The three-dimensional structure of myenteric neurons in the guinea-pig ileum.

Myenteric neurons of the guinea-pig ileum were intracellularly filled with the fluorescent dye Lucifer Yellow, optically sectioned with a confocal microscope and volume reconstructed to recreate 3-D images of the cells. The resulting images provide information not evident from regular microscopy. The somata varied in cross-section from flat-oval to nearly circular, and their surface membranes were marked by invaginations and protrusions significantly increasing the surface area of the somatic membrane. The neurons could be divided into four morphological classes: Dogiel type I, Dogiel type II, filamentous, and intermediate. There was no clear correlation between cell class and the shape of the soma in cross-section. The dendritic processes of all the neurons studied extended in an orad-caudad or circumferential direction of the bowel wall. When the filled neurons were viewed edge-on, the spatial arrangement of the processes was confined to a plane that had a thickness less than the thickness of the parent soma. The broad, short dendrites of Dogiel type I neurons were oval or nearly circular in cross-section. Directly measured quantitative data were obtained for the volume and surface area of the somata and visible processes. The structural details reported herein are likely to have important implications regarding the functional properties of individual enteric ganglion neurons and circuits of enteric ganglion neurons.

Visualization of interstitial cells of Cajal in the mouse colon by vital staining.

Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 microm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3-6 main processes whose mean length was 56.3 microm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P<0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues.

Phorbol esters alter the morphology of cultured guinea-pig myenteric glia via a protein kinase C-independent mechanism.

Cultures of myenteric ganglia from adult guinea-pigs were used to study the influence of neuroactive substances on glial cells by monitoring changes in their morphology. The following substances had no effect on glial morphology: adenosine, ATP, carbachol, glutamate, bradykinin, isoprenaline, prostaglandin E2, sodium nitroprusside and lipopolysaccharide. The only substances found to affect glial morphology were phorbol esters, and in particular phorbol 12-myrisate 13-acetate (PMA), which acted at the nM range. Glial cells, which were normally polygonal, assumed a stellate shape within 30-60 min after the addition of PMA. Protein kinase C (PKC) inhibitors did not block this effect, and PKC activators did not mimic it. The effect of PMA was also not mediated by changes in the intracellular concentrations of either Ca2+, H+ or cyclic AMP. Dye coupling among glial cells was blocked by PMA. The phorbol ester-mediated effect on glial structure may have profound influence on neuronal organization and function.

Nitric oxide-containing nerves in bowel segments of patients with Hirschsprung's disease.

To assess the role of nerves that synthesize nitric oxide (NO) in Hirschsprung's Disease (HD), the authors studied the distribution of the enzyme NADPH diaphorase (NADPHd) in normal and diseased bowel segments. In the proximal (ganglionic) segment of the colon, NADPHd-positive neurons were present in both myenteric and submucosal plexuses. In the distal involved colonic segments from HD patients, the typical pattern of the neuronal network was completely missing in the regions of the two plexuses; instead, only disorganized NADPHd-positive nerve fibers were present and NADPHd-reactive neurons were absent. Mucosal NO synthase activity was 2.76 +/- 0.38 nmol/g/min in the proximal segment and only 0.83 +/- 0.49 nmol/g/min in the distal segment (P < .05, N = 3).