controlling GABAergic signaling.

TRPC6 expression in neurons is differentially regulated by NR2A- and NR2B-containing NMDA receptors.

The expression of transient receptor potential canonical 6 (TRPC6) in central nervous system (CNS) is important for neuronal functions and certain neural disorders. However, the regulatory mechanism of TRPC6 expression in neurons is still obscure. In this study, we show that TRPC6 expression in the primary cultured cortical neurons is bidirectionally regulated by glutamate. Activation of NR2A-containing NMDARs induces TRPC6 transcription through a calcineurin-dependent pathway. In contrast, activation of NR2B-containing NMDARs causes TRPC6 degradation through calpain. Thus, TRPC6 expression in neurons is regulated by glutamate in a bidirectional manner that is dependent on NR2A and NR2B.

Effects of intra-accumbal administration of dopamine and ionotropic glutamate receptor drugs on delay discounting performance in rats.

Nucleus accumbens core (NAcc) has been implicated in impulsive choice, as measured in delay discounting. The role of dopamine (DA) in impulsive choice has received considerable attention, whereas glutamate (Glu) has recently been shown to be an important mediator of discounting. However, research has not examined how DA or Glu receptors in NAcc mediate different aspects of delay discounting performance, that is, (a) sensitivity to reinforcer magnitude and (b) sensitivity to delayed reinforcement. Adult male Sprague-Dawley rats were first trained in a delay discounting task, in which the delay to a large magnitude food reinforcer increased across blocks of trials. Following behavioral training, rats received bilateral implantation of guide cannulas into NAcc. Half of the rats (n = 12) received infusions of the DA-selective ligands SKF 38393 (D1-like agonist: 0.03 or 0.1 μg), SCH 23390 (D1-like antagonist: 0.3 or 1.0 μg), quinpirole (D2-like agonist: 0.3 or 1.0 μg), and eticlopride (D2-like antagonist: 0.3 or 1.0 μg). The other half received infusions of the ionotropic Glu ligands MK-801 (NMDA uncompetitive antagonist: 0.3 or 1.0 μg), AP-5 (NMDA competitive antagonist: 0.3 or 1.0 μg), ifenprodil (noncompetitive antagonist at NR2B-containing NMDA receptors: 0.3 or 1.0 μg), and CNQX (AMPA competitive antagonist: 0.2 or 0.5 μg). Results showed that SCH 23390 (0.3 μg) decreased sensitivity to reinforcer magnitude without altering impulsive choice, whereas ifenprodil (1.0 μg) decreased sensitivity to delayed reinforcement (i.e., impulsive choice). The current results show that DA and NMDA receptors in NAcc mediate distinct aspects of discounting performance. (PsycINFO Database Record

NMDA Receptors Contribute to Retrograde Synaptic Transmission from Ganglion Cell Photoreceptors to Dopaminergic Amacrine Cells.

Recently, a line of evidence has demonstrated that the vertebrate retina possesses a novel retrograde signaling pathway. In this pathway, phototransduction is initiated by the photopigment melanopsin, which is expressed in a small population of retinal ganglion cells. These ganglion cell photoreceptors then signal to dopaminergic amacrine cells (DACs) through glutamatergic synapses, influencing visual light adaptation. We have previously demonstrated that in Mg2+-containing solution, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate this glutamatergic transmission. Here, we demonstrate that removing extracellular Mg2+ enhances melanopsin-based DAC light responses at membrane potentials more negative than −40 mV. Melanopsin-based responses in Mg2+-free solution were profoundly suppressed by the selective N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5. In addition, application of NMDA to the retina produced excitatory inward currents in DACs. These data strongly suggest that DACs express functional NMDA receptors. We further found that in the presence of Mg2+, D-AP5 reduced the peak amplitude of melanopsin-based DAC responses by ~70% when the cells were held at their resting membrane potential (−50 mV), indicating that NMDA receptors are likely to contribute to retrograde signal transmission to DACs under physiological conditions. Moreover, our data show that melanopsin-based NMDA-receptor-mediated responses in DACs are suppressed by antagonists specific to either the NR2A or NR2B receptor subtype. Immunohistochemical results show that NR2A and NR2B subunits are expressed on DAC somata and processes. These results suggest that DACs express functional NMDA receptors containing both NR2A and NR2B subunits. Collectively, our data reveal that, along with AMPA receptors, NR2A- and NR2B-containing NMDA receptors mediate retrograde signal transmission from ganglion cell photoreceptors to DACs.

Perturbing NR2B-PSD-95 interaction relieves neuropathic pain by inactivating CaMKII-CREB signaling.

Neuropathic pain is characterized by central sensitization. The interaction between N-methyl-D-aspartate receptors (NMDARs) and postsynaptic density protein-95 (PSD-95) plays a major role in central sensitization. Here, we aimed to investigate the analgesic effect of disruption of the interaction between NMDAR and PSD-95. Chronic dorsal root ganglia compression model rats were used to mimic sciatica. Thermal hyperalgesia and mechanical allodynia were evaluated. The expression of spinal phospho-NR2B, PSD-95, calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element binding protein (CREB) was measured using western blotting. A mimetic peptide Myr-NR2B9c was injected intrathecally to disrupt the interaction between PSD-95 and NR2B and detected by coimmunoprecipitation. Chronic dorsal root ganglia compression surgery induced thermal hyperalgesia and mechanical allodynia, and upregulated pain-related proteins such as phospho-NR2B, PSD-95, CaMKII, and CREB expressions in the spinal cord. Myr-NR2B9c disrupted the interaction between NR2B-containing NMDARs and PSD-95 in the spinal cord. Intrathecal administration of Myr-NR2B9c attenuated neuropathic pain behaviors and downregulated the expressions of phospho-NR2B, PSD-95, CaMKII, and CREB in the spinal cord. The present study indicates that dissociation of NR2B-containing NMDARs from PSD-95 inactivates CaMKII and CREB signaling and relieves pain.