Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret.

Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret. The NMDA subtype of glutamate receptor is known to exhibit marked changes in subunit composition and functional properties during neural development. The prevailing idea is that NMDA receptor-mediated synaptic responses decrease in duration after the peak of cortical plasticity in rodents. Accordingly, it is believed that shortening of the NMDA receptor-mediated current underlies the developmental reduction of ocular dominance plasticity. However, some previous evidence actually suggests that the duration of NMDA receptor currents decreases before the peak of plasticity. In the present study, we have examined the time course of NMDA receptor changes and how they correlate with the critical period of ocular dominance plasticity in the visual cortex of a highly binocular animal, the ferret. The expression of NMDA receptor subunits NR1, NR2A, and NR2B was examined in animals ranging in age from postnatal day 16 to adult using Western blotting. Functional properties of NMDA receptors in layer IV cortical neurons were studied using whole cell patch-clamp techniques in an in vitro slice preparation of ferret primary visual cortex. We observed a remarkable increase in NR1 and NR2A, but not NR2B, expression after eye opening. The NMDA receptor-mediated synaptic currents showed an abrupt decrease in decay time concurrent with the increase in NR2A subunit expression. Importantly, these changes occurred in parallel with increased ocular dominance plasticity reported in the ferret. In conclusion, molecular changes leading to decreased duration of the NMDA receptor excitatory postsynaptic current may be a requirement for the onset, rather than the end, of the critical period of ocular dominance plasticity.     

Genetic variants of the NMDA receptor influence cortical excitability and plasticity in humans

N-methyl-d-aspartate (NMDA) receptors play crucial roles in glutamate-mediated synaptic transmission and plasticity and are involved in a variety of brain functions. Specific single nucleotide polymorphisms (SNPs) in the genes encoding NMDA receptor subunits have been associated with some neuropsychiatric disorders involving altered glutamate transmission, but how these polymorphisms impact on synaptic function in humans is unknown. Here, the role of NMDA receptors in the control of cortical excitability and plasticity was explored by comparing the response to single, paired, and repetitive transcranial magnetic stimulations of the motor cortex in 77 healthy subjects carrying specific allelic variants of the NR1 subunit gene (GRIN1 rs4880213 and rs6293) or of the NR2B subunit gene (GRIN2B rs7301328, rs3764028, and rs1805247). Our results showed that individuals homozygous for the T allele in the rs4880213 GRIN1 SNP had reduced intracortical inhibition, as expected for enhanced glutamatergic excitation in these subjects. Furthermore, individuals carrying the G allele in the rs1805247 GRIN2B SNP show greater intracortical facilitation and greater long-term potentiation-like cortical plasticity after intermittent -burst stimulation. Our results provide novel insights into the function of NMDA receptors in the human brain and might contribute to the clarification of the synaptic bases of severe neuropsychiatric disorders associated with defective glutamate transmission.     

Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo

Sensory experience is crucial in the refinement of synaptic connections in the brain during development. It has been suggested that some forms of experience-dependent synaptic plasticity in vivo are associated with changes in the complement of postsynaptic glutamate receptors, although direct evidence has been lacking. Here we show that visual experience triggers the rapid synaptic insertion of new NMDA receptors in visual cortex. The new receptors have a higher proportion of NR2A subunits and, as a consequence, different functional properties. This effect of experience requires NMDA receptor activation and protein synthesis. Thus, rapid regulation of postsynaptic glutamate receptors is one mechanism for developmental plasticity in the brain. Changes in NMDA receptor expression provide a mechanism by which brief sensory experience can regulate the properties of NMDA receptor-dependent plasticity in visual cortex.     

Co-regulation of ocular dominance plasticity and NMDA receptor subunit expression in glutamic acid decarboxylase-65 knock-out mice

Experience can shape cortical circuits, especially during critical periods for plasticity. In visual cortex, imbalance of activity from the two eyes during the critical period shifts ocular dominance (OD) towards the more active eye. Inhibitory circuits are crucial in this process: OD plasticity is absent in GAD65KO mice that show diminished inhibition. This defect can be rescued by application of benzodiazepines, which increase GABAergic signalling. However, it is unknown how such changes in inhibition might disrupt and then restore OD plasticity. Since NMDA dependent synaptic plasticity mechanisms are also known to contribute to OD plasticity, we investigated whether NMDA receptor levels and function are also altered in GAD65KO. There are reduced NR2A levels and slower NMDA currents in visual cortex of GAD65KO mice. Application of benzodiazepines, which rescues OD plasticity, also increases NR2A levels. Thus it appears as if OD plasticity can be restored by adding a critical amount of excitatory transmission through NR2A-containing NMDA receptors. Together, these observations can unify competing ideas of how OD plasticity is regulated: changes in either inhibition or excitation would engage homeostatic mechanisms that converge to regulate NMDA receptors, thereby enabling plasticity mechanisms and also ensuring circuit stability.     

Distinct populations of NMDA receptors at subcortical and cortical inputs to principal cells of the lateral amygdala

Fear conditioning involves the transmission of sensory stimuli to the amygdala from the thalamus and cortex. These input synapses are prime candidates for sites of plasticity critical to the learning in fear conditioning. Because N-methyl-D-aspartate (NMDA)-dependent mechanisms have been implicated in fear learning, we investigated the contribution of NMDA receptors to synaptic transmission at putative cortical and thalamic inputs using visualized whole cell recording in amygdala brain slices. Whereas NMDA receptors are present at both of these pathways, differences were observed. First, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-mediated component of the synaptic response, relative to the NMDA component, is smaller at thalamic than cortical input synapses. Second, thalamic NMDA responses are more sensitive to Mg2+. These findings suggest that there are distinct populations of NMDA receptors at cortical and thalamic inputs to the lateral amygdala. Differences such as these might underlie unique contributions of the two pathways to fear conditioning.     

Development of human visual cortex: a balance between excitatory and inhibitory plasticity mechanisms

Formation of neural circuitry in the developing visual cortex is shaped by experience during the critical period. A number of mechanisms, including N-methyl-D-aspartate (NMDA) receptor activation and gamma-aminobutyric acid (GABA)-mediated inhibition, are crucial in determining onset and closure of the critical period for visual plasticity. Animal models have shown that a threshold level of tonic inhibition must be reached for critical period plasticity to occur and that NMDA receptors contribute to Hebbian synaptic plasticity in the developing visual cortex. There are a number of developmental changes in these glutamatergic and GABAergic mechanisms that have been linked to plasticity; however, those changes have been shown only in animal models, and their development in the human visual cortex is not known. We have addressed this question by studying the expression of the major glutamatergic receptors, GABA(A) receptors, and glutamic acid decarboxylase (GAD) isoforms during the first 6 years of postnatal development of human visual cortex. There are significant changes in the expression of these proteins during postnatal development of human visual cortex. The time course of the changes is quite prolonged and suggests that it may set the pace for the prolonged critical period in human visual development. The changes also affect the nature of spatial and temporal integration in visual cortical neurons and thereby contribute to the maturation of visual functions.     

Ca2+/calmodulin signaling in NMDA-induced synaptic plasticity

Repeated experiences induce a synaptic plasticity in neurons that can be very long lasting. The neurotransmitter, glutamate, acting through N-methyl-D-aspartate (NMDA) receptors is integrally involved in eliciting persistent changes in synaptic function resulting in learning and memory. The permeability of NMDA receptors to Ca2+ implies the close involvement of Ca2+ and the Ca2+-binding protein, calmodulin, in NMDA-induced synaptic plasticity. A notable example of NMDA-induced synaptic plasticity is long-term potentiation in the hippocampal CA1 region. The involvement of Ca2+ and calmodulin in the induction and expression of LTP has been intensively investigated and documented. Less well studied are neurochemical adaptations in another example of NMDA-induced synaptic plasticity, stimulant-induced behavioral sensitization. Although amphetamine and cocaine increase synaptic monoamines, glutamate is involved in the induction and expression of the sensitization. Activating NMDA receptors in dopamine midbrain cell bodies is required for inducing stimulant sensitization, implying a role for Ca2+ in this plasticity. The purpose of this review is to examine the role of Ca2+ and calmodulin in two examples of NMDA-based plasticity, LTP, and stimulant-induced behavioral sensitization. There are similarities in the neuroadaptations, although the role of Ca2+ and calmodulin has not been thoroughly investigated in the stimulant-induced plasticity.     

Effects of stress and adrenalectomy on activity-regulated cytoskeleton protein (Arc) gene expression

Activity-regulated cytoskeletal-associated protein (Arc) is an effector immediate early gene induced by novelty and involved in consolidation of long-term memory. Since activation of glucocorticoid receptors is a prerequisite for memory consolidation, we therefore aimed to study the effect of acute restraint stress on Arc gene expression in adrenalectomized rats. Acute stress produced a significant increase in Arc gene expression in the medial prefrontal cortex, but not in the parietal cortex or in the pyramidal cell layer of the hippocampus. The basal level of Arc mRNA in adrenalectomized animals was high in the medial prefrontal cortex and unaffected by acute stress in these animals. These data are consistent with the role of Arc as an integrative modulator of synaptic plasticity by emphasizing the potential role of stress and glucocorticoids in the control of Arc gene expression.     

Amyloid-beta at sublethal level impairs BDNF-induced arc expression in cortical neurons

Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive dysfunction that probably due to a deficit in synaptic plasticity. One member of neurotrophins, brain-derived neurotrophic factor (BDNF), is known to be involved in the hippocampal long-term potentiation (LTP), a cellular model for learning and memory. Moreover, activity-regulated cytoskeleton-associated gene (Arc), an immediate early gene, is found to be a downstream effector of the BDNF signaling cascade. Inhibition of Arc protein synthesis impairs both the maintenance of LTP and the consolidation of long-term memory. In addition, the formation of senile plaques is a pathological feature in AD and mainly consists of the deposition of amyloid-beta (Abeta), a proteolytic product of amyloid precursor protein. Several studies concerning neurobehavioral performance have suggested that Abeta at sublethal levels interfere with the signaling cascades critical for synaptic plasticity and thus lead to the cognitive impairment in early stage of AD. Whether the BDNF-mediated Arc synthesis is impaired by sublethal Abeta in early AD is still unclear. Therefore, in the present study, primary cultures of neonatal rat cortical neurons were used to evaluate the effect of sublethal Abeta on the BDNF-induced Arc protein expression. Consistent with the literature, Arc, an indicator of synaptic plasticity, was induced by BDNF (25 ng/ml) in both dose- and time-dependent manners. After treating cultures with sublethal Abeta (5 microM), a significant suppression was observed on the level of BDNF-induced Arc protein expression. This result indicates that Abeta at sublethal level impairs the BDNF-mediated signaling in cortical neurons and thus underlies the deficits of synaptic plasticity occurred at the early stage of AD before significant neuronal loss.     

Early APV chronic blocked alters experience-dependent plasticity of auditory spatial representation in rat auditory cortical neurons

Development of auditory function can be affected by environment and experience. In this study, we investigated whether the NMDA receptor mediates the plasticity of auditory spatial representation during development of the rat auditory cortex. We found that early auditory experience significantly increased the auditory spatial sensitivity of A1 neurons and induced training-dependent plasticity. Implantation of Elvax-APV in the auditory cortex gradually reduced the auditory spatial sensitivity of A1 neurons and blocked the auditory spatial plasticity induced by early auditory experience. These results indicate that the NMDA receptor has a key role in experience-dependent plasticity of auditory cortical circuits immediately after birth.     

Regulation of NMDA receptor trafficking by amyloid-beta

Amyloid-beta peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-beta reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-beta promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-beta by treating neurons with a gamma-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-beta application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-beta-dependent endocytosis of NMDA receptors required the alpha-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-beta can cause synaptic dysfunction and contribute to Alzheimer disease pathology.     

Protein kinase CK2 modulates synaptic plasticity by modification of synaptic NMDA receptors in the hippocampus

Synaptic plasticity is the foundation of learning and memory. The protein kinase CK2 phosphorylates many proteins related to synaptic plasticity, but whether it is directly involved in it has not been clarified. Here, we examined the role of CK2 in synaptic plasticity in hippocampal slices using the CK2 selective inhibitors 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and 4,5,6,7-tetrabromobenzotriazole (TBB). These significantly inhibited N -methyl- d -aspartate (NMDA) receptor-dependent long-term potentiation (LTP). DRB also inhibited NMDA receptor-mediated synaptic transmission, while leaving NMDA receptor-independent LTP unaffected. NMDA receptors thus appear to be the primary targets of CK2. Although both long-term depression (LTD) and LTP are induced by the influx of Ca²⁺ through NMDA receptors, surprisingly, LTD was not affected by CK2 inhibitors. We postulated that the LTP-selective modulation by CK2 is due to selective modulation of NMDA receptors, and tested two hypotheses concerning the modulation of NMDA receptors: (i) CK2 selectively modulates NR2A subunits possibly related to LTP, but not NR2B subunits possibly related to LTD; and (ii) CK2 selectively affects synaptic but not extrasynaptic NMDA receptors whose activation is sufficient to induce LTD. DRB decreased NMDA receptor-mediated synaptic transmission in the presence of selective NR2A subunit antagonist. The former hypothesis thus appears unlikely to be correct. However, DRB decreased synaptic NMDA receptor responses in cultured hippocampal neurons without affecting extrasynaptic NMDA receptor current. These findings support the latter hypothesis, that CK2 selectively affects LTP by selective modification of synaptic NMDA receptors in a receptor-location-specific manner.     

Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS

The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.     

Post-synaptic density-93 mediates tyrosine-phosphorylation of the N-methyl-D-aspartate receptors

Src family protein kinases (SFKs) -mediated tyrosine-phosphorylation regulates N-methyl-d-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that post-synaptic density (PSD) -93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4-phorbol 12-myristate 13-acetate in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn.     

Effects of agonists and antagonists of NMDA and ACh receptors on plasticity of bat auditory system elicited by fear conditioning

In big brown bats, tone-specific plastic changes [best frequency (BF) shifts] of cortical and collicular neurons can be evoked by auditory fear conditioning, repetitive acoustic stimuli or cortical electric stimulation. It has been shown that acetylcholine (ACh) plays an important role in evoking large long-term cortical BF shifts. However, the role of N-methyl-d-aspartate (NMDA) receptors in evoking BF shifts has not yet been studied. We found 1) NMDA applied to the auditory cortex (AC) or inferior colliculus (IC) augmented the auditory responses, as ACh did, whereas 2-amino-5-phosphovalerate (APV), an antagonist of NMDA receptors, reduced the auditory responses, as atropine did; 2) although any of these four drugs did not evoke BF shifts, they influenced the development of the long-term cortical and short-term collicular BF shifts elicited by conditioning; 3) like ACh, NMDA augmented the cortical and collicular BF shifts regardless of whether it was applied to the AC or IC; 4) endogenous ACh of the AC and IC is necessary to produce the long-term cortical and short-term collicular BF shifts; 5) blockade of collicular NMDA receptors by APV abolished the development of the collicular BF shift and made the cortical BF shift small and short-term; 6) blockade of cortical NMDA receptors by APV reduced the cortical and collicular BF shifts and made the cortical BF shift short-term; and 7) conditioning with NMDA + atropine applied to the AC evoked the small, short-term cortical BF shift, whereas conditioning with APV + ACh applied to the AC evoked the small, but long-term cortical BF shift.     

Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles

NR2A and NR2B are the predominant NR2 NMDA receptor subunits expressed in cortex and hippocampus. The relative expression level of NR2A and NR2B is regulated developmentally and these two subunits have been suggested to play distinct roles in long-term synaptic plasticity. We have used patch-clamp recording of recombinant NMDA receptors expressed in HEK293 cells to characterize the activation properties of both NR1/NR2A and NR1/NR2B receptors. Recordings from outside-out patches that contain a single active channel show that NR2A-containing receptors have a higher probability of opening at least once in response to a brief synaptic-like pulse of glutamate than NR2B-containing receptors (NR2A, 0.80; NR2B, 0.56), a higher peak open probability (NR2A, 0.50; NR2B, 0.12), and a higher open probability within an activation (NR2A, 0.67; NR2B, 0.37). Analysis of the sequence of single-channel open and closed intervals shows that both NR2A- and NR2B-containing receptors undergo multiple conformational changes prior to opening of the channel, with at least one of these steps being faster for NR2A than NR2B. These distinct properties produce profoundly different temporal signalling profiles for NR2A- and NR2B-containing receptors. Simulations of synaptic responses demonstrate that at low frequencies typically used to induce long-term depression (LTD; 1 Hz), NR1/NR2B makes a larger contribution to total charge transfer and therefore calcium influx than NR1/NR2A. However, under high-frequency tetanic stimulation (100 Hz; > 100 ms) typically used to induce long-term potentiation (LTP), the charge transfer mediated by NR1/NR2A considerably exceeds that of NR1/NR2B.     

Balanced tone-evoked synaptic excitation and inhibition in mouse auditory cortex

The recent characterization of excitatory and inhibitory synaptic receptive fields in rat auditory cortex laid the basis for further investigation of the roles of synaptic excitation and inhibition in cortical computation and plasticity. The mouse is an increasingly important model system because of the wide range of genetic tools available for it. Here we present the first in vivo whole-cell voltage-clamp measurements of synaptic excitation and inhibition in the mouse cortex. We find that a substantial population of auditory cortical neurons receives balanced synaptic excitation and inhibition, whose amplitude ratios and relative time courses remain approximately constant across tone frequency. We conclude that the synaptic mechanisms underlying tone-evoked auditory cortical responses in mice closely resemble those in rats, supporting the mouse as a suitable model for synaptic processing in auditory cortex.     

大鼠听皮质NMDA受体亚单位NR2B蛋白质的年龄-依赖性表达

目的：研究sD大鼠生后发育过程中,听皮质神经元NMDA受体亚单位NR2B蛋白质的表达规律。方法：采用免疫组织化学反应和蛋白质印迹技术,分别检测生后1、2、3周和成年动物听皮质神经元NMDA受体亚单位NR2B蛋白质的表达。结果：NR2B阳性神经元在生后第1周密度最高,随着生后周龄增长,NR2B阳性神经元密度递减．3周龄后降至成年动物的低表达水平。结论：大鼠生后发育过程巾．听皮质NR2B亚单位蛋白质呈现年龄-依赖性表达,此结果与mRNA水平上的表达趋势一致。     

Brain-derived neurotrophic factor activation of extracellular signal-regulated kinase is autonomous from the dominant extrasynaptic NMDA receptor extracellular signal-regulated kinase shutoff pathway

NMDA receptors bidirectionally modulate extracellular signal-regulated kinase (ERK) through the coupling of synaptic NMDA receptors to an ERK activation pathway that is opposed by a dominant ERK shutoff pathway thought to be coupled to extrasynaptic NMDA receptors. In the present study, synaptic NMDA receptor activation of ERK in rat cortical cultures was partially inhibited by the highly selective NR2B antagonist Ro25-6981 (Ro) and the less selective NR2A antagonist NVP-AAM077 (NVP). When Ro and NVP were added together, inhibition appeared additive and equal to that observed with the NMDA open-channel blocker MK-801. Consistent with a selective coupling of extrasynaptic NMDA receptors to the dominant ERK shutoff pathway, pre-block of synaptic NMDA receptors with MK-801 did not alter the inhibitory effect of bath-applied NMDA on ERK activity. Lastly, in contrast to a complete block of synaptic NMDA receptor activation of ERK by extrasynaptic NMDA receptors, activation of extrasynaptic NMDA receptors had no effect upon ERK activation by brain-derived neurotrophic factor. These results suggest that the synaptic NMDA receptor ERK activation pathway is coupled to both NR2A and NR2B containing receptors, and that the extrasynaptic NMDA receptor ERK inhibitory pathway is not a non-selective global ERK shutoff.