Autoradiographic analysis of benzodiazepine receptors in mutant mice with cerebellar defects.

[3H]Flunitrazepam binding sites were analysed in the cerebellar cortex, deep cerebellar and vestibular nuclei of Purkinje cell deficit (pcd) mutant mice which have an almost complete postnatal loss of Purkinje cells, and weaver mutant mice, in which there is a postnatal degeneration of granule cells. Increases in [3H]flunitrazepam binding site densities were found in the molecular and granule cell layer of weaver mutant mice, whereas decreases were observed in pcd mutant mice when compared to wildtype 'control' mice. Apparently unaltered benzodiazepine receptor densities were found in the flocculus of pcd mutant mice, whereas increases were seen in the flocculus of weaver mutant mice. The densities of benzodiazepine binding sites in the medial and lateral deep cerebellar nuclei of both mutants significantly exceeded those in control mice. Significant increases in flunitrazepam binding sites were also found in the superior and spinal nucleus of the vestibular complex of pcd mice as compared to wildtype. In the weaver mutants the benzodiazepine receptor density is enhanced in the superior and medial vestibular nucleus. Apparently unaltered numbers of such receptors compared to the wildtype control group were found in the remaining vestibular nuclei of both mutants.

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.

Electrophysiological evidence for cholinoceptive neurons in the medial vestibular nucleus: studies on rat brain stem in vitro.

Electrophysiological studies were performed to determine whether or not cholinoceptive neurons are present in the rat medial vestibular nucleus (MVN) using brainstem slice preparations. Fifty-three MVN neurons, whose activities were extracellularly recorded, fired spikes spontaneously and regularly with an interspike interval of 180 +/- 27 ms (mean +/- S.E.M.) and a coefficient of variation of 0.11 +/- 0.02. Intracellularly recorded neurons also exhibited similar spontaneous and regular generation of action potentials. Carbachol dose-dependently increased the spontaneous firing, although the firing rate was decreased in a few neurons. The addition of atropine reduced the firing rate, and dose-dependently attenuated the carbachol-induced excitation of the neurons. In a low Ca2+ and high Mg2+ medium, carbachol also increased the firing rate. These results indicate that the MVN contains neurons with spontaneous and regular firing, and that the excitability of these neurons is regulated by a cholinergic muscarinic mechanism.

Serotonin-like immunoreactive fibers in the medial dorsal accessory group and the anterior fornical nucleus of the magnocellular neurosecretory system in the rat.

The area lateral to the stria medullaris and the fornix at the level of the interventricular foramen, an area known to be occupied by the medial dorsal accessory group and the anterior fornical nucleus of the magnocellular neurosecretory system, was found to be studded with serotonin (5-HT)-like immunoreactive (LI) varicosities. Double immunostaining with antisera against 5-HT and oxytocin (OXY) proved that the 5-HT-LI varicosities were in close proximity to the OXY-LI neurons in the medial dorsal accessory group and the anterior fornical nucleus. These nuclei were distributed more densely with 5-HT-LI varicosities than other cell groups in the magnocellular neurosecretory system.

Vestibulospinal pathway in rabbit includes GABA-synthesizing neurons.

When Fast blue is injected into the rabbit spinal cord it is retrogradely transported into nerve cell bodies located in the medial and the descending vestibular nuclei. Approximately 50% of the Fast blue-positive cells also contain glutamic acid decarboxylase-like immunoreactivity. These neurons presumably synthesize gamma-aminobutyric acid (GABA) and the rabbit vestibulospinal pathway therefore contains a substantial inhibitory component.

Comparisons of the immunocytochemical localization of choline acetyltransferase in the vestibular nuclei of the monkey and rat.

Immunocytochemical studies of the brainstem were done in the squirrel monkey and rat using the same polyclonal antisera for choline acetyltransferase (ChAT). Cells immunoreactive for ChAT (ChATir) were evident in large numbers in visceral and motor cranial nerve nuclei in both species, but virtually no ChATir cells were seen in the vestibular nuclear complex of the rat. In the monkey ChATir cells were distributed in caudal parts of the medial (MVN) and in dorsal parts of the inferior (IVN) vestibular nuclei. Only a few immunoreactive cells were seen in the rostral MVN and none were found in cell group f of the IVN. Nearly all cells of group z and x, which do not receive primary vestibular afferents, were immunoreactive to ChAT. None of the cells in the superior and lateral vestibular nuclei, cell group y, the infracerebellar nucleus or the interstitial nucleus of the vestibular nerve were immunoreactive for ChAT. Cells immunoreactive to ChAT were present in large numbers in the rostral part of the nucleus prepositus in the monkey, but not in the rat. The relatively small number and distribution of ChATir cells in the MVN suggested they could constitute only a small fraction of the MVN neurons that contribute to a massive commissural system. Significant differences in cholinergic vestibular neurons appear to exist between the rat and the monkey.

Immunohistochemical localization of gamma-aminobutyric acid- and aspartate-containing neurons in the guinea pig vestibular nuclei.

The immunohistochemical distributions of gamma-aminobutyric acid (GABA)- and aspartate-containing neurons were studied in the guinea pig vestibular nuclei using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered throughout all regions of the vestibular nuclei. The largest number of these cells was found in the medial nucleus. Intraventricular injection of colchicine markedly increased GABA-like immunoreactivity in these cell bodies. GABA-containing terminals were distributed throughout all 4 subdivisions of the nuclei, with the richest localization found around the floor of the fourth ventricle. Various sized aspartate-containing neurons were noted in the vestibular nuclei and small cells were present in the superior, medial and lateral nucleus. Medium-sized cells were observed throughout the vestibular nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.

Ontogenesis of serotoninergic nuclei in the rat stem.

The development of central serotoninergic neurons has been investigated with immunohistochemical techniques using the indirect peroxidase-antiperoxidase (PAP) method in 16-and 19-day-old rat embryos, in 1, 10 and 26 days old young and in adult animals. Immunoreactive neurons were present on embryonic day 16 in the subventricular area of the brain stem. First the countour of nucleus raphe dorsalis became distinct in the subventricular cell mass of the lower midbrain. In the ventral part of the tegmentum, cells were grouped along the midline in bilateral columns from which the nucleus centralis superior, the nucleus raphe pontis and the nuclei pontis differentiated. These nuclei were well defined in the newborn on either side of the midline, and the nucleus centralis superior and nucleus raphe pontis were fused on the midline in 10-day old rat. In the ventral part of the pons and medulla, a bilateral cell mass was also found along the midline. A number of immunoreactive cells moving off the midline constituted the nucleus raphe magnus which was formed on 19. embryonis day. Another contingent of immunoreactive cells remained at the midline and formed the nuclei raphe obscurus and pallidus. In newborn rat, these nuclei were well separated from the nucleus raphe magnus. They would later fuse on the midline, whereas the nucleus raphe magnus would remain a bilateral structure.

Demonstration of taurine-like immunoreactive structures in the rat brain.

An antiserum was raised in a rabbit by immunization with taurine bound to bovine serum albumin. The antibody was purified by passage over an immunoabsorbent column (formyl-cellulofine-taurine) and it did not cross-react significantly with glutamate, aspartate, glycine, GABA (0.4%), glutamine, proline, cysteine, beta-alanine, cysteic acid, carnosine or homocarnosine in enzyme-linked immunosorbent assays and nitrocellulose paper immunoblots. Immunocytochemical studies employing the peroxidase-antiperoxidase immunohistochemical technique revealed that many cerebellar Purkinje cells showed taurine-like immunoreactivity. Labelled axons could be followed within the white matter up to the deep cerebellar nuclei, where numerous puncta were observed. Immunoelectron microscopic examination revealed that labelled puncta were presynaptic terminals, and axo-dendritic or axo-somatic symmetrical synapses were observed on deep cerebellar nuclear neurons.

Neuropeptides and gamma-aminobutyric acid in the vestibular nuclei of the rat: an immunohistochemical analysis. I. Distribution.

The distribution of substance P, Leu-enkephalin and gamma-aminobutyric acid (GABA) containing structures in the rat vestibular nuclei were investigated by means of an indirect immunofluorescent method using specific antisera to substance P, Leu-enkephalin and glutamic acid decarboxylase (GAD), respectively. Numerous positive neurons and fibers containing these three substances were found in the medial vestibular nucleus. Most of them were situated in the caudal part of the nucleus and those in the rostral part were concentrated dorsally. In the descending vestibular nucleus, a large number of substance P, Leu-enkephalin and GAD containing neurons were evenly distributed among longitudinally directing fiber bundles. A number of positive fibers with these substances were also observed. The lateral vestibular nucleus contained numerous coarse GAD-immunoreactive fibers surrounding Deiters' neurons, while substance P-immunoreactive and Leu-enkephalin-immunoreactive fibers were rather poorly distributed in this nucleus as well as in the superior vestibular nucleus.

Distribution of muscarinic receptors in the caudal cerebellum and electrosensory lateral line lobe of gymnotiform fish.

Muscarinic binding sites were found in the electrosensory lateral line lobe (ELLL) and vestibulo cerebellum (LC) of certain gymnotid fish; these binding sites were not present in significant numbers in the corpus cerebelli. Autoradiography of [3H]quinuclidinylbenzilate and [3H]propylbenzylcholine mustard binding confirmed these results and also demonstrated that, within the ELLL the region with muscarinic binding sites was coextensive with the region of cholinergic input. We did not find any evidence for nicotinic receptors (alpha-bungarotoxin binding) in ELLL, LC, or corpus cerebelli.

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase.

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain.

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.

[Vestibular pharmacology (author's transl)].

The paper deals with recent advances in anatomical, physiological, biochemical and pharmacological studies on the vestibular system. Fiber connections and characteristics of neurons in the vestibular nuclei were discussed in relation to regulation from other structures such as the cerebellum, oculomotor nuclei, reticular formation and spinal cord. In addition, a review was made of the actions on the vestibular system of antivertigo drugs including anticholinergic and antihistamine drugs, tranquilizers and barbiturates. All the obtained data show that the vestibular nuclei are devoid of noradrenergic, dopaminergic and serotonergic innervation, in contrast to other brainstem nuclei. Acetylcholine is apparently a neurotransmitter in the afferent transmission from the vestibular nerve to the vestibular nuclei. Antivertigo drugs produce modification in the neuron activity in the medial and lateral vestibular nuclei.

The brain-specific S-100 protein on neuronal cell membranes.

The S-100 protein has been localized to the neuronal plasma membranes of isolated Deiters' neurons by fluorescence microscopy using fluorescein-conjugated antiserum to the protein and by immunoelectron microscopy using peroxidase-conjugated anti-S-100 antiserum. In the present study this is shown also by incubating neurons with Sepharose 4B or methylacrylate spherules to which were coupled anti-S-100 antibodies. The specificity of the antiserum is discussed in the text. The technique described can be used to study the topography of antigenic characteristics of nerve cells by using antisera insolubilized on spherules of suitable size.

Projections to lateral vestibular nucleus from cerebellar climbing fiber zones.

1. The olivary projections to the lateral vestibular nucleus (LVN), direct and excitatory through climbing fiber collaterals and indirect and inhibitory through climbing fiber activated Purkinje cells, were investigated in cats with the spinal cord transected at C3 sparing only the contralateral ventral funiculus. In this preparation all spinocerebellar paths are interrupted except the ventral spino-olivocerebellar paths (VF-SOCPs). Three FV-SOCPs responding with different latencies and receptive fields on limb nerve stimulation project to different sagittal zones in the anterior lobe vermis: the a-zone medially and the b-zone laterally in the vermis and the c1-zone in the extreme lateral part of the vermis and the medial part of the pars intermedia. 2. The EPSPs evoked through climbing fiber collaterals and the following IPSPs evoked through climbing fiber activated Purkinje cells were recorded intracellularly from LVN neurons on limb nerve stimulation. Simultaneously the climbing fiber responses evoked in Purkinje cells located in the a-, b- and c1-zones were recorded as positive potentials from the cerebellar surface. 3. Three groups of LVN neurons were distinguished: X neurons (about 20%) which did not receive excitation or inhibition from the olivary paths, aCF neurons (about 5%) which received excitation and inhibition from the olivary path projecting to the a-zone, and bCF neurons (about 75%) which received excitation and inhibition from the olivary path projecting to the b-zone. No LVN neurons were related to the c1-zone. 4. The aCF and bCF neurons occurred intermingled throughout the LVN. The X neurons occurred predominantly in its ventral part. 5. The findings are discussed in relation to a hypothesis of cerebellar organization.

Climbing fiber microzones in cerebellar vermis and their projection to different groups of cells in the lateral vestibular nucleus.

1. The projections from one of the paths (b-VF-SOCP) in the ventral spino-olivocerebellar system to the cortical b-zone located in the lateral part of the anterior lobe vermis and to the lateral vestibular nucleus (LVN) have been studied in cats with the spinal cord transected at C3 sparing only the contralateral ventral funiculus. The projection to the b-zone was studied by recording climbing fiber responses in single Purkinje cells on stimulation of limb nerves. The projections to the LVN, direct through climbing fiber collaterals and indirect through Purkinje cells, were studied by recordings EPSPs and IPSPs in LVN neurons. 2. The Purkinje cells in the b-zone were arbitrarily divided into five groups with different inputs and occupying different microzones each with a width of about 200 micron. On passing medially across the b-zone the microzones had the following input characteristics: 1. activation exclusively from hindlimb nerves, 2. short-latency activation from hindlimb and long-latency activation from forelimb nerves, 3. short-latency activation from hindlimb and forelimb nerves, 4. short-latency activation from forelimb and long-latency activation from hindlimb nerves, and 5. activation exclusively from forelimb nerves. 3. The five microzones projected to different groups of LVN neurons which occurred intermingled throughout the nucleus. The LVN neurons inhibited from a certain microzone were activated by the collaterals of the climbing fibers projecting to that microzone. 4. The organization of the spino-olivo-cerebello-vestibulo-spinal path is discussed. It is suggested that the microzone and collection of subcortical neurons represent the basic computational unit of the cerebellum.

A nonpeptide morphine-like compound: immunocytochemical localization in the mouse brain.

A nonpeptide morphine-like compound (MLC) which cross reacts with morphine-specific antibodies has been localized with the use of immunocytochemistry. This morphine-like compound is found in neuronal perikarya or processes (or both) in nuclei related to vestibular, cerebellar, and raphe systems.