A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Enteroendocrine profile of α-transducin and α-gustducin immunoreactive cells in the chicken (Gallus domesticus) gastrointestinal tract.

The enteroendocrine profile and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) protein subunits, were studied in the gastrointestinal (GI) tract of the chicken (Gallus domesticus) using double labeling immunohistochemistry. Gαtran or Gαgust immunoreactivity was observed in enteroendocrine cells (EEC) expressing different peptides throughout the entire GI tract with different density. In the proventriculus tubular gland, Gαtran or Gαgust/gastrin (GAS) immunoreactive (-IR) cells were more abundant than Gαtran/or Gαgust containing glucagon-like peptide-1 (GLP-1) or peptide YY (PYY), whereas only few Gαtran or Gαgust cells co-stored ghrelin (GHR) or 5-hydroxytryptamine (5-HT). In the pyloric mucosa, many Gαtran or Gαgust-IR cells co-expressed GAS or GHR, with less Gαtran or Gαgust cells containing GLP-1, PYY, or 5-HT. In the small intestine, a considerable subset of Gαtran or Gαgust-IR cells co-expressed 5-HT in the villi of the duodenum and ileum, PYY in the villi of the jejunum, CCK or GLP-1 in the villi of the ileum, and GHR in the duodenum crypts. In the large intestine, many Gαtran or Gαgust-IR cells contained 5-HT or GLP-1 in the villi of the rectum, whereas some Gαtran/Gαgust-IR cells co-expressed PYY- or CCK-, and few Gαtran/Gαgust-IR cells were positive for GHR-IR. In the cecum, several Gαtran or Gαgust-IR cells were IR for 5-HT. Finally, many Gαtran/Gαgust cells containing 5-HT were observed in the villi and crypts of the cloaca, whereas there were few Gαtran or Gαgust/CCK-IR cells. The demonstration that Gα-subunits are expressed in the chicken GI enteroendocrine system supports the involvement of taste signaling machinery in the chicken chemosensing processes.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea.

BACKGROUND AND PURPOSE: Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. EXPERIMENTAL APPROACH: NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. KEY RESULTS: alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. CONCLUSIONS AND IMPLICATIONS: In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

Downregulation of neuronal vasoactive intestinal polypeptide in Parkinson's disease and chronic constipation.

BACKGROUND: Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinson's disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls. METHODS: Twenty-nine PD/CC and 10 Rome III-defined CC patients were enrolled. Twenty asymptomatic age-sex matched subjects served as controls. Colonic transit time measurement and conventional anorectal manometry were evaluated in PD/CC and CC patients. Colonoscopy was performed in all three groups. Colonic submucosal whole mounts from PD/CC, CC, and controls were processed for immunohistochemistry with antibodies for vasoactive intestinal polypeptide (VIP) and peripheral choline acetyltransferase, markers for functionally distinct submucosal neurons. The mRNA expression of VIP and its receptors were also assessed. KEY RESULTS: Four subgroups of PD/CC patients were identified: delayed colonic transit plus altered anorectal manometry (65%); delayed colonic transit (13%); altered manometric pattern (13%); and no transit and manometric impairment (9%). There were no differences in the number of neurons/ganglion between PD/CC vs CC or vs controls. A reduced number of submucosal neurons containing VIP immunoreactivity was found in PD/CC vs controls (P<.05). VIP, VIPR1, and VIPR2 mRNA expression was significantly reduced in PD/CC vs CC and controls (P<.05). CONCLUSIONS AND INFERENCES: Colonic motor and rectal sensory functions are impaired in most PD/CC patients. These abnormalities are associated with a decreased VIP expression in submucosal neurons. Both sensory-motor abnormalities and neurally mediated motor and secretory mechanisms are likely to contribute to PD/CC pathophysiology.

Enteroendocrine cells: a review of their role in brain-gut communication.

BACKGROUND: Specialized endoderm-derived epithelial cells, that is, enteroendocrine cells (EECs), are widely distributed throughout the gastrointestinal (GI) tract. Enteroendocrine cells form the largest endocrine organ in the body and play a key role in the control of GI secretion and motility, the regulation of food intake, postprandial glucose levels and metabolism. EECs sense luminal content and release signaling molecules that can enter the circulation to act as classic hormones on distant targets, act locally on neighboring cells and on distinct neuronal pathways including enteric and extrinsic neurons. Recent studies have shed light on EEC sensory transmission by showing direct connections between EECs and the nervous system via axon-like processes that form a well-defined neuroepithelial circuits through which EECs can directly communicate with the neurons innervating the GI tract to initiate appropriate functional responses. PURPOSE: This review will highlight the role played by the EECs in the complex and integrated sensory information responses, and discuss the new findings regarding EECs in the brain-gut axis bidirectional communication.

Distribution of α-transducin and α-gustducin immunoreactive cells in the chicken (Gallus domesticus) gastrointestinal tract.

The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated ("bottle-" or "pear-like") or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract.

Effect of N-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats.

Intestinal ischemia impairs gastrointestinal motility. The aims of this study were to investigate the effect of intestinal ischemia on gastrointestinal transit and on the expression of enteric transmitters in the rat, and whether the glutamate N-methyl-d-aspartate receptors influence these effects. Ischemia (1 h), induced by occluding the superior mesenteric artery, was followed by 0 or 24 h of reperfusion. Normal and sham-operated rats served as controls. Serosal blood flow was measured with laser Doppler flow meter. Gastrointestinal transit was measured as time of appearance of a marker in fecal pellets. Immunohistochemistry was used to evaluate the number of neurons immunoreactive for neuronal nitric oxide synthase (NOS) or vasoactive intestinal polypeptide and the density of substance P immunoreactive fibers in the myenteric plexus. The N-methyl-d-aspartate receptors antagonist, (+)-5-methyl-10,11-dihydro-5HT-[a,b] cyclohepten-5,10-imine (MK-801) (1 mg/kg i.v.) or the NOS inhibitor, N-nitro-l-arginine (10 mg/kg i.v.) was administered prior to ischemia. Serosal blood flow was decreased by 70% during ischemia, but it was not altered in sham-operated rats. Gastrointestinal transit was significantly prolonged in ischemic/reperfused rats compared with controls. There was a significant increase in the number of vasoactive intestinal polypeptide and neuronal nitric oxide synthase immunoreactive neurons, and a marked decrease of substance P immunoreactive fibers in ischemia followed by 24 h of reperfusion animals compared with controls. These alterations were not observed in ischemia without reperfusion. A significant delay of gastrointestinal transit and increase of vasoactive intestinal polypeptide neurons were also observed in sham-operated rats. The changes in transmitter expression and gastrointestinal transit in ischemic/reperfused rats were prevented by pre-treatment with the NOS inhibitor, N-nitro-l-arginine or the N-methyl-d-aspartate receptors antagonist, MK-801. This study suggests an involvement of the glutamatergic system and its interaction with nitric oxide in intestinal ischemia/reperfusion. Ischemia/reperfusion might induce local release of glutamate that activates N-methyl-d-aspartate receptors leading to increased production of nitric oxide and adaptive changes in enteric transmitters that might contribute to gastrointestinal dysmotility.

Identification of galanin receptor 1 on excitatory motor neurons in the guinea pig ileum.

Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1-16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1-16 (0.3-3 micromol L(-1)) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 micromol L(-1)). By contrast, galanin 1-16 was ineffective when added to the intermediate or caudal compartment up to 3 micromol L(-1). GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.

Activation of µ opioid receptors modulates inflammation in acute experimental colitis.

BACKGROUND: µ opioid receptors (µORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral µORs ameliorates the acute and delayed phase of colitis. METHODS: C57BL/6J mice were treated with 3% dextran sodium sulfate (DSS) in water, 5 days with or without the peripherally acting µOR agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or with DAMGO+µOR antagonist at day 2-5, then euthanized. Other mice received DSS followed by water for 4 weeks, or DSS with DAMGO starting at day 2 of DSS for 2 or 3 weeks followed by water, then euthanized at 4 weeks. Disease activity index (DAI), histological damage, and myeloperoxidase assay (MPO), as index of neutrophil infiltration, were evaluated. Cytokines and µOR mRNAs were measured with RT-PCR, and nuclear factor-kB (NF-kB), the antiapoptotic factor Bcl-xL, and caspase 3 and 7 with Western blot. KEY RESULTS: DSS induced acute colitis with elevated DAI, tissue damage, apoptosis and increased MPO, cytokines, µOR mRNA, and NF-kB. DAMGO significantly reduced DAI, inflammatory indexes, cytokines, caspases, and NF-kB, and upregulated Bcl-xL, effects prevented by µOR antagonist. In DSS mice plus 4 weeks of water, DAI, NF-kB, and µOR were normal, whereas MPO, histological damage, and cytokines were still elevated; DAMGO did not reduce inflammation, and did not upregulate Bcl-xL. CONCLUSIONS & INFERENCES: µOR activation ameliorated the acute but not the delayed phase of DSS colitis by reducing cytokines, likely through activation of the antiapoptotic factor, Bcl-xL, and suppression of NF-kB, a potentiator of inflammation.

Developmentally regulated expression of alpha- and beta-calcitonin gene-related peptide mRNA and calcitonin gene-related peptide immunoreactivity in the rat inferior olive.

Immunohistochemical methods have revealed the transient neonatal expression of calcitonin gene-related peptide (CGRP) in olivocerebellar compartments, and it has been hypothesized that this peptide plays a role in the development of olivocerebellar connectivity. Furthermore, the distribution of the CGRP binding sites in the cerebellar cortex also favors this hypothesis. In this study, the pattern of postnatal expression of alpha- and beta-CGRP mRNAs in the inferior olive (IO) complex was analyzed using in situ hybridization histochemistry with RNA probes complementary to specific sequences of alpha- and beta-CGRP mRNAs, and the results were compared with the pattern of CGRP immunoreactivity. High levels of alpha-CGRP mRNA expression were found in specific subnuclei of the IO complex, i.e., the medial part of the dorsal fold of the dorsal accessory olive, the beta nucleus, the dorsal cap, the caudal third of the medial accessory olive, and the rostral part of the dorso-medial cell column; in the same subnuclei beta-CGRP mRNA was detected. The olivary distribution of the two CGRP mRNA coincided with that of CGRP immunoreactivity. The expressions of alpha-CGRP mRNA and CGRP immunoreactivity were restricted to the first 2 postnatal weeks, the peak being reached at the end of the first week; beta-CGRP mRNA was transiently expressed in the same olivary compartments, but only from postnatal day 6 to 9. In general, the alpha-CGRP signal was also more intense than the beta-CGRP signal. The present findings indicate that the alpha- and beta-CGRP mRNA expression in the olivary complex is under developmental control and restricted to specific olivocerebellar compartments. The data provide a basis for the transient expression of a CGRP olivocerebellar compartment and further support the hypothesis of a role for CGRP in the complex postnatal cerebellar phenomena of connectivity reshaping and synapse stabilization.

Cellular sites of expression of the neurokinin-1 receptor in the rat gastrointestinal tract.

In the digestive system, substance P is an excitatory transmitter to muscle, a putative excitatory neuro-neuronal transmitter, a vasodilator, and a mediator in inflammatory processes. Many of the biological effects of substance P are mediated by a high-affinity interaction with the tachykinin receptor neurokinin-1. The aim of the present study was to identify the sites of expression of this receptor in the rat stomach and intestine by immunohistochemistry with a polyclonal antiserum raised to the intracellular C-terminal portion of the rat neurokinin-1 receptor. Neurokinin-1 receptor immunoreactivity is present in a large population of enteric neurons. The relative density of these neurons along the gut is colon > ileum >> stomach. In the intestine, stained neurons have a smooth cell body with processes that can be followed within and between plexuses, and make close approaches to other neuronal cells, but do not appear to project outside the plexuses, suggesting that they are interneurons. In the stomach, neurokinin-1 receptor-immunoreactive neurons are infrequent and have a poorly defined and irregular shape. Neurokinin-1 receptor immunoreactivity is also localized to numerous non-neuronal cells in the inner portion of the circular muscle layer of the small intestine, which have the appearance of small dark smooth muscle cells or interstitial cells of Cajal. These cells are postulated to form a "stretch-sensitive" system with the deep muscular plexus and thus constitute an important site of regulation of muscle activity. Double labeling immunofluorescence was used to simultaneously localize neurokinin-1 receptor and substance P/tachykinin immunoreactivities. These experiments demonstrate that in the enteric plexuses, substance P/tachykinin-immunoreactive varicose fibers encircle the cell bodies of most neurokinin-1 receptor-containing neurons, and in the inner portion of the circular muscle layer of the small intestine they lie close to neurokinin-1 receptor-immunoreactive non-neuronal cells. In addition, some enteric neurons express both neurokinin-1 receptor and substance P/tachykinin immunoreactivities. The present study provides strong evidence that the neurokinin-1 receptor is the tachykinin receptor mediating the actions of substance P on enteric neurons and smooth muscle.

Distribution of calcitonin gene-related peptide immunoreactivity in relation to the rat central somatosensory projection.

The distribution of the neuropeptide calcitonin gene-related peptide (CGRP) was studied in relation to the known subcortical somatosensory pathways and contiguous systems in the central nervous system (CNS) of rats by using peroxidase histochemical methods in order to relate zones of immunoreactivity (IR) to cytoarchitecture. CGRP is the most ubiquitous peptide found to date in sensory ganglion cells: principally small and medium-size neurons emitting thin axons inferred to be largely nociceptive in function on the basis of the peripheral distribution of their terminals. Its apparent absence in sympathetic axons provides an especially useful sensory marker. The distribution of CGRP-IR axons displays remarkable selectivity at each level of the CNS. The trigeminal root distributes axons primarily to the pericornual layers (laminae I and II) of spinal V nucleus caudalis and to subnucleus oralis, evading the subnucleus interpolaris and contributing only few axons to principal V. Although there are only a few CGRP-IR somata at each level, heavily labeled axon trajectories can be traced to the nuclei of the solitary tract, the parabrachial nuclei, several sectors of the caudal medial thalamus, and the central nucleus of the amygdala. A sector of labeled neuron somata lies contiguous to each of these axon terminal zones, the largest of which is a thalamic nucleus containing cells of distinctive dendritic architecture extending from the periaqueductal gray across the posterior group nuclei to the peripeduncular nucleus, forming a linear array at the mesodiencephalic junction. The relation of CGRP-IR axonal distribution to spinothalamic, visceral, and gustatory systems is discussed in the context of a specialized "chemosensory" component of the thin-fiber somatosensory system.

Neurokinin 1 receptor expression in the rat retina.

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the gamma-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine tells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina.

Neurons containing calcitonin gene-related peptide in the parabrachial nucleus project to the central nucleus of the amygdala.

The source, distribution, and morphology of axons displaying calcitonin gene-related peptide (CGRP) immunoreactivity in the central amygdaloid nucleus of the adult rat were investigated with immunohistochemical techniques, both alone and in combination with retrograde transport of fluorescent tracers. An extremely dense plexus of CGRP-immunoreactive axons is differentially concentrated within the lateral capsular and lateral central subdivisions of the central nucleus, and much lighter concentrations of labeled fibers are present in the rostral part of the medial subdivision. No immunoreactive neurons were observed in the central nucleus in any of the experimental animals. The immunoreactive axons characteristically form prominent pericellular terminal arborizations surrounding unlabeled neurons. The number of cells receiving this dense input increases at caudal levels of the central nucleus. Retrograde label of central nucleus neurons by dye transport from injections into the pontine parabrachial nucleus and the nucleus of the tractus solitarius combined with CGRP immunohistochemistry established that many neurons in the central nucleus which receive dense pericellular innervation from CGRP-immunoreactive axons are projecting caudally to the parabrachial nucleus or, to a lesser extent, to the nucleus tractus solitarii. Central amygdaloid injections of rhodamine-labeled microspheres or fluorogold followed by immunohistochemical localization of cellular CGRP immunoreactivity revealed that the central amygdaloid CGRP fiber plexus originates bilaterally from the parabrachial nucleus. These multipolar CGRP-containing neurons are preferentially concentrated in the external medial and external lateral subnuclei, in the ventral aspect of the parabrachial nucleus. These results relating central amygdaloid CGRP to ascending and descending brainstem pathways, taken together with the extreme density of the fiber plexus, strongly suggest the relevance of the CGRP input to central nucleus function in cardiovascular and other autonomic regulation.

Peripheral patterns of calcitonin-gene-related peptide general somatic sensory innervation: cutaneous and deep terminations.

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.

Nitric oxide producing neurons in the monkey and human digestive system.

Nitric oxide has been proposed as an inhibitory transmitter molecule that plays a role in muscle relaxation and vasodilation in the gastrointestinal tract. The present study analyzes the distribution of nitric-oxide-producing neurons in the monkey and human digestive system by means of nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. This histochemical method is reliable and convenient for the visualization of neuronal nitric-oxide synthase, the enzyme responsible for nitric-oxide generation. In the gastrointestinal tract, nitric-oxide-synthase-related diaphorase activity was present in nerve fibers running throughout the muscular layer (circular > longitudinal) and in numerous ganglion cells and processes in the myenteric plexus of monkeys and humans. Labelled ganglion cells and fibers also were observed in the submucous plexus, although they were much less numerous than those seen in the myenteric plexus. In the submucosa, a few positive fibers were seen around blood vessels. In the mucosa, stained fibers were sparse at the base of the villi and crypts, whereas they were quite abundant in the muscularis mucosae, especially in the small intestine and colon. In the gallbladder (human), labelling was found in ganglion cells and processes of the innermost and outermost ganglionated plexuses. Stained fibers also were distributed to the muscular layer and, less abundantly, to the mucosa and vasculature. Labelled fibers were more abundant in the sphincter of Oddi (human) than in the gallbladder. In the monkey and human pancreas, nicotinamide-adenine-dinucleotide-diaphorase staining was seen mainly in ganglion cells and fibers of intrapancreatic ganglia, and in processes running among acini, around ducts and in the stroma. A moderate density of stained fibers also was distributed to the vasculature, whereas the islets showed few positive processes. Finally, double label experiments performed in the pancreas showed that the vast majority of neurons producing nitric oxide are immunoreactive for vasoactive intestinal peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin 2A receptor is expressed by enteric neurons, and by interstitial cells of Cajal and enterochromaffin-like cells of the gastrointestinal tract.

Somatostatin exerts multiple effects by activating distinct G protein-coupled receptors. Here we report the cellular sites of expression of the somatostatin subtype 2A (sst2A) receptor in the rat enteric nervous system by using a C-terminus-specific, affinity-purified antiserum and immunohistochemistry. Antibody specificity was confirmed by the cell surface staining of human embryonic kidney 293 cells expressing the sst2A receptor, the lack of staining of cells expressing the somatostatin subtype 2B receptor, and the abolition of staining by preincubating the antiserum with the C-terminus peptide used for immunization, SSt2A(361-369). The SSt2A receptor antibody recognized a broad 80 kDa band on Western blots of membranes prepared from cells transfected with sst2A receptor cDNA; following receptor membrane deglycosylation, the antibody detected an additional 40 kDa band. In the enteric nervous system, the sst2A antibody primarily stained neurons of the myenteric and submucosal plexuses, and abundant fibers distributed to the muscle, mucosa, and vasculature. Immunoreactive staining was also observed in non-neuronal cells, including presumed interstitial cells of Cajal of the intestine and enterochromaffin-like cells of the stomach. Fibers expressing sst2A receptor immunoreactivity were often in close proximity to D cells of the gastric and intestinal mucosa. Colocalization of somatostatin and sst2A receptor immunoreactivities was not observed in endocrine cells nor in enteric neurons. Double-label immunohistochemistry revealed colocalization of sst2A and vasoactive intestinal peptide immunoreactivities in enteric neurons. The multiple types of cells expressing the sst2A receptor, including enteric neurons and non-neuronal structures, in addition to the relationship between somatostatin and sst2A receptor elements, provide evidence that the sst2A receptor mediates somatostatin effects in the gastrointestinal tract via neuronal and paracrine pathways.

Cellular localization of Pan-trk immunoreactivity and trkC mRNA in the enteric nervous system.

The members of the trk family of tyrosine receptor kinases, trkA, trkB, and trkC, are the functional receptors for neurotrophins, a family of related neurotrophic factors. In this study, we investigated 1) the distribution of neurotrophin receptors in the developing and adult rat digestive tract with a pan-trk antibody that recognizes all known trks and 2) the cellular localization of trk-encoding mRNAs in the adult gut with single-stranded RNA probes specific for trkA, trkB, and trkC. In the developing myenteric plexus, trk immunoreactivity was present at embryonic day (ED) 14. Cells and fibers immunoreactive for trk could be visualized in the myenteric plexus at ED 16. At this age, dense staining was found in thick bundles of fibers in proximity to the myenteric plexus in the longitudinal muscle and in association with blood vessels in the mesentery. At ED 18, trk immunoreactivity was also seen in thin processes running from the myenteric plexus into the circular muscle, and in fibers and cells in intrapancreatic ganglia. By ED 20, immunoreactive staining was quite dense in both the myenteric and submucosal plexuses. At birth, virtually all enteric ganglia displayed strong trk immunoreactivity; the intensity of the staining at this age made it difficult to discern individual cells. During postnatal development, there was a decrease in cell body staining and an increase in the density of trk-containing fibers that became widely distributed to the gut wall and pancreas. The adult pattern of trk immunoreactivity was established between postnatal days 5 and 10. In adults, trk immunoreactivity was found in numerous enteric and intrapancreatic ganglion cells and in dense networks of fibers innervating all the layers of the gut, the pancreas, and vasculature. The trkC mRNA was expressed in adult enteric ganglion cells of both the myenteric and submucous plexus. By contrast, the trkA and trkB mRNAs could not be detected in enteric ganglia. All three trk mRNAs were expressed in dorsal root ganglia, which were used as positive controls. The density and wide distribution of trk immunoreactivity together with its persistence in adulthood support the concept that neurotrophins play a broad role in the digestive system from development through adult life, perhaps being involved in differentiation, phenotypic expression, and tissue maintenance. The presence of trkC mRNA in enteric neurons along with recent evidence that neurotrophin-3 plays a role in the development of the enteric nervous system suggest that trkC and neurotrophin-3 are a major neurotrophin system in the gastrointestinal tract.

Abdominal surgery induces mu opioid receptor endocytosis in enteric neurons of the guinea-pig ileum.

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.

Etorphine increases the number of mu-opioid receptor-positive cells in the cerebral cortex.

Imunocytochemical techniques were used to determine whether agonist-induced activation of mu-opioid receptors alters the number and distribution of mu-opioid receptor-positive cells in the rat cerebral cortex. In untreated rats, mu-opioid receptor immunoreactivity was localized to neuronal perikarya and dendrites and to neuropilar punctate structures. mu-Opioid receptor-positive neurons were mostly in layers II and III and exhibited a bipolar or bitufted morphology. In rats treated with the mu-opioid receptor agonist etorphine (0.1mg/kg intraperitoneally) and perfused after different survival periods, there was an enhancement of immunostaining for mu-opioid receptors observed at 15min, reaching a maximum at 60min, and which returned to normal at 480min. Etorphine-induced effects included an increase in the intensity of cellular and neuropil staining; statistical analysis showed that the number of mu-opioid receptor-positive cells in etorphine-treated groups was significantly higher than in controls or saline-treated rats. In animals that received both etorphine and the mu-opioid receptor antagonist naloxone, the pattern of mu-opioid receptors immunoreactivity was similar to that of untreated animals. This study shows that the number of mu-opioid receptor-positive cells is significantly increased following etorphine treatment and suggests that agonist treatment may be exploited to increased immunostaining of mu-opioid receptors and also of other G-protein coupled receptors.