Protein kinase C in central vestibular,cerebellar, and precerebellar pathways of the rat

The protein kinase C family of enzymes is composed of at least ten different isoforms that display a variety of distinct biochemical specificities. Many of these isoforms are highly expressed in brain, and some show regional specificity in their distribution, suggesting that they may serve specific functions. By using immunocytochemistry to localize the βI, βII, γ, or δ isoforms of protein kinase C in the central vestibular system of the adult rat, we found the vestibular ganglion and its peripheral and central processes of the eighth nerve to be heavily labeled with protein kinase C βI immunoreactivity. Labeled axons and terminals were also found in all four vestibular nuclei. Some neurons of the vestibular ganglion were weakly stained with the antibody to protein kinase C βII, as were scattered axons in the eighth nerve, and scattered axons and terminals were found in all four vestibular nuclei among weakly labeled neurons. A few axons in the vestibular portion of the eighth nerve were labeled with protein kinase C γ immunoreactivity, and neurons of the spinal, lateral, and superior vestibular nuclei were heavily decorated with synapses, presumably derived from Purkinje neurons, which were also strongly immunoreactive. Neurons of the medial vestibular nucleus were not as heavily innervated. With the antibody to protein kinase C δ, we found scattered, weakly immunoreactive neurons in the vestibular portion of the eighth nerve. Myelinated fiber bundles of the spinal vestibular nucleus contained moderate numbers of labeled axons, and the other vestibular nuclei were well innervated by protein kinase C δ axons and terminals. Most of these probably derive from Purkinje cells, which were labeled in longitudinal bands interspersed with bands of labeled basket cells. These data suggest that particular protein kinase C isoforms play specific roles in vestibular and cerebellar function. J. Comp. Neurol. 385:26–42, 1997. © 1997 Wiley-Liss, Inc.     

Hexokinase I Messenger RNA in the Rat Central Nervous System

Hexokinase I (ATP: -hexose 6-phosphotransferase, EC 2.7.1.1) is the first enzyme required in the metabolism of glucose in the central nervous system and plays a major role in regulation of the cerebral glycolytic rate. The distribution of hexokinase I mRNA was examined throughout the central nervous system of the rat by use of oligonucleotide probes and in situ hybridization histochemistry. In the rhinencephalon, strong hexokinase I mRNA labeling was demonstrated in the glomerular, mitral, internal granular, and internal plexiform layers, whereas the olfactory nerve, external plexiform, and subependymal layers and ependyma were devoid of labeling. Within the telencephalon, strong labeling was present in all layers (with the exception of the molecular layer) of the cerebral cortex, in the septum, in CA1-4 and dentate gyrus of the hippocampus, and in several amygdaloid nuclei. There was only weak labeling in the nucleus accumbens and caudate putamen. In the diencephalon, there was in general a strong labeling in the epithalamus, in several thalamic nuclei, including the anteriodorsal, anterioventral, anteriomedial, reticular, paravetricular, intermediodorsal, anteriomedial, interanteriomedial, rhomboid, reuniens, and parafascicular thalamic nuclei. Several hypothalamic regions, including the subfornical organ, the medial preoptic area, the suprachiasmatic, supraoptic, paraventricular, dorsomedial, ventromedial nuclei, and the zona incerta, were strongly labeled. In the mesencephalon, there was particularly strong labeling in the pars compacta and reticulata of the substantia nigra, central gray, and red nucleus, in the Darkschewitsch nucleus, and in the medial accessory oculomotor nucleus. In the rhombencephalon, there was strong hybridization in all raphe nuclei, pontine, tegmental, lateral parabrachial, olivary nuclei, and several cranial motor nuclei. All neurons of the locus ceruleus were heavily labeled. Very strong labeling was present in Purkinje and granular cells of the cerebellar cortex. Neurons of the medulla oblongata area postrema, nucleus tractus solitarius, reticular nucleus, nucleus cuneatus and several motor nuclei were strongly labeled. In the spinal cord, labeled cells were present in all laminae, and also neurons of the dorsal root ganglion were heavily labeled. Hexokinase I mRNA was also demonstrated in the epithelium lining the choroid plexus. In the E15 fetus, very strong labeling was seen in the liver, heart, and trigeminal ganglion, with less intense labeling in the brain and other tissues having more moderate labeling. Administration of 2% saline as drinking water resulted in a marked increase in hexokinase I mRNA in the magnocellular neurons of the supraoptic and paraventricular nuclei. In summary, the results show extensive neuronal distribution of hexokinase I mRNA with regional differences in the expression pattern.     

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon's horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.     

Immunoperoxidase labelling of rat brain sections with sera from patients with paraneoplastic cerebellar degeneration and systemic neoplasia

Sera from patients with systemic cancer found by immunofluorescence staining to have antibodies to human cerebellar cell populations were reacted with vibratome sections of rat cerebellum and examined by peroxidase-antiperoxidase (PAP) methods. Seven patients with clinically or pathologically confirmed paraneoplastic cerebellar degeneration and two neurologically normal patients with high titers of anticerebellar antibodies were studied. Sera from all antibody-positive patients, but not from controls, produced intense staining of brain sections. Sera from patients with ovarian adenocarcinoma reacted predominantly with Purkinje cells and neurons within brainstem nuclei. Sera from patients with oat cell carcinoma and one patient with ductal carcinoma of the breast produced nuclear and cytoplasmic staining of neurons throughout the central nervous system. Serum from a patient with Hodgkin's disease labeled the peripheries of Purkinje cells and Golgi II cells. Serum from a patient with mixed mesodermal sarcoma of the ovary labeled Purkinje cells, basket cells, and scattered astrocytes. Staining of extraneural tissues was not observed. This study confirms the presence of antineural antibodies in patients with systemic neoplasia with and without paraneoplastic cerebellar degeneration and suggests that the antigens recognized by this antibody response may vary with the associated neoplasm.     

Cell proliferation in the lamprey central nervous system

After spinal cord transection, axons regenerate both in larval and adult lampreys. It is not known to what degree cells proliferate, even in the uninjured animal. Therefore, we have determined the prevalence of mitosis in the lamprey central nervous system (CNS). Bromodeoxyuridine (BrdU) was injected and incorporated for 4 hours into 2- to 5-year-old larvae, animals undergoing metamorphosis, and young adults. Labeled cells were counted in the rhombencephalon (where most supraspinal projecting neurons are located) and spinal cord. A mitotic index (MI) was calculated as the percentage of nuclei that were labeled. There was a seasonal variation in mitotic activity, with higher MIs occurring in summer. Within the summer, there was an additional transient spike in mitosis, especially in the rhombencephalon. There was no correlation between age and MI within the range of developmental stages examined. Baseline MIs in the rhombencephalon and spinal cord were approximately 0.15% and 0.20%, respectively. In most animals, the highest mitotic rates in both the rhombencephalon and spinal cord were seen in the ependyma, but many labeled cells were found in nonependymal regions as well. During the summer spike, almost all of the additional mitosis in the rhombencephalon was in the ependyma, but this finding was not true in the spinal cord. Many BrdU-labeled cells in the spinal cord and rhombencephalon were also stained by monoclonal antibodies specific for lamprey glial keratin but were never labeled by anti-neurofilament antibodies. These results suggest that (1) neurogenesis is uncommon in the lamprey CNS; (2) during most of the year, baseline gliogenesis occurs mainly in the ependyma with substantial contribution by nonependymal areas. During the summer, a spike of mitotic activity occurs in the ependyma of the rhombencephalon and throughout the spinal cord.     

Type B monoamine-oxidase-containing cells and fibers in the cat hypothalamus demonstrated by an improved enzyme histochemical method

The present study, using a diaminobenzidine (DAB)-coupled peroxidation method, examined the distribution and morphological characteristics of neuronal structures containing type B monoamine oxidase (MAO-B) in the cat hypothalamus. Large and intensely stained, distinctive MAO-B-positive cells, multipolar and with long dendritic arbors, were principally distributed in the ventral hypothalamus extending from A7 to A12.5 of the Horsley-Clarke plane. These cells were located caudally in the ventral surface of the brain including the tuberomamillary nucleus (TM) and the region surrounding the mamillary nuclei. Rostrally, they were aggregated in the area surrounding the fornix, particularly in the lateral perifornical region, and dispersed in the anterior mamillary nucleus, lateral hypothalamic area (HLA), and the medial tip of the entopeduncular nucleus. The most rostral positive cell group was identified in a narrow space between the optic tract and the entopeduncular nucleus at the A12.5 level. In addition to these large cells, the present study disclosed the presence of "small" to "very small" MAO-B-positive cells in the area surrounding the mamillary recess and the lateral part of the caudal arcuate nucleus. Distinct MAO-B-stained fibers were identified in all regions of the hypothalamus. A large number of thick labeled fibers were observed in the ventral hypothalamus including the TM and premamillary nucleus and posterior and lateral hypothalamic areas. A dense network of MAO-B-positive terminal-like fibers was observed in the dorsomedial nucleus where very small labeled cells were scattered. Many intensely stained thick and straight fibers were seen running ventrolaterally in the anterior part of the HLA and in the narrow space between the entopeduncular nucleus and optic tract. In the area of the tuber cinereum and the ventral part of the HLA, there were many positive fibers cut transversely, possibly projecting to the more anterior parts of the brain such as the diagonal band of Broca or septal nuclei.     

Regional distribution of the cells expressing glycine receptor β subunit mRNA in the rat brain

The expression of mRNA of the β subunit of the glycine receptor was investigated in the rat by in situ hybridization histochemistry using an oligonucleotide probe specific to the sequence of the β subunit. Neurons expressing β subunit mRNA were widely and abundantly distributed in the rat brain from the olfactory bulb to the spinal cord. The pattern of distribution of cells containing β subunit mRNA in the lower brainstem was very similar to that of cells containinga₁ subunit mRNA. In addition, β subunit mRNA was strongly expressed by the neurons of the cerebral cortex, hippocampal formation and diencephalon as well as by the Purkinje cells wherea₁ subunit mRNA expression is rare. These findings indicated that the glycine receptor is heterogeneous. The sites where strong labeling was noted were as follows. In the forebrain and diencephalon, strongly labeled neurons were abundant in the olfactory region, hippocampal formation, cerebral cortex, and thalamus. In the hippocampal formation, neurons in the subiculum, pyramidal cells in Ammon's horn, and neurons in the polymorphic layer of the dentate gyrus were strongly labeled. In the thalamus, the anterodorsal, reticular, parafascicular, and the subthalamic nuclei were strongly labeled. In the brainstem, the red nucleus, almost all of the motor neurons in the cranial motor nuclei innervating striated muscles, the trigeminal mesencephalic nucleus, the ventral tegmental nucleus of Gudden, and the pontine nucleus were strongly labeled. In the cerebellum, Purkinje cells in the Purkinje cell layer and all of the cerebellar nuclei were strongly labeled.     

Central innervation of the rat ependyma and subcommissural organ with special reference to ascending serotoninergic projections from the raphe nuclei

The subcommissural organ (SCO) and the cerebral ependyma receive serotoninergic innervation, but little is known about their origin in the raphe nuclei. Application of the retrograde tracer cholera toxin subunit B (ChB) in the third ventricle resulted in uptake in ependymal axons and backfilling of perikarya in the dorsomedian part of the dorsal raphe nucleus, immediately under the caudal aqueduct. By using dual staining with antisera against serotonin and ChB, a portion of the retrogradely labeled neurons was observed to co-store serotonin. Phaseolus vulgaris–leucoagglutinin (PHA-L) was injected into different raphe nuclei to fill the neurons in the same areas where the retrogradely labeled neurons were found. PHA-L injection in the midline of the dorsal raphe nucleus gave rise to ascending axonal processes in the mesencephalic central gray, from where they entered the periventricular strata and the third ventricular ependyma. In the cerebral ependyma, large numbers of positive fibers were consistently found in the ventral part of the lateral ventricles and in the dorsal part of the third ventricle. A large number of PHA-L-immunoreactive fibers were observed in the hypendymal layer of the lateral part of the SCO. Terminal fibers near the ependymal cells were also observed. In all cases, the PHA-L injections labeled innervating fibers both within the ependyma and in the SCO, whereas injections into the median raphe nucleus or in other raphe nuclei (i.e., the raphe pallidus and the raphe pontis) labeled fibers neither in the SCO nor in the ependyma. This study shows that a specific group of predominantly serotoninergic neurons innervates both the ependyma and the SCO and is probably involved in cerebrospinal fluid regulation. J. Comp. Neurol. 384:556–568, 1997. © 1997 Wiley-Liss, Inc.     

The tuberoinfundibular system of the rat as demonstrated by immunohistochemical localization of retrogradely transported wheat germ agglutinin (WGA) from the median eminence

Injections of horseradish peroxidase (HRP) were made iontophoretically in the vestibular nuclear complex and the prepositus hypoglossal nucleus (PH) in cats. Differential localization of labeled Purkinje cells due to the retrograde axonal transport of HRP was studied in the flocculus. It was found that there existed three-dimensional zones which were perpendicular to the long axis of the folia in the ipsilateral flocculus; Purkinje cells projecting to the superior vestibular nucleus were distributed in the rostral zone, those to the medial vestibular nucleus in the middle zone. Specific projection-zones to the inferior vestibular nucleus, lateral vestibular nucleus and the PH were not found. Only a small number of labeled Purkinje cells were scattered in the flocculus after injection of HRP in them.     

The rotational behavior model: asymmetry in the effects of unilateral 6-OHDA lesions of the substantia nigra in rats

Three cats received large injections in the pontine nuclei of horseradish peroxidase labeled wheat germ agglutinin. Pontocerebellar axons were stained throughout their length and dense terminal label was present in the granular layer. The cerebellar nuclei, however, contained only a few scattered labeled fibers without a consistent distribution from case to case. If nuclear collaterals from pontocerebellar fibers exist, they appear to be very few and can be expected to give only a very small contribution to the excitatory input to the cerebellar nuclei.     

Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis: abundant expression in telencephalic neurons

The distribution of chelecystokinin B (CCK-B) receptors in the Mastomys brain was studied using Northern blot analysis and in situ hybridization technique. By Northern blot analysis using³²P-labeled cDNA probe, the cortex had the highest hybridization signal of CCK-B receptor mRNA in the brain. The olfactory bulb and hippocampus showed a moderate level of signals. In situ hybridization using³⁵S-labeled cRNA probes revealed a wide and region-specific distribution of CCK-B receptor mRNA in the telencephalon. Throughout the cerebral cortex, labeled cells were found in all layers, with higher intensities in layers II, V and VI. Pyramidal cells of the layer II of the piriform cortex showed the highest level of signals in the brain. In the hippocampus, most of the pyramidal cells of the Ammon's horn were labeled, although labeled cells were not detected in other layers. Distinct signals were also detected in the various amygdaloid nuclei, caudate-putamen, reticular thalamic nucleus, hypothalamic ventromedial nucleus and inferior colliculus. This distribution pattern may further support the prominent existence of CCK-B receptors in the brain particularly in the telencephalon.     

Purkinje cell neuroaxonal dystrophy similar to nervous mutant mice phenotype in two sibling kittens

Three 4-month-old kittens from the same litter were presented, two of which were exhibiting cerebellar signs. Euthanasia was requested. No cerebellum atrophy was disclosed on necropsy. General cerebellar anatomy was normal, including the thickness of the cortical layers, myelination, and neurons of the deep cerebellar nuclei. In the ataxic cat vermis, Purkinje cells were lacking along broad parasagittal bands symmetrically disposed relative to the midline. Many Purkinje cells were also lacking in the hemispheres. The nodulus and the flocculus were normal. Surviving Purkinje cells had frequent main dendrite swellings visible with anti-calbindin and anti-microtubule associated protein. In affected regions, calbindin and phosphorylated neurofilaments immunesera stained numerous axonal torpedoes located in the granular layer and the folial white matter. They were also present in processes of the deep cerebellar nuclei and lateral vestibular nucleus. Loss of synaptic endings onto the neurons of these nuclei was evident. Hypertrophied Purkinje cell recurrent axons and enhanced retrograde synaptic endings were present in the granular layer. Bergmann glia was strongly labeled by anti-GFAP, but no abnormal supplementary fibers were seen. None of these alterations were present in the normal sister. However, abnormal vacuolation of the Purkinje cell main dendrites was evident in all three cats, but not in six unrelated control cats that were 3–6 months old. The inferior olive and pontine nuclei were also normal. The two ataxic cats had a primary Purkinje cell degeneration that shared many common features with the abnormal Purkinje cells of the nervous mutant mouse.     

Transferrin receptors in rat central nervous system. An immunocytochemical study

Using an immunocytochemical method we demonstrated the presence of TfR on adult rat neurons, particularly in the cerebral cortex and brain stem. The monoclonal antibody (mab) against rat TfR (clone OX 26) stained neurons of all cortical layers and in the brain stem where the reaction was most evident. Purkinje cells in the cerebellum and scattered neurons in the gray matter of the cervical spinal cord were weakly stained. Choroid plexus cells also reacted with the mab against TfR whereas oligodendrocytes in the cerebral white matter were faintly outlined by the mab. The presence of TfR on endothelial cells of brain capillaries was here confirmed.     

Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat cerebellum

Voltage-gated K(+) (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum.     

Localization of gene expression of calreticulin in the brain of adult mouse.

The localization of gene expression of calreticulin, a calcium-binding protein in the endoplasmic reticulum, was examined throughout the entire brain of adult mice by in situ hybridization. Calreticulin mRNA is expressed widely and heterogeneously in discrete neurons throughout the brain, but the white matters expressed it weekly or faintly. In the olfactory bulb, the mRNA is expresses moderately in the mitral cells, but weakly in the periglomerular cells and internal granule cells. In the cerebrum, the gene is expressed intensely in the piriform cortex, but weakly in neocortex, the entorhinal cortex and the amygdaloid nuclei. In the hippocampal formation, calreticulin mRNA is expressed intensely in the CA1-CA3 regions but less intensely in the granule cells of the dentate gyrus. The caudate-putamen, thalamic and hypothalamic nuclei, and mammillary nuclei express the mRNA weakly or faintly. In the mesencephalon, pons and medulla, moderate expression of the mRNA is detected in the pontine nuclei and the locus ceruleus. Weak expression of the mRNA is detected in several discrete nuclei and zones such as the substantia nigra, the superior colliculus and the central gray. Expression signals of calreticulin mRNA are faint in the inferior olive. In the cerebellum, calreticulin mRNA is expressed moderately in the Purkinje cells whereas no significant expression is detected in the granule cells. The plexus choroideus of the lateral, third and fourth ventriculi express calreticulin mRNA intensely although no distinct expression of the mRNA is discerned in the ependyma.     

Temporal and spatial expression of NGF receptor mRNA during postnatal rat brain development analyzed by in situ hybridization.

The expression of nerve growth factor (NGF) receptor mRNA was examined in the rat brain during postnatal development using in situ hybridization. Cells expressing NGF receptor mRNA were detected in the basal forebrain at all ages examined, with a peak in expression at 2 weeks of age. NGF receptor mRNA was further demonstrated to be expressed transiently in several brainstem nuclei. Expression of NGF receptor mRNA was high at postnatal day (P) 1 and 1 week of age in the facial and abducens nuclei, but was undetectable in the facial nucleus by 2 weeks of age. In the abducens nucleus, a few labeled cells were still present at 2 weeks of age, but absent by 3 weeks. In the cerebellum, a strong signal was present at P1 and 1 week of age which clearly diminished by 2 weeks and disappeared by 3 weeks of age. The labeled cells in the cerebellum had the size and morphology of developing Purkinje cells. These data suggest that the population of NGF-responsive cells in the brain is more widespread during development than in the adult, and that the trophic requirements of specific brain regions are altered with maturity.     

NADPH-Diaphorase in the central nervous system of the tench (tinca tinca L., 1758)

The distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (ND)–positive cells and fibers in the tench central nervous system was mapped by using a direct histochemical method. This enzyme was observed in specific cell populations throughout all main divisions of the tench brain. In the telencephalon, we found strongly labeled olfactory fibers, as well as positive cells and fibers in the area ventralis of the telencephalic lobes. Positive staining was observed in the following diencephalic nuclei: nucleus preopticus magnocellularis pars magnocellularis, nucleus recessus lateralis, nucleus recessus posterioris, nucleus posterior tuberis, and nucleus diffusus torus lateralis, as well as small cells with a diffuse distribution surrounding the diencephalic ventricle. In the mesencephalon, heavily stained ND-positive neurons were observed in the nucleus fasciculi longitudinalis medialis, nucleus nervi oculomotorius, and nucleus nervi trochlearis. In the hindbrain the most evident staining was observed as large neurons located in the nuclei of the cranial nerves, scattered positive cells located between the negative fibers of the cranial nerves, and in the nucleus fasciculi solitari. Finally, in the spinal cord, ND-positive cells and fibers were mainly located in the ventral horn. This distribution of ND labeling in the brain of the tench is significantly different from previous data on ND activity in the brain of terrestrial vertebrates and does not correlate with the presence and distribution patterns of several neurotransmitters and neuroactive substances in the teleost brain. © 1995 Wiley-Liss, Inc.