Vanilloid receptor TRPV1-positive sensory afferents in the mouse ankle and knee joints

TRPV1, a cation channel on sensory nerves sensitive to heat and capsaicin, plays an important role in the transduction of noxious stimuli to the spinal cord. It is expressed by neurons in dorsal root ganglia (DRG) that may also express neuropeptides, which are important for the development of inflammation. Mice with genetic deletion of TRPV1 have been used to study the involvement of this receptor in the mediation of pain and inflammation in animal models of arthritis. However, the expression of TRPV1 in the mouse articular afferents has not been studied. We here provide numerical data on expression of TRPV1 in an identified population of sensory afferents to the mouse L3–L5 DRG that innervate joints, in comparison with that from bladder and skin. A combination of tracing and immunocytochemistry revealed that TRPV1-positive fibers innervate the mouse knee and ankle. At the level of DRG, ~40% of articular afferents from these joints express TRPV1 and the majority of them are peptidergic, as revealed by simultaneous immunostaining for the neuropeptide calcitonin gene-related peptide. These findings are consistent with the idea that activation of TRPV1 in peripheral axons of joint afferents may mediate the synovial release of neuropeptides in arthritis

Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain

Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (Ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (Glial Fibrillary Acidic Protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (Adjuvant-Induced Arthritis, AIA), and neuropathic pain (Partial Sciatic Nerve Ligation, PSNL). We found that i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naïve WT mice, ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7–14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7–14 days after AIA, and 7 days after PSNL, iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14–21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain

Increased expression of CGRP in sensory afferents of arthritic mice – effect of genetic deletion of the vanilloid receptor TRPV1

The neuropeptide calcitonin gene-related peptide (CGRP), expressed by nociceptive sensory afferents in joints, is an important mediator in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release CGRP from their central and/or peripheral axons, suggesting a functional link between CGRP and the capsaicin receptor TRPV1. The expression of both TRPV1 and CGRP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release CGRP remains unclear. We here wanted to ascertain whether the increase in the number of CGRP-positive primary afferents during arthritis may be affected by genetic deletion of TRPV1. For this, we quantified the expression of CGRP in primary afferent neurons in DRG in wild type mice (WT) vs. TRPV1-KO mice with adjuvant-induced arthritis (AIA), using immunohistochemistry. We found that the fraction of DRG neurons that were immunopositive for CGRP 1) was higher in naïve TRPV1-KO mice than in naïve WT mice, 2) increased progressively 3–21 days after induction of AIA, and 3) this increase was bilateral but significantly greater on the CFA-injected side than on the IFA-injected side in TRPV1-KO mice. The increased expression of CGRP in AIA may reflect a phenotypic switch of primary afferents from non-peptidergic to peptidergic and the larger increase in TRPV1-KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit

Effect of genetic deletion of the vanilloid receptor TRPV1 on the expression of Substance P in sensory neurons of mice with adjuvant-induced arthritis

The neuropeptide Substance P (SP), expressed by nociceptive sensory afferents in joints, plays an important role in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release SP from their central and peripheral axons, suggesting a functional link between SP and the capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1). The expression of both TRPV1 and SP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release SP is not completely understood. We here wanted to ascertain whether the increase in the number of SP-positive primary afferents in arthritis may be affected by genetic deletion of TRPV1. For this, we used immunohistochemistry to quantify the expression of SP in primary afferent neurons in wild type mice (WT) vs. TRPV1-knockout (KO) mice with adjuvant-induced arthritis (AIA). We found that the expression of SP in DRG 1) increased significantly over naïve level in both WT and KO mice 3 weeks after AIA, 2) was significantly higher in KO mice than in WT mice in naïve mice and 2-3 weeks after AIA, 3) was significantly higheron the side of AIA than on the contralateral, vehicle-injected side at all time points in WT mice, but not in KO mice, and 4) increased predominantly in small-size neurons in KO mice and in small- and medium-size neurons in WT mice. Since the size distribution of SP-positive DRG neurons in arthritic TRPV1-KO mice was not significantly different from that in naïve mice, we speculate that the increased expression of SP is unlikely to reflect recruitment of A-fiber primary afferents and that the higher expression of SP in KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit

Synaptic interactions between primary afferent terminals and GABA and nitric oxide-synthesizing neurons in superficial laminae of the rat spinal cord

The superficial laminae (I and II) of the spinal dorsal horn receive small caliber primary afferent fibers responsive to noxious stimulation, and contain local circuit neurons that modulate afferent input. Many of these neurons are GABAergic; about a third of these also synthesize nitric oxide. We identified three main morphological types of primary afferent terminals in superficial laminae after injections of a tracer selective for small caliber afferents into the sciatic nerve of rats. The relative densities of the three types varied through the dorsoventral extent of laminae I and II. Synaptic contacts of each type with GABA- and nitric oxide synthase (NOS)-containing dendrites and axon terminals were determined by preembedding and postembedding immunocytochemistry. Nonglomerular primary afferent terminals, likely to originate from peptidergic unmyelinated fibers, were not seen in synaptic contact with either GABA- or NOS-containing neurons. Primary afferent terminals at the center of type 1 glomeruli (C1) and at the center of type 2 glomeruli (C2) are likely to originate from unmyelinated and small myelinated fibers, respectively. GABAergic terminals contacted more C2 than C1 terminals, suggesting more effective presynaptic inhibition of C2 terminals. Many GABAergic terminals were also positive for NOS, but all GABAergic terminals presynaptic to primary afferent terminals were negative for NOS. Only C2 terminals established frequent synapses with NOS-positive dendrites. (ABSTRACT TRUNCATED AT 250 WORDS

Synaptic Localization of Nitric Oxide Synthase and Soluble Guanylyl Cyclase in the Hippocampus

Functional evidence suggests that nitric oxide released from CA1 pyramidal cells can act as a retrograde messenger to mediate hippocampal long-term potentiation, but the failure to find neuronal nitric oxide synthase (NOS-I) in the dendritic spines of these cells has cast doubt on this suggestion. We hypothesized that NOS-I may be in spines but in a form inaccessible to antibody when using standard histological fixation procedures. Supporting this hypothesis, we found that after a weak fixation protocol shown previously to enhance staining of synaptic proteins, CA1 pyramidal cells exhibit clear immunoreactivity for NOS-I. Confocal microscopy revealed that numerous dendritic spines in the stratum radiatum contained the NR2 subunit of the NMDA receptor and the adaptor protein postsynaptic density-95, and a subset of these spines also contained NOS-I. Quantitative studies showed that only approximately 8% of synaptic puncta (identified by synaptophysin staining) were associated with NOS-I, and approximately 9% contained the beta subunit of soluble guanylyl cyclase (sGC), a major target of NO. However, the majority of NOS-I-positive synaptic puncta was associated with sGC and vice versa. Postembedding immunogold electron microscopy showed that NOS-I concentrates just inside the postsynaptic plasma membrane of asymmetric axospinous synapses in the stratum radiatum of CA1, whereas sGCbeta concentrates just inside the presynaptic membrane. Together, these findings support the possibility that NO may act as a retrograde messenger to help mediate homosynaptic plasticity in a subpopulation of synapses in the stratum radiatum of CA1

Primary Afferent Terminals in Spinal Cord Express Presynaptic AMPA Receptors

Larger dorsal root ganglion neurons are stained by an antibody for the C terminus of glutamate receptor subunit 2 (GluR2) and GluR3 (GluR2/3) rather than by an antibody for GluR4. In dorsal roots, anti-GluR2/3 stains predominantly myelinated fibers; anti-GluR4 or anti-GluR2/4 stains predominantly unmyelinated fibers. In the dorsal horn, puncta immunopositive for synaptophysin and GluR2/3 are predominantly in laminas III and IV, whereas puncta immunopositive for synaptophysin and GluR4 or GluR2/4 are predominantly in laminas I and II. Puncta immunopositive for GluR2/3 costain with the B subunit of cholera toxin, whereas puncta immunopositive for GluR2/4 costain with isolectin B4 after injections of these tracers in the sciatic nerve. No puncta costain with calcitonin gene-related peptide and AMPA receptor subunits. Electron microscopy indicates that AMPA receptor-immunopositive terminals are more numerous than suggested by confocal microscopy. Of all synapses in which immunostaining is presynaptic, postsynaptic, or both, the percentage of presynaptic immunostain is approximately 70% with anti-GluR4 or anti-GluR2/4 (in laminas I-III), 25-30% with anti-GluR2/3 (in laminas III and IV), and 5% with anti-GluR2 (in laminas I-III). Because of fixation constraints, the types of immunostained terminals could be identified only on the basis of morphological characteristics. Many terminals immunostained for GluR2/3, GluR4, or GluR2/4 have morphological features of endings of primary afferents. Terminals with morphological characteristics of presumed GABAergic terminals are also immunostained with anti-GluR2/4 and anti-GluR4 in laminas I and II and with anti-GluR2/3 in laminas III and IV. The conspicuous and selective expression of presynaptic AMPA receptor subunits may contribute to the characteristic physiological profile of different classes of primary afferents and suggests an important mechanism for the modulation of transmitter release by terminals of both myelinated and unmyelinated primary afferents

Interaction between GRIP and Liprin-α/SYD2 Is Required for AMPA Receptor Targeting

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-α/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-α and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-α is important for postsynaptic as well as presynaptic maturation

CRIPT, a Novel Postsynaptic Protein that Binds to the Third PDZ Domain of PSD-95/SAP90

AbstractThe 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

Interaction between Liprin-α and GIT1 Is Required for AMPA Receptor Targeting

Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes