Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses

Channel properties and synaptic targeting of N-methyl-D-aspartate (NMDA) receptors determine their importance in synaptic transmission, long-term synaptic plasticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel properties and synaptic localization, we analyzed gene-targeted mice expressing C-terminally truncated NR2B subunits (NR2B(DeltaC/DeltaC) mice; Sprengel et al. [1998] Cell 92:279-89). Because homozygous NR2B(DeltaC/DeltaC) mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifenprodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B(DeltaC/DeltaC) mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B(DeltaC/DeltaC) mice, although their single channel conductance was unchanged. To study effects on synaptic targeting, we determined the fraction of synaptically localized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B(DeltaC/DeltaC) mice, the synaptic NMDA receptor fraction was drastically reduced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B(DeltaC/DeltaC) mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses.     

Differences in excitatory transmission between thalamic and cortical afferents to single spiny efferent neurons of rat dorsal striatum

The striatum is crucially involved in motor and cognitive function, and receives significant glutamate input from the cortex and thalamus. The corticostriatal pathway arises from diverse regions of the cortex and is thought to provide information to the basal ganglia from which motor actions are selected and modified. The thalamostriatal pathway arises from specific thalamic nuclei and is involved in attention and possibly strategy switching. Despite these fundamental functional differences, direct comparisons of the properties of these pathways are lacking. N‐methyl‐d ‐aspartate (NMDA) receptors at synapses powerfully affect postsynaptic processing, and incorporation of different NR2 subunits into NMDA receptors has profound effects on the pharmacological and biophysical properties of the receptor. Utilization of different NMDA receptors at thalamostriatal and corticostriatal synapses could allow for afferent‐specific differences in information processing. We used a novel rat brain slice preparation preserving corticostriatal and thalamostriatal pathways to medium spiny neurons to examine the properties of NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs) recorded using the whole‐cell, patch‐clamp technique. Within the same neuron, the NMDA/non‐NMDA ratio is greater for excitatory responses evoked from the thalamostriatal pathway than for those evoked from the corticostriatal pathway. In addition, reversal potentials and decay kinetics of the NMDA receptor‐mediated EPSCs suggest that the thalamostriatal synapse is more distant on the dendritic arbor. Finally, results obtained with antagonists specific for NR2B‐containing NMDA receptors imply that NMDA receptors at corticostriatal synapses contain more NR2B subunits. These synapse‐specific differences in NMDA receptor content and pharmacology provide potential differential sites of action for NMDA receptor subtype‐specific antagonists proposed for the treatment of Parkinson’s disease.     

Synaptic localization of NMDA receptor subunits in the rat retina

The distribution and synaptic clustering of N-methyl-D-aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD-95, PSD-93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2; subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals.     

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR1C2' were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits delta 1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the delta 1/2 subunits are mostly localized on amacrine cell processes.     

Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons

Immunoblot analysis, using antibodies against distinct N-methyl-d -aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.     

Development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos and rat pups at birth

We studied the development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos at the 18th day of gestation (E18) and rat pups at birth (P0). Between 7 and 14 days in vitro (DIV), large increases in the amplitudes and frequencies of the spontaneous excitatory postsynaptic currents (EPSCs) of both cultured E18 and P0 neurons were observed. The EPSCs of E18 neurons were mediated primarily by alpha-amino-3-hydroxy-5-methyl-4-iso-xazole-propionic acid (AMPA) receptors at 7 DIV and by both N-methyl-D-aspartate (NMDA) and AMPA receptors at 14 DIV. Consistently, immunostaining indicated significant increases in the proportion of the clusters of NR1, an NMDA receptor subunit, which were associated with the accumulation of synaptophysin, a presynaptic marker, in cultured E18 neurons between 7 and 14 DIV. The proportion of NR1 clusters residing in synaptic regions and the proportion of synapses that colocalized with NR1 clusters in 7-day-old P0 neurons were not different statistically from those found in 7-day-old E18 neurons. However, cultured P0 neurons at 7 DIV displayed clear EPSCs mediated by NMDA receptors. Our results suggest that the targeting of NMDA receptors to synaptic regions lag behind the synaptic clustering of AMPA receptors during the in vitro development of cultured rat E18 cortical neurons. The results further suggest that the cortical neurons at P0 differ from those at E19 in certain cellular properties; as a result, the currents mediated by the synaptic NMDA receptors in 7-day-old P0 neurons are larger than those mediated by the synaptic NMDA receptors in 7-day-old E18 neurons.     

The MUPP1-SynGAPalpha protein complex does not mediate activity-induced LTP

At excitatory synapses of hippocampal neurons, the multi-PDZ domain scaffolding protein, MUPP1, assembles the NR2B subunit of the NMDA receptor (NMDAR), Ca2+-calmodulin kinase (CamKII), and the alpha1 isoform of the postsynaptic density GTPase activating protein, SynGAP (SynGAPalpha). In order to evaluate the role of this complex in excitatory synaptic neurotransmission we specifically disrupted MUPP1-SynGAPalpha interactions in CA1 neurons of acute hippocampal slices using intracellular perfusion with peptides derived from SynGAPalpha-MUPP1 binding domains. Disruption of the interaction between MUPP1 and SynGAPalpha with two complementary peptides derived from SynGAP and MUPP1 mutual binding sites resulted in enhancement of excitatory postsynaptic currents (EPSCs). This potentiation did not occlude pairing-induced long-term potentiation (LTP); indeed the amplitude of postsynaptic responses of activity-potentiated synapses was further increased. Pre-potentiation of excitatory synapses with theta burst stimulations did not modify the MUPP1-SynGAPalpha-dependent enhancement of EPSCs. Our data suggest that MUPP1-SynGAPalpha complex dissociation triggers a mechanism for AMPAR enhancement that is distinct from activity-induced LTP.     

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs

The N-methyl-D-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A-D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4-P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.     

Estradiol potentiation of NR2B‐dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation

The hormone, 17β‐estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR‐dependent long‐term potentiation, and improves hippocampus‐dependent working memory. Smith and McMahon ( 2006 J Neurosci 26:8517–8522) reported that the larger NMDAR‐mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B‐containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B‐dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2‐induced increase in NR2B‐dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR‐dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B‐containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state. © 2010 Wiley‐Liss, Inc.     

Calpain-mediated regulation of NMDA receptor structure and function

Calpains have been previously shown to regulate AMPA receptor properties by producing partial truncation of the C-terminal domains of several receptor subunits. We now report that NMDA receptor subunits, in particular NR2 subunits, are also subjected to calpain-mediated truncation. Treatment of synaptic membranes with calpain I resulted in truncation of both NR1 and NR2 subunits, with the appearance of NR2 species with lower mol.wt. than native subunits, but still recognized by antibodies directed at the C-terminal domain. This treatment did not modify the binding of several ligands of the NMDA receptors, such as glutamate, glycine or TCP. Incubation of thin frozen-thawed brain sections with calcium resulted in calpain-mediated selective degradation of NR2 subunits, as truncation into smaller fragments was totally blocked by calpain inhibitors. Under the same conditions, TCP binding to sections was decreased by about 50%, an effect also blocked by calpain inhibitors. Treatment of hippocampal slices in culture with the excitotoxin, kainic acid, also produced calpain-mediated truncation of the C-terminal domain of NR2 but not NR1 subunits of the NMDA receptors. The results indicate that calpain activation produces several modifications of NMDA receptors, including the truncation of the C-terminal domain of NR2 subunits, and changes in channel binding properties. They suggest that calpain-mediated regulation of NMDA receptors might represent a feed-back regulation of the receptors which could be used to limit receptor activation.     

Calpain-mediated truncation of rat brain AMPA receptors increases their Triton X-100 solubility

Previous studies have indicated that calpain activation results in the truncation of the C-terminal domains of AMPA and NMDA receptor subunits. The present study determined the distribution of the truncated species of the subunits between Triton-soluble and -insoluble fractions. Western blots were performed with various antibodies to quantify the amounts of the various species of GluR1, GluR2, GluR3 and NR2B subunits. The results indicate that calpain activation decreased the amount of all the intact subunits in Triton-insoluble fractions. Calpain-generated truncated forms of GluR1 and GluR2, but not NR2B, were absent in these fractions, and were recovered in Triton-soluble fractions. These findings suggest that calpain-mediated truncation of AMPA but not NMDA receptor C-terminal domains results in modifications of the interactions between the receptors and postsynaptic densities, and that this mechanism could be involved in activity-dependent changes in the subcellular distribution of AMPA receptors.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

Expression of NR2 receptor subunit in rat somatic sensory cortex: synaptic distribution and colocalization with NR1 and PSD-95.

Functional N-methyl-D-aspartate (NMDA) receptors comprise heteromeric combinations of NR1 and NR2 subunits. In the present study, we employed light and electron microscopic immunocytochemistry to study the expression of NR2A and NR2B (NR2A/B) protein in somatic sensory cortex of adult rats. To relate this distribution to that of NR1 and to the NMDA receptor anchoring protein PSD-95, we documented extensive cellular colocalization of NR2A/B with NR1 at the light microscopic level. In contrast, PSD-95 exhibited little somatic staining, being restricted mainly to dendrites and neuropil. We employed postembedding immunocytochemistry to study the ultrastructural expression of NR2A/B. Labeling in neuronal perikarya was associated with rough endoplasmic reticulum and Golgi apparatus; in dendrites, gold particles labeled microtubules. The preponderance of labeling was associated with asymmetric synapses. Double immunolabeling revealed that NR2 colocalized in many synapses with NR1 and with PSD-95. Quantitative measurements revealed that density of gold particles coding for both NR2 and PSD-95 was highest just inside the postsynaptic membrane. Tangentially along the membrane, gold particles were concentrated at the synaptic specialization. These data provide structural evidence in neocortex for heteromeric NMDA receptors anchored at the postsynaptic membrane.     

Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of γ-aminobutyric acid type A (GABA_A) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA_A receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABA_A receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABA_A receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The α2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface–associated staining is therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABA_A receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210, 1998. © 1998 Wiley-Liss, Inc.     

Activity dependent localization of synaptic NMDA receptors in spinal neurons

In cultured spinal neurons, NMDA receptors are absent from excitatory synapses under basal conditions, but can be made to appear at excitatory synapses following blockade of excitatory synaptic activity. The activity dependent synaptic localization of NMDA receptors is critically dependent on both the gradual, global accumulation of the NR2A and NR2B subunits and on a rapid, surface redistribution phase that is primed by the accumulation of NR2A and NR2B and inhibited by synaptic activity. Global changes in NR2A and NR2B accumulation and heterogeneous increases in synaptic NMDA receptor localization can also result from inhibitors of proteasomal processing, from manipulations of proteasomal subunit composition and from media conditioned by neurons undergoing synaptic scaling. While proteasomal processing is a mechanism shared with AMPA receptor scaling in cultured spinal neurons, diffusible factors, heterogeneity, and a rapid surface redistribution phase appear to be unique to activity dependent synaptic NMDA receptor localization.     

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer.

N-methyl-d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca2+ and sensitive to Mg2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca2+-impermeable and Mg2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca2+ permeability and Mg2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.     

A novel form of low-frequency hippocampal mossy fiber plasticity induced by bimodal mGlu1 receptor signaling

Mossy fiber synapses act as the critical mediators of highly dynamic communication between hippocampal granule cells in the dentate gyrus and CA3 pyramidal neurons. Excitatory synaptic strength at mossy fiber to CA3 pyramidal cell synapses is potentiated rapidly and reversibly by brief trains of low-frequency stimulation of mossy fiber axons. We show that slight modifications to the pattern of stimulation convert this short-term potentiation into prolonged synaptic strengthening lasting tens of minutes in rodent hippocampal slices. This low-frequency potentiation of mossy fiber EPSCs requires postsynaptic mGlu1 receptors for induction but is expressed presynaptically as an increased release probability and therefore impacts both AMPA and NMDA components of the mossy fiber EPSC. A nonconventional signaling pathway initiated by mGlu1 receptors contributes to induction of plasticity, because EPSC potentiation was prevented by a tyrosine kinase inhibitor and only partially reduced by guanosine 5'-O-(2-thiodiphosphate). A slowly reversible state of enhanced synaptic efficacy could serve as a mechanism for altering the integrative properties of this synapse within a relatively broad temporal window.     

NMDA receptor subunits GluRε1, GluRε3 and GluRζ1 are enriched at the mossy fibre–granule cell synapse in the adult mouse cerebellum

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRepsilon1 (NR2A), GluRepsilon3 (NR2C), and GluRzeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRepsilon1, GluRepsilon3, and GluRzeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRzeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluRzeta1 mRNA at high levels and high levels of GluRzeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRepsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRepsilon subunits in synaptic localization of GluRzeta1.     

Association of AMPA receptors with a subset of glutamate receptor-interacting protein in vivo.

The NMDA and AMPA classes of ionotropic glutamate receptors are concentrated at postsynaptic sites in excitatory synapses. NMDA receptors interact via their NR2 subunits with PSD-95/SAP90 family proteins, whereas AMPA receptors bind via their GluR2/3 subunits to glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), and protein interacting with C kinase 1 (PICK1). We report here a novel cDNA (termed ABP-L/GRIP2) that is virtually identical to ABP except for additional GRIP-like sequences at the N-terminal and C-terminal ends. Like GRIP (which we now term GRIP1), ABP-L/GRIP2 contains a seventh PDZ domain at its C terminus. Using antibodies that recognize both these proteins, we examined the subcellular localization of GRIP1 and ABP-L/GRIP2 (collectively termed GRIP) and their biochemical association with AMPA receptors. Immunogold electron microscopy revealed the presence of GRIP at excitatory synapses and also at nonsynaptic membranes and within intracellular compartments. The association of native GRIP and AMPA receptors was confirmed biochemically by coimmunoprecipitation from rat brain extracts. A majority of detergent-extractable GluR2/3 was complexed with GRIP in the brain. However, only approximately half of GRIP was associated with AMPA receptors. Unexpectedly, immunocytochemistry of cultured hippocampal neurons and rat brain at the light microscopic level showed enrichment of GRIP in GABAergic neurons and in GABAergic nerve terminals. Thus GRIP is associated with inhibitory as well as excitatory synapses. Collectively, these findings support a role for GRIP in the synaptic anchoring of AMPA receptors but also suggest that GRIP has additional functions unrelated to the binding of AMPA receptors.     

Prenatal exposure to morphine alters kinetic properties of NMDA receptor-mediated synaptic currents in the hippocampus of rat offspring

Whole-cell patch-clamp recordings of pharmacologically isolated N-methyl-D-asparate (NMDA) receptor-mediated evoked excitatory postsynaptic currents (EPSCs) were made, to study whether prenatal exposure to morphine affected functional properties of synaptic NMDA receptors in hippocampal slices of 2-week-old rat offspring from morphine-addicted mothers. The saturated amplitude of synaptic NMDA receptor-mediated EPSCs from morphine-treated offspring was about twofold larger than that from vehicle-control offspring. The apparent dissociation constant (Kd) values of NMDA receptors for Mg2+ at 0 mV were 7.5 +/- 1.4 and 7.9 +/- 1.3 mM in slices from vehicle-control and morphine-treated offspring, respectively. In addition, no distinguishable changes in the voltage-dependent nature and the reversal potential of NMDA receptors occurred in morphine-treated offspring, suggesting no alterations of Mg2+ blockade and ion selectivity to NMDA receptors. The 10-90% rise times of NMDA receptor-mediated EPSCs in morphine-treated offspring became longer than those in vehicle-control offspring. The decay of NMDA receptor-mediated EPSCs in both morphine-treated and vehicle-control offspring could be described by the sum of a fast and a slow exponential function. The slow, but not fast, decay times of synaptic NMDA receptor-mediated currents in morphine-treated offspring became slower than those in vehicle-control offspring. Collectively, these results suggest that prenatal exposure to morphine altered kinetic properties of synaptic NMDA receptors in hippocampal CA1 pyramidal neurons of rat offspring during early life. The extended duration of synaptic NMDA receptor-mediated currents presumably provided more Ca2+ entry through NMDA receptors in morphine-treated offspring, and its further prolongation by depolarization in such young offspring strengthened NMDA receptor-dependent functions. Thus, in light of pathophysiological implications within the central nervous system of morphine-treated offspring during early life, the present study may provide important insights and serve as a basis for therapeutic intervention in conditions under which NMDA receptors become abnormal.