Neural and glial phenotypic expression by neural crest cells in culture: effects of control and presumptive aganglionic bowel from ls/ls mice.

The enteric nervous system is formed by cells that migrate to the bowel from the neural crest. Previous experiments have established that avian crest cells in vitro will colonize explants of murine bowel and there give rise to neurons. It has been proposed that phenotypic expression by the crest-derived precursors of enteric neurons and glia is critically influenced by the microenvironment these cells encounter within the gut. To test this hypothesis, quail crest cells were cocultured with explants of control or presumptive aganglionic bowel from the ls/ls mutant mouse, and the effects of the enteric tissue on five phenotypic markers of crest cell development were followed. Aganglionosis develops in the terminal region of the colon of the ls/ls mouse because viable crest-derived neural and glial precursors fail to colonize this tissue. Expression of the phenotypic markers in the cocultures was compared with that in cultures of crest alone, crest plus neural tube, and gut grown alone. The markers examined were melanogenesis and immunostaining with antisera to 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) and the monoclonal antibodies, NC-1 and GlN1. Explants of control, but not presumptive aganglionic ls/ls gut were found to increase the incidence of the expression of 5-HT and NC-1 immunoreactivities; moreover, especially near the gut, the assumption of a neuronal morphology by 5-HT-, NC-1-, and GlN1-immunoreactive cells was also increased. Coincidence of expression of 5-HT with NC-1 and GlN1 immunoreactivities was observed. The effect of the bowel was selective in that the expression of TH immunoreactivity, which is not a marker of mature enteric neurons, was reduced rather than enhanced. The effect of enteric explants on crest cell development was specific in that it was not mimicked by explants of metanephros, which inhibited expression of 5-HT immunoreactivity and the acquisition of a neuritic form by NC-1-immunoreactive cells. It is concluded that the enteric microenvironment affects the phenotypic expression of subsets of crest cells and that this action of the bowel is manifested in vitro. The inability of presumptive aganglionic gut from ls/ls mice to influence neural phenotypic expression may be due to the failure of this tissue to produce putative factor(s) required for the effect or to the inability of the crest-derived precursor cells to migrate into the abnormal enteric tissue.

Vesicular localization of immunoreactive [Met5]enkephalin in the globus pallidus.

The distribution of enkephalin immunoreactivity in the neuropil of globus pallidus was analyzed with a quick-freezing, postembedment-staining technique for light and electron microscopic immunocytochemistry. Fluorescence images of ultrathin sections on glass slides, obtained with a silicon-intensified-tube (type) video camera, revealed staining in the form of scattered fluorescent dots, each 200-400 nm in diameter. Colloidal gold staining under the electron microscope was associated with 80- to 100-nm vesicles of average electron density, widely dispersed in the neuropil, with usually one and no more than four vesicles in individual sectioned neuronal processes. Analysis of fluorescence images in paired serial sections of thicknesses varying from 25 to 150 nm proved that the 200- to 400-nm dots of fluorescence came from smaller structures, presumably the 80- to 100-nm vesicles. Enkephalinergic vesicles in the globus pallidus were nearly always found in what appeared to be axons of passage and were only infrequently associated with synaptic profiles.

Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell.

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.

Ultrastructural localization of gamma amino butyric acid immunoreactivity in B cells of the rat pancreas.

The purpose of this study was to determine the ultrastructural localization of gamma amino butyric acid (GABA) within the B cell of isolated rat islets, particularly with respect to the B cell secretion granules. GABA immunoreactivity was localized ultrastructurally, with colloidal gold, in the B cells and absent in the A cells and D cells. Quantitative analysis of the colloidal gold particle distribution in the B cell revealed 29.5 +/- 5.2 gold particles/micron 2 in the nuclei, 29.3 +/- -6.9 gold particles/micron 2 in the mitochondria and 4.9 +/- 1.0 gold particles/micron 2 in the secretion granules. Particle density in the remainder of the cytoplasm was 41.9 +/- 4.1 particles/micron 2. The paucity of gamma amino butyric acid in the B cell secretion granules differs from observations on gamma amino butyric acidergic neurons, where there is an accumulation of gamma amino butyric acid within the neurosecretory vesicles. These findings indicate that if gamma amino butyric acid is released from the islet, then it is by a nongranular mechanism. In addition, the results are compatible with the hypotheses that gamma amino butyric acid within the B cell functions in the regulation of insulin biosynthesis, and/or functions as an alternative energy source for the B cell through the gamma amino butyric acid shunt.

Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence.

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.

Co-localization of neurophysin- and enkephalin-like immunoreactivity in cat pituitary.

Enkephalin and neurophysin immunoreactivity have been co-localized in terminals of frozen-dried cat posterior pituitary, using two methods of immunocytochemistry--the protein A-gold procedure and the PAP method. Absorption controls show reduced staining in all cases. Intermediate lobe cells are negative using the enkephalin and neurophysin antisera, but with alpha-MSH antiserum, posterior lobe terminals are negative and intermediate lobe cells are positive. The data are compatible with the hypothesis that inhibition of release of oxytocin and vasopressin by the pituitary opioid system is accomplished by an autoregulatory mechanism in which the release of enkephalin with oxytocin or vasopressin serves to inhibit release of the neurohormones.

Ultrastructural localization of neurophysin-like immunoreactivity in the rat posterior pituitary. An alternative method using frozen-dried tissue fixed with OsO4 vapor.

Electron microscopic identification of elements containing neurophysin-like immunoreactivity can be accomplished in rat posterior pituitary that has been frozen-dried and fixed with OsO4 vapor. Alternating serial ultrathin sections are placed on grids and glass slides. The sections on the slides are stained for neurophysin using immunofluorescence histochemistry, and the resultant images are superimposed on electron micrographs from the adjacent sections. The method provides several advantages for the localization of neuropeptide immunoreactivity in nervous tissue.

Preparation of biological tissues for electron microscopy by freeze-drying.

A dependable method for freeze-drying tissues for electron microscopy has been developed. Thin slices of fresh tissue were frozen by bringing them into direct contact with a polished copper bar at liquid nitrogen temperature. The tissue was transferred to a copper specimen block equipped with a thermocouple and heating circuit for accurate control of the environmental temperature of the tissue, and evacuated in a glass freeze-drier using clean high vacuum techniques for keeping the system free of hydrocarbons. The tissue was dried by increasing the temperature of the specimen block 10 degrees C each hour while monitoring the rate of water removal from the tissue with a partial pressure analyzer. The dry tissue was fixed with OsO4 vapor, vacuum embedded in a low viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Tissue processed in this manner exhibits excellent morphological preservation at both cellular and organellar levels without prefixation or the use of cryoprotective agents. The results of the experiments using the partial pressure analyzer indicate that small blocks of tissue can be dried in a short time at low temperature.