Labeled Dorsal Root Ganglion Neurons Research Articles

Mapping Somatosensory Afferent Circuitry to Bone Identifies Neurotrophic Signals Required for Fracture Healing.

The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aβ-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.

Serotonin-Mediated Activation of Serotonin Receptor Type 1 Oppositely Modulates Voltage-Gated Calcium Channel Currents in Rat Sensory Neurons Innervating Hindlimb Muscle.

Serotonin (5-HT) is a multifaceted neurotransmitter that has been described to play a role as a peripheral inflammatory mediator when released in ischemic or injured muscle. Dorsal root ganglia (DRG) neurons are key sensors of noxious stimuli that are released under inflammatory conditions or mechanical stress. Little information is available on the specific 5-HT receptor subtypes expressed in primary afferents that help regulate reflex pressor responses. In the present study, the whole-cell patch-clamp technique was employed to examine the modulation of voltage-gated calcium channel (CaV) 2.2 currents by 5-HT and to identify the 5-HT receptor subtype(s) mediating this response in acutely dissociated rat DRG neurons innervating triceps surae muscle. Our results indicate that exposure of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons to 5-HT can exert three modulatory effects on CaV currents: high inhibition, low inhibition, and enhancement. Both 5-HT-mediated inhibition responses were blocked after pretreatment with pertussis toxin (PTX), indicating that 5-HT receptors are coupled to CaV2.2 via Gα i/o protein subunits. Application of selective serotonin receptor type 1 (5-HT1) agonists revealed that modulation of CaV2.2 currents occurs primarily after 5-HT1A receptor subtype stimulation and minimally from 5-HT1D activation. Finally, the intrathecal administration of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), significantly (P < 0.05) decreased the pressor response induced by intra-arterial administration of lactic acid. This suggests that 5-HT1A receptors are expressed presynaptically on primary afferent neurons innervating triceps surae muscle. Our findings indicate that preferential stimulation of 5-HT1 receptors, expressed on thin fiber muscle afferents, serves to regulate the reflex pressor response to metabolic stimuli. SIGNIFICANCE STATEMENT: The monoamine serotonin (5-HT), released under ischemic conditions, can contribute to the development of inflammation that negatively affects the exercise pressor reflex. The 5-HT receptor subtype and signaling pathway that underlies calcium channel modulation in dorsal root ganglia afferents, innervating hindlimb muscles, are unknown. We show that 5-HT can either block (primarily via serotonin receptor type 1 (5-HT1)A subtypes) or enhance voltage-gated calcium channel (CaV2.2) currents. Our findings suggest 5-HT exhibits receptor subtype selectivity, providing a complexity of cellular responses.

Anti-RANKL antibodies decrease CGRP expression in dorsal root ganglion neurons innervating injured lumbar intervertebral discs in rats.

Nuclear factor-κB (NF-κB), receptor activator of NF-κB (RANK), and RANK ligand (RANKL) are transcriptional regulators of inflammatory cytokines. RANKL expression in dorsal root ganglion (DRG) neurons is elevated in animal models of pain or intervertebral disc herniation. We sought to evaluate the effect of anti-RANKL antibodies on sensory nerves innervating injured intervertebral discs. We labeled DRG neurons innervating L5-6 discs with FluoroGold (FG). The L5-6 discs of 36 rats were punctured using a 23-gage needle and 18 rats underwent sham surgery without disc puncture. The puncture group was evenly subdivided into a group in which 10 μl saline was administered to the injured disc and a group in which 10 μl of anti-RANKL antibody was administered. Seven and 14 days postsurgery, DRGs at L2 level were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP). The proportion of CGRP-immunoreactive (IR) DRG neurons of all FG-positive neurons was determined. Amount of tumor necrosis factor (TNF)-α and interleukin(IL)-6 was measured within the intervertebral discs in each group at 7 and 14 days after surgery using an enzyme-linked immunosorbent assay (ELISA). The proportion of CGRP-IR DRG neurons to total FG-labeled neurons innervating injured intervertebral discs and amount of TNF-α and IL-6 in the injured discs in the saline control group was significantly increased compared with that found in rats from the sham surgery group (P < 0.05). However, application of anti-RANKL antibody to the injured discs significantly decreased the proportion of CGRP-IR DRG neurons to total FG-labeled neurons and amount of TNF-α and IL-6 in the injured discs (P < 0.05). TNF-α and IL-6 in the injured discs increased and CGRP expression increased in DRG neurons innervating injured discs, and antibodies to RANKL could suppress this increased TNF-α, IL-6, and CGRP expression. RANKL may be a therapeutic target for pain control in patients with lumbar disc degeneration.

Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freund’s adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4− while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1μM) produced a transient (1min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery.

Upregulation of NaV1.7 in Dorsal Root Ganglia After Intervertebral Disc Injury in Rats

Animal study. To investigate pain-related expression of NaV1.7 in dorsal root ganglia (DRG) innervating intervertebral discs. The pathophysiology of discogenic low back pain is not fully understood. Prostaglandins and cytokines produced by degenerated discs can cause pain, but nonsteroidal anti-inflammatory and steroid medications are often ineffective at pain reduction. Tetrodotoxin-sensitive, voltage-gated sodium (NaV) channels are associated with sensory transmission in primary sensory nerves, and the NaV1.7 channel has emerged as an attractive analgesic target. The purpose of this study was to investigate pain-related expression of NaV1.7 in DRG innervating intervertebral discs. Using a rodent model of disc puncture, we labeled DRG neurons innervating L5-L6 discs with FluoroGold neurotracer (n = 20). Half of the rats (n = 10) underwent intervertebral disc puncture using a 23-gauge needle (puncture group), and the other half underwent non-puncture sham surgery (non-puncture group). Seven and 14 days after surgery, DRGs from the L1 to L6 levels were harvested, sectioned, and immunostained for NaV1.7, and the proportion of NaV1.7-immunoreactive DRG neurons was evaluated. NaV1.7 was expressed in DRG neurons innervating intervertebral discs from L1 to L5. The ratio of NaV1.7-expressing DRG neurons to total FG-labeled neurons was 7.2% and 7.6% at 1 and 2 weeks after surgery, respectively, in the non-puncture group and 16.2% and 16.3% at 1 and 2 weeks, respectively, in the puncture group. The upregulation of NaV1.7 after puncture was significant at both 1 and 2 weeks after surgery (P < 0.01). We found that disc injury increases NaV1.7 expression in DRG neurons innervating injured discs. NaV1.7 may be a therapeutic target for pain control in patients with lumbar disc degeneration.

IκB kinase β inhibitor downregulates pain-related neuropeptide production in the sensory neurons innervating injured lumbar intervertebral discs in the dorsal root ganglia of rats

Background contextNuclear factor-κB (NF-κB) is an essential gene transcriptional regulator of inflammatory cytokines, and it plays important roles in numerous conditions, including inflammatory and neuropathic pain, especially when discogenic pain is involved. Phosphorylation of IκB protein through IκB kinase (IKK) is the first step in the activation of NF-κB activation and the upregulation of NF-κB–responsive genes. PurposeTo investigate whether IKK inhibition alters the properties of pain-related neuropeptides in the rat lumbar degenerative intervertebral disc (IVD) model. Study designRetrograde neurotracing and immunofluorescent investigation of pain-related neuropeptide (calcitonin gene–related peptide [CGRP]) in the sensory innervation of injured lumbar IVD in rat dorsal root ganglia (DRGs). MethodsForty female Sprague-Dawley rats were equally divided into four groups: naive, sham, and two agent-treated groups (vehicle [saline] group and anti-IKKβ [IMD-0560, IKKβ inhibitor] group). The L5–L6 IVDs of the agent-treated rats were exposed and injured by repeated punctures. The retrograde neurotracer Fluoro-Gold (FG) and corresponding treatment agents were intradiscally applied. In the sham group, FG alone was applied onto uninjured IVDs. One week later, L1–L3 DRGs were harvested and immunolabeled for CGRP as a pain marker. The proportions of FG-labeled CGRP-immunoreactive (-ir) DRG neurons were assessed. ResultsFluoro-Gold–labeled DRG neurons were almost equally prevalent at each DRG level. The proportions of FG-labeled CGRP-ir DRG neurons in the two agent-treated groups were significantly increased in comparison with those in the naive and the sham groups (p<.05) and were significantly decreased in the anti-IKKβ group in comparison with that in the vehicle group (p<.05). ConclusionsThe neuropeptide CGRP as a pain marker was upregulated in DRG neurons innervating the injured IVDs, and intradiscal inhibition of IKKβ significantly suppressed CGRP production in the DRG neurons innervating the rat IVD, suggesting the possible analgesic effect of IKKβ inhibition in discogenic pain.

Disk Injury in Rats Produces Persistent Increases in Pain-Related Neuropeptides in Dorsal Root Ganglia and Spinal Cord Glia but Only Transient Increases in Inflammatory Mediators

Immunohistological analysis in an injured intervertebral disk (IVD) model. To elucidate and compare in rats the behavior of the sensory nervous system and inflammatory mediators in experimentally injured IVDs. Multiple human and animal studies have verified the presence of sensory nerve fibers in IVDs or investigated the behavior of inflammatory mediators in injured IVDs, but no in vivo study to date has examined the relationship between the 2. Eight-week-old female rats were used. In the disk-injured group, L5/L6 disks were injured with a 24-gauge needle; simultaneously, the neurotracer Fluoro-gold was injected into the L5/L6 IVD. The L5/L6 IVD dorsal root ganglia (DRGs) from the L1 to L6 levels, and the spinal cord was resected at several time points after surgery. Nerve growth factor, tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in the IVDs were quantified using enzyme-linked immunosorbent assay. DRGs were immunostained for calcitonin gene-related peptide, and spinal cord sections were immunostained for ionized calcium-binding adaptor molecule-1 and glial fibrillary acidic protein. Nerve growth factor, and TNF-α levels (through 1 week) and IL-6 levels (through 4 days) were significantly higher in the disk-injured group than in the noninjured group (P < 0.05). However, starting at 2 weeks (nerve growth factor and TNF-α) or 1 week (IL-6), the differences in inflammatory mediator levels between the 2 groups no longer were significant. In contrast, the percentage of calcitonin gene-related peptide-immunoreactive neurons among Fluoro-gold-labeled DRG neurons, and the numbers of ionized calcium-binding adaptor molecule-1-immunoreactive microglia and glial fibrillary acidic protein-immunoreactive astrocytes in the spinal dorsal horn remained significantly higher in the injured group than in the noninjured group at all-time points (P < 0.05). Disk injury in rats produces persistent increases in neuropeptides in DRGs and glia in the spinal cord, but only transient increases in inflammatory mediators in IVDs.

Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats

We recently reported that reactive oxygen species (ROS) plays an excitatory role in modulation of the exercise pressor reflex (EPR) in normal rats. In this study, we further tested two independent hypotheses: 1) ROS interacts with EPR-related ionotropic receptors such as the purinergic receptors (P(2)) and transient receptor potential vanilloid 1 receptors (TRPV1) to indirectly modulate the EPR function; 2) ROS directly affects excitability of muscle afferents by modulating the voltage-gated sodium (Na(v)) channels. To test the first hypothesis, we performed animal experiments to investigate the effect of the SOD mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) on the pressor response to hindlimb intra-arterial (IA) injection of either α,β-methylene ATP (a P(2X) agonist) or capsaicin (a TRPV1 agonist) in decerebrate rats. To test the second hypothesis, we used the patch-clamp technique to determine the effect of ROS on Na(v) channels on the soma of muscle afferents. We also performed local microinjection of a sodium channel blocker, tetrodotoxin (TTX), into ipsilateral L4/L5 dorsal root ganglia (DRGs) to investigate whether the blockade of Na(v) channels by TTX affects the EPR function. We found that Tempol did not affect the pressor response to injection of either capsaicin or α,β-methylene ATP but significantly decreased the Na(v) current in small and medium-sized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons. A membrane-permeant superoxide dismutase, polyethylene glycol (PEG)-SOD, had an effect on the Na(v) current in these neurons similar to that of Tempol. Microinjection of TTX into L4/L5 DRGs dramatically attenuated the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots. These data suggest that ROS modulates the EPR by affecting the activity of the Na(v) channels on muscle afferents.

Mechanisms underlying purinergic P2X3 receptor-mediated mechanical allodynia induced in diabetic rats.

BackgroundDiabetic neuropathy is a common neuropathy associated with paresthaesia and pain. The mechanisms underlying the painful conditions are not well understood. The aim of this study is to investigate the participation of purinergic P2X3 receptors in painful diabetic neuropathy.ResultsDiabetes was induced by an intraperitoneal injection of streptozotocin (STZ). We showed that mechanical allodynia was induced two weeks after a STZ injection and lasted for at least another seven weeks. The mechanical allodynia was significantly attenuated by peripheral administration of the P2X receptor antagonists, PPADS or TNP-ATP. DiI was subcutaneously injected into the rat hindpaw to label hindpaw-innervated dorsal root ganglion (DRG) neurons. ATP activated fast-inactivating P2X3 receptor-mediated currents in the labeled DRG neurons were studied. ATP responses in STZ-treated rats were ~2-fold larger than those in control rats. Furthermore, the expression of P2X3 receptor proteins in the plasma membrane of L4-6 DRGs of STZ rats was significantly enhanced while the total expression of P2X3 receptors remained unaltered.ConclusionsThese results indicate that a large enhancement of P2X3 receptor activity and an increase in the membrane expression of P2X3 receptors contribute to the development of chronic pain in STZ-induced diabetic rats and suggest a possible target for the treatment of diabetic neuropathic pain.

Sympathetic innervation of the ileocecal junction in horses

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 +/- 8% and 93 +/- 12% of the neurons innervating the ICJ expressed TH- and DBH-IR, respectively. In the CaudMG 90 +/- 15% and 94 +/- 5% of ICJ innervating neurons were TH- and DBH-IR, respectively. Sympathetic (TH-IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS-, CGRP-, and SP-IR nerve cells were observed. Dense networks of CGRP-, nNOS-, and SP-IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP-IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ.

Efficacy of fluorescent tracers in retrograde labeling of cutaneous afferent neurons in the rat

Background In the present study, the labeling efficacy of tracers Fluoro-ruby (FR), Fluoro-emerald (FE), True Blue (TB), Fluoro-Gold (FG), Diamidino Yellow (DY) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) to retrogradely label the cutaneous afferent neurons in the rat was examined. Methods The proximal stump of the transected sural nerve was exposed for 1 hour either to one of the examined dyes (FR, FE, TB, FG, DY and DiI group) in single labeling experiments, or to mixtures of two dyes (TB-FG, FG-DiI, TB-DY and TB-DiI group) in double labeling experiments ( n = 5 for each group). After 10 days, dorsal root ganglia (DRGs) L3-S1 were harvested, cut to 20 μm thick longitudinal sections and all labeled neurons were counted. Results The average numbers of labeled DRG neurons in FR group (1063 ± 158; mean ± SD) and FE group (1067 ± 203) were statistically significantly lower than those in TB group (2831 ± 379), FG group (2802 ± 134), DY group (2888 ± 262) or DiI group (2900 ± 278) ( p < 0.05). In double labeling experiments, the average number of double labeled neurons in TB-DY group (2208 ± 207) was statistically significantly lower than those in TB-FG group (2775 ± 316), FG-DiI group (2921 ± 419), or TB-DiI group (2805 ± 179) ( p < 0.05). Conclusions Among examined tracers, TB, FG, DY and DiI, have the highest and similar labeling efficacy for retrograde labeling of cutaneous afferent neurons in the rat. The tracers TB, DiI and FG effectively label the same neuronal population in double labeling, therefore, their combinations are most suitable for double retrograde labeling studies of cutaneous afferent neurons in the rat.

Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav 1.8 and TRPV1, in the Rat Dorsal Root Ganglion

Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30% to 100% of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25% and 4.0% (w/v) and five picric acid concentrations between 0.0% and 0.8% (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Na(v)1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary antiserum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Na(v)1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69% of neurons express Na(v)1.8, and 77% of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Up-regulation of p55 TNF alpha-receptor in dorsal root ganglia neurons following lumbar facet joint injury in rats

The rat L5/6 facet joint is multisegmentally innervated from the L1 to L6 dorsal root ganglia (DRG). Tumor necrosis factor (TNF) is a known mediator of inflammation. It has been reported that satellite cells are activated, produce TNF and surround DRG neurons innervating L5/6 facet joints after facet injury. In the current study, changes in TNF receptor (p55) expression in DRG neurons innervating the L5/6 facet joint following facet joint injury were investigated in rats using a retrograde neurotransport method followed by immunohistochemistry. Twenty rats were used for this study. Two crystals of Fluorogold (FG; neurotracer) were applied into the L5/6 facet joint. Seven days after surgery, the dorsal portion of the capsule was cut in the injured group (injured group n = 10). No injury was performed in the non-injured group (n = 10). Fourteen days after the first application of FG, bilateral DRGs from T13 to L6 levels were resected and sectioned. They were subsequently processed for p55 immunohistochemistry. The number of FG labeled neurons and number of FG labeled p55-immunoreactive (IR) neurons were counted. FG labeled DRG neurons innervating the L5/6 facet joint were distributed from ipsilateral L1 to L6 levels. Of FG labeled neurons, the ratio of DRG neurons immunoreactive for p55 in the injured group (50%) was significantly higher than that in the non-injured group (13%). The ratio of p55-IR neurons of FG labeled DRG neurons was significantly higher in total L1 and L2 DRGs than that in total L3, 4, 5 and 6 DRGs in the injured group (L1 and 2 DRG, 67%; L3, 4, 5 and 6 DRG, 37%, percentages of the total number of p55-IR neurons at L1 and L2 level or L3-6 level/the total number of FG-labeled neurons at L1 and L2 level or L3-6 level). These data suggest that up-regulation of p55 in DRG neurons may be involved in the sensory transmission from facet joint injury. Regulation of p55 in DRG neurons innervating the facet joint was different between upper DRG innervated via the paravertebral sympathetic trunks and lower DRG innervated via other direct routes.

Expression of TRPV1 in sensory and sympathetic neurons innervating kidney

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand-gated cation channel activated by capsaicin, heat, protons and cytosolic lipids. TRPV1 is expressed primarilyin sensory nerves and functions as a molecular integrator for multiple types of sensory input. Recent reports suggest that TRPV1 plays an important role in regulating salt sensitivity during development of hypertension. Our study determined the expression of TRPV1 in the neurons supplying the kidney, which regulates salt balance. We used Fast Blue (FB) to label retrogradely sympathetic and sensory neurons innervating the kidney. In T12 to L4 segments, 4% and 6% or neurons were labeled by FB in dorsal root ganglia (DRG) and paravertebral ganglia (PVG) respectively. Labeled neurons were not found in celiac or nodose ganglia. We used antibodies raised against CGRP and tyrosine hydroxylase (TH) to mark sensory and sympathetic neurons respectively. Immunocytochemical studies showed that TRPV1 was expressed in FB-CGRP labeled DRG neurons and in FB-TH labeled PVG neurons. Whole cell recordings revealed that capsaicin caused in inward current in FB labeled DRG neurons. FB labeled neurons in PVG did not respond to capsaicin. Our data indicate that TRPV1 is expressed in DRG and PVG neurons supplying the kidney. The electrophysiological properties of TRPV1 in PVG differ from that in DRG.

Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability.

The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.

Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord.

Exposure of the lumbar spinal cord of rats to X-rays 3 days after birth results in changes in the composition of central glia. Shortly after irradiation, there is both retardation of central myelin formation and a loss of integrity of the astrocyte-derived glia limitans on the dorsal surface of the cord. Subsequently, Schwann cells invade, undergo division and myelinate axons in the dorsal funiculi in the irradiated region of the cord, creating there an environment similar to that of peripheral nerve. The present study was undertaken to compare the ability of lesioned dorsal root axons to grow back into the altered glial environments that exist within the spinal cord after irradiation. This regrowth was assessed by injecting Fluoro-Gold into the spinal cord and subsequently examining neurons in the dorsal root ganglia (DRG) for the presence of this label. Numbers of retrogradely labeled neurons were counted in the DRG in both injured and contralateral non-injured sides. Non-irradiated control rats had almost no labeled DRG neurons on the injured side, whereas Fluoro-Gold labeled neurons were observed in substantial numbers in the DRG on the injured side of irradiated rats. There was a definite trend in the data, indicating that the longer the interval between irradiation and root injury, the greater the number of labeled neurons. Since the Fluoro-Gold labeling technique does not allow for visualization of the labeled axons within the spinal cord, a few animals were used to assess anterograde labeling with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP/HRP) from the dorsal root into the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)