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Objectives Recent observations demonstrated the expression of the insulin receptor (InsR) and its functional interaction with the transient receptor potential vanilloid type 1 receptor (TRPV1) in sensory ganglion neurons. Because sensory nerves are implicated in pancreatic inflammatory processes, we studied the colocalization of the InsR with TRPV1 and proinflammatory neuropeptides in spinal and vagal pancreatic afferent neurons. Methods Immunohistochemistry and quantitative morphometry were used to analyze the expression of TRPV1, InsR, substance P (SP), and calcitonin gene-related peptide (CGRP) in retrogradely labeled pancreatic dorsal root ganglion (DRG) and nodose ganglion (NG) neurons. Results The proportions of retrogradely labeled pancreatic TRPV1-, InsR-, SP-, and CGRP-immunoreactive neurons amounted to 68%, 48%, 33%, and 54% in DRGs and 64%, 49%, 40%, and 25% in the NGs. Of the labeled DRG and NG neurons, 23% and 35% showed both TRPV1 and InsR immunoreactivity. Colocalization of the InsR with SP or CGRP was demonstrated in 14% and 28% of pancreatic DRG and 24% and 8% of pancreatic NG neurons. Conclusions The present findings provide morphological basis for possible functional interactions among the nociceptive ion channel TRPV1, the InsR, and the proinflammatory neuropeptides SP and CGRP expressed by pancreatic DRG and NG neurons.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth. ispartof: AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY vol:318 issue:1 pages:G53-G65 ispartof: location:United States status: published

High frequency spontaneous activity in injured primary afferents has been proposed as a pathological mechanism of neuropathic pain following nerve injury. Although spinal infusion of glial cell line-derived neurotrophic factor reduces the activity of injured myelinated A-fiber neurons after fifth lumbar (L5) spinal nerve ligation in rats, the implicated molecular mechanism remains undetermined. The fast-inactivating transient A-type potassium current (IA) is an important determinant of neuronal excitability, and five voltage-gated potassium channel (Kv) alpha-subunits, Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, display IA in heterologous expression systems. Here, we examined the effect of spinal glial cell line-derived neurotrophic factor infusion on IA and the expression of these five Kv mRNAs in injured A-fiber neurons using the in vitro patch clamp technique and in situ hybridization histochemistry. Glial cell line-derived neurotrophic factor infusion reversed axotomy-induced reduction of the rheobase, elongation of first spike duration, and depolarization of the resting membrane potential. L5 spinal nerve ligation significantly reduced the current density of IA and glial cell line-derived neurotrophic factor treatment reversed the reduction. Among the examined Kv mRNAs, only the change in Kv4.1-expression was parallel with the change in IA after spinal nerve ligation and glial cell line-derived neurotrophic factor treatment. These findings suggest that glial cell line-derived neurotrophic factor should reduce the hyperexcitability of injured A-fiber primary afferents by IA recurrence. Among the five IA-related Kv channels, Kv4.1 should be a key channel, which account for this IA recurrence.

Gastric acid is of paramount importance for digestion and protection from pathogens but, at the same time, is a threat to the integrity of the mucosa in the upper gastrointestinal tract and may give rise to pain if inflammation or ulceration ensues. Luminal acidity in the colon is determined by lactate production and microbial transformation of carbohydrates to short chain fatty acids as well as formation of ammonia. The pH in the oesophagus, stomach and intestine is surveyed by a network of acid sensors among which acid-sensing ion channels (ASICs) and acid-sensitive members of transient receptor potential ion channels take a special place. In the gut, ASICs (ASIC1, ASIC2, ASIC3) are primarily expressed by the peripheral axons of vagal and spinal afferent neurons and are responsible for distinct proton-gated currents in these neurons. ASICs survey moderate decreases in extracellular pH and through these properties contribute to a protective blood flow increase in the face of mucosal acid challenge. Importantly, experimental studies provide increasing evidence that ASICs contribute to gastric acid hypersensitivity and pain under conditions of gastritis and peptic ulceration but also participate in colonic hypersensitivity to mechanical stimuli (distension) under conditions of irritation that are not necessarily associated with overt inflammation. These functional implications and their upregulation by inflammatory and non-inflammatory pathologies make ASICs potential targets to manage visceral hypersensitivity and pain associated with functional gastrointestinal disorders.This article is part of the Special Issue entitled ‘Acid-Sensing Ion Channels in the Nervous System’.

Calcitonin gene–related peptide (CGRP) is a 37-amino acid peptide found primarily in the C and Aδ sensory fibers arising from the dorsal root and trigeminal ganglia, as well as the central nervous system. Calcitonin gene–related peptide was found to play important roles in cardiovascular, digestive, and sensory functions. Although the vasodilatory properties of CGRP are well documented, its somatosensory function regarding modulation of neuronal sensitization and of enhanced pain has received considerable attention recently. Growing evidence indicates that CGRP plays a key role in the development of peripheral sensitization and the associated enhanced pain. Calcitonin gene–related peptide is implicated in the development of neurogenic inflammation and it is upregulated in conditions of inflammatory and neuropathic pain. It is most likely that CGRP facilitates nociceptive transmission and contributes to the development and maintenance of a sensitized, hyperresponsive state not only of the primary afferent sensory neurons but also of the second-order pain transmission neurons within the central nervous system, thus contributing to central sensitization as well. The maintenance of a sensitized neuronal condition is believed to be an important factor underlying migraine. Recent successful clinical studies have shown that blocking the function of CGRP can alleviate migraine. However, the mechanisms through which CGRP may contribute to migraine are still not fully understood. We reviewed the role of CGRP in primary afferents, the dorsal root ganglion, and in the trigeminal system as well as its role in peripheral and central sensitization and its potential contribution to pain processing and to migraine.

Elevated excitability of primary afferent neurons underlies chronic pain in patients with functional or inflammatory bowel diseases. Recent studies have established an essential role for an enhanced transient receptor potential vanilloid subtype 1 (TRPV1) signaling in mediating peripheral hyperalgesia in inflammatory conditions. Since colocalization of Toll-like receptor 4 (TLR4) and TRPV1 has been observed in primary afferents including the trigeminal sensory neurons and the dorsal root ganglion neurons, we test the hypothesis that TLR4 might regulate the expression and function of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis using the TLR4-deficient and the wild-type C57 mice. Despite having a higher disease activity index following administration of 2,4,6-trinitrobenzene sulfate, the TLR4-deficient mice showed less inflammatory infiltration in the colon than the wild-type mice. Increased expression of TLR4 and TRPV1 as well as increased density of capsaicin-induced TRPV1 current was observed in L4-S2 dorsal root ganglion neurons of the wild-type colitis mice till two weeks post 2,4,6-trinitrobenzene sulfate treatment. In comparison, the TLR4-deficient colitis mice had lower TRPV1 expression and TRPV1 current density in dorsal root ganglion neurons with lower abdominal withdrawal response scores during noxious colonic distensions. In the wild type but not in the TLR4-deficient dorsal root ganglion neurons, acute administration of the TLR4 agonist lipopolysaccharide increased the capsaicin-evoked TRPV1 current. In addition, we found that the canonical signaling downstream of TLR4 was activated in 2,4,6-trinitrobenzene sulfate-induced colitis in the wild type but not in the TLR4-deficient mice. These results indicate that TLR4 may play a major role in regulation of TRPV1 signaling and peripheral hyperalgesia in inflammatory conditions.

Recent studies have analyzed the role of nitric oxide (NO) in pain modulation in several models of sciatic nerve injury. In the present study we have investigated NO production in lumbar dorsal root ganglia (DRG) and spinal cord (SC) over time after sciatic nerve cut. Neuronal nitric oxide synthase (nNOS)-like immunoreactivity (LI) was also determined, since the expression and activity of the enzyme do not always correlate. Nerve section induced a progressive increase in NO production in the ipsilateral L4-5 DRGs and the corresponding levels in the SC in a pattern that correlated with nNOS-LI; this increase was gradual after 7 days of survival time, more pronounced after 14 days, with the highest values detected 28 days after axotomy. This peak was followed by a progressive decrease, reaching control values 42 days after the lesion. The present study shows that nNOS upregulation is related to an increased NO production and release. The temporal pattern of NO production parallels the one observed for the expression of the enzyme, suggesting that the induction of nNOS synthesis yields a protein that is functional and highly active. Fil: Coronel, Maria Florencia. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Defagot, Maria C.. Universidad Austral; Argentina Fil: Musolino, Patricia L.. Universidad Austral; Argentina Fil: Villar, Marcelo Jose. Universidad Austral; Argentina

Bone marrow mesenchymal stem cells (MSCs) preferentially migrate to the injured hemisphere when administered intravenously to rats with traumatic or ischemic brain injuries. In this study, we have investigated the localization of MSCs injected into the lumbar-4 dorsal root ganglion (L4-DRG) of rats with a sciatic nerve single ligature nerve constriction (SLNC). MSCs were isolated by their adherence to plastic, cultured until confluence and labelled with Hoechst. Animals with a unilateral injection of MSCs were subjected to an ipsilateral, bilateral or contralateral SLNC. After 9 days, they were perfused and the lumbar DRGs were dissected out, cut in a cryostat and observed with a fluorescence microscope. Large numbers of Hoechst-positive cells were observed in the injected L4-DRG, distributed around primary afferent neurons, resembling the anatomical localization of glial cells. In animals with an ipsilateral SLNC, some cells were detected in the ipsilateral L3, L5 or L6-DRGs but not in the contralateral ganglia. In animals with a bilateral lesion, MSCs migrated to both the ipsilateral and contralateral DRGs whereas in animals with a contralateral ligature, MSCs migrated to the contralateral DRGs. These results suggest that MSCs preferentially engraft in DRGs hosting primary sensory neurons affected by a lesion of their peripheral branches. Further studies should be carried out in order to elucidate the molecular mechanisms involved in this migration and homing, in order to evaluate the possible use of MSCs as a new therapeutic strategy for the treatment of peripheral nerve neuropathies. Fil: Coronel, Maria Florencia. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Musolino, Patricia Leonor. Universidad Austral; Argentina Fil: Villar, Marcelo José. Universidad Austral; Argentina

ATP-sensitive potassium (K(ATP)) currents were examined in dorsal root ganglion neurons from neuropathic and control rats using whole-cell voltage clamp recordings. K(ATP) channel openers (diazoxide and pinacidil) enhanced, and the blocker glibenclamide inhibited an outward current in control neurons in a manner dependent on the pipette ATP concentration. Analysis of reversal potentials showed that this current is carried by K(+) ions. Outward current in cells from rats with peripheral nerve injury was not sensitive to modulators of K(ATP) channels. Gabapentin, a putative K(ATP) channel opener, had minimal effect on currents in either group of neurons. We conclude that normal primary afferent neurons express K(ATP) channels that conduct current which is eliminated by peripheral nerve injury. Gabapentin does not affect this current significantly.

The arrangement between astrocytes and the primary afferent neurons of the mesencephalic trigeminal nucleus (Me5) was analyzed in a light and electron microscopy immunocytochemistry study using anti-GFAP antibodies. It appears that each Me5 neuron is almost entirely sheathed with astrocytic processes radiating out from two or more astrocytes. Ultrastructural observations confirmed the embracement of Me5 neurons by astrocytic processes that only allowed some synaptic contacts on the neuronal surface. The possible functional significance of this intimate morphological relationship for the mesencephalic trigeminal neurons is discussed.