Lateral olivocochlear neurons contain both enkephalin and dynorphin immunoreactivities: immunocytochemical co-localization studies.

Antibodies to methionine enkephalin and to dynorphin B were used in an immunocytochemical study examining co-containment of enkephalins and dynorphins in olivocochlear neurons in the guinea pig lateral superior olive. Two methods of sequential co-localization were employed: one using primary antibodies from different species, the second using elution of antibodies. Co-localization of enkephalin-like and dynorphin-like immunoreactivities was found in lateral olivocochlear neurons, suggesting co-containment of enkephalins and dynorphins in this projection pathway from the lateral superior olive to the organ of Corti.

Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex.

Polyclonal antibodies were made in rabbits against glycine conjugated to bovine serum albumin with glutaraldehyde and were used for immunocytochemical studies in the cochlear nucleus and superior olivary nucleus of the guinea-pig. Antibodies selective for glycine were prepared by affinity chromatography. By dot-blot analysis this preparation showed a strong recognition of glycine conjugates and relatively little recognition of conjugates of most other amino acids tested. However, there was a significant reaction with conjugates of alanine and beta-alanine, and this cross-reaction could not be removed by affinity chromatography without eliminating the preparation's recognition of glycine. The affinity-purified preparation showed only a weak recognition of conjugates of gamma-aminobutyrate (GABA) which was detectable at high concentrations of primary antibody. Immunocytochemical studies showed several intensely staining cell bodies in the cochlear nucleus and superior olivary complex. Most immunoreactive cell bodies in the cochlear nucleus were in the dorsal cochlear nucleus, being present in both the superficial and deep layers. Scattered immunoreactive cells were present in the ventral cochlear nucleus. Intense staining of cell bodies was seen in the medial nucleus of the trapezoid body, and these cells appear to correspond to the principal cells of that nucleus. Punctate labelling, suggestive of immunoreactive presynaptic terminals, was also apparent, particularly in the ventral cochlear nucleus and lateral superior olive. In the ventral cochlear nucleus, immunoreactive puncta were found around unlabeled cell bodies, at times nearly covering the perimeter of the cell. A population of glycine-immunoreactive cell bodies in the superficial dorsal cochlear nucleus also labeled with anti-GABA antibodies as determined through double-labeling studies. However, glycine-positive cells in the deep dorsal cochlear nucleus were not labeled with anti-GABA antibodies, and some populations of GABA-positive cells in the superficial layers were not labeled with anti-glycine antibodies. In the hippocampus intense staining of cell bodies and puncta was seen with anti-GABA antibodies while essentially no staining was seen with anti-glycine antibodies. These results suggest that anti-glycine antibodies can be useful for immunocytochemical identification of glycinergic neurons. From this study several populations of putative glycinergic neurons are identified in the auditory nuclei of the brain stem using these antibodies. Some populations of GABA-containing neurons also contain high levels of glycine or a related molecule.

Immunocytochemical characterization of glutamate dehydrogenase in the cerebellum of the rat.

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.

Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA) was determined in the cochlear nucleus of the guinea pig using affinity-purified antibodies made against GABA conjugated to bovine serum albumin. Light microscopic immunocytochemistry shows immunoreactive puncta, which appear to be GABA-positive presynaptic terminals, distributed throughout the cochlear nucleus. In the ventral cochlear nucleus, these puncta are often found around unlabeled neuronal cell bodies. While occasional labeled small cells are found in the ventral cochlear nucleus, most GABA-immunoreactive cell bodies are present in the superficial layers of the dorsal cochlear nucleus. Based on size and shape, immunoreactive cells in the dorsal cochlear nucleus are divided into 3 classes: medium round cells with diameters averaging 16 microns, small round cells with average diameters of 9 microns and small flattened cells with major and minor diameters averaging 11 and 6 microns, respectively. Labeled fusiform and granule cells are not seen. A similar distribution of label was seen using antibodies against glutamic acid decarboxylase. Electron microscopic immunocytochemistry of the anteroventral cochlear nucleus shows GABA immunoreactive boutons containing oval/pleomorphic synaptic vesicles on cell bodies and dendrites. Other major classes of terminals, including those with small round, large round and flattened synaptic vesicles are unlabeled.

Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor.

The distribution and morphology of glycinergic synapses in the cochlear nucleus were investigated using monoclonal antibodies against the glycine receptor. Glycine receptor immunoreactivity was seen on somas and proximal processes of most cells in all divisions of the cochlear nucleus; distribution of label in neuropil was denser in the dorsal cochlear nucleus and granule cell cap than in the ventral cochlear nucleus. At the ultrastructural level, glycine receptor immunoreactivity was specifically distributed postsynaptically to terminals that contained flattened vesicles in the guinea pig anteroventral cochlear nucleus. These studies show that the immunocytochemical localization of the glycine receptor can provide a means of identifying and characterizing glycinergic synapses throughout the central nervous system.

Localization of dynorphin B-like and alpha-neoendorphin-like immunoreactivities in the guinea pig organ of Corti.

Antiserum to dynorphin B and antiserum to alpha-neoendorphin were used in an immunocytochemical examination of the guinea pig organ of Corti. Immunoreactive staining for these two proenkephalin B (prodynorphin)-derived peptides was seen in the lateral system of olivocochlear efferents in the organ of Corti: the inner spiral bundle, the tunnel spiral bundle and by the bases of inner hair cells. Immunoreactive staining with both antisera was also seen in efferent terminals on outer hair cells at or above the level of the nucleus, which may represent terminals of either the lateral or the medial system. No immunoreactive staining was seen in tunnel crossing fibers and at bases of outer hair cells corresponding to the medial system of efferents. The staining seen with antiserum to dynorphin B and to alpha-neoendorphin has similar distribution to that seen with antisera to methionine enkephalin; there may be co-localization of these neuropeptides in the lateral system of efferents. Choline acetyltransferase-like immunoreactivity (co-localized with enkephalin-like immunoreactivity in the lateral system in the brainstem) and glutamic acid decarboxylase (GAD)-like immunoreactivity have also been found in olivocochlear efferents. Further studies will be necessary to determine if the dynorphins are co-localized with other neurotransmitter candidates and what their interactions may be.

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.