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.

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.

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.

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.

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.

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.

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.

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.

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.

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.

Ultrastructural and immunocytochemical characterization of primary afferent terminals in the rat cuneate nucleus.

The cuneate nucleus is a relay center for somatosensory information by receiving tactile and proprioceptive inputs from primary afferent fibers that ascend in the dorsal funiculus. The morphology, synaptic contacts, and neurochemical content of primary afferent terminals in the cuneate nucleus of rats were investigated by combining anterograde transport of horseradish peroxidase conjugated to wheat-germ agglutinin or to cholera toxin (injected in cervical dorsal root ganglia) with postembedding immunogold labeling for glutamate and GABA. Both tracers gave similar results. Two types of terminals were labeled: type I terminals were irregularly shaped, had a mean area of 4.0 microns 2, synapsed on several dendrites, and were contacted by other terminals, some of which were GABA positive. Type II terminals were dome-shaped, had a mean area of 2.18 microns 2, and made synaptic contact on a single dendrite. All the anterogradely labeled terminals (interpreted as endings of primary afferents) were enriched in glutamate but not in GABA. The finding that identified primary afferent terminals are enriched in glutamate with respect to other tissue profiles strongly suggests a neurotransmitter role for glutamate in this afferent pathway to the rat cuneate nucleus.

Ultrastructural and immunocytochemical characterization of terminals of postsynaptic ascending dorsal column fibers in the rat cuneate nucleus.

The morphology, synaptic contacts, and neurotransmitter enrichment of postsynaptic dorsal column terminals in the cuneate nucleus of rats were investigated and compared with those of identified primary afferents. For this purpose, anterograde transport of horseradish peroxidase-based tracers injected in the spinal cord was combined with postembedding immunogold labeling for glutamate and gamma-aminobutyric acid (GABA). Anterogradely labeled postsynaptic dorsal column terminals were morphologically homogeneous: they were small (mean area = 1.37 microns 2) and dome-shaped, contacted single dendritic shafts or cell bodies, and were not involved in axoaxonic synapses. The majority of them were not enriched in glutamate or GABA immunoreactivity compared with other tissue components. Their morphology, size, and neurotransmitter content thus differed from that of primary afferents. These differences are consistent with distinct functional roles for the two main afferent systems ascending to the cuneate nucleus.

Neurons in rat hippocampus that synthesize nitric oxide.

We studied the distribution and light- and electron-microscopic morphology of neurons in the hippocampal formation containing nitric oxide synthase (NOS), and thus likely to release nitric oxide, a freely diffusible neuromediator implicated in long-term potentiation. Only a small fraction of hippocampal neurons contained NOS or its marker, NADPH diaphorase. Most of the positive neurons were in the pyramidal layer of the subiculum, stratum radiatum of Ammon's horn, and subgranular zone of the dentate gyrus. Positive neurons were also conspicuous in the molecular layer of the dentate gyrus and in the pyramidal layer of CA3, sparse in the pyramidal layer of CA2 and CA1, and almost absent from presubiculum and parasubiculum. Numerous positive fibers were seen, especially in stratum radiatum and stratum lacunosum-moleculare of Ammon's horn. Double staining experiments demonstrated that nearly all NADPH diaphorase-positive neurons in the hippocampus also contained gamma-aminobutyric acid. On the basis of their morphology, distribution, and inhibitory neurotransmitter content, most NOS-positive cells in the hippocampus are probably local circuit neurons. These data suggest that nitric oxide in CA1 may function as a paracrine agent, rather than a spatially precise messenger, in long-term potentiation.

Amino acid immunocytochemistry of primary afferent terminals in the rat dorsal horn.

We combined transganglionic tracing methods with postembedding electron microscopic immunocytochemistry to determine whether identified primary afferent fibers terminating in spinal laminae I-IV may use glutamate and aspartate as neurotransmitters. Sciatic injections of wheat-germ agglutinin conjugated to horseradish peroxidase labeled fine afferent fibers with terminals in laminae I-II of the lumbar spinal cord, whereas injections of the B subunit of cholera toxin conjugated to horseradish peroxidase labeled primary afferent terminals in deeper laminae. Many labeled primary afferent terminals in superficial laminae were involved in glomerular synaptic arrangements; others established nonglomerular contacts. Most glomerular arrangements were clearly immunopositive for glutamate, compared with dendrites, astrocytes, or terminals immunopositive for gamma-aminobutyric acid (GABA). The degree of enrichment varied in labeled terminals of different morphological types. Aspartate was enriched, though to a lesser degree than glutamate, in labeled central terminals of glomeruli in superficial laminae. Labeled primary afferent terminals in laminae III-IV were immunopositive for glutamate, though at lower levels than glomerular terminals in superficial laminae. Aspartate was not enriched in these terminals compared with dendrites, glia, and GABA-positive terminals. These results support a neurotransmitter role for glutamate in primary afferents to the dorsal horn. Quantitative differences in the content of glutamate in identified primary afferent terminals may be related to functional differences. Enrichment of aspartate in terminals in superficial but not deep laminae is compatible with a role for this amino acid in sustained, NMDA-mediated phenomena characteristic of activity in fine caliber afferents.

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.

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.

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.

Central release of tracer after noxious stimulation of the skin suggests non-synaptic signaling by unmyelinated fibers.

Injury to a peripheral nerve causes central changes of various nature and complexity reflecting activation of multiple signaling mechanisms. In a previous study we reported that nerve lesion triggers central release of a tracer, wheatgerm-agglutinin conjugated to horse-radish peroxidase, by unmyelinated fibers in the spinal cord. The released tracer occupies the space between nerve terminals and dendrites without extending into the synaptic cleft. We interpreted this to suggest release of unidentified endogenous factor(s) at nonsynaptic sites, which may contribute to the signaling of peripheral injury to the central nervous system. For such signaling to occur, a message must first be communicated along the axon. This message may depend on axonal transport and/or altered electrical activity. In pilot experiments we observed that application of tetrodotoxin (to block impulse conduction) to the intact nerve did not result in tracer release. We hypothesized that the message might be the sustained discharge of C fibers that occurs after injury. We show here that selective activation of C fibers (by applying mustard oil to the hindlimb of anesthetized rats) causes central release of tracer previously transported from the sciatic nerve to superficial laminae of the dorsal horn.

Peripheral injury and anterograde transport of wheat germ agglutinin-horse radish peroxidase to the spinal cord.

Previous observations have revealed labeling in the extracellular space surrounding boutons and unmyelinated fibers in superficial laminae of the spinal cord after injection of the tracer wheat germ agglutinin conjugated to horseradish peroxidase in dorsal root ganglia. The degree of extracellular labeling appeared related to the extent of the damage to the ganglia at the time of the injection. To determine whether injury might produce extracellular labeling, we investigated the effects of unilateral nerve crush or transection on spinal labeling after bilateral injections of the tracer into sciatic nerves. Confirming previous reports, labeling was confined to small dorsal root ganglion cells and to spinal laminae I and II, suggesting a selective affinity of this tracer for unmyelinated fibers. Labeling of both ganglion neurons and superficial spinal laminae was increased on the injured side, probably as a result of increased efficiency of receptor-mediated endocytosis. Electron microscopical observations revealed that the tracer was largely confined to unmyelinated dorsal root fibers bilaterally; a higher percentage of these fibers were labeled on the injured side. In the dorsal horn, the tracer was predominantly within unmyelinated axons and their terminals on the control side, whereas most of the labeling was extracellular and transneuronal on the injured side. The extracellular labeling surrounded unmyelinated fibers and their terminals in the spinal cord, but was excluded from the synaptic cleft. The demonstration that injury is accompanied by significantly increased release of this tracer from the terminals of unmyelinated fibers into the extracellular space suggests that endogenous substances may be released after peripheral lesions as a central signal of injury.