Selective expression of Narp in primary nociceptive neurons: role in microglia/macrophage activation following nerve injury.

Neuronal activity regulated pentraxin (Narp) is a secreted protein implicated in regulating synaptic plasticity via its association with the extracellular surface of AMPA receptors. We found robust Narp immunostaining in dorsal root ganglia (DRG) that is largely restricted to small diameter neurons, and in the superficial layers of the dorsal horn of the spinal cord. In double staining studies of DRG, we found that Narp is expressed in both IB4- and CGRP-positive neurons, markers of distinct populations of nociceptive neurons. Although a panel of standard pain behavioral assays were unaffected by Narp deletion, we found that Narp knockout mice displayed an exaggerated microglia/macrophage response in the dorsal horn of the spinal cord to sciatic nerve transection 3days after surgery compared with wild type mice. As other members of the pentraxin family have been implicated in regulating innate immunity, these findings suggest that Narp, and perhaps other neuronal pentraxins, also regulate inflammation in the nervous system.

Organization of NMDA receptors at extrasynaptic locations.

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors.

Activity-dependent secretion of neuronal activity regulated pentraxin from vasopressin neurons into the systemic circulation.

Neuronal activity regulated pentraxin (Narp) is a secreted, synaptic protein that has been implicated in modulating synaptic transmission. However, it is unclear how Narp secretion is regulated. Since we noted prominent Narp immunostaining in vasopressin neurons of the hypothalamus and in the posterior pituitary, we assessed whether it, like vasopressin, is released into the systemic circulation in an activity-dependent fashion. Consistent with this hypothesis, electron microscopic studies of the posterior pituitary demonstrated that Narp is located in secretory vesicles containing vasopressin. Using affinity chromatography, we detected Narp in plasma and found that these levels are markedly decreased by hypophysectomy. In addition, we confirmed that injection of a viral Narp construct into the hypothalamus restores plasma Narp levels in Narp knockout mice. In checking for activity-dependent secretion of Narp from the posterior pituitary, we found that several stimuli known to trigger vasopressin release, i.e. hypovolemia, dehydration and endotoxin, elevate plasma Narp levels. Taken together, these findings provide compelling evidence that Narp is secreted from vasopressin neurons in an activity-dependent fashion.

Synapses with a segmented, completely partitioned postsynaptic density express more AMPA receptors than other axospinous synaptic junctions.

Axospinous perforated synapses of one morphological subtype exhibit multiple transmission zones, each one being formed by an axon terminal protrusion apposing a postsynaptic density (PSD) segment and separated from others by complete spine partitions. Such segmented, completely partitioned (SCP) synapses have been implicated in synaptic plasticity and postulated to be exceptionally efficacious. The present study explored the validity of this supposition. Postembedding immunogold electron microscopy was used for quantifying the postsynaptic AMPA receptor (AMPAR) expression, which is widely regarded as a major determinant of synaptic efficacy. Various subtypes of axospinous synapses were examined in the rat CA1 stratum radiatum. The results showed that the number of immunogold particles for AMPARs in SCP synapses markedly and significantly exceeded that in other perforated subtypes (by 101% on the average) and in nonperforated immunopositive synapses (by 1086%). Moreover, the particle number per single PSD segment, each of which also contained NMDA receptors, was significantly higher than that per nonperforated PSD (by 485%). SCP synapses also exhibited a higher particle density per unit PSD area, as well as a larger overall PSD area as compared with other synaptic subtypes. Analysis of covariance revealed that the high AMPAR expression in SCP synapses was related to the segmented PSD configuration, not only to the PSD size. Moreover, the subpopulations of SCP and other perforated synapses with either overlapping or equal PSD sizes differed in AMPAR content and concentration, with both measures being significantly higher in SCP synapses. Thus, the elevated AMPAR expression in SCP synapses is associated with the presence of separate PSD segments, not only with their large PSD area. These findings are consistent with the idea that SCP synapses have a relatively greater efficacy and may support maximal levels of synaptic enhancement characteristic of certain forms of synaptic plasticity such as the early LTP phase.

Early events in the trafficking of N-methyl-D-aspartate (NMDA) receptors.

The N-methyl-D-aspartate (NMDA) receptor plays a central role at excitatory synapses where it has been implicated in multiple functions associated with synaptic plasticity. While this receptor has been intensely studied with respect to its physiology and pharmacology, its cell-biological properties, such as subunit assembly, post-translational processing and trafficking in neurons, are only beginning to be addressed. Critical to many of the functions of the NMDA receptor are the multiple proteins with which it interacts. While these interactions have been most thoroughly studied with respect to the receptor at the synapse, the same proteins may also interact with the receptor much earlier in its biosynthetic pathway and play important roles in receptor trafficking from the endoplasmic reticulum to the synapse.

Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells.

Sensory (hair) cells of the inner ear contain two specialized areas of membrane delivery. The first, located at the cell base, is the afferent synapse where rapid delivery of synaptic vesicles is required to convey information about auditory signals with exceedingly high temporal precision. The second area is at the apex. To accommodate the continuous movement of stereocilia and facilitate their repair, recycling of membrane components is required. Intense vesicular traffic is restricted to a narrow band of cytoplasm around the cuticular plate, which anchors stereocilia. Our previous analyses showed that SNARE proteins (syntaxin 1A/SNAP25/VAMP1) are concentrated at both poles of hair cells, consistent with their involvement in membrane delivery at both locations. To investigate further the molecules involved in membrane delivery at these two sites, we constructed a two-hybrid library of the organ of Corti and probed it with syntaxin 1A. Here we report the cloning of a novel syntaxin-binding protein that is concentrated in a previously uncharacterized organelle at the apex of inner hair cells.

Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway.

The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

Glutamate receptor targeting in the postsynaptic spine involves mechanisms that are independent of myosin Va.

Targeting of glutamate receptors (GluRs) to synapses involves rapid movement of intracellular receptors. This occurs in forms of synaptic upregulation of receptors, such as long-term potentiation. Thus, many GluRs are retained in a cytoplasmic pool in dendrites, and are transported to synapses for upregulation, presumably via motor proteins such as myosins travelling along cytoskeletal elements that extend up into the spine. In this ultrastructural immunogold study of the cerebellar cortex, we compared synapses between normal rats/mice and dilute lethal mutant mice. These mutant mice lack myosin Va, which has been implicated in protein trafficking at synapses. The postsynaptic spine in the cerebellum lacks the inositol trisphosphate receptor (IP3R) -laden reticular tubules that are found in normal mice and rats (Takagishi et al., Neurosci. Lett., 1996, 215, 169). Thus, we tested the hypothesis that myosin Va is necessary for transport of GluRs and associated proteins to spine synapses. We found that these spines retain a normal distribution of (i) GluRs (delta 1/2, GluR2/3 and mGluR1alpha), (ii) at least one associated MAGUK (membrane-associated guanylate kinase) protein, (iii) Homer (which interacts with mGluR1alpha and IP3Rs), (iv) the actin cytoskeleton, (v) the reticulum-associated protein BiP, and (vi) the motor-associated protein, dynein light chain. Thus, while myosin Va may maintain the IP3R-laden reticulum in the spine for proper calcium regulation, other mechanisms must be involved in the delivery of GluRs and associated proteins to synapses. Other possible mechanisms include diffusion along the extrasynaptic membrane and delivery via other motors running along the spine's actin cytoskeleton.

Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea.

PDZ-domain containing proteins of the MAGUK (membrane-associated guanylate kinase) family target, anchor, and cluster receptors and channels to subcellular sites. Among the MAGUK proteins, the members of the PSD-95 family (MAGUKs: PSD-95, PSD-93, SAP-97, and SAP-102) target and anchor glutamate receptors to the synaptic terminals. Associations of glutamate receptors with MAGUKs have been described in the brain but not in the cochlea. In this study, RT-PCR, immunofluorescence microscopy, and immunoelectron microscopy were used to investigate the presence and distribution of MAGUK proteins in the organ of Corti. The presence of the mRNA for PSD-95, PSD-93, SAP-97, and SAP-102 in the organ of Corti was confirmed by RT-PCR. Immunocytochemistry using a "pan-MAGUK" antibody, which recognizes all four MAGUK proteins, and selective antibodies against these proteins revealed that all four MAGUKs are present within the base of inner hair cells while all except SAP-97 are found within the base of the outer hair cells. In addition, PSD-93 and PSD-95 are found in postsynaptic afferent terminals on inner hair cells, while postsynaptic afferent terminals on outer hair cells have PSD-93.

PSD-93 knock-out mice reveal that neuronal MAGUKs are not required for development or function of parallel fiber synapses in cerebellum.

Membrane-associated guanylate kinases (MAGUKs) are abundant postsynaptic density (PSD)-95/discs large/zona occludens-1 (PDZ)-containing proteins that can assemble receptors and associated signaling enzymes at sites of cell-cell contact, including synapses. PSD-93, a postsynaptic neuronal MAGUK, has three PDZ domains that can bind to specific ion channels, including NMDA delta2 type glutamate receptors, as well as Shaker and inward rectifier type K(+) channels, and can mediate clustering of these channels in heterologous cells. Genetic analyses of Drosophila show that MAGUKs play critical roles in synaptic development because mutations of discs large disrupt the subsynaptic reticulum and block postsynaptic clustering of Shaker K(+) channels. It is uncertain whether MAGUKs play an essential role in the development of central synapses. There are four neuronal MAGUKs with overlapping expression patterns in the mammalian brain; however, we find PSD-93 is the only MAGUK expressed in cerebellar Purkinje neurons. Therefore, we targeted disruption of PSD-93 in mouse. Despite the absence of MAGUK immunoreactivity in Purkinje neurons from the knock-outs, these mice have no structural or functional abnormality in cerebellum. Both the dendritic architecture and the postsynaptic localization of PSD-93 interacting proteins remain intact at light and electron microscopic levels in the knock-outs. Postsynaptic Purkinje cell responses, monosynaptic climbing fiber innervation, and cerebellar-dependent behaviors are also normal. Our data demonstrate that MAGUK proteins of the PSD-93/95 family are not essential for development of certain central synapses but may instead participate in specialized aspects of synaptic signaling and plasticity.

Differential distribution of glutamate receptors in the cochlear nuclei.

Glutamate receptors are the major excitatory neurotransmitter receptors of the mammalian central nervous system, and include AMPA, kainate, delta, NMDA, and metabotropic types. In the cochlear nucleus (CN), the AMPA receptor subunits GluR2-4 are found in major kinds of neurons, while GluR1 subunit distribution is more restricted. GluR2 is low in the anteroventral CN, suggesting that many AMPA receptors here are calcium-permeable. Delta receptors are most prevalent in cartwheel cells in the dorsal CN. Of the NMDA receptors, NR1 is widespread while the NR2 subunits show more restricted distributions. Of the metabotropic glutamate receptors, mGluR1alpha is most prevalent in the dorsal CN, and mGluR2 is concentrated in Golgi cells and unipolar brush cells. AMPA receptors in endbulb synapses in the anteroventral CN are mainly GluR3+4 complexes: probably an adaptation for rapid auditory neurotransmission. Glutamate receptors are differentially distributed in synapses of fusiform cells of the dorsal CN; GluR4 and mGluR1alpha are present only at basal dendrite synapses (auditory nerve), while other glutamate receptors occupy both apical and basal synapses. Analysis of cytoplasmic distribution suggests that a selective targeting mechanism may restrict movement of GluR4 and mGluR1alpha to basal dendrites, although other targeting mechanisms may be present.

A developmental change in NMDA receptor-associated proteins at hippocampal synapses.

The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.

Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

Characterization of the glutamate receptor-interacting proteins GRIP1 and GRIP2.

The molecular mechanisms underlying the targeting and localization of glutamate receptors at postsynaptic sites is poorly understood. Recently, we have identified a PDZ domain-containing protein, glutamate receptor-interacting protein 1 (GRIP1), which specifically binds to the C termini of AMPA receptor subunits and may be involved in the synaptic targeting of these receptors. Here, we report the cloning of GRIP2, a homolog of GRIP1, and the characterization of the GRIP1 and GRIP2 proteins in the rat CNS. GRIP1 and GRIP2 are approximately 130 kDa proteins that are highly enriched in brain. GRIP1 and GRIP2 are widely expressed in brain, with the highest levels found in the cerebral cortex, hippocampus, and olfactory bulb. Biochemical studies show that GRIP1 and GRIP2 are enriched in synaptic plasma membrane and postsynaptic density fractions. GRIP1 is expressed early in embryonic development before the expression of AMPA receptors and peaks in expression at postnatal day 8-10. In contrast, GRIP2 is expressed relatively late in development and parallels the expression of AMPA receptors. Immunohistochemistry using the GRIP1 antibodies demonstrated that GRIP1 is expressed in neurons in a somatodendritic staining pattern. At the ultrastructural level, DAB and immunogold electromicroscopy studies showed that GRIP1 was enriched in dendritic spines near the postsynaptic density and was expressed in dendritic shafts and in peri-Golgi regions in the neuronal soma. GRIP1 appeared to be clustered at both glutamatergic and GABAergic synapses. These results suggest that GRIP1 and GRIP2 are AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses. GRIP1 also may play functional roles at both excitatory and inhibitory synapses, as well as in early neuronal development.

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.

Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp.

Narp (neuronal activity-regulated pentraxin) is a secreted immediate-early gene (IEG) regulated by synaptic activity in brain. In this study, we demonstrate that Narp possesses several properties that make it likely to play a key role in excitatory synaptogenesis. Narp is shown to be selectively enriched at excitatory synapses on neurons from both the hippocampus and spinal cord. Overexpression of recombinant Narp increases the number of excitatory but not inhibitory synapses in cultured spinal neurons. In transfected HEK 293T cells, Narp interacts with itself, forming large surface clusters that coaggregate AMPA receptor subunits. Moreover, Narp-expressing HEK 293T cells can induce the aggregation of neuronal AMPA receptors. These studies support a model in which Narp functions as an extracellular aggregating factor for AMPA receptors.

Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.

To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.