vasoactive-intestinal-peptide and 3-3--dioctadecylindocarbocyanine

The present study identified and characterised myenteric neurones involved in the innervation of the gastric mucosa. We applied retrograde neuronal tracing methods by using the dye DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorat) in combination with the immunohistochemical demonstration of choline acetyltransferase (ChAT), enkephalin (ENK), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). This method showed distinct neurochemical coding of DiI-labelled neurones with projections to the mucosa (mucosa neurones): ChAT/- (indicating the presence of ChAT only, 32%), ChAT/NPY/ +/- VIP (22%), NOS/NPY/ +/- VIP (19%), ChAT/SP/ +/- ENK (12%), NOS/- (indicating the presence of NOS only, 8%), or ChAT/ENK (4.6%). DiI-labelled mucosa neurones did not contain calretinin, serotonin, or somatostatin. All ChAT population had primarily ascending projections, whereas the NOS populations had mainly descending projections. Both were further classified as longitudinally and circumferentially projecting neurones, the latter having projection preferences towards the lesser or greater curvature. All subpopulations exhibited projection preferences. Nitrergic projections primarily arose from cell bodies located at the lesser curvature. ChAT/- projections, which dominated the cholinergic pathway, mainly arose from cell bodies located at the greater curvature. The other major cholinergic pathway with the code ChAT/NPY/ +/- VIP consisted of neurones located mainly at the lesser curvature. The results suggest specific coding of gastric myenteric neurones with projections to the mucosa. Polarised projections consisted of ascending cholinergic and descending nitrergic neurones; the additional presence of NPY/VIP was a prominent feature in both pathways. Chemical coding, polarity, and projection preferences of enteric pathways to the gastric mucosa are remarkably different from those of other regions in the gut.

Topics: Animals; Axonal Transport; Axons; Carbocyanines; Choline O-Acetyltransferase; Enkephalins; Fluorescent Dyes; Gastric Mucosa; Guinea Pigs; Immunohistochemistry; Myenteric Plexus; Neurons; Neuropeptide Y; Nitric Oxide Synthase; Substance P; Vasoactive Intestinal Peptide

1. Neuronal retrograde tracing with the dye DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in combination with immunohistochemical detection of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), were used to identify the innervation of the mucosa of the guinea-pig proximal colon by submucosal neurones. Ussing chamber experiments were performed to measure changes in short circuit current (delta Isc) evoked by electrical stimulation of the oral or anal end of the preparation. 2. The tracing studies revealed that the mucosa was primarily innervated by descending neurones (78%); the vast majority of these were VIP positive (85%). The numerically smaller ascending pathway (13%) was predominantly ChAT positive (69%). A small population (9%) of DiI-labelled neurones projected circumferentially. 3. Ussing chamber experiments revealed that oral electrical stimulation induced a significantly larger delta Isc than anal stimulation. The VIP antagonist VIP(6-28) significantly reduced only orally induced delta Isc. Anally induced delta Isc were significantly more atropine sensitive that orally induced delta Isc. Tissue incubation with carbachol or VIP significantly potentiated delta Isc induced by VIP and carbachol, respectively, indicating cross-potentiation. 4. This study provides the first functional demonstration of polarized innervation patterns from submucosal neurones to enteric mucosa. The ascending ChAT and descending VIP pathways suggest the existence of reflexes resulting in preferential release of VIP or acetylcholine. The distinct pathways might favour the observed cross-potentiation of cholinergic and VIPergic mediated secretion.

Topics: Action Potentials; Affinity Labels; Animals; Carbocyanines; Choline O-Acetyltransferase; Colon; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Electric Stimulation; Evoked Potentials; Guinea Pigs; Hormone Antagonists; Immunohistochemistry; Intestinal Mucosa; Neurons; Sensitivity and Specificity; Vasoactive Intestinal Peptide

In order to obtain a detailed understanding of the chemical identity of callosal neurons and of their synaptic targets during development of the rat, a technique was developed combining anterograde and retrograde transport of the carbocyanine dye, DiI, previously applied in living or fixed tissue with conventional immunocytochemistry for peptides. It is reported here that photoconversion of the fluorescent DiI label to a stable diaminobenzidine reaction product is fully compatible with the application of the most widely used immunocytochemical techniques peroxidase-antiperoxidase (PAP) or avidin-biotin (ABC) on the same tissue section, for correlated light and electron microscopic studies. Advantages of this double-labeling procedure over previously described techniques which permit concurrent visualization of projection systems and chemically defined neuronal elements are discussed.

Topics: Animals; Carbocyanines; Corpus Callosum; Fluorescent Dyes; Immunohistochemistry; Methods; Microscopy, Electron; Neurons; p-Dimethylaminoazobenzene; Rats; Rats, Wistar; Somatostatin; Vasoactive Intestinal Peptide

Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa.

Topics: Animals; Calbindins; Carbocyanines; Guinea Pigs; Ileum; Immunohistochemistry; Intestinal Mucosa; Microscopy, Fluorescence; Myenteric Plexus; Neural Pathways; Neurons; Neuropeptide Y; S100 Calcium Binding Protein G; Substance P; Vasoactive Intestinal Peptide