. Thermosensitive transient receptor potential channels in vagal afferent neurons of the mouse. Am J Physiol Gastrointest Liver Physiol 286: G983-G991, 2004. First published January 15, 2003 10.1152/ajpgi.00441.2003.-A number of transient receptor potential (TRP) channels has recently been shown to mediate cutaneous thermosensitivity. Sensitivity to warm and cool stimuli has been demonstrated in both human and animal gastrointestinal tract; however, the molecular mechanisms that underlie this have not been determined. Vagal afferent neurons with cell bodies in the nodose ganglion are known to mediate nonnociceptive sensation from the upper gut. In this study, isolated cultured nodose ganglion from the mouse neurons showed changes in cytoplasmic-free Ca 2ϩ concentrations over a range of temperatures, as well as to icilin (a TRPM8 and TRPN1 agonist) and capsaicin (a TRPV1 agonist). RT-PCR was used to show the presence of six temperature-sensitive TRP channel transcripts (TRPV1-4, TRPN1, and TRPM8) in whole nodose ganglia. In addition, RT-PCR of single nodose cell bodies, which had been retrogradely labeled from the upper gut, detected transcripts for TRPV1, TRPV2, TRPV4, TRPN1, and TRPM8 in a proportion of cells. Immunohistochemical labeling detected TRPV1 and TRPV2 proteins in nodose ganglia. The presence of TRP channel transcripts and proteins was also detected in cells within several regions of the gastrointestinal tract. Our results reveal that TRP channels are present in subsets of vagal afferent neurons that project to the stomach and may confer temperature sensitivity on these cells. primary afferent neurons; nodose ganglion; stomach; retrograde labeling ACCURATE DETECTION OF TEMPERATURE is required for thermal homeostasis and for avoiding damage by excessively high or low temperatures. Recent studies (13,20,24,25) have identified key molecular components of neuronal temperature sensitivity in brain slices from the preoptic area/anterior hypothalamus (POAH) and in cultured dorsal root ganglion neurons. These studies have shown that several temperature-activated, nonselective cation channels cause generator potentials and firing behavior in thermally sensitive neurons. In cultured dorsal root ganglion cells, warming and cooling can evoke significant changes in cytoplasmic-free Ca 2ϩ concentrations ([Ca 2ϩ ] cyt ) (7, 31). The cloning and functional reconstitution in heterologous cell types of transient receptor potentials (TRP)V1 (VR1), TRPV2 (VRL-1), TRPV3 and TRPV4 (TRPO4, VR-OAC), TRPN1 (ANKTM1), and TRPM8 (CMR1) have suggested that the increase in [Ca 2ϩ ] cyt in response to thermal stimuli may be mediated by this group of Ca 2ϩ permeable TRP channels (1,2,9,17,23,29,30,35).Thermosensitive afferent nerve fibers have been identified projecting to the esophagus, stomach, duodenum, and rectum both in animals and humans (5,6,34,42). In the feline vagus nerves, three types of thermosensitive unmyelinated fibers can be distinguished by cold (10 -36°C), warm (39 -50°C), and mixed (10 -35°C and 40 -50°C) te...

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs) are the two putative mechanoreceptors that the vagus nerve supplies to gastrointestinal smooth muscle. To examine whether neurotrophin-4 (NT-4)-deficient mice, which have only 45% of the normal number of nodose ganglion neurons, exhibit selective losses of these endings and potentially provide a model for assessing their functional roles, we inventoried IGLEs and IMAs in the gut wall. Vagal afferents were labeled by nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase, and a standardized sampling protocol was used to map the terminals in the stomach, duodenum, and ileum. NT-4 mutants had a substantial organ-specific reduction of IGLEs; whereas the morphologies and densities of both IGLEs and IMAs in the stomach were similar to wild-type patterns, IGLEs were largely absent in the small intestine (90 and 81% losses in duodenum and ileum, respectively). Meal pattern analyses revealed that NT-4 mutants had increased meal durations with solid food and increased meal sizes with liquid food. However, daily total food intake and body weight remained normal because of compensatory changes in other meal parameters. These findings indicate that NT-4 knock-out mice have a selective vagal afferent loss and suggest that intestinal IGLEs (1) may participate in short-term satiety, probably by conveying feedback about intestinal distension or transit to the brain, (2) are not essential for long-term control of feeding and body weight, and (3) play different roles in regulation of solid and liquid diet intake.

The extrinsic efferent innervation of the distal colon and rectum of the guinea pig was compared, by using retrograde tracing combined with immunohistochemistry. Application of the carbocyanine tracer DiI to the rectum filled significantly greater numbers of extrinsic neurons than similar injections into the distal colon. Approximately three-fourths of all filled neurons from either location were either sympathetic or parasympathetic; the rest were spinal sensory neurons. Nerve cell bodies in sympathetic prevertebral ganglia labelled from the two regions were similar in number. Both regions were innervated by sympathetic neurons in paravertebral ganglia; however, the rectum received much more input from this source than the colon. The rectum received significantly more input from pelvic ganglia than the colon. The rectum also received direct innervation from two groups of neurons in the spinal cord. Neurons located in the spinal parasympathetic nucleus in segment S2 and S3 were labelled by DiI injected into the rectal wall. Similar numbers of neurons, located in intermediolateral cell column and dorsal commissural nucleus of lumbar segments, also projected directly to rectum, but not colon. The great majority (>80%) of retrogradely labelled nerve cell bodies in sympathetic ganglia were immunoreactive for tyrosine hydroxylase. In pelvic ganglia, retrogradely labelled neurons contained choline acetyltransferase and/or nitric oxide synthase or tyrosine hydroxylase. Although the rectum and colon in this species are continuous and macroscopically indistinguishable, they have significantly different patterns of extrinsic efferent innervation, presumably reflecting their different functions.

Interrelationships among concentrations and maturation of intramuscular collagen, serum concentration of hydroxyproline and testosterone and meat tenderness were determined in growing bulls and steers. Sixty-four Charolais X Angus bulls were assigned to sex treatment groups (intact or castrate) and slaughter groups (9, 12, 15 or 18 mo of age). Animals were bled at 30-min intervals via intrajugular catheters between 0600 and 1400 beginning 48 h before slaughter. Serum concentrations of testosterone were determined in each sample from bulls and from four samples from steers; serum hydroxyproline was determined in the last sample from both sexes. Testosterone mean values for the collection period were calculated. Samples of the longissimus, semitendinosus and infraspinatus muscles secured within 45 min postmortem were analyzed for intramuscular collagen concentration, percent soluble collagen and collagen thermal shrinkage temperature. Tenderness of loin steaks was determined by Warner-Bratzler shear test. Serum concentrations of hydroxyproline and testosterone were higher (P less than .01) in bulls than steers. Age effects were noted for both hydroxyproline (P less than .01) and testosterone (P less than .06). Total intramuscular collagen was greater (P less than .01) in bulls than steers and was different (P less than .01) among muscles, but the muscle differences were not uniform over all ages (P less than .05). Percent soluble collagen declined (P less than .01) with age and was different (P less than .01) among muscles. Interaction of age and muscle (P less than .01) and age and sex (P less than .05) also were noted for percent soluble collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the stem cell niche is crucial for understanding the factors that regulate these cells. Rodent enteric neural crest-derived stem cells have previously been isolated by flow cytometry and culture of cell suspensions from the outer smooth muscle layers or the entire gut wall from postnatal and adult animals. Such cell suspensions contain a mixture of cell types, including smooth muscle, fibroblasts and cells associated with the vasculature and extrinsic innervation. Thus these preparations may be contaminated by stem cells associated with extrinsic sensory and autonomic nerves and by other types of stem cell that reside in the gut. Here we describe a different approach, similar to that recently used for infant human gut, to obtain enteric ganglionderived cells, with properties of neural progenitor cells, using isolated myenteric ganglia from postnatal rat ileum. Myenteric ganglia were separated from the gut wall, dispersed and resulting cell dissociates were plated in non-adherent culture conditions with EGF and FGF-2. Under these conditions neurospherelike bodies (NLB) developed. Cells in NLB incorporated BrdU and expressed the stem cell marker nestin but not the pan-neuronal marker PGP 9.5. Upon growth factor withdrawal some BrdU-immunopositive cells assumed the morphology of neurons and expressed PGP 9.5; others were flattened and expressed the glial cell marker GFAP. This work therefore provides evidence that neural crest-derived progenitors in the postnatal rat gut are located in the myenteric plexus, and shows that these cells can be expanded and differentiated in NLB in vitro.