Colocalization of calbindin and GABA in medial nucleus of the trapezoid body of the rat.

Using immunocytochemical methods, both calbindin and GABA were found to be colocalized in the somas of all the cells of the medial nucleus of the trapezoid body (NMTB) of the rat auditory system. In the lateral superior olive (LSO), calbindin was also found in the terminals but not in the cells. Some terminal labelling was found in the medial superior olive (MSO). GABA was also found in the somas of some cells in both LSO and MSO, but most of the labelling was in terminals. In the rat, calbindin appears to be more involved in a pathway that detects interaural intensity differences.

Immunohistochemical evidence for GABAergic cell bodies in the medial nucleus of the trapezoid body and in the lateral vestibular nucleus in the guinea pig brainstem.

The presence of gamma-aminobutyric acid (GABA) in two brainstem nuclei is demonstrated by using a pre-embedding immunohistochemical procedure followed by staining intensification. Firstly, immunoreactivity was found in numerous cell bodies and profiles of the medial nucleus of the trapezoid body (MNTB). Secondly, numerous neurons including giant Deiters' cells, terminals and fibers were strongly labelled within the lateral vestibular nucleus (LVN). These observations suggest that the inhibitory part of the efferent innervation of outer hair cells in the cochlea can originate from the MNTB, and that GABAergic neurons in the LVN may contribute to information processing within this nucleus.

Extracellular matrix of the superior olivary nuclei in the dog.

The extracellular matrix around nerve cell bodies in canine lateral and medial superior olivary nuclei was examined by conventional electron microscopy, Golgi impregnation and histochemical techniques. Each neuron is surrounded by a region of myelin-free neuropil embedded amongst the myelinated fibres of the trapezoid body. In the myelin-free neuropil there are astrocytes, axons, synaptic boutons and extracellular matrix. The extracellular matrix fills the spaces between slender axons near the terminals, synaptic boutons and glial processes, but not the synaptic cleft. Golgi impregnation selectively stains the perineuronal nets which cover some of all of the nerve cell bodies and dendrites. The Golgi-EM method revealed that the impregnated profiles of the nets are restricted to the extracellular matrix. Synaptic boutons are situated in the holes of the perineuronal nets. Peanut (PNA) and soybean (SBA) agglutinins bound the extracellular matrix but not the synaptic boutons, glial processes, nerve cell bodies or basal lamina of blood capillaries. Light microscopic immunohistochemistry of the glial fibrillary acidic protein (GFAP) and S-100 protein did not stain a layer corresponding to the extracellular matrix and synapses but showed an intensely positive reaction immediately outside this layer. These data suggest the existence of a unique microenvironments associated with glycoconjugates around nerve cell bodies in canine superior olivary nuclei.

A study of proteins in the auditory system of rabbits using two-dimensional gels: identification of glial fibrillary acidic protein and vitamin D-dependent calcium binding protein.

Two-dimensional gel electrophoresis and computerized optical densitometry were employed to compare the relative content of proteins across major auditory brain regions in rabbits. Areas examined included the dorsal and ventral cochlear nuclei which receive the primary afferents from the organ of Corti, the lateral superior olivary nucleus which has strong reciprocal relationships with the cochlear nucleus, and the successively more rostral projections of the auditory pathways to inferior colliculus, medial geniculate and auditory cortex. Twelve proteins demonstrated significant decreases and 5 proteins significant increases in content at successively more rostral levels of the auditory system, including 2 proteins which were highly localized to the cochlear nuclei and 2 proteins greatest in amounts in the auditory cortex. One protein which was localized to the cochlear nuclei and lateral superior olive (molecular weight (MW) = 50.3, isoelectric point (pI) = 5.7) was identified as the glial fibrillary acidic protein by reaction of specific antisera on blots. Antisera to the vitamin D-dependent calcium binding protein reacted specifically with one protein (MW = 27.2, pI = 4.8) which was greatest in amount in the lateral superior olive (LSO) versus other auditory regions examined. The significance of these findings rests in the potential for identifying specific markers for cellular elements that are important in auditory function and which might be lost as a consequence of developmental abnormalities or other traumas.

Demonstration of GABA immunoreactive cells in the inferior olive of baboons (Papio papio and Papio anubis).

The distribution of gamma-aminobutyric acid (GABA)-like immunoreactivity was studied in semithin sections through the inferior olivary complex in two baboon species. About 5% of the olivary neurons were GABA-immunoreactive. The GABA-immunoreactive neurons differed from the large majority of olivary neurons by their smaller size and their lower contents of aspartate, as judged by analysis of alternate sections labelled with an aspartate antiserum. The present observations raise the possibility that in primates the GABAergic modulation of the activity of the climbing fibre system is effected not only by the previously described input from the cerebellar nuclei and other extrinsic sources, but that intrinsic neurons also participate.

Lectin histochemical study of lipopigments with special regard to neuronal ceroid-lipofuscinosis. Results with concanavalin A.

Concanavalin A (ConA) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. The nature of the mannose bearing compound was twofold. One part was soluble in modified chloroform-methanol-water mixture (10:10:3) corresponding possibly to the oligosaccharyl diphosphodolichol (oligo-PP-Dol) described to be increased in LPs especially of inherited types. The second part, most probably a glycoprotein (GP), was entirely resistant to various extraction procedures. The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) and in the myocardium but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The "protein type variant" of the storage material (Elleder 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being extremely low or entirely absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detectable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the dog model of NCL lipid bound mannose clearly predominated, the GP component being concentrated in the cytoplasm and on the periphery od some storage granules. The nature of the GP component, a new finding of LP analysis, is discussed. The metabolic relationship between the two components is uncertain. Neither could be identified as the component resposible for autofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

[Homologous cerebral atrophy and ischemic insults in progeria adultorum]. / Homologe zerebrale Atrophie und ischämische Insulte bei Progeria adultorum.

Neuropathological findings obtained from a female patient, 49 years of age, with progeria adultorum (Werner's syndrome) are described in this paper. Vascular sclerosis with multiple ischaemic infarctions, cerebral atrophy, and vascular myelopathy were the most strongly pronounced CNS findings. Also recorded were severe lipofuscin depositions from the cortex, dentate nucleus, and olivae as well as amylaceous bodies in the insula of Reil and the hippocampus. Neurofibrillary tangles or plaques were not recordable. The central nervous system proved somewhat comparable to other organs, in that mesenchymal regressive changes were the main features.

Immunoreactive corticotropin releasing factor in adult and developing rat cerebellum: its presence in climbing and mossy fibres.

Corticotropin Releasing Factor (CRF) immunoreactivity was localized within the rat central nervous system with immunocytochemical methods, using a highly specific anti-serum to rat-CRF. In adult rats abundant CRF immunoreactivity (CRF-ir) was found in perikarya of the inferior olivary nucleus, in climbing fibres in the molecular layer, and mossy fibres in the granular layer of the cerebellum and in the cerebellar nuclei. CRF-ir climbing fibres were found in a longitudinal zonal pattern throughout the molecular layer. In developing cerebellum only a few CRF-positive fibres were seen on postnatal day 8. From day 8 onwards a gradual increase in CRF-ir was found in both the molecular and granular layer with formation of CRF-positive nests around Purkinje cellbodies starting at day 12. Subsequent outgrowth into the molecular layer could be followed, leading to an adult level of CRF-ir in the cerebellum at approximately day 22 postnatally. CRF-ir in mossy fibres has as adult appearance from approximately day 18. The temporal and morphological changes of CRF-ir in fibres in the cerebellum of immature rats followed closely the morphological development of climbing and mossy fibres. The present findings indicate that CRF (or related substances) are already present in both climbing and mossy fibres during their outgrowth into the cerebellum.

Substance P-like immunoreactive retinal terminals found in two retinorecipient areas of the Japanese monkey.

Using peroxidase-antiperoxidase immunocytochemistry, we demonstrated a dense accumulation of substance P-like (SP) immunoreactive terminals in the internal layer of the pregeniculate nucleus and in the pretectal olivary nucleus of the Japanese monkey. These distributions of immunostaining were similar to those for retinofugal terminals in the same nuclei. Bilateral eye enucleation markedly decreased SP immunoreactivity in the nuclei. The reduced immunoreactivity probably reflected the loss of SP-containing retinal ganglion cells that sent axons to the pregeniculate and pretectal olivary nuclei.

Neural connections identified with PHA-L anterograde and HRP retrograde tract-tracing techniques.

This paper describes a method of identifying specific input and output elements of a two-neuron projection pathway. Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde tract-tracing was used in combination with the retrograde transport of horseradish peroxidase (HRP) to demonstrate connections between small groups of neurons in the brainstem auditory system. Specifically, the projection from cochlear nucleus to olivary neurons that project to the cochlea were demonstrated. The first neuron in this pathway (the cochlear nucleus neuron) was anterogradely labelled with PHA-L and could be traced via labelled axons and terminals to the second neuron (the olivocochlear neuron) whose soma was labelled with HRP after cochlear injection.

A new combination of WGA-HRP anterograde tracing and GABA immunocytochemistry applied to afferents of the cat inferior olive at the ultrastructural level.

In order to identify cerebellar terminals in the cat inferior olive which contain gamma-aminobutyric acid (GABA), a technique was developed combining anterograde transport of wheatgerm agglutinine-conjugated horseradish peroxidase (WGA-HRP) with gold-immunocytochemistry. With this technique both the HRP reaction product and the immunogold labelling can be visualized in a single ultrathin section. Our results suggest that most, if not all of the WGA-HRP-labelled cerebellar terminals in the rostral medial accessory olive (MAO) and the rostral principal olive (PO) are GABAergic. In an additional experiment the GABAergic innervation of the rostral MAO was studied in combination with WGA-HRP anterograde tracing from the rostral mesencephalon. In this case the WGA-HRP-labelled terminals were never found to be GABA-positive.

Corticotropin-releasing factor in cerebellar afferent systems: a combined immunohistochemistry and retrograde transport study.

The flocculus and paraflocculus of cat and sheep cerebellum were studied with immunohistochemical methods, using antisera to corticotropin-releasing factor (CRF). CRF immunoreactivity was present within 3 populations of varicose nerve fibers. One population of CRF-immunoreactive (CRF-IR) fibers appeared to appose Purkinje cell somata and to follow their dendrites into the molecular layer. This arrangement suggested they were climbing fibers. A second group of CRF-IR profiles reminiscent of mossy fibers was widely distributed throughout the granule cell layer. A third population of CRF-IR fibers was present as a beaded plexus lying parallel to the pial surface, above and subadjacent to the Purkinje cell layer. The fibers of this plexus extended into the Purkinje cell layer and surrounded these somata. The source of some of the CRF-IR fibers within the flocculus and paraflocculus was determined by a retrograde axonal transport study utilizing the fluorescent tracer Fast blue (FB) in combination with the immunohistochemical localization of CRF. It was determined that CRF-IR perikarya within the inferior olivary nucleus gave rise to a population of climbing fibers within those lobules. Furthermore, all divisions of the inferior olive were found to contain CRF-IR somata. This latter finding suggests the potential for CRF-IR climbing fiber projections from the inferior olive to other regions of the cerebellar cortex. The existence of CRF-IR mossy fibers and fibers within the ganglionic plexus suggests the possibility of CRF-IR afferent projections from other regions of the brain stem to the flocculus and paraflocculus.

Origin, distribution and organization of the serotoninergic innervation in the inferior olivary complex of the rat.

The serotoninergic innervation of the inferior olivary complex of the rat was studied using a specific immunohistochemical technique. Fibers and varicosities positive for serotonin were present throughout the nucleus. The densest varicosities were found in the lateral portion of the dorsal accessory olive and the caudal portion of the medial accessory olive. The rostral medial accessory olive and the principal olive were sparsely populated with labeled elements. Ultrastructurally, labeled profiles were found in close opposition to small dendrites and to olivary cell bodies, but they did not display any synaptic specialization. Labeled perikaria were found in the periolivary regions, some of them located laterally to the olivary complex are responsible for the serotoninergic innervation of the dorsal accessory olive; some others located dorsally and medially in the nucleus raphe obscurus and raphe pallidus were responsible for the innervation of the medial accessory olive.

Immunoreactivity to calcitonin gene-related peptide in the superior olivary complex and cochlea of cat and rat.

In both cat and rat, the cells of origin, axons, and terminals of the lateral olivocochlear system exhibit immunoreactivity to antisera to calcitonin gene-related peptide (CGRP). In the cat, immunoreactive neurons in the brainstem are located in the hilus of the lateral superior olivary nucleus and around its margins. In the rat, immunoreactive neurons are located within the lateral superior olivary nucleus proper. In both species, immunoreactivity in the cochlear duct is limited to the region beneath the inner hair cells. Immunoreactive axons in the cochlear nucleus could not be traced to their source but may arise as collaterals of the lateral olivocochlear system. No other components of the brainstem auditory system react to any extent with the antisera.

The dentato-olivary projection in the rat as a presumptive GABAergic link in the olivo-cerebello-olivary loop. An ultrastructural study.

It is here shown that autoradiographically labelled axon terminals of the dentato-olivary projection form a heterogeneous population. However, a majority of them constitute an even class of synapses, characterized by their small axonal size, their content in pleimorphic vesicles, and the establishment of symmetric synapses on small dendrites, about 5% of which are linked through a gap junction. The same material, used for immunocytochemistry of GABA with the postembedding technique, discloses that a majority of boutons with cytological features similar to the dentato-olivary terminals are GABA-immunoreactive, especially those synapsing on dendrites linked by gap junctions. The cerebello-olivary projection, despite its heterogeneity, thus appears as part of the GABAergic system which governs the synaptic modulation of the electrotonic coupling between olivary neurons.

Quantitative distribution of the glycine receptor in the auditory brain stem of the gerbil.

The concentration and relative distribution of glycine receptors were determined for gerbil brain stem auditory nuclei using 3H-strychnine and quantitative autoradiographic techniques. Significant binding was observed in the anteroventral cochlear nucleus, the dorsal cochlear nucleus, the lateral superior olivary nucleus, and the inferior colliculus. A non-uniform distribution of binding was seen in 3 of these nuclei, such that the greatest concentration of glycine receptors was located in the high-frequency regions. An analysis of neuron soma density suggested that the amount of post-synaptic membrane could partially explain the distribution of receptor.

Development of glycine receptor distribution in the lateral superior olive of the gerbil.

The concentration and relative distribution of glycine receptors were determined for the gerbil lateral superior olive at several postnatal ages. Quantitative autoradiographic techniques revealed significant 3H-strychnine binding to all regions of the nucleus from 4 to 90 d. However, during the first 20 d, a nonuniform distribution of binding emerged, such that the greatest concentration of receptor was found in the high-frequency region of the nucleus. An analysis of neuron packing density showed an approximate 2-fold distribution along the same axis at all ages. This evidence is consistent with an elimination of glycine receptors in the ventral region of the lateral superior olive as maturation progresses.

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.

Corticotropin-releasing factor mRNA is expressed in the inferior olives of rodents and primates.

Immunohistochemical studies have suggested that corticotropin-releasing factor (CRF) was a transmitter of the olivocerebellar projection. We used in situ hybridization histochemistry with a 35S-labeled oligodeoxyribonucleotide probe for CRF mRNA to show that inferior olivary neurons of rats, baboons, and humans synthesize CRF.

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain.

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.