Peptide-containing neurons remain unaffected after intestinal autotransplantation: an experimental study in the piglet

The gut is richly supplied with peptide-containing nervous elements. In the present immunocytochemical study the origin, occurrence and topographical distribution of nerves containing vasoactive intestinal polypeptide (VIP), enkephalin, substance P (SP), somatostatin, neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), gastrin-releasing peptide (GRP) and galanin were investigated in the porcine small intestine. In order to study the origin (extrinsic or intrinsic) of the nerve fibers, specimens from autotransplanted and extrinsically denervated jejunum were examined. Furthermore, possible changes in the distribution of intrinsic neurons after extrinsic denervation were studied. In the control jejunum each nerve fiber population had its own characteristic topographic distribution. There was no overt difference in distribution pattern of peptide-containing nerve fibers and cell bodies between the transplanted and the control segment except that NPY-, SP- and CGRP-containing nerve fibers disappeared around blood vessels. Thus VIP-, somatostatin-, GRP-, enkephalin- and galanin-containing nerve fibers were visibly unchanged in the transplanted segment. The results support the view that the peptide-containing nerve fibers are mainly intrinsic in origin except the NPY-, SP- or CGRP-containing perivascular nerve fibers which are extrinsic to the gut wall. In addition, the results of the present study suggest that transplantation and extrinsic denervation have no major effect on the distribution pattern of the intrinsic neuronal systems.     

Increased active stress generation of denervated rat intestinal smooth muscle: functional analysis of muscarinic receptor population

The effects of denervation on the active stress production by the longitudinal muscle (LM) layer of rat jejunum were examined. Extrinsic and myenteric denervation of a segment of rat jejunum was accomplished by the serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). Isolated muscle contraction experiments revealed that the LM of the jejunum taken from rats treated with BAC 15 days before developed significantly increased active stress in response to bethanechol and carbachol, but not in response to potassium chloride. No change in -log EC50 values of any of the agonists was observed in the denervated LM layer, although a significant increase in the slope of the carbachol and bethanechol concentration-response curves was observed in the denervated LM. Schild analysis of several muscarinic antagonists revealed a 3-fold increase in the apparent dissociation constant of the M2 antagonist methoctramine in BAC-treated LM. These results suggest that the increased responsiveness of the denervated LM may originate in the muscarinic receptor population of the myocytes.     

Projections of pelvic autonomic neurons within the lower bowel of the male rat: an anterograde labelling study

The tissues of the large intestine which receive an innervation by neurons of the major pelvic ganglia were identified following in vivo and in vitro anterograde labelling with the lipophilic tracer 1,1'didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the male rat. The primary target in the gut of major pelvic ganglion neurons is the myenteric plexus of the distal colon and the rectum. The serosal ganglia, on the surface of the most distal region of the rectum and the circular muscle of the distal colon and rectum were less densely innervated. The pelvic ganglia do not innervate the longitudinal muscle, submucosal blood vessels, submucosal plexus, or mucosa. The pelvic supply reaches the bowel via two groups of rectal nerves and branches of the penile nerves. All of these connections also carry the axons of viscerofugal neurons from the bowel, some of which have terminal axons in the major pelvic ganglia. Finally, the different nerves supplied different targets. In particular, while the rectal nerves carried pelvic axons supplying the myenteric plexus, circular muscle, and serosal ganglia, the penile nerves only innervated the serosal ganglia. In addition, the two groups of rectal nerves innervated slightly different regions of the bowel and provided different projection patterns. However, successful in vivo labelling was achieved in only 6/12 animals and while all in vitro experiments resulted in successful labelling, it was clear that only a proportion of pelvic projections in any given nerve were labelled. These studies have shown that the major pelvic ganglia are primarily involved in the control of motility, but not of vascular and secretomotor functions. Thus pelvic neurons do not innervate the same range of target tissues within the bowel as the prevertebral ganglia. This study has also shown that the different pathways to the gut from the major pelvic ganglia innervate different tissues, suggesting that the autonomic innervation of the gut is not homogeneous along its length.     

Morphological and immunohistochemical identification of neurons and their targets in the guinea-pig duodenum

Nerve circuits within the proximal duodenum were investigated using a combination of immunohistochemistry for individual neuron markers and lesion of intrinsic nerve pathways to determine axon projections. Cell shapes and axonal projections were also studied in cells that had been injected with a marker substance. Several major neuron populations were identified. Calbindin immunoreactivity occurred in a population of myenteric nerve cells with Dogiel type II morphology. These had axons that projected to other myenteric ganglia, to the circular muscle and to the mucosa. All were immunoreactive for the synthesizing enzyme for acetylcholine, choline acetyltransferase, and some were also immunoreactive for calretinin. Myenteric neurons with nitric oxide synthase immunoreactivity projected anally to the circular muscle. These were also immunoreactive for vasoactive intestinal peptide, and proportions of them had enkephalin and/or neuropeptide Y immunoreactivity. It is suggested that they are inhibitory motor neurons to the circular muscle. A very few (about 2%) of nitric oxide synthase-immunoreactive neurons had choline acetyltransferase immunoreactivity. Tachykinin (substance P)-immunoreactive nerve cells were numerous in the myenteric plexus. Some of these projected orally to the circular muscle and are concluded to be excitatory motor neurons. Others projected to the tertiary plexus which innervates the longitudinal muscle and others provided terminals in the myenteric plexus. Two groups of descending interneurons were identified, one with somatostatin immunoreactivity and one with vasoactive intestinal peptide immunoreactivity. The two most common nerve cells in submucous ganglia were neuropeptide Y- and vasoactive intestinal peptide-immunoreactive nerve cells. Both provided innervation of the mucosa. There was also a population of calretinin-immunoreactive submucous neurons that innervated the mucosal glands, but not the villi. Comparison with the ileum reveals similarities in the chemistries and projections of neurons. Differences include the almost complete absence of nitric oxide synthase immunoreactivity from vasoactive intestinal peptide-immunoreactive interneurons in the duodenum, the projection of calbindin-immunoreactive Dogiel type II neurons to the circular muscle and the absence of tachykinin-immunoreactivity from these neurons.     

Functional changes in nonadrenergic, noncholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats

This experiment was designed to determine mechanisms of change in nonadrenergic, noncholinergic (NANC) inhibitory neurons in the ileum after small bowel transplantation (SBT) in the rat and whether nitric oxide (NO) serves as an important NANC inhibitory neurotransmitter in the rat ileum. Eight groups of rats (N > or =8 rats/group) were studied: neurally intact unoperated controls; rats one week after anesthesia and sham celiotomy; and separate groups one and eight weeks after either 40 min of cold ischemia of the jejunoileum, combined jejunal and ileal intestinal transection/reanastomosis, or orthotopic SBT of the entire jejunoileum. Contractile activity was evaluated in full-thickness ileal circular muscle strips under isometric conditions. Spontaneous activity did not differ among groups. In all groups, exogenous NO, NG-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), and methylene blue (soluble guanylate cyclase inhibitor) had no effect on spontaneous activity, while 8-bromocyclic guanosine monophosphate (8Br-cGMP) inhibited contractile activity in all groups. Low frequency (2-10 Hz) electrical field stimulation (EFS) inhibited contractile activity only in control and SBT groups; L-NMMA and methylene blue did not alter the response to EFS in any group. These results suggest that each aspect of the SBT procedure, ischemia/reperfusion injury, disruption of enteric neural continuity by intestinal transection, and extrinsic denervation, alter function of enteric ileal inhibitory neurons separately early (one week) after operation. NO, a known inhibitory neurotransmitter in other gut regions, does not affect ileal circular muscle in neurally intact tissue nor mediate functional changes in inhibitory nerve function nor smooth muscle contractility after SBT.     

Pharmacokinetics of prednisolone transfer to breast milk

A possible interaction of salmon-calcitonin with opioid systems was studied in isolated tissues. Neurogenic contractions were elicited by electrical stimulation in guinea-pig ileum myenteric plexus-longitudinal muscle strips, rabbit vas deferens and mouse vas deferens. Bremazocine inhibited neurogenic contractions in all three tissues (presumably through kappa-receptors) [D-Pen2, D-Pen5]enkephalin and [Met5]enkephalin inhibited contractions in mouse vas deferens (presumably through delta-receptors), and [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) inhibited contractions in guinea-pig ileum and mouse vas deferens (presumably through mu-receptors). All inhibitory effects were concentration-dependent. Salmon-calcitonin 0.1 IU/ml increased the effect of bremazocine in guinea-pig ileum and rabbit vas deferens and also increased the effects of [D-Pen2, D-Pen5]enkephalin and [Met5]enkephalin in mouse vas deferens. In contrast, salmon-calcitonin up to 0.4 IU/ml did not change the effect of bremazocine in mouse vas deferens and the effect of DAMGO in guinea-pig ileum and mouse vas deferens. It is concluded that salmon-calcitonin enhances agonist effects at opioid kappa- and delta- but not at opioid mu-receptors. The level of this interaction remains to be elucidated. The interaction may be the basis of the analgesic effect of salmon-calcitonin in vivo.     

The effects of heating with ultrasound on knee joint displacement

The effect of prostaglandin E2 (PGE2) on the activity-related expression of the proto-oncogene c-fos in specific populations of enteric neurons was investigated. Segments of guinea-pig ileum were incubated in vitro in the presence or absence of PGE2, and whole mounts of the myenteric and submucosal plexus were prepared for immunocytochemical localization of Fos, VIP and NPY. Control tissues exhibited a low number of Fos-immunoreactive (Fos-IR) neurons (7 +/- 2% of total). Incubation of the tissues with 10-1000 nM PGE2 for 30 min caused a concentration-dependent increase in Fos-IR submucosal neurons (maximum at 100 nM; 39 +/- 6%), which was not inhibited by TTX. PGE2 did not evoke an increase in Fos-IR myenteric neurons. In double labeling experiments, Fos colocalized exclusively with VIP in the submucosal plexus, and not with NPY. Exposure of stripped segments of guinea pig ileum in Ussing chambers to 100 nM PGE2 evoked an increase in short circuit current (20 +/- 7 microA/cm2), of which the initial rapid phase could be abolished by TTX, and not by atropine and hexamethonium. It is concluded that PGE2 can activate VIP non-cholinergic secretomotor neurons.     

Immunolocalization of leukemia inhibitory factor in normal and denervated human muscle

The cytokine leukemia inhibitory factor (LIF) stimulates myoblast proliferation in vitro and vivo and is neurotrophic for motor neurons. In experimentally reinnervated muscle, exogenous LIF application increases muscle mass through myofiber hypertrophy. The goal of this study was to evaluate possible sources of endogenous LIF in human muscle, and whether LIF immunoreactivity (-IR) was detectable in specific myofiber types and/or re-expressed in human denervated muscle. Our study shows that LIF-IR is constitutively detectable in type I myofibers of normal human muscle. In acute and chronically denervated and reinnervated human muscle, LIF-IR is found in all type I myofibers and in addition in some atrophic and almost all angulated atrophic type II myofibers.     

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.     

The effect of denervated muscle and Schwann cells on axon collateral sprouting

The effects of denervated muscle and Schwann cells on collateral sprouting from peripheral nerve were studied in the peroneal and tibial nerves of 48 Sprague-Dawley rats. Three groups were prepared. In group MSW (muscle-Schwann cell-window), the peroneal nerves were transected 3 mm below the sciatic bifurcation. The proximal stumps were sealed in a blocked tube to prevent regeneration and the distal stumps were implanted into denervated muscle cells that were wrapped around the ipsilateral tibial nerve, which had a window of perineurium resected. Schwann cells from the ipsilateral sural nerve were implanted into the muscle. Group MS (muscle-Schwann cell) was similar to group MSW, except that the tibial nerve perineurium was kept intact. In group MW (muscle-window), the muscle was prepared without Schwann cells and the tibial nerve perineurium was windowed. S-100 immunostain was used to identify the Schwann cells surviving 1 week after transplantation. After 16 weeks of regeneration, horseradish peroxidase tracer was used to label motor neurons and sensory neurons reinnervating the peroneal nerve. Myelinated axons of the reinnervated peroneal nerves were quantified with the Bioquant OS/2 computer system (R&M Biometrics, Nashville, TN). A mean of 169 motor neurons in group MSW, 64 in group MW, and 26 in group MS reinnervated the peroneal nerve. In the dorsal root ganglion, the mean number of labeled sensory neurons was 1,283 in group MSW, 947 in group MS, and 615 in group MW. The mean number of myelinated axons in the reinnervated peroneal nerve was 1,659 in group MSW, 359 in group MS, and 348 in group MW. Reinnervated anterolateral compartment muscles in group MSW were significantly heavier than those in group MS or MW. This study demonstrates that the transplantation of denervated muscle and Schwann cells promotes motor and sensory nerve collateral sprouting through a perineurial window.     

Recovery of muscle after different periods of denervation and treatments

Three aspects of reinnervation and recovery of skeletal muscle following various periods of denervation were investigated: (1) the effect of duration of denervation; (2) the effect of hyperthyroidism on recovery; and (3) whether the muscle or the nerve limits recovery. The rat medial gastrocnemius (MG) nerve was cut and then resutured after 0, 3, 7, 21, or 56 days. In a second group of animals, the MG muscle was denervated and, in addition, the animal received triiodothyronine (T3) supplementation during reinnervation. The third group of animals had the denervated MG muscle reinnervated by a larger number of newly transected foreign axons. The force produced by the reinnervated muscle depends on the period that the muscle was denervated. Recovery was impaired when the period of denervation exceeded 7 days. T3 treatment did not benefit the return of force production, nor did providing the muscle with a larger number of newly transected axons.     

The role of acetylcholine receptors and acetylcholinesterase activity in the development of denervation supersensitivity

Strips of muscle from innervated and denervated rat hemidiaphragm were tested for sensitivity to acetylcholine and to carbachol. For both agonists, denervation (6-8 days) produced notable supersensitivity. However, the increase in sensitivity to acetylcholine (ca. 600-fold) was much greater than that to carbachol (ca. 51-fold). Denervation also produced an increase in [3H]alpha-bungarotoxin binding (ca. 20-fold), presumably indicative of an increase in the number of acetylcholine receptors. In addition to causing increases in tissue sensitivity and receptor number, denervation caused a marked loss of acetylcholinesterase activity (ca. 70%) and a modest loss of butyrylcholinesterase activity (ca. 20%). When innervated muscle was pretreated with eserine (5 X 10(-5) M), there was a loss of acetylcholinesterase activity (ca. 86%) and butyrylcholinesterase activity (ca. 36%). Simultaneously, there was an increase in tissue sensitivity to acetylcholine (ca. 26-fold). When denervated muscle was pretreated with eserine, there was no loss of enzyme activity beyond that caused by denervation. Furthermore, eserine pretreatment did not increase denervated muscle sensitivity to acetylcholine. The data suggest that both an increase in acetylcholine receptors and a decrease in acetylcholinesterase activity contribute to the phenomenon of denervation supersensitivity.     

Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase

Tenascin is a large oligomeric glycoprotein of the extracellular matrix. Its location is innervated muscle tissues. We investigated immunohistologically, using two monoclonal antibodies (mah) against Tenascin, biopsied denervated human muscle of children and adults. Tenascin was present in the interstitial space among denervated muscle fibres. Accumulation of Tenascin in denervated adult muscle tissue was frequent, accumulation in denervated muscle tissue of children was sparse and weak. The two antibodies reacted correspondingly. Tenascin was not only found in the vicinity of atrophic muscle fibres, but also close to normally sized fibres, suggesting an early stage of denervation even before reduction of muscle fibre volume.     

Regional differences in the nitrergic innervation between the proximal and the distal colon in rats

BACKGROUND & AIMS: Functional differences in the inhibitory neural pathway between the proximal and the distal colon are unknown. METHODS: We investigated the nonadrenergic, noncholinergic (NANC) relaxation, nitric oxide synthase (NOS) synthesis, and NOS messenger RNA (mRNA) expression of the myenteric plexus in the proximal and the distal colon in rats. RESULTS: Transmural nerve stimulation of the neuromuscular preparations from the proximal colon showed greater NANC relaxations than those from the distal colon. NANC relaxations were abolished by the NO biosynthesis inhibitor (NG-nitro-L-arginine methyl ester) in the proximal and the distal colon, suggesting mediation by NO released from the myenteric plexus. The average number of NOS-immunoreactive cells was significantly higher in the tissue from the proximal colon than in the tissue from the distal colon. Western and Northern blot analyses showed a higher density of the immunoreactive NOS band and the NOS mRNA band in the tissue from the proximal colon than in that from the distal colon. CONCLUSIONS: These observations indicate that the number of NOS-containing neurons and the NOS activity are increased in the myenteric plexus of the proximal colon compared with the distal colon, resulting in greater NANC relaxation in the proximal colon. These findings may explain the physiological role of the proximal colon as an organ for fecal storage and absorption of excess fluid.     

Motor reconnection between the damaged cervical cord and the denervated biceps muscle using an autologous peripheral nerve segment in the adult marmoset]

Our research group is studying, in the primate (marmoset), the conditions of an anatomical and functional reconstruction of the spinal cord and of its motor connections, following a focal spinal lesion. In this attempt to repair the damaged neuronal circuitry, we used long segments of autologous peripheral nerves joining the injured cervical spinal cord to an aneural region of the denervated biceps brachialis muscle (7 marmosets) or to the musculocutaneous nerve (6 marmosets). After retrograde tracing (HRP) and histochemical studies of the muscle, we found that a great number of neurons, located mostly in the ventral part of the grey matter extended axons into the peripheral nerve graft. Some of these labelled neurons were motoneurons, which could established functionnal neuromuscular junctions. The muscle regeneration was effective but slower than already known in rat studies.     

Analysis of fiber types in the pigeon's metapatagialis muscle. II. Effects of denervation

The pigeon's metapatagialis muscle consists of three slips, two twitch and one tonic, and these slips are distinguishable at the gross anatomical level. Comparative studies of denervation are facilitated because the two fiber types are under the same mechanical forces, can be denervated as one muscle, and can be distinguished after denervation. Both fiber types atrophied after denervation, with the twitch fibers having a more variable response. Pathological alterations observed by light microscopy suggested that the twitch fibers were more affected by denervation than the tonus fibers. Ultrastructurally, both fiber types showed the same changes, with the twitch fibers again being more consistently altered. Proliferation of the transverse tubular system and sarcoplasmic reticulum were more marked in the tonus than twitch fibers, and the sarcoplasmic reticulum proliferated prior to the transverse tubules. Filament and fibril degeneration, peripheral and central degeneration, lysosomes and their derivatives, and satellite cell proliferation were common to both fiber types. Contracture knots were common to the denervated fibers, and were suggested to be characteristic of degenerating fibers. Degenerating motor end plates were observed, and most neurons in the fibers were naked, lacking myelin sheaths. The results are discussed in relation to the function of the neuron in maintaining the muscle, and the possibility of denervation inducing a transformation of tonic to twitch fibers.     

Electrophysiological and pharmacological responses of chronically denervated lower esophageal sphincter of the opossum

BACKGROUND & AIMS: Achalasia is characterized by loss of myenteric neurons and incomplete relaxation of the lower esophageal sphincter (LES). The aim of this study was to develop an achalasia model in the opossum using the surfactant benzyldimethyltetradecylammonium chloride (BAC). This study further characterizes the achalasia model. METHODS: BAC or saline was injected circumferentially into the LES of 14 adult opossums. Eight months after injection, manometry, isolated muscle bath studies, electrical field stimulation, and histochemical analysis were performed. RESULTS: Manometrically, the LES of BAC-treated opossums showed higher pressures (38.7 +/- 12 mm Hg vs. 17 +/- 3.0 mm Hg) and reduced esophageal body contraction amplitudes (4.2 +/- 3 mm Hg vs. 27.4 +/- 12 mm Hg). Isolated muscle strips challenged with carbachol and sodium nitroprusside contracted and relaxed similarly to controls. Electrical field stimulation failed to induce relaxation in BAC-treated tissue but did induce contraction. Contractile responses were markedly reduced by tetrodotoxin and atropine in BAC-treated animals and controls. An altered nitric oxide system was shown by the lack of response to L-arginine and N omega-nitro-L-arginine. Histology showed loss of myenteric neurons and increased cholinergic nerve bundles. CONCLUSIONS: Loss of NO inhibitory myenteric neurons markedly reduces the relaxation of the LES, and histology and pharmacological responses suggest a proliferation of cholinergic nerves into the LES contributing to the static elevated pressures of the amyenteric LES.     

Evidence for an inhibition by endogenous galanin of neurogenic cutaneous vasodilatation in the pigeon

The effect of high affinity galanin antagonist M35 on neurogenic cutaneous vasodilatation has been studied in the pigeon using a Laser Doppler Imager. Cutaneous application of mustard oil or antidromic electrical stimulation of a cutaneous nerve produced a small increase in skin blood flow. Close arterial injection of M35 prior to chemical or electrical stimulation resulted in a marked augmentation of the vasodilatory response. This effect was abolished by chronic denervation. The results suggest a nerve-mediated inhibitory effect of endogenous galanin on neurogenic cutaneous vasodilatation in the pigeon skin and provide the first experimental evidence for an inhibitory local regulatory function of cutaneous sensory nerves at least in the avian skin.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D28, and s-100. Cultured cells or tissues were incubated with 10 microM BODIPY forskolin for 30 min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D28 (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.