Richard A. Altschuler

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 preparations 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.

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.

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.

Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat

Most of the early studies on the immunohistochemical distribution of enkephalin pentapeptide-like immunoreactivity used antisera that stained both proenkephalin- and prodynorphin-containing neurons. The present study used the peroxidase-antiperoxidase method, thick Vibratome sections and antisera specific for the carboxyl termini of [Met]enkephalin, [Met]enkephalyl-Arg6-Phe7, [Met]enkephalyl-Arg6-Gly7-Leu8, and metorphamide and for BAM 22P in order to obtain a detailed description of the distribution of authentic proenkephalin-containing perikarya and nerve processes. The peroxidase-antiperoxidase reaction product was intensified by the selective deposition of silver crystals in order to display the morphology of proenkephalin-containing neurons with great fidelity. The results indicate that the magnocellular perikarya in the supraoptic and paraventricular nuclei contain prodynorphin rather than proenkephalin as had been suggested by earlier investigators. The coarse fibers in the internal zone of the median eminence and the granule cell-mossy fiber pathway in the hippocampus also contain prodynorphin rather than proenkephalin. The number of proenkephalin-containing perikarya and/or the density of proenkephalin-containing nerve terminals in several other areas of the brain, e.g. the substantia nigra, the central amygdaloid nucleus, the periaqueductal gray and the parabrachial nuclei, were overestimated by earlier investigators. The distribution of authentic proenkephalin-containing perikarya and nerve processes is, despite these errors, similar to the distribution of enkephalin pentapeptide-like immunoreactivity described by earlier investigators. Proenkephalin-containing perikarya were identified for the first time in the medial and lateral habenular nuclei of the adult rat. Antisera specific for [Met]enkephalin, [Met]enkephalyl-Arg6-Phe7, [Met]enkephalyl-Arg6-Gly7-Leu8 and BAM 22P stain perikarya and nerve terminals with a similar distribution. The metorphamide antiserum also stains the same perikarya and nerve terminals; however, it also stains magnocellular perikarya in the zona incerta and the lateral hypothalamus that are not stained by any of the other proenkephalin-specific antisera.

Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea

Olivocochlear fibers have been demonstrated to have acetylcholinesterase-positive staining both in brainstem and cochlea. Olivocochlear fibres in the cochlea have also been determined to contain enkephalin-like immunoreactivity. In this study, we first determined the source of olivocochlear fibers in the guinea-pig using horseradish peroxidase and wheat germ agglutinin in retrograde transport studies. These cells were then examined for enkephalin-like immunoreactivity followed by acetylcholinesterase staining on the same sections to determine which cells and fibers showed staining for both. It was found that cells in the guinea-pig lateral superior olive that project to the cochlea have both enkephalin-like immunoreactivity staining and acetylcholinesterase-positive staining. Cells in other areas giving rise to olivocochlear fibers showed only acetylcholinesterase staining. These results suggest that there is co-localization of enkephalin and acetylcholine in a population of olivocochlear cells and fibers.

Glutamic acid decarboxylase immunoreactivity of olivocochlear neurons in the organ of Corti of guinea pig and rat

The distribution of glutamic acid decarboxylase (GAD)-like immunoreactivity in the organ of Corti of guinea pig and rat was studied under the light microscope. Indirect immunohistochemical techniques were used. Cochleae were first incubated with a specific antiserum to rat brain GAD and then stained through an avidin-biotin-horseradish peroxidase (HRP) procedure. GAD-like immunoreactivity was visualized as staining with HRP reaction product. Surface preparations were prepared from the immunoreacted cochleae. GAD-like immunoreactivity was found in the inner spiral bundle, tunnel spiral bundle, tunnel crossing fibers, outer hair cell synaptic regions and outer spiral bundles. Little staining was seen in the basal turn. Most of the immunoreactivity was seen in the third and lower fourth turn of the guinea pig cochlea, but even there many efferent fibers and endings were unstained. It is concluded that GAD-like immunoreactivity is present in a subpopulation of cochlear efferents that contains elements from both the medial and the lateral olivocochlear system. Future studies are needed to determine whether this subpopulation is GABA-ergic (i.e. uses gamma-aminobutyric acid as a neurotransmitter) and/or cholinergic.

GABA visualized by immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons

Antiserum raised against GABA coupled with glutaraldehyde to bovine serum albumin was applied to the guinea pig cochlea. Immunoreactivity was visualized as horseradish peroxidase reaction product in surface preparations of the organ of Corti using immunocytochemical techniques. Bright-field, differential interference contrast and video-enhanced contrast light microscopy were used. GABA-like immunoreactivity was found in axons and endings of efferent neurons in all turns of the cochlear spiral, but predominantly in the third turn and first half of the fourth turn. In these apical turns, immunoreactivity was seen in the efferent components: inner spiral bundle, tunnel spiral bundle, tunnel-crossing fibers, large nerve endings synapsing on outer hair cell bases, nerve endings high up on outer hair cells, nerve endings or varicosities close to outer hair cells, and outer spiral fibers. Some immunoreactive large nerve endings at outer hair cells were found in the apical half of the fourth turn. This study shows that axons and endings of efferent neurons in the organ of Corti of guinea pig contain GABA-like immunoreactivity with a distribution similar to that of GAD-like immunoreactivity as shown in a previous study. In both studies, many efferent nerve axons and endings were unstained, even in regions of maximal density of immunoreactivity in the apical turns. The evidence indicates that a subpopulation of efferent neurons projecting to the organ of Corti is GABAergic and very likely different from the lateral and the medial olivocochlear efferent systems.

Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea

The immunocytochemical localization of the enzyme choline acetyltransferase (ChAT) was examined in the guinea pig organ of Corti to determine if both lateral and medial systems of efferents would show immunoreactive labeling for this specific enzyme marker of cholinergic neurons. Cochleae were also examined after lesion of efferents to determine if ChAT-like immunoreactivity is confined to efferents. ChAT-like immunoreactivity was seen in the inner spiral bundle, tunnel spiral bundle and by the bases of inner hair cells corresponding to the lateral system of efferents. ChAT-like immunoreactivity was also seen in crossing fibers and puncta at the bases and by the nuclei of outer hair cells corresponding to the medial system of efferents. With the use of video enhanced contrast microscopy more than 9 ChAT-like immunoreactive puncta at the bases of outer hair cells could be resolved. In cochleae examined 6 weeks after ipsilateral lesion of efferents, no ChAT-like immunoreactivity was observed. These results add strong evidence that acetylcholine is a transmitter of both the medial and lateral systems of efferents.

Neurotransmitter-related immunocytochemistry of the organ of Corti.

The principles of immunocytochemistry were outlined in 1942 by Coons et al. and in the 1970s immunocytochemistry emerged as a powerful method for identifying structures and tracing pathways in the nervous system. It now plays a fundamental role in the neuroanatomical and histochemical analysis of the central nervous system. The first immunocytochemical studies of the mammalian cochlea were reported in 1980, from three different laboratories. Since then many studies on cochlear immunocytochemistry have been carried out, concerned with questions about neurotransmitter candidates or about structural proteins. This review describes immunoreactivity of enkephalin, choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), gamma-aminobutyric acid (GABA), aspartate aminotransferase (AATase) and glutaminase (GLNase) in the organ of Corti. ChAT is the enzyme that catalyzes the synthesis of acetylcholine (ACh). GAD is the terminal enzyme in the biosynthesis of the inhibitory neurotransmitter GABA. AATase and GLNase are two enzymes involved in the metabolism of the excitatory neurotransmitter candidates aspartate and glutamate. We have much relied on surface preparations of the organ of Corti. We have also used cryostat sectioning of the cochlea, particularly when there was a need to apply a number of different antisera to comparable preparations from one and the same cochlea. We have used immunofluorescence and immunoperoxidase procedures. Immunoperoxidase procedures have given us better signal noise ratio for specific immunoreactivity (in surface preparations) than has immunofluorescence. Occasionally, to achieve maximal resolution of surface preparations in light microscopy studies, we have used enhanced contrast video display. We have found immunoreactivity in efferent fibers in the organ of Corti following the application of antisera to enkephalin, ChAT, GAD, GABA, AATase and GLNase. Most of these different antisera give different distributions of immunoreactivity and other antisera have evoked no immunoreactivity in the organ of Corti. To the best of our knowledge, the cells of origin of efferent axons and terminals in the organ of Corti are located in the brainstem. Originally described as crossed and uncrossed olivocochlear neurons, these efferents have recently been classified into a medial and a lateral system predominantly innervating, respectively, the outer hair cell region and the inner hair cell region. However, our findings on the distribution of GAD- and GABA-like immunoreactivity indicate that there may be more than two different systems of efferents in the organ of Corti, as previously suggested by Schwartz and Ryan (1983).

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus

There is considerable evidence that pathways of the hippocampus use an excitatory amino acid as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.