Cholinergic and VIPergic innervation in cerebral arteries: a sequential double-labeling immunohistochemical study.

The possible co-localization of choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) in the nerve fibers of cat cerebral arteries was examined by a sequential double-labeling immunohistochemical method. Diaminobenzidine and tetramethylbenzidine were used as chromogens to distinguish ChAT (protein) and VIP (peptide) immunoreactivities. Since available fixatives often did not provide simultaneous preservation of optimal protein and peptide immunoreactivities, a new fixative, buffered periodate-paraformaldehyde-picric acid-formaldehyde-lysine (PPPFL), was formulated and tested. PPPFL fixative is more reliable for simultaneously preserving ChAT and VIP immunoreactivities than were periodate-lysine-paraformaldehyde (PLP) fixative, Zamboni's fixative, or 2% paraformaldehyde solution alone. Using PPPFL as fixative, both ChAT immunoreactive (ChAT-I) and VIP-immunoreactive (VIP-I) fibers in cerebral arteries appeared as bundle and fine fibers. Most ChAT-I and VIP-I fibers were separate. Portions of ChAT-I and VIP-I fibers often ran closely in parallel or across each other. Overlaying of VIP-I on ChAT-I fibers and relay connections between them were also observed. These morphological data suggest the potential functional interactions between cholinergic and VIPergic innervations. In less than 5% of the fibers examined did ChAT and VIP immunoreactivities appear to be co-localized. These data therefore do not support the hypothesis that acetylcholine and VIP are co-localized in most fibers innervating the cerebral arterial wall.

Purification and subunit composition of a cholinergic synaptic vesicle glycoprotein, phosphointermediate-forming ATPase.

A glycoprotein ATPase in cholinergic synaptic vesicles of Torpedo electric organ was solubilized with octa-ethylene glycol dodecyl ether detergent. Study of potential stabilizing factors identified crude brain phosphatidylserine, glycerol, dithiothreitol, and protease inhibitors as of value in maintaining activity. The ATPase was purified from the solubilized, stabilized material by glycerol density gradient band sedimentation velocity ultracentrifugation, and hydroxylapatite, wheat germ lectin affinity, and size exclusion chromatographies. The pure ATPase had a specific activity of about 37 mumol ATP hydrolyzed/min/mg protein. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified material typically exhibited three polypeptides of molecular masses 110, 104, and 98 kilodaltons (kDa) and a fourth diffuse polypeptide of 60 kDa. This composition suggests that the ATPase is a member of the P-type, or phosphointermediate-forming, family, but it was shown to be distinct from the ouabain-sensitive Na+,K+- and CA2+-stimulated Mg2+-ATPases. The purified vesicle enzyme was rapidly phosphorylated by [gamma-32P]ATP on about 14% of the subunits with molecular weights of 98,000-110,000. About 16% of the ATPase was phosphorylated in whole-vesicle ghosts in a manner consistent with formation of a phosphointermediate, thus confirming the P-type nature of this enzyme.

Neurofilament-like pattern of reactivity in human foetal PNS and spinal cord following immunostaining with polyclonal anti-glial fibrillary acidic protein antibodies.

The intermediate filament protein, glial fibrillary acidic protein (GFAP), is widely used as a cell-specific marker molecule for immunocytochemical identification of astrocyte lineages in cell culture, in tissues during development, and in tissues undergoing pathological changes. This study demonstrates that a reaction pattern of two commercially available polyclonal anti-GFAP antibodies shows extensive similarity to the pattern of reactivity obtained with monoclonal antibodies to neurofilaments in the PNS and spinal cord of human embryos and foetuses, at 5 to 12 weeks of gestation. The polyclonal antibodies to GFAP labelled populations of neurons and their processes in the PNS and in the spinal cord. Monoclonal antibodies to GFAP only labelled glial cells in the spinal cord. Neurofilament adsorption of one of the anti-GFAP antisera abolished the neurofilament-like reaction pattern, while the structures also labelled with monoclonal antibodies to GFAP remained immunostained. The results presented may question previously published data obtained with these and possibly other polyclonal anti-GFAP antibodies.

Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase and gamma-aminobutyrate immunoreactivity.

The transmitters acetylcholine and gamma-aminobutyrate (GABA) play critical roles in the formation of receptive-field properties of retinal ganglion cells. In rabbit retina, cholinergic amacrine and displaced amacrine cells were identified by immunohistochemical staining for the enzyme choline acetyltransferase and by their avid accumulation of the fluorescent dye 4',6-diamidino-2-phenylindole. Several GABA-immunoreactive and glutamate decarboxylase-immunoreactive cell types, including a prominent population of small, round amacrine and displaced amacrine cells, were also identified. Double-label experiments demonstrated that all amacrine and displaced amacrine cells that prominently accumulate 4',6-diamidino-2-phenylindole contain GABA and glutamate decarboxylase immunoreactivity. However, not all GABA-immunoreactive cells accumulate this dye. Quantitative analysis of the ganglion cell layer of whole mount preparations of the retina showed that choline acetyltransferase-immunoreactive cells and the majority of GABA-immunoreactive cells have a small, round shape and similar cell density profiles that parallel that of displaced amacrine cells. These studies establish that cholinergic cells are a major subpopulation of GABA-immunoreactive amacrine and displaced amacrine cells. The role these cells have in the formation of ganglion cell receptive-field properties may be parsimoniously explained by an excitatory postsynaptic action mediated by acetylcholine and an inhibitory presynaptic action mediated by GABA.

Catecholaminergic parasympathetic efferents within the dorsal motor nucleus of the vagus in the rat: a quantitative analysis.

Catecholaminergic vagal motor neurones were identified within the dorsal motor nucleus of the vagus, by retrograde tracing of True blue from the stomach followed by immunocytochemistry using antibodies directed against tyrosine hydroxylase. Presumed dopaminergic efferents were largely confined to caudal regions, where they averaged as much as 30% of the labelled efferents. Most but not all of these were also identified on the basis of acetylcholinesterase histochemistry.

Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip.

The distribution and origin of perivascular acetylcholinesterase-active and vasoactive intestinal polypeptide-immunoreactive nerve fibers were studied in the rat lower lip by means of acetylcholinesterase histochemistry and vasoactive intestinal polypeptide immunohistochemistry. The perivascular nerve fibers stained intensely with both histochemical techniques and were widely distributed on small arteries and arterioles of the lower lip, especially in the transitional zone between the hairy skin and the mucous membrane. The distributions of the two types of fibers were very similar and most of them showed overlapping coloration, on consecutive staining for vasoactive intestinal polypeptide and acetylcholinesterase. Both acetylcholinesterase-positive and vasoactive intestinal polypeptide-immunoreactive fibers were completely lost on removal of the otic ganglion, while they were not affected by sympathetic ganglion removal or sensory nerve sectioning. In the otic ganglion, most cells exhibited acetylcholinesterase activity, and about 60% of the cells showed light to heavy vasoactive intestinal polypeptide immunoreactivity. These findings indicate that vessels in the rat lip are innervated by parasympathetic fibers originating from the otic ganglion and support the view that vasoactive intestinal polypeptide is present in cholinergic neurons. This may suggest the possible control by the parasympathetic nervous system of cutaneous blood vessels through vasoactive intestinal polypeptide-containing cholinergic neurons, in general or at least in the facial area.

The localization of central cholinergic neurons.

Over the past decade our understanding of the localization of central cholinergic neurons has greatly increased. Interest in these systems has also intensified due to the involvement of cholinergic mechanisms in Alzheimer's disease. The distribution of central cholinergic neurons is reviewed, focusing on recent work in experimental animals. The pharmacohistochemical procedure for acetylcholinesterase and the development of antibodies to choline acetyltransferase are two of the major technical advances that have shaped our knowledge of the distribution of central cholinergic neurons. The results, advantages and limitations of both techniques are discussed. A discussion of the phenomenon of coexistence of acetylcholine with neuroactive peptides in central neurons is also included.

Distribution of neuropeptide Y-like immunoreactivity and its relationship to FMRF-amide-like immunoreactivity in the sixth lumbar and first sacral spinal cord segments of the rat.

The present study was aimed at describing the distribution of neuropeptide Y (NPY)-like immunoreactivity in the sixth lumbar (L6) and first sacral segments (S1) of the rat spinal cord, comparing this distribution to that of FMRF-amide-like immunoreactivity and determining whether NPY- and FMRF-amide-like immunoreactivities are present in the same neurons in the dorsal gray commissure (DGC) in L6 and S1 of the rat spinal cord. For distribution studies tissue from colchicine-treated animals was processed according to the peroxidase-antiperoxidase technique using anti-NPY as the primary antiserum. For co-localization studies serial 5-micron sections were processed for immunofluorescence. Adjacent sections were incubated with either anti-NPY or anti-FMRF-amide as the primary antiserum. The number of immunoreactive cells per section was counted and each section was photographed. The sections were then restained with the other antiserum (i.e., tissue first stained with anti-NPY was stained with anti-FMRF-amide and vice versa), the number of cells per section was recounted, and the sections were rephotographed. NPY-like immunoreactive cells and fibers were identified in the DGC, sacral parasympathetic nucleus, substantia gelatinosa, marginal zone, nucleus proprius, and ventral horn. Every cell in the DGC that contained NPY-like immunoreactivity was found also to contain FMRF-amide-like immunoreactivity, and the distribution of NPY-like immunoreactive fibers was found to be similar, although denser than FMRF-amide-like immunoreactive fibers. The distribution of NPY-like immunoreactivity in L6 and S1 of the rat spinal cord suggests that an NPY-like peptide may be involved in regulation of pelvic viscera, processing of primary afferent information, and motor regulation of pelvic muscles. The presence of NPY- and FMRF-amide-like immunoreactivities in the same neurons in the DGC together with the lack of bona fide FMRF-amide in the rat central nervous system, the presence of NPY in the rat central nervous system, and the cross-reactivity of anti-FMRF-amide with NPY support the hypothesis that the FMRF-amide antiserum recognizes an NPY-like peptide in the rat spinal cord.

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.