Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

Origin of sensory and autonomic innervation of the rat temporomandibular joint: A retrograde axonal tracing study with the fluorescent dye fast blue

Previous studies that have used retrograde axonal tracers (horseradish peroxidase alone or conjugated with wheat germ agglutinin) have shown that the temporomandibular joint (TMJ) is supplied with nerve fibers originating mainly from the trigeminal ganglion, in addition to other sensory and sympathetic ganglia. The existence of nerve fibers in the TMJ originating from the trigeminal mesencephalic nucleus is unclear, and the possible innervation by parasympathetic nerve fibers has not been determined. In the present work, the retrograde axonal tracer, fast blue, was used to elucidate these questions and re-evaluated the literature data. The tracer was deposited in the supradiscal articular space of the rat TMJ, and an extensive morphometric analysis was performed of the labeled perikaryal profiles located in sensory and autonomic ganglia. This methodology permitted us to observe labeled small perikaryal profiles in the trigeminal ganglion, clustered mainly in the posterior-lateral region of the dorsal, medial and ventral thirds of horizontal sections, with some located in the anterior-lateral region of the ventral third. Sensory perikarya were also labeled in the dorsal root ganglia from C2 to C5. No labeled perikaryal profiles were found in the trigeminal mesencephalic nucleus. on the other hand, autonomic labeled perikaryal profiles were distributed in the sympathetic superior cervical and stellate ganglia, and parasympathetic otic ganglion. Our results confirmed those of previous studies and also demonstrated that: (i) there is a distribution pattern of labeled perikaryal profiles in the trigeminal ganglion; (ii) some perikaryal profiles located in the otic ganglion were labeled; and (iii) the trigeminal mesencephalic nucleus did not show any retrogradely labeled perikaryal profiles.

The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K+ channels. Crotalphine (0.2 or 5 mu g/kg, orally; 0.0006 mu g/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of delta-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of kappa-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K+ channel blocker. The results suggest that peripheral delta-opioid and kappa-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain. Behavioural Pharmacology 23:14-24 (C) 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

RGMa and RGMb expression pattern during chicken development suggest unexpected roles for these repulsive guidance molecules in notochord formation, somitogenesis, and myogenesis

Background: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues. Results: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo. Conclusions: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development. Developmental Dynamics, 2012. (C) 2012 Wiley Periodicals, Inc.

The severity of neural invasion is associated with shortened survival in colon cancer

PURPOSE Neural invasion (NI) is a histopathologic feature of colon cancer that receives little consideration. Therefore, we conducted a morphologic and functional characterization of NI in colon cancer. EXPERIMENTAL DESIGN NI was investigated in 673 patients with colon cancer. Localization and severity of NI was determined and related to patient's prognosis and survival. The neuro-affinity of colon cancer cells (HT29, HCT-116, SW620, and DLD-1) was compared with pancreatic cancer (T3M4 and SU86.86) and rectal cancer cells (CMT-93) in the in vitro three-dimensional (3D)-neural-migration assay and analyzed via live-cell imaging. Immunoreactivity of the neuroplasticity marker GAP-43, and the neurotrophic-chemoattractant factors Artemin and nerve growth factor (NGF), was quantified in colon cancer and pancreatic cancer nerves. Dorsal root ganglia of newborn rats were exposed to supernatants of colon cancer, rectal cancer, and pancreatic cancer cells and neurite density was determined. RESULTS NI was detected in 210 of 673 patients (31.2%). Although increasing NI severity scores were associated with a significantly poorer survival, presence of NI was not an independent prognostic factor in colon cancer. In the 3D migration assay, colon cancer and rectal cancer cells showed much less neurite-targeted migration when compared with pancreatic cancer cells. Supernatants of pancreatic cancer and rectal cancer cells induced a much higher neurite density than those of colon cancer cells. Accordingly, NGF, Artemin, and GAP-43 were much more pronounced in nerves in pancreatic cancer than in colon cancer. CONCLUSION NI is not an independent prognostic factor in colon cancer. The lack of a considerable biologic affinity between colon cancer cells and neurons, the low expression profile of colonic nerves for chemoattractant molecules, and the absence of a major neuroplasticity in colon cancer may explain the low prevalence and impact of NI in colon cancer.

Immunohistochemical demonstration of lumbar intervertebral disc innervation in the dog.

Low back pain is a common ailment in dogs, particularly in specific breeds such as the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplates and meninges have been associated with this condition. Yet, in spite of all diagnostic efforts, the origin of pain remains obscure in a substantial proportion of all cases. A further structure often being involved in vertebral column disorders is the intervertebral disc. The presence of nerves, however, is a precondition for pain sensation and, consequently, structures lacking innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of the intervertebral disc in man, rabbit and rat. With regard to the dog, however, the extent of intervertebral disc innervation is still being disputed. The goal of the present study, therefore, was to substantiate and expand current knowledge of intervertebral disc innervation. Protein gene product (PGP) 9.5 was used for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This general marker revealed nerve fibres to be confined to the periphery of the intervertebral discs. These results indicate that even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.

Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: A putative mediator of cholecystokinin-induced satiety

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the central nervous system. Recent studies have pointed to a role for CART-derived peptides in inhibiting feeding behavior. Although these actions have generally been attributed to hypothalamic CART, it remains to be determined whether additional CART pathways exist that link signals from the gastrointestinal tract to the central control of food intake. In the present study, we have investigated the presence of CART in the rat vagus nerve and nodose ganglion. In the viscerosensory nodose ganglion, half of the neuron profiles expressed CART and its predicted peptide, as determined by in situ hybridization and immunohistochemistry. CART expression was markedly attenuated after vagotomy, but no modulation was observed after food restriction or high-fat regimes. A large proportion of CART-labeled neuron profiles also expressed cholecystokinin A receptor mRNA. CART-peptide-like immunoreactivity was transported in the vagus nerve and found in a dense fiber plexus in the nucleus tractus solitarii. Studies on CART in the spinal somatosensory system revealed strong immunostaining of the dorsal horn but only a small number of stained cell bodies in dorsal root ganglia. The present results suggest that CART-derived peptides are present in vagal afferent neurons sensitive to cholecystokinin, suggesting that the role of these peptides in feeding may be explained partly by mediating postprandial satiety effects of cholecystokinin.

Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system

Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology.

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

bcl-2 transgene expression can protect neurons against developmental and induced cell death.

The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.

The Ets domain transcription factor Erm distinguishes rat satellite glia from Schwann cells and is regulated in satellite cells by neuregulin signaling

Distinct glial cell types of the vertebrate peripheral nervous system (PNS) are derived from the neural crest. Here we show that the expression of the Ets domain transcription factor Erm distinguishes satellite glia from Schwann cells beginning early in rat PNS development. In developing dorsal root ganglia (DRG), Erm is present both in presumptive satellite glia and in neurons. In contrast, Erm is not detectable at any developmental stage in Schwann cells in peripheral nerves. In addition, Erm is downregulated in DRG-derived glia adopting Schwann cell traits in culture. Thus, Erm is the first described transcription factor expressed in satellite glia but not in Schwann cells. In culture, the Neuregulin1 (NRG1) isoform GGF2 maintains Erm expression in presumptive satellite cells and reinduces Erm expression in DRG-derived glia but not in Schwann cells from sciatic nerve. These data demonstrate that there are intrinsic differences between these glial subtypes in their response to NRG1 signaling. In neural crest cultures, Erm-positive progenitor cells give rise to two distinct glial subtypes: Erm-positive, Oct-6-negative satellite glia in response to GGF2, and Erm-negative, Oct-6-positive Schwann cells in the presence of serum and the adenylate cyclase activator forskolin. Thus, Erm-positive neural crest-derived progenitor cells and presumptive satellite glia are able to acquire Schwann cell features. Given the in vivo expression of Erm in peripheral ganglia, we suggest that ganglionic Erm-positive cells may be precursors of Schwann cells.

Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo

The ciliary neurotrophic factor alpha-receptor(CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf -/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.