Vanilloid receptor TRPV1-mediated phosphorylation of ERK in murine adjuvant arthritis.

OBJECTIVE: The vanilloid receptor transient receptor potential vanilloid 1 (TRPV1), expressed by sensory neurons that innervate joints, is implicated in arthritis but the mechanisms are not fully understood. One possibility is that downstream effects of activation of TRPV1 are mediated by the extracellularly-regulated kinase (ERK). ERK is phosphorylated (p-ERK) in sensory neurons in response to noxious stimuli and its inhibition has been found to be antinociceptive in several pain models. We here wanted to ascertain whether TRPV1 may contribute to the pain hypersensitivity and inflammation of arthritis via an ERK-mediated pathway. METHODS: We used a model of adjuvant-induced arthritis (AIA) of the ankle and investigated the changes in expression of p-ERK in sensory afferent neurons in dorsal root ganglia (DRG) and spinal dorsal horn of TRPV1-knockout (KO) mice, compared to wild-type (WT) mice of the same genetic background, using multiple immunofluorescence. RESULTS: Two to three weeks after inducing AIA in mice, the number of neurons in DRG and spinal cord that expressed p-ERK was significantly higher on the side of AIA than on the contralateral, vehicle-injected side. The fraction of p-ERK-positive neurons in the DRG that also expressed TRPV1 was increased, indicating that activation of ERK occurred preferentially in TRPV1-positive neurons. Moreover, TRPV1-KO mice had reduced activation of ERK in sensory neurons, compared to WT mice. These changes in expression of p-ERK correlated with changes in pain behavior and joint histopathology: TRPV1-KO mice had reduced nociceptive behavior and severity of arthritis, compared to WT mice. CONCLUSION: Our results support the idea that activation of ERK in primary afferent neurons is mediated, at least in part, by TRPV1. In the absence of TRPV1, the signs of arthralgia and histopathology in the mouse model of AIA are reduced. We conclude that TRPV1, expressed by neurons in the articular afferent pathway, contributes to the pathogenesis of arthritis via an ERK-mediated pathway.

Acute inflammatory and neuropathic pain in Lewis and Fischer rats.

Inbred, histocompatible Lewis and Fischer 344 rats (LEW and FIS) have been used to identify an inverse relationship between hypothalamic-pituitary-adrenal (HPA) axis activity and susceptibility to autoimmune and chronic inflammatory disorders, with LEW showing blunted HPA axis activity and increased susceptibility toward the development of autoimmunity and chronic inflammation, and FIS showing the opposite relationship. In the present study, LEW and FIS were used to determine the relationship between HPA axis function and acute inflammatory pain (carrageenan-induced hindpaw inflammation) and neuropathic pain (partial sciatic nerve ligation; PSNL). The results showed that carrageenan-induced thermal and mechanical allodynia and hyperalgesia were greater in FIS than in LEW. Similarly, FIS showed more carrageenan-induced hindpaw swelling and higher levels of myeloperoxidase (a measure of neutrophils) in the carrageenan-inflamed hindpaw. After PSNL, LEW showed a profound mechanical allodynia and hyperalgesia, whereas mechanical sensitivity in FIS was unaltered. However, FIS, but not LEW, developed thermal allodynia and hyperalgesia after PSNL. These results provide strong evidence for a positive relationship between HPA axis activity and acute inflammatory pain. The results also support a relationship between HPA axis activity and neuropathic pain, but the relationship is complex and may depend on the pain assay.

Vanilloid receptor VR1-positive afferents are distributed differently at different levels of the rat lumbar spinal cord.

The vanilloid receptor VR1 renders a group of primary afferents that express it sensitive to noxious heat and capsaicin, and is thus an important marker for nociceptors. We use double immunofluorescence and confocal microscopy to show that the density of VR1-positive fibers and boutons in the dorsal horn increases progressively from spinal segments L4 to L6 and that the colocalization of VR1 with the neuropeptide substance P (SP) in lamina I and along the lateral collateral path, where the majority of visceral afferents terminate, is negligible at L4, but substantial at L6. We conclude that VR1 is expressed by visceral afferents to the lower lumbar spinal cord in the rat, which also express SP.

Biochemical and morphological characterization of an intracellular membrane compartment containing AMPA receptors.

AMPA receptors cycle rapidly in and out of the postsynaptic membrane, while NMDA receptors are relatively immobile. Changing the distribution of AMPA receptors between intracellular and surface synaptic pools is an important means of controlling synaptic strength. However, little is known about the intracellular membrane compartments of neurons that contain AMPA receptors. Here we describe biochemical and morphological characteristics of an intracellular pool of AMPA receptors in rat brain. By velocity gradient centrifugation of microsomal light membranes from rat brain, we identified a membrane fraction enriched for AMPA receptor subunits GluR2/3 but lacking NMDA receptors. This membrane compartment sedimented more slowly than synaptosomes but faster than synaptic vesicles and cofractionated with GRIP, PICK-1 and syntaxin-13. Morphological examination of this fraction revealed round and tubular vesicles ranging from approximately 50 to 300 nm in diameter. Immunocytochemistry of cultured hippocampal neurons showed that a significant portion of AMPA receptors colocalized with syntaxin-13 (a SNARE protein associated with tubulovesicular recycling endosomes) and with transferrin receptors. Taken together, these results suggest that a pool of intracellular GluR2/3 resides in a syntaxin 13-positive tubulovesicular membrane compartment, which might serve as a reservoir for the dendritic recycling of AMPA receptors.

Kainate receptors in primary afferents to the rat gracile nucleus.

In a previous work, we demonstrated that under weak paraformaldehyde fixation, kainate receptors (KR) (GluR5/6/7) are expressed in primary afferent terminals in superficial dorsal horn. We extended our study to primary afferents to the gracile nucleus; immunostaining for GluR5/6/7 in weakly fixed sections was in puncta of variable size. In double-stained sections, the majority of immunostained puncta colocalized with synaptophysin. Because of their large size and relations with smaller puncta, single-stained for synaptophysin, these terminals were presumed to be of dorsal column primary afferents. This was confirmed by anterograde labeling with cholera toxin B and with electron microscopy, which showed that GluR5/6/7 was present in terminals with morphology of primary afferents. These observations demonstrate that expression of presynaptic KR is a general feature of primary afferents with different functional properties.

A role for the cytoskeleton-associated protein palladin in neurite outgrowth.

The outgrowth of neurites is a critical step in neuronal maturation, and it is well established that the actin cytoskeleton is involved in this process. Investigators from our laboratory recently described a novel protein named palladin, which has been shown to play an essential role in organizing the actin cytoskeleton in cultured fibroblasts. We investigated the expression of palladin in the developing rat brain by Western blot and found that the E18 brain contained a unique variant of palladin that is significantly smaller (approximately 85 kDa) than the common form found in other developing tissues (90-92 kDa). Because the expression of a tissue-specific isoform suggests the possibility of a cell type-specific function, we investigated the localization and function of palladin in cultured cortical neurons. Palladin was found preferentially targeted to the developing axon but not the dendrites and was strongly localized to the axonal growth cone. When palladin expression was attenuated by transfection with antisense constructs in both the B35 neuroblastoma cell line and in primary cortical neurons, a reduction in the expression of palladin resulted in a failure of neurite outgrowth. These results implicate palladin as a critical component of the developing nervous system, with an important role in axonal extension.

Palladin is expressed preferentially in excitatory terminals in the rat central nervous system.

Palladin is a recently described intracellular protein associated with the actin cytoskeleton and cell adhesion in fibroblasts. In Western and Northern blot analyses, palladin expression is ubiquitous in embryonic mice, but it is down-regulated dramatically in most adult tissues. Significant amounts of palladin persist in the brain of adult rodents, as assessed by Western blot analysis. With this work, we extend preliminary observations and determine the overall distribution and subcellular location of palladin throughout the rat brain. In sagittal and coronal sections of the central nervous system, immunostain for palladin is present throughout the brain and spinal cord, but not uniformly. The densest regions of immunostain include the olfactory bulb, cerebral and cerebellar cortex, hippocampus, amygdala, superior colliculus, and superficial laminae of the spinal dorsal horn. Because immunostain characteristically is punctate, we performed double staining for palladin and the presynaptic marker synaptophysin. Confocal microscopy showed that palladin-immunopositive puncta are also immunopositive for synaptophysin; the proportion of synaptophysin-immunopositive puncta that also stained for palladin ranged from 100% of mossy fiber terminals in field CA3 of the hippocampus and in the cerebellar cortex to 60--70% of terminals in the cerebral cortex, striatum, and spinal dorsal horn. The presence of palladin in synaptic terminals was confirmed by electron microscopy. Because immunostained terminals commonly establish asymmetric synapses, the selectivity of palladin expression in synaptic terminals was tested by double staining for palladin and gamma-aminobutyric acid. The modest level of colocalization in this material at both the light microscopic and electron microscopic levels suggests a selectivity of palladin for terminals that release excitatory neurotransmitters. As concomitant work in cell cultures has shown that palladin participates in axonal development and migration, the present results suggest that palladin persists at excitatory synapses of the adult nervous system.

Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superficial laminae of the rat dorsal horn.

Terminals in the rat spinal cord that express the vanilloid receptor VR1 are from small and medium dorsal root ganglion (DRG) neurons and appear prominent in lamina I and inner lamina II. Because primary afferents from these neurons can be myelinated or unmyelinated and their terminals in these laminae can be of various morphological and functional types, we undertook this study to identify the type(s) of VR1-positive afferent fibers and terminals. In the DRG, many small and medium-sized neurons are immunopositive. Under electron microscopy, dorsal root afferents that are immunopositive for VR1 are predominantly unmyelinated. Large numbers of VR1-positive terminals in lamina I are of the nonglomerular type and may contain dense core vesicles. VR1 immunoreactivity in terminals in lamina I is in good agreement with data on noxious, heat-sensitive neurons in the dorsal horn. Two types of glomerular afferent terminals in lamina II also are immunopositive for VR1. In both laminae, most VR1-positive terminals are distinct from substance P-positive terminals. However, the immunoreactivity in lamina II also is prominent in dendrites that are contacted by primary afferent endings. Because we also observed patchy immunostaining in cell bodies in lamina II, this unexpected result may reflect synthesis of VR1 by neurons in this lamina. However, because dorsal rhizotomy abolishes VR1 staining in both laminae I and II, it is suggested that the expression and intracellular dynamics of VR1 in lamina II neurons are controlled by presynaptic input.

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord.

Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms.

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

Laminar organization of the NMDA receptor complex within the postsynaptic density.

The NR2 subunit is an essential component of the NMDA receptor. Recent biochemical research has identified a number of molecules that can bind directly or indirectly to its cytoplasmic tail. These postsynaptic density (PSD) proteins play a role in intracellular signal transduction, and are implicated in synaptic plasticity and memory mechanisms. We performed systematic electron microscopic immunogold analysis in rat neocortex to determine the spatial organization of NR2, in relation to six other proteins thought to be involved in the NMDA receptor complex. Peak concentrations of each protein were within the PSD but in different "layers" of the density. In the axodendritic axis, gold particles coding for PSD-95 lay an average of 12 nm cytoplasmic to the extracellular face of the plasma membrane, very close to the C terminal of NR2. Nitric oxide synthase lay 18 nm inside the membrane; the scaffolding proteins guanylate kinase-associated protein and Shank lay 24-26 nm inside the membrane; and CRIPT and dynein light chain, proteins that may link the complex to cytoskeletal elements, lay on the cytoplasmic side of the PSD, 29-32 nm inside the plasma membrane and extending into the spine cytoplasm. The supramolecular organization of these molecules may modulate intracellular transduction of NMDA-mediated signals.

SAP97 concentrates at the postsynaptic density in cerebral cortex.

SAP97, a PDZ-containing protein, is reported to concentrate in axon terminals, where its function remains unknown. Using highly specific new antibodies, we show that SAP97 in rat cerebral cortex is associated with heteromeric AMPA receptors via a selective biochemical interaction between SAP97 and the GluR1 subunit. Using light and electron microscopic immunocytochemistry, we demonstrate cellular and synaptic colocalization of SAP97 and GluR1, and show that SAP97 concentrates at synapses that contain GluR1 but not necessarily GluR2 or GluR3. Using quantitative postembedding immunogold electron microscopy, we find that SAP97 is at highest concentration within the postsynaptic density of asymmetric synapses. These data suggest that SAP97 may help to anchor GluR1-containing AMPA receptors at the synapse. As a multifunctional scaffolding protein, SAP97 may organize components of AMPA-related intracellular signalling pathways, including those associated with calcium-permeable homomeric GluR1 channels.

Nitric oxide synthase-containing projections to the ventrobasal thalamus in the rat.

Microiontophoretic studies of thalamic neurons suggests that nitric oxide (NO) plays an important role in mediating somatosensory transmission. The thalamus contains few nitric oxide synthase (NOS)-immunoreactive neurons; thus, the major source of thalamic NO is presumably from NOS-positive axons of extrathalamic origin. The cells of origin of these putative NOS-containing pathways to the ventrobasal thalamus were investigated in rats by combining retrograde tracing with immunocytochemistry for NOS. The location and morphology of double-labeled neurons was compared with that of single-labeled neurons. The most significant sources of NOS-containing afferents to the thalamus were found to be the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei. NOS-immunoreactive neurons in these cholinergic nuclei project bilaterally to the thalamus, most strongly ipsilaterally. The thalamus appears to be a major target of PPN, since even selective thalamic injections result in retrograde labeling of at least one third of its NOS-immunoreactive neurons. A significant number of NOS-negative neurons in both the PPN and LDT also project to the thalamus. Minor sources of NOS-containing thalamic afferents include the lateral hypothalamus, the dorsal, median and pontine raphe nuclei, the parabrachial nuclei, and the pontomedullary reticular formation. In all these structures, NOS-negative thalamopetal neurons greatly outnumber the NOS-positive ones. Ascending sensory pathways to the thalamus, including those from the sensory trigeminal nuclei, the dorsal column nuclei, and the spinal cord, as well as the auditory and vestibular centers, arise exclusively from NOS-negative neurons. The major NOS-positive projections are implicated in affective and alerting systems, supporting that NO may act to modulate attentiveness in thalamic relay nuclei.

Characterization of glutamate receptor interacting protein-immunopositive neurons in cerebellum and cerebral cortex of the albino rat.

Glutamate receptor interacting protein (GRIP) binds to the C-terminus of the glutamate receptor 2 (GluR2) subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in vitro and may play an important role in the synaptic organization of these receptors. To determine the distribution of GRIP in vivo, GRIP was localized immunocytochemically in cerebellum and cerebral cortex of adult Sprague-Dawley rats. In the cerebellar cortex, GRIP staining was prominent in perikarya and proximal dendrites of Purkinje cells, whereas Golgi cells were stained more weakly. Double labeling revealed that GRIP and GluR2 were colocalized in Purkinje cells but not in Golgi cells. In the cerebral cortex, GRIP-stained dendrites and somata of nonpyramidal neurons were scattered throughout cortical layers, whereas pyramidal cells were only weakly immunopositive. GRIP was especially prominent in a subset of GluR2-containing cells that also expressed a high level of GluR1. The large majority of strongly GRIP-positive cells in neocortex were immunopositive for gamma-aminobutyric acid (GABA), including the overwhelming majority of calbindin-positive cells in superficial cortical layers, most of the parvalbumin-positive cells, and half of the calretinin-positive interneurons. Staining in the neuropil became more punctate after antigen was unmasked with proteinase K. Electron microscopic localization in the cerebral cortex by postembedding immunogold showed that somatic GRIP was associated with rough endoplasmic reticulum and Golgi apparatus. GRIP was seen over the postsynaptic density of axospinous and axodendritic asymmetric synapses and at high levels in dendrites of GABA-positive neurons. The present data support a role for GRIP in anchoring AMPA receptors and suggest that GRIP trafficking may be especially active in GABAergic neurons.

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.

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.

Metabotropic glutamate receptors in superficial laminae of the rat dorsal horn.

Light and electron microscopic immunocytochemistry were employed here to show the distribution of metabotropic glutamate receptors (mGluRs) mGluR2/3 and mGluR5 in laminae I and II of the dorsal horn, to identify their pre- and postsynaptic location, and to test colocalization with gamma-aminobutyric acid (GABA). mGluR2/3 was mainly in the inner part of lamina II; mGluR5 was mainly in laminae I and II. Electron microscopy showed that both mGluR2/3 and mGluR5 were in perikarya, dendrites, and vesicle-containing profiles. Two main morphological types of primary afferent terminals can be distinguished in the superficial laminae: C1, likely to be endings of unmyelinated fibers, and C2, of small myelinated fibers. Quantitative data show that only a small fraction of C2s stained for either receptor; more common were immunopositive dendrites postsynaptic to these terminals, and most common were appositions between C2s and mGluR5 immunopositive dendrites. Vesicle-containing profiles were characteristically apposed to primary afferent terminals, mainly C2s. Immunopositivity for mGluRs, especially mGluR2/3, was present in vesicle-containing profiles apposed to C2, none to C1, and about half of the profiles immunostained for either receptor were also stained for GABA. The presence of presynaptic and postsynaptic mGluRs in both inhibitory and excitatory interneurons may contribute to complex processing of fast and slow responses to peripheral input in superficial laminae. As selective agonists of mGluRs may modulate GABA release, the present demonstration of mGluRs in GABAergic terminals of presumed interneurons suggests that facilitatory effects may involve a mechanism of disinhibition.

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90.

The synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses.

Colocalization of GABA and glycine in the rat dorsal column nuclei.

About half the neurons in the rat dorsal column nuclei were immunopositive for glycine or for GABA; these were smaller than immunonegative neurons. In double-stained material, 29% of stained neurons were immunopositive for GABA only, and 42% for both antigens. The results resemble those reported for spinal cord laminae that receive fast-conducting primary afferents, and suggest that glycine is an inhibitory neurotransmitter in the dorsal column nuclei.