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.     

Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones

We investigated the role N -methyl- d -aspartate (NMDA) receptor subunits play in shaping excitatory synaptic currents in cultures of cerebellar granule cells (CGCs) from NR2A knockout (NR2A−/−) and wild-type (+/+) mice. Cultures were maintained in a condition that facilitates the occurrence of functional synapses, allowing us to record NMDA-miniature excitatory postsynaptic currents (mEPSCs) in addition to NMDA receptor-mediated whole-cell currents at three ages in vitro . Whole-cell NMDA current density decreased with development in both strains though currents from NR2A−/− neurones demonstrated greater sensitivity to CP101 606, an NR2B subunit specific blocker. Sensitivity to Mg²⁺ blockade decreased with age in vitro in +/+ but not in NR2A−/− CGCs. Immunocytochemistry revealed that dendrites and somas displayed distinct NR1 and NR2A subunit clusters which became increasingly colocalized in +/+ neurones. Qualitatively the overall NR2B subunit staining pattern was similar in +/+ and NR2A−/− neurones throughout development, suggesting that the NR2B subunit distribution is not mediated by the NR2A subunit. In addition, staining with markers for excitatory synapses showed that expression of NR2A subunit (but not NR2B) increases at both synaptic and extrasynaptic sites in +/+ neurones during development. In parallel, NMDA-mEPSCs were faster in +/+ compared with NR2A−/− neurones at all time points studied, suggesting that the NR2A subunit begins to replace NR2B-rich NMDA receptors even at early stages of development. Many NR2A−/− neurones were devoid of NMDA-mEPSCs at the later time point, and transfection of the NR2A subunit in these neurones restored fast decay and the occurrence of NMDA-mEPSCs. Taken together, our results indicate that the NR2A subunit is mainly responsible for the developmental changes observed in the maturation of excitatory synapses.     

Virally mediated knock-down of NR2 subunits ipsilateral to the deprived eye blocks ocular dominance plasticity

NMDA receptors (NMDARs) are important in developmental plasticity in the visual cortex. The NR2A and NR2B subunits of this receptor develop with different time courses, suggesting that they play different roles in plasticity. To understand the role of the NR2B subunit, we knocked-down NR2B gene expression in visual cortex by injecting a recombinant adenovirus containing an antisense NR2B oligonucleotide. To assess knock-down, we injected the recombinant adenovirus into the right visual cortex of rats (p22) or mice (p30). Eight days later we perfused the animals and processed the visual cortex for NMDAR subunit immunoreactivity (IR). NR2B-IR was depleted dramatically in the neuropil near the injection. Depletion was more modest in the neuronal somata. Surprisingly, NR2A-IR was also reduced, but NR1-IR was not reduced. To assess the functional effects of depletion, we measured ocular dominance plasticity with monocular deprivation (MD). We compared mice receiving the NR2B antisense virus with mice receiving virus containing only the GFP sequence and mice receiving no injection. All injections were between p26 and p29 in the right cortex and bilateral recordings were performed 6–8 days later. Animals receiving the antisense virus lost plasticity if the right eye was deprived. If the left eye was deprived, the cortex was normally plastic bilaterally. Injection of control virus had no effect on plasticity. The data indicate that ocular dominance plasticity requires normal NMDARs in the hemisphere ipsilateral to the deprived eye but not in the hemisphere contralateral to the deprived eye.     

Glutamate-induced currents reveal three functionally distinct NMDA receptor populations in rat dorsal horn - effects of peripheral nerve lesion and inflammation

The importance of the N-methyl-D-aspartate (NMDA) receptor in various painful conditions is well established. The effects of peripheral nerve lesion or joint inflammation, as models of different pain states, on NMDA receptor-mediated currents and NMDA receptor subunit mRNA expression were therefore studied in acutely dissociated neurones from the rat spinal cord dorsal horn. In the neuronal population from control rats, all four NR2 subunits and both NR1 splice variants assayed were detected. A majority of neurones expressed mRNA for more than one NR2 subunit, and some neurones expressed all four NR2 subunits as well as both NR1 splice variants. The NR2B subunit was the most commonly expressed, while the NR2C was the rarest. Following nerve lesion, fewer neurones expressed NR2A compared to the control. The dose-response curve for glutamate-evoked NMDA receptor-mediated currents in the neurones was best described by a three-component fit, suggesting that three functionally distinct NMDA receptor populations are present in the dorsal horn. Minor changes in the dose-response curve after nerve lesion could not be ascribed with certainty to the lesion. Changes in other parameters of NMDA receptor-mediated currents were observed neither after nerve lesion nor after joint inflammation.In summary, the present work demonstrates that single dorsal horn neurones express mRNA for several NMDA receptor subunits. The glutamate dose-response curves indicate that there are three major types of NMDA receptors present in dorsal horn neurones. We also report a reduced expression of NR2A following peripheral nerve lesion.     

Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy

To elucidate characteristic changes in the N -methyl- d -aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2–3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and d , l -2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.     

NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.     

Reelin signaling facilitates maturation of CA1 glutamatergic synapses

Reelin signaling through the low-density lipoprotein receptor family members, apoliproprotein E receptor 2 (apoER2) and very-low-density lipoprotein receptor (VLDLR), plays a pivotal role in dictating neuronal lamination during embryonic brain development. Recent evidence suggests that this signaling system also plays a role in the postnatal brain to modulate synaptic transmission, plasticity, and cognitive behavior, mostly likely due to a functional coupling with N-methyl-d-aspartate (NMDA) receptors. In this study, we investigated the effects of reelin on the maturation of CA1 glutamatergic function using electrophysiological and biochemical approaches. In cultured hippocampal slices, reelin treatment increased the amplitude of AMPAR-mediated miniature excitatory postsynaptic currents and the evoked AMPA/NMDA receptor current ratios. In addition, reelin treatment also reduced the number of silent synapses, facilitated a developmental switch from NR2B to NR2A of NMDARs, and increased surface expression of AMPARs in CA1 tissue. In cultured hippocampal neurons from reeler embryos, reduced numbers of AMPAR subunit GluR1 and NMDAR subunit NR1 clustering were observed compared with those obtained from wild-type embryos. Supplementing reelin in the reeler culture obliterated these genotypic differences. These results demonstrate that reelin- and lipoprotein receptor-mediated signaling may operate during developmental maturation of hippocampal glutamatergic function and thus represent a potential important mechanism for controlling synaptic strength and plasticity in the postnatal hippocampus.     

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.     

Fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the epsilon2 (NR2B) subunit

The N-methyl-D-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the epsilon2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although epsilon2(-/-) mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the epsilon2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from epsilon2(-/-) neurons expressed an NMDA receptor-mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg(2+) or AP5. Whole cell currents from epsilon2(-/-) neurons were also more sensitive to block by low concentrations of Zn(2+) and much less sensitive to the epsilon2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile from epsilon2(-/-) neurons are consistent with the expression of zeta1/epsilon1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the epsilon2 subunit. Thus epsilon1 can substitute for the epsilon2 subunit at synapses and epsilon2 is not required for targeting of NMDA receptors to the postsynaptic membrane.     

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.     

Emergence of brain-derived neurotrophic factor-induced postsynaptic potentiation of NMDA currents during the postnatal maturation of the Kolliker-Fuse nucleus of rat

The Kölliker–Fuse nucleus (KF) contributes essentially to respiratory pattern formation and adaptation of breathing to afferent information. Systems physiology suggests that these KF functions depend on NMDA receptors (NMDA-R). Recent investigations revealed postnatal changes in the modulation of glutamatergic neurotransmission by brain-derived neurotrophic factor (BDNF) in the KF. Therefore, we investigated postnatal changes in NMDA-R subunit composition and postsynaptic modulation of NMDA-R-mediated currents by BDNF in KF slice preparations derived from three age groups (neonatal: postnatal day (P) 1–5; intermediate: P6–13; juvenile: P14–21). Immunohistochemistry showed a developmental up-regulation of the NR2D subunit. This correlated with a developmental increase in decay time of NMDA currents and a decline of desensitization in response to repetitive exogenous NMDA applications. Thus, developmental up-regulation of the NR2D subunit, which reduces the Mg²⁺ block of NMDA-R, causes these specific changes in NMDA current characteristics. This may determine the NMDA-R-dependent function of the mature KF in the control of respiratory phase transition. Subsequent experiments revealed that bath-application of BDNF progressively potentiated these repetitively evoked NMDA currents only in intermediate and juvenile age groups. Pharmacological inhibition of protein kinase C (PKC), as a downstream component of the BDNF–tyrosine kinase B receptor (trkB) signalling, prevented BDNF-induced potentiation of NMDA currents. BDNF-induced potentiation of NMDA currents in later developmental stages might be essential for synaptic plasticity during the adaptation of the breathing pattern in response to peripheral/central commands. The lack of plasticity in neonatal neurones strengthens the hypothesis that the respiratory network becomes permissive for activity-dependent plasticity with ongoing postnatal development.     

PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat

We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro . Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro ( DIV) 7–8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for flag-tagged NR2A subunit clusters than for flag-tagged NR2B subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits.     

Effect of Conantokin G on NMDA receptor-mediated spontaneous EPSCs in cultured cortical neurons

Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to N-methyl-D-aspartate (NMDA) receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. This study was designed to evaluate the action of Con G on NMDA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13-19 days in vitro) using the whole cell patch-clamp technique. Con G inhibited NMDA receptor-mediated sEPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed after application of Con G in the presence of high (10 microM) and nominal (no added) concentrations of glycine was not different at 13 days in vitro (DIV). Furthermore, similar results were obtained with experiments on Con G-induced inhibition of NMDA-evoked whole cell currents. These results indicate that glycine concentrations do not have a direct effect on Con G-induced inhibition of NMDA currents. In addition, age dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.     

Genetic enhancement of inflammatory pain by forebrain NR2B overexpression

N-methyl-D-aspartate (NMDA) receptors contribute to many brain functions. We studied the effect of forebrain-targeted overexpression of the NMDA receptor subunit NR2B on the response of mice to tissue injury and inflammation. Transgenic mice exhibited prominent NR2B expression and enhanced NMDA receptor-mediated synaptic responses in two pain-related forebrain areas, the anterior cingulate cortex and insular cortex, but not in the spinal cord. Although transgenic and wild type mice were indistinguishable in tests of acute pain, transgenic mice exhibited enhanced responsiveness to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant. Genetic modification of forebrain NMDA receptors can therefore influence pain perception, which suggests that forebrain-selective NMDA receptor antagonists, including NR2B-selective agents, may be useful analgesics for persistent pain.     

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.     

Changes in NMDA receptor expression in auditory cortex after learning

Extensive practice on auditory learning tasks dramatically alters the functional organization and response properties of neurons in the auditory cortex. The cellular mechanisms responsible for this auditory learning-induced cortical plasticity are unclear; however, changes in synaptic function involving NMDA receptors have been strongly implicated. To test this hypothesis, we measured the change in gene expression of NMDA receptors and associated proteins in the auditory cortex of adult rats trained to perform an auditory identification task. NMDA receptor 2A and 2B gene expression in auditory cortex decreased significantly as auditory discrimination improved whereas expression of Arc, an immediate early gene involved in memory stabilization, increased. These results suggest that changes in NMDA receptors 2A and 2B and Arc enhance synaptic plasticity, thereby facilitating experience-dependent cortical remodeling and auditory learning.     

Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition

Activation of the tyrosine kinase Src potentiates NMDA-receptor currents, which is thought to be necessary for induction of hippocampal long-term potentiation. Although the carboxy(C)-terminal domain of the NR2A subunit contains potential tyrosine phosphorylation sites, the mechanisms by which Src modulates synaptic plasticity and NMDA receptor currents is not fully understood. Here we present evidence from NR1 mutants and splice variants that Src potentiates NMDA-receptor currents by reducing the tonic inhibition of receptors composed of NR1 and NR2A subunits by extracellular zinc. Using site-directed mutagenesis, we have identified three C-terminal tyrosine residues of NR2A that are required for Src's modulation of the zinc sensitivity of NMDA receptors. Our data link two modulatory sites of NMDA receptors that were previously thought to be independent.     

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.     

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.