NR2A-containing NMDA Receptors Research Articles

Dual Role of NMDAR Containing NR2A and NR2B Subunits in Alzheimer's Disease.

Alzheimer's disease (AD) is the main cause of dementia worldwide. Given that learning and memory are impaired in this pathology, NMDA receptors (NMDARs) appear as key players in the onset and progression of the disease. NMDARs are glutamate receptors, mainly located at the post-synapse, which regulate voltage-dependent influx of calcium into the neurons. They are heterotetramers, and there are different subunits that can be part of the receptors, which are usually composed of two obligatory GluN1 subunits plus either two NR2A or two NR2B subunits. NR2A are mostly located at the synapse, and their activation is involved in the expression of pro-survival genes. Conversely, NR2B are mainly extrasynaptic, and their activation has been related to cell death and neurodegeneration. Thus, activation of NR2A and/or inactivation of NR2B-containing NMDARS has been proposed as a therapeutic strategy to treat AD. Here, we wanted to investigate the main differences between both subunits signalling in neuronal primary cultures of the cortex and hippocampus. It has been observed that Aβ induces a significant increase in calcium release and also in MAPK phosphorylation signalling in NR2B-containing NMDAR in cortical and hippocampal neurons. However, while NR2A-containing NMDAR decreases neuronal death and favours cell viability after Aβ treatment, NR2B-containing NMDAR shows higher levels of cytotoxicity and low levels of neuronal survival. Finally, it has been detected that NMDAR has no effect on pTau axonal transport. The present results demonstrate a different role between GluNA and GluNB subunits in neurodegenerative diseases such as Alzheimer's.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Phencyclidine Disrupts Neural Coordination and Cognitive Control by Dysregulating Translation

BackgroundPhencyclidine (PCP) causes psychosis, is abused with increasing frequency, and was extensively used in antipsychotic drug discovery. PCP discoordinates hippocampal ensemble action potential discharge and impairs cognitive control in rats, but how this uncompetitive NMDA receptor (NMDAR) antagonist impairs cognition remains unknown. MethodsThe effects of PCP were investigated on hippocampal CA1 ensemble action potential discharge in vivo in urethane-anesthetized rats and during awake behavior in mice, on synaptic responses in ex vivo mouse hippocampus slices, in mice on a hippocampus-dependent active place avoidance task that requires cognitive control, and on activating the molecular machinery of translation in acute hippocampus slices. Mechanistic causality was assessed by comparing the PCP effects with the effects of inhibitors of protein synthesis, group I metabotropic glutamate receptors (mGluR1/5), and subunit-selective NMDARs. ResultsConsistent with ionotropic actions, PCP discoordinated CA1 ensemble action potential discharge. PCP caused hyperactivity and impaired active place avoidance, despite the rodents having learned the task before PCP administration. Consistent with metabotropic actions, PCP exaggerated protein synthesis–dependent DHPG-induced mGluR1/5-stimulated long-term synaptic depression. Pretreatment with anisomycin or the mGluR1/5 antagonist MPEP, both of which repress translation, prevented PCP-induced discoordination and the cognitive and sensorimotor impairments. PCP as well as the NR2A-containing NMDAR antagonist NVP-AAM077 unbalanced translation that engages the Akt, mTOR (mechanistic target of rapamycin), and 4EBP1 translation machinery and increased protein synthesis, whereas the NR2B-containing antagonist Ro25-6981 did not. ConclusionsPCP dysregulates translation, acting through NR2A-containing NMDAR subtypes, recruiting mGluR1/5 signaling pathways, and leading to neural discoordination that is central to the cognitive and sensorimotor impairments.

Design and synthesis of novel GluN2A NMDAR positive allosteric modulators via scaffold hopping strategy as anti-stroke therapeutic agents.

NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo . Activation of NR2A-containing NMDA receptors promotes neuronal survival and exerts a neuroprotective action, whereas over activating GluN2B-containing receptor results in excitotoxicity, increasing neuronal apoptosis. Our previous study has identified Npam 43 as a NMDAR positive allosteric modulators. However, the cis-trans isomerization impedes the development of Npam 43 as potential neuroprotective agents. To discover more potent and selective GluN2A NMDAR positive allosteric modulators, 38 derivatives were synthesized and evaluated their neuroprotective effect on glutamate-exposed PC-12 cells. The allosteric activities of compounds were evaluated using calcium imaging approaches. Among them, compound 5c exhibit GluN1/2A selectivity over GluN1/2B and show neuroprotective activity in vitro and in vivo. This study reported a series of GluN1/2A positive allosteric modulators as neuroprotective agents, and provided a potential opportunity to discover new drugs for stroke treatment.

Neonatal blockade of NR2A-containing NMDA receptor induces schizophrenia-related behaviors in adult rats

It is known that N-methyl-D-aspartate (NMDA) receptor is essential for early brain development. NMDA receptor blockade during neonatal period causes various abnormal behaviors in later life. In the developing brain, NR2A- and NR2B-containing NMDA receptors show different expression patterns. However, the functions of these NMDA receptors in brain development are unknown. Therefore, we investigated the effects of pharmacological inactivation of NR2A- and/or NR2B-containing NMDA receptors on various behaviors in adulthood. We postnatally treated rats with an NR2A-preferring (PEAQX), an NR2B-selective (ifenprodil), or a nonselective NMDA receptor blocker (MK-801). Interestingly, neonatal treatment with PEAQX or MK-801 caused significant decrease in spontaneous alternation in the Y-maze test. In addition, PEAQX or MK-801 treatment increased startle response to acoustic stimuli. Neonatal PEAQX treatment also induced hypersensitivity to locomotor-stimulating effect of MK-801. On the other hand, neonatal ifenprodil treatment did not cause these behavioral alterations. Furthermore, PEAQX-treated but not ifenprodil-treated rats exhibited deformity of the hippocampal CA1 area, while neonatal NMDA receptor blockade did not alter the cell density in the hippocampus. In conclusion, our results suggest that the NR2A-containing NMDA receptor plays important roles in early brain development in rats, and hypofunction of this subunit during developmental period induces schizophrenia-related behaviors in adulthood.

Developmental stage-specific involvement of NMDA receptor hypofunction in risk formation for psychiatric disorders

Successful maturation of the brain relies on complex interactions between various neurotransmitter signaling. Glutamate, a major excitatory neurotransmitter in mammalian central nervous system, and an ionotropic glutamate receptor subtype, N-methyl-D-aspartate (NMDA) receptor, play essential roles in brain development. It has been reported that animals treated with an NMDA receptor antagonist during neonatal period show various behavioral abnormalities relevant to neurodevelopmental disorders in later life. We previously found that NMDA receptor blockade during neonatal period, but not adulthood, caused significant cognitive impairment in rats. Thus, the adverse effects of NMDA receptor blockade are dependent on the time when the treatment is administered. We further investigated the precise timing that NMDA receptors are involved in the development of cognitive functions, and the mechanism underlying the vulnerability of the immature brain to NMDA receptor blockade. In this symposium, we discuss the importance of the second postnatal week on the development of hippocampus-dependent learning in rats. We also discuss the involvement of NR2A-containing NMDA receptors in the formation of schizophrenia-related behavioral changes induced by neonatal NMDA receptor blockade.

Neonatal blockade of NR2A-containing but not NR2B-containing NMDA receptor induces spatial working memory deficits in adult rats

The immature brain is highly sensitive to disturbances in the functioning of N-methyl-d-aspartate (NMDA) receptor in rodents, and blockade of the receptor during postnatal brain development period causes schizophrenia-like behavior in adulthood. During the postnatal period, NR2A- and NR2B-containing NMDA receptors are highly expressed, and these two subunits show different expression patterns in the brain. However, the functions of these two NMDA receptors are unknown. In this study, we treated rats with an NR2A-preferring NMDA receptor antagonist (PEAQX, 10 mg/kg), an NR2B-selective NMDA receptor antagonist (ifenprodil, 7.5 mg/kg), or a nonselective blocker of the NMDA receptor (MK-801, 0.4 mg/kg) during the neonatal period. Rats neonatally treated with MK-801 or PEAQX showed spatial working memory deficits in the Y-maze test. PEAQX-treated rats also showed greater reactivity to acoustic stimuli and hypersensitivity to acute MK-801 challenge. However, ifenprodil treatment did not cause any detectable behavioral changes. These results suggest that the NR2A-containing NMDA receptor is indispensable for proper brain development in rats, and functional disturbances in this subunit impair hippocampus-dependent spatial working memory in adulthood.

N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia.

Although stroke is very often the cause of death worldwide, the burden of ischemic and hemorrhagic stroke varies between regions and over time regarding differences in prognosis, prevalence of risk factors, and treatment strategies. Excitotoxicity, oxidative stress, dysfunction of the blood-brain barrier, neuroinflammation, and lysosomal membrane permeabilization, sequentially lead to the progressive death of neurons. In this process, protein kinases-related checkpoints tightly regulate N-methyl-D-aspartate (NMDA) receptor signaling pathways. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca2+ overload and ultimately neuronal death. Thus, reduced expression of postsynaptic density-95 protein and increased protein S-nitrosylation in neurons is responsible for neuronal vulnerability in cerebral ischemia. In this chapter death-associated protein kinases, cyclin-dependent kinase 5, endoplasmic reticulum stress-induced protein kinases, hyperhomocysteinemia-related NMDA receptor overactivation, ephrin-B-dependent amplification of NMDA-evoked neuronal excitotoxicity and lysosomocentric hypothesis have been discussed.Consequently, ample evidences have demonstrated that enhancing extrasynaptic NMDA receptor activity triggers cell death after stroke. In this context, considering the dual roles of NMDA receptors in both promoting neuronal survival and mediating neuronal damage, selective augmentation of NR2A-containing NMDA receptor activation in the presence of NR2B antagonist may constitute a promising therapy for stroke.

Inhibition of NR2A reduces calcitonin gene-related peptide gene expression induced by cortical spreading depression in rat amygdala

Despite robust evidence on the role of calcitonin gene-related peptide (CGRP) in migraine via both central and peripheral actions, relatively less is known about how CGRP in the limbic system is involved in migraine progression. This study investigated whether CGRP production machinery exists in the two key limbic regions including hippocampus and amygdala using cortical spreading depression (CSD) as a model of migraine and whether such alteration by CSD is sensitive to N-methyl-d-aspartate (NMDA) receptor regulation in rats. A single or repetitive CSD was induced by topical application of KCl and monitored using electrophysiological methods. The NR2A-containing NMDA receptor antagonist, NVP-AAM077, or its vehicle, was perfused into the contralateral cerebroventricular ventricle of rat. Quantitative PCR was used to measure CGRP mRNA levels in the ipsilateral and contralateral hippocampus and amygdala after CSD events and compared to respective sham treatments. The results showed that neither a single CSD nor repetitive CSD affected CGRP mRNA levels in both the contralateral and ipsilateral hippocampus at 24 h post CSD induction. Differently, significant elevation of CGRP gene expression was observed in the ipsilateral amygdala at 24 h post multiple CSD, but not contralateral side, and not post-single CSD. Further results showed that the CSD-induced CGRP gene expression in the amygdala was markedly reduced by NVP-AAM077 and this reduction corresponded to a reduced cortical susceptibility to CSD in rats. We conclude that repetitive CSD events induce CGRP gene expression in amygdala, which is sensitive to NR2A regulation.

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.

Oscillatory Synchronous Inhibition in the Basolateral Amygdala and its Primary Dependence on NR2A-containing NMDA Receptors

Synchronous, rhythmic firing of GABAergic interneurons is a fundamental mechanism underlying the generation of brain oscillations, and evidence suggests that NMDA receptors (NMDARs) play a key role in oscillatory activity by regulating the activity of interneurons. Consistent with this, derangement of brain rhythms in certain neuropsychiatric disorders, notably schizophrenia and autism, is associated with NMDAR hypofunction and loss of inhibitory interneurons. In the basolateral amygdala (BLA)—dysfunction of which is involved in a host of neuropsychiatric diseases—, principal neurons display spontaneous, rhythmic “bursts” of inhibitory activity, which could potentially be involved in the orchestration of oscillations in the BLA network; here, we investigated the role of NMDARs in these inhibitory oscillations. Rhythmic bursts of spontaneous IPSCs (0.5 Hz average burst frequency) recorded from rat BLA principal cells were blocked or significantly suppressed by D-AP5, and could be driven by NMDAR activation alone. BLA interneurons generated spontaneous bursts of suprathreshold EPSCs at a similar frequency, which were also blocked or reduced by D-AP5. PEAQX (GluN2A-NMDAR antagonist; 0.4 μM) or Ro-25-6981 (GluN2B-NMDAR antagonist; 5 μM) suppressed the IPSC and EPSC bursts; suppression by PEAQX was significantly greater than that by Ro-25-6981. Immunohistochemical labeling revealed the presence of both GluN2A- and GluN2B-NMDARs on GABAergic BLA interneurons, while, functionally, GluN2A-NMDARs have the dominant role, as suggested by a greater reduction of NMDA-evoked currents by PEAQX versus Ro-25-6981. Entrainment of BLA principal neurons in an oscillatory generation of inhibitory activity depends primarily on activation of GluN2A-NMDARs, and interneuronal GluN2A-NMDARs may play a significant role.

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.

Deficient striatal adaptation in aminergic and glutamatergic neurotransmission is associated with tardive dyskinesia in non-human primates exposed to antipsychotic drugs

Tardive dyskinesia (TD) is a potentially disabling condition encompassing all delayed, persistent, and often irreversible abnormal involuntary movements arising in a fraction of subjects during long-term exposure to centrally acting dopamine receptor-blocking agents such as antipsychotic drugs and metoclopramide. However, the pathogenesis of TD has proved complex and remains elusive. To investigate the mechanism underlying the development of TD, we have chronically exposed 17 Cebus apella monkeys to typical (11) or atypical (6) antipsychotic drugs. Six additional monkeys were used as controls. Using autoradiography, Western blot and in situ hybridization techniques, we compared neurochemical components of the dopamine, serotonin, and glutamate neurotransmitter systems modulating striatal activity in monkeys chronically exposed to haloperidol and clozapine. Five (5) out of 11 monkeys treated with haloperidol develop TD, whereas none of the monkeys treated with clozapine develop TD. Haloperidol treatment significantly upregulated the levels of serotonin 5-HT2A receptor, NR2A-containing NMDA receptors, and tyrosine hydroxylase contents in the monkey putamen, whereas clozapine regulated putamen NMDA receptor levels and tyrosine hydroxylase contents, and 5-HT2A and dopamine transporter outside the putamen. Comparisons of neurochemical alterations between dyskinetic and non dyskinetic animals within the haloperidol-treated group indicate that modulations of 5-HT2A, metabotropic glutamate type 5, NR2A- and NR2B-containing NMDA receptors, and vesicular monoamine transporter type 2 levels were restricted to the non dyskinetic group. The foregoing results suggest that TD is associated with complex deficient adaptation in aminergic and glutamatergic neurotransmission in the striatum of non-human primates chronically exposed to antipsychotic drugs.

Extrasynaptic NMDA receptor dependent long-term potentiation of hippocampal CA1 pyramidal neurons

In the adult mouse hippocampus, NMDA receptors (NMDARs) of CA1 neurons play an important role in the synaptic plasticity. The location of NMDARs can determine their roles in the induction of long-term potentiation (LTP). However, the extrasynaptic NMDARs (ES-NMDARs) dependent LTP haven’t been reported. Here, through the use of a 5-Hz stimulation and MK-801 (an irreversible antagonist of NMDARs) in the CA1 neurons of adult mice hippocampal slices, synaptic NMDARs were selectively inhibited and NMDAR-mediated excitatory postsynaptic currents were not recovered. We found that a robust LTP was induced by 3-train 100-Hz stimulation when the synaptic NMDARs and extrasynaptic NR2B containing NMDARs were blocked, but not in the any of the following conditions: blocking of all NMDARs (synaptic and extrasynaptic), blocking of the synaptic NMDARs, and blocking of the synaptic NMDARs and extrasynaptic NR2A-containing NMDARs. The results indicate that this LTP is ES-NMDARs dependent, and NR2B-containing ES-NMDARs modulates the threshold of LTP induction.

Making of a Synapse: Recurrent Roles of Drebrin A at Excitatory Synapses Throughout Life.

Mature excitatory synapses are composed of more than 1500 proteins postsynaptically and hundreds more that operate presynaptically. Among them, drebrin is an F-actin-binding protein that increases noticeably during juvenile synaptogenesis. Electron microscopic analysis reveals that drebrin is highly enriched specifically on the postsynaptic side of excitatory synapses. Since dendritic spines are structures specialized for excitatory synaptic transmission, the function of drebrin was probed by analyzing the ultrastructural characteristics of dendritic spines of animals with genetic deletion of drebrin A (DAKO), the adult isoform of drebrin. Electron microscopic analyses revealed that these brains are surprisingly intact, in that axo-spinous synaptic junctions are well-formed and not significantly altered in number. This normal ultrastructure may be because drebrin E, the alternate embryonic isoform, compensates for the genetic deletion of drebrin A. However, DAKO results in the loss of homeostatic plasticity of N-methyl-D-aspartate receptors (NMDARs). The NMDAR activation-dependent trafficking of the NR2A subunit-containing NMDARs from dendritic shafts into spine head cytoplasm is greatly diminished within brains of DAKO. Conversely, within brains of wild-type rodents, spines respond to NMDAR blockade with influx of F-actin, drebrin A, and NR2A subunits of NMDARs. These observations indicate that drebrin A facilitates the trafficking of NMDAR cargos in an F-actin-dependent manner to mediate homeostatic plasticity. Analysis of the brains of transgenic mice used as models of Alzheimer's disease (AD) reveals that the loss of drebrin from dendritic spines predates the emergence of synaptic dysfunction and cognitive impairment, suggesting that this form of homeostatic plasticity contributes toward cognition. Two studies suggest that the nature of drebrin's interaction with NMDARs is dependent on the receptor's subunit composition. Drebrin A can be found co-clustering with NR2B-containing NMDARs at the plasma membrane, while NR2A-containing NMDARs co-traffic into the spine cytoplasm but do not co-cluster at the plasma membrane. Most recently, we encountered a physiological condition that supports this idea. When adolescent female rats are reared under a condition of restricted food access and ad libitum wheel access, they paradoxically become excessive runners, choosing to run, even during the limited hours of food availability. This behavioral pattern is termed activity-based anorexia (ABA) and has served as an animal model for anorexia nervosa. Those animals that exhibit the greatest ABA vulnerability, in that they lose the most amount of body weight and run with greatest exuberance to the point of risking their lives, exhibit the highest levels of NR2B-NMDARs and drebrin at the postsynaptic membrane of hippocampal pyramidal neurons. Those animals that exhibit the greatest resilience to ABA, in that they run minimally under such condition, thereby losing minimal amount of weight, exhibit the highest level of NR2A-NMDARs in the spine cytoplasm and lowest levels of drebrin at the postsynaptic membrane. This pattern suggests that drebrin has dual roles: retention of NR2A-NMDARs in the reserve pool and trafficking of NR2B-NMDARs to the postsynaptic membrane, ultimately contributing to an individual's reactivity to stress. Altogether, these observations indicate that drebrin is a protein that is important for synaptic plasticity and deserves the attention of neuroscientists studying the neurobiological basis of cognition and stress reactivity.

NR2A-Containing NMDARs in the Prefrontal Cortex Are Required for Working Memory and Associated with Age-Related Cognitive Decline.

Working memory, the ability to hold information "in mind," requires persistent activity of pyramidal neurons in prefrontal cortex (PFC) mediated by NMDA receptor (NMDAR) activation. NMDAR loss in PFC may account for working memory impairments in aging and psychiatric disease. Our studies demonstrate that NMDARs containing the NR2A subunit, but not the NR2B subunit, are required for working memory and that loss of NR2A predicts severity of age-related working memory impairment. The importance of NR2A to working memory is likely due its abundant contribution to pyramidal neuron activity and location at synaptic sites in PFC. This information is useful in designing new therapies to treat working memory impairments by enhancing the function of NR2A-containing NMDARs.

MicroRNA-139-5p negatively regulates NR2A-containing NMDA receptor in the rat pilocarpine model and patients with temporal lobe epilepsy.

Regulation of N-methyl-d-aspartate (NMDA) subunits NR2A and NR2B expression during status epilepticus (SE) remains incompletely understood. Here we explored the role of brain-enriched microRNA (miR)-139-5p in this process. miRNA microarray was performed to examine changes in miRNA expression in the rat pilocarpine model following NMDA-receptor blockade. The dynamic expression patterns of miR-139-5p, NR2A, and NR2B levels were measured in rats during the three phases of temporal lobe epilepsy (TLE) development using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Similar expression methods were applied to hippocampi obtained from patients with TLE and from normal controls. Moreover, miR-139-5p agomir and antagomir were utilized to explore the role of miR-139-5p in determining NMDA-receptor subunit expression patterns. We identified 18 miRNAs that were significantly altered in the rat pilocarpine model following NMDA-receptor blockade. Of these, miR-139-5p was significantly up-regulated and Grin2A was predicted as its potential putative target. In patients with TLE, miR-139-5p expression was significantly down-regulated, whereas NR2A and NR2B levels were significantly up-regulated. In the rat model of SE, miR-139-5p expression was down-regulated while NR2A was up-regulated in the acute and chronic phases, but not in the latent phase. NR2B expression was up-regulated during the three phases of TLE development. Overexpression of miR-139-5p decreased, whereas depletion of miR-139-5p enhanced the expression levels of NR2A, but not NR2B, induced by pilocarpine treatment. Of interest, NMDA nonselective antagonist and NR2A selective antagonist enhanced miR-139-5p levels suppressed by pilocarpine treatment, whereas the NR2B selective antagonist was ineffective. These findings elucidate the potential role of miR-139-5p in NMDA-receptor involvement in TLE development and may provide novel therapeutic targets for the future treatment of TLE.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

NR2A contributes to genesis and propagation of cortical spreading depression in rats.

Cortical spreading depression (CSD) is a transient propagating excitation of synaptic activity followed by depression, which is implicated in migraine. Increasing evidence points to an essential role of NR2A-containing NMDA receptors in CSD propagation in vitro; however, whether these receptors mediate CSD genesis in vivo requires clarification and the role of NR2A on CSD propagation is still under debate. Using in vivo CSD in rats with electrophysiology and in vitro CSD in chick retina with intrinsic optical imaging, we addressed the role of NR2A in CSD. We demonstrated that NVP-AAM077, a potent antagonist for NR2A-containing receptors, perfused through microdialysis probes, markedly reduced cortex susceptibility to CSD, but also reduced magnitude of CSD genesis in rats. Additionally, NVP-AAM077 at 0.3 nmol perfused into the contralateral ventricle, considerably suppressed the magnitude of CSD propagation wave and propagation rate in rats. This reduction in CSD propagation was also observed with TCN-201, a negative allosteric modulator selective for NR2A, at 3 μM, in the chick retina. Our data provides strong evidence that NR2A subunit contributes to CSD genesis and propagation, suggesting drugs selectively antagonizing NR2A-containing receptors might constitute a highly specific strategy treating CSD associated migraine with a likely better safety profile.

Activation of GPR30 attenuates chronic pain-related anxiety in ovariectomized mice

Estrogen regulates neuroendocrine and inflammatory processes that play critical roles in neuroinflammation, anxiety, and chronic pain. Patients suffering from chronic pain often complain of anxiety. However, limited information is available regarding the neural circuitry of chronic pain-related anxiety and the related function of estrogen. Hindpaw injection of complete Freund's adjuvant (CFA) and chronic constriction injury (CCI) of the sciatic nerve induced notable pain sensitization and anxiety-like behavior in ovariectomized (OVX) mice. We found that the level of G-protein-coupled receptor 30 (GPR30), a membrane estrogen receptor, was significantly increased in the basolateral amygdala (BLA) of ovariectomized (OVX) mice suffering from chronic inflammatory and neuropathic pain. Subcutaneous injection or BLA local infusion of the GPR30 agonist G1 significantly reduced anxiety-like behavior in CFA-injected and CCI-OVX mice; however, this treatment did not alter the nociceptive threshold. GPR30 knock down by shRNA in the BLA of OVX mice inhibited the anxiolytic effects of GPR30 activation. G1 administration reversed the upregulation of GluR1 subunit in AMPA and NR2A-containing NMDA receptors and the downregulation of GABAA receptors in the BLA of CFA-injected and CCI-OVX mice. Electrophysiological recording revealed that GPR30 activation could prevent imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected OVX mice. In conclusion, GPR30 activation induced anxiolytic effects but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of GPR30 were partially due to maintaining the balance between excitatory and inhibitory transmissions in the BLA.