Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA. J. Comp. Neurol. 382:247-259, 1997. © 1997 Wiley-Liss, Inc.     

Constitutive expression of heat shock protein HSP25 in the central nervous system of the developing and adult mouse

Immunohistochemistry and in situ hybridization have been used to survey constitutive heat shock protein (HSP)25 expression in the brain and spinal cord of the developing and adult mouse. The data reveal both transient and sustained patterns of expression and demonstrate robust differences between mice and rats. During development, HSP25 is transiently expressed in neurons of the inferior colliculus, various thalamic subnuclei, and the majority of Purkinje cells in the cerebellum. Sustained expression into adulthood is seen in neurons of the cranial nerve nuclei, spinal cord motoneurons, median preoptic nucleus, and a subset of Purkinje cells. Differences in HSP25 expression between adult rats and mice include the somatic motor nuclei innervating the extraocular muscles, which are HSP25 immunoreactive only in the rat. Similar differences in HSP25 expression are seen during the development of the inferior colliculus, thalamus, and cerebellum, where expression is restricted to mice.     

Expression of s-laminin and laminin in the developing rat central nervous system.

The extracellular matrix component, s-laminin, is a homologue of the B1 subunit of laminin. S-laminin is concentrated in the synaptic cleft at the neuromuscular junction and contains a site that is adhesive for motor neurons, suggesting that it may influence neuromuscular development. To ascertain whether s-laminin may also play roles in the genesis of the central nervous system, we have examined its expression in the brain and spinal cord of embryonic and postnatal rats. S-laminin was not detectable in synapse-rich areas of adults. However, s-laminin was present in discrete subsets of three laminin-containing structures: (1) In the developing cerebral cortex, laminin and s-laminin were expressed in the subplate, a transient layer through which neuroblasts migrate and cortical afferents grow. Both laminin and s-laminin disappeared as embryogenesis proceeded; however, laminin was more widely distributed and present longer than s-laminin. (2) In the developing spinal cord, laminin was present throughout the pia. In contrast, s-laminin was concentrated in the pia that overlies the floor plate, a region in which extracellular cues have been postulated to guide growing axons. (3) In central capillaries, s-laminin appeared perinatally, an interval during which the blood-brain barrier matures. In contrast, laminin was present in capillary walls of both embryos and adults. To extend our immunohistochemical results, we used biochemical methods to characterize s-laminin in brain. We found that authentic s-laminin mRNA is present in the embryonic brain, but that brain-derived s-laminin differs (perhaps by a posttranslational modification) from that derived from nonneural tissues. We also used tissue culture methods to show that glia are capable of synthesizing "brain-like" s-laminin, and of assembling it into an extracellular matrix. Thus, glia may be one cellular source of s-laminin in brain. Together, these results demonstrate that s-laminin is present in the developing central nervous system, and raise the possibility that this molecule may influence developmental processes.     

Distribution of immunoreactive dynorphin in the central nervous system of the rat

A widespread distribution of immunoreactive dynorphin (ir-Dyn) in rat brain and spinal cord was demonstrated by means of a highly specific radioimmunoassay. The highest concentrations of ir-Dyn (greater than 399 pg/mg protein) were found in hypothalamic nuclei, i.e. the premamillary, anterior hypothalamic and dorsomedial nuclei and median eminence. Relatively high concentrations of ir-Dyn (between 320 and 399 pg/mg protein) were found in other hypothalamic nuclei such as the medial and lateral preoptic, perifornical, suprachiasmatic, ventromedial nuclei and in the medulla oblongata in the area postrema and in the nucleus of the solitary tract (commissural part). Moderate levels of ir-Dyn (between 140 and 320 pg/mg protein) were found in most diencephalic areas other than the hypothalamic nuclei and further nuclei in the medulla oblongata, in the mesencephalon, pons and spinal cord. Low to moderate levels of ir-Dyn were found in the telencephalon, with lowest levels (less than 140 pg/mg protein) found in the cerebral cortex, olfactory bulb, dorsal septal nucleus, medial amygdaloid nucleus, caudate-putamen, superior collicle, cerebellum and certain areas of the reticular formation.     

Distribution of GABA immunoreactivity in the central and peripheral nervous system of amphioxus (Branchiostoma lanceolatum pallas)

On the basis of labeling with an anti-γ-aminobutyric acid (GABA) antibody, we report for the first time the presence and distribution of GABA-immunoreactive cells in the central and peripheral nervous system of amphioxus. In the nerve cord, there is a large dorsorostral group of cerebrospinal-fluid-contacting (CSFc) cells at the caudal end of the brain vesicle that gives rise to a large ventral commissure and neuropilar region. In the middle and caudal region of the brain, numerous commissural and CSFc neurons are situated below the region of large dorsal cells. In the spinal cord, several types of GABA-immunoreactive neurons of different size, appearance, and distribution were observed. In the dorsalmost region, very small commissural cells are scattered regularly along the cord. More ventrally in the cord, GABAergic neurons, both of commissural and CSFc cell types, form segmental groups, but scattered cells are observed throughout. These cells give rise to dense longitudinal fascicles of GABAergic fibers and to scattered commissural fibers. The caudal ampulla lacks GABAergic cells and fibers. Some of the fibers of the most rostral and caudal peripheral (sensory) nerves, as well as some sensory cells of the rostral and caudal epidermis, are GABA immunoreactive. The significance of these results for the understanding of the evolution of GABAergic systems of vertebrates is discussed. J. Comp. Neurol. 401:293–307, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system

Intracellular recordings were obtained from granule cells of the dentate gyrus in mouse hippocampal slices maintained in vitro. All the spikes observed in standard Krebs solution had a short duration and were tetrodotoxin (TTX)-sensitive. When elicited by synaptic activation or by direct electrical stimulation of the cells, these fast sodium spikes were followed by a brief spike afterhyperpolarization. In contrast, antidromic spikes elicited by electrical stimulation of the hilum as well as spikes arising at the end of hyperpolarizing current pulses passed through the recording microelectrode were followed by depolarizing afterpotentials (DAPs). These DAPs were reversed into brief spike afterhyperpolarizations by depolarization of the cells. After substitution of calcium (Ca) by barium (Ba) or after introduction of tetraethylammonium (TEA) in the bath, the fast spike repolarization became slower and the brief spike afterhyperpolarizations were abolished, suggesting that they involved fast K conductances. Slow spikes and long-lasting depolarizations were also elicited in granule cells in the presence of Ba or TEA. Since these slow events were left unaffected by TTX and were selectively abolished by the Ca channel blockers cobalt or cadmium, they are likely to be mediated by voltage-dependent Ca conductances, unmasked by the reduction of the fast K conductances.     

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors.     

Distribution of neurotensin-containing neurons in the central nervous system of the pigeon and the chicken

Neurotensin is widely located in neurons of the central and peripheral nervous systems among mammalian species. To obtain a comparative evaluation, we examined the distribution of neurotensin-containing cell bodies and fibers in the central nervous system of the pigeon and the chicken. The pattern of localization of neurotensin immunoreactivity was similar in the two species. Abundant accumulations of neurotensin-containing cell bodies were found in the dorsolateral corticoid area, the piriform cortex, the parahippocampal area, the medial part of the frontal neostriatum, the lateral part of the caudal neostriatum, nucleus accumbens, the bed nucleus of the stria terminalis, ventral paleostriatum, the preoptic area, the ventromedial hypothalamic nucleus, the inferior hypothalamic nucleus, the infundibular hypothalamic nucleus, and the mammillary nuclei. Extremely dense networks of neurotensin-containing fibers were found in the pallial commissure, the lateral septal nucleus, the preoptic area, the periventricular gray around the third ventricle, the dorsalis hypothalamic area, the hypothalamic nuclei, the parabrachial nucleus, the locus ceruleus, and the dorsal vagal complex. Major differences of immunoreactivity between the two species were as follows. 1) The chicken neurohypophysis contained an extremely large accumulation of immunoreactive fibers, but there were few in the median eminence. The reverse was found in the pigeon. 2) The optic tectum in the pigeon contained immuroreactive cells and fibers in layers 2 and 4, but no immunoreactivity was seen in the chicken optic tectum. 3) The cerebellar cortex in the pigeon contained a small number of immunoreactive fibers, whereas that in the chicken did not. 4) The pigeon spinal cord contained immunoreactive neurons in the subependymal layer, but the chicken spinal cord did not. Our observations suggest the presence of a very wide network of neurotensin-containing neurons in the avian brain and spinal cord, which is also the case in mammals. © 1996 Wiley Liss, Inc.     

Distribution of the iron-regulating protein hepcidin in the murine central nervous system

Iron serves as an essential trace element for all body tissues, including the central nervous system (CNS). Because iron deficiency as well as iron overload is known to cause damage to the mammalian brain, the maintenance of iron homeostasis is crucial. It has been discovered recently that hepcidin plays an essential role in iron metabolism outside the CNS. A defect in hepcidin expression is responsible for iron accumulation and mice over-expressing hepcidin die postnatally by a severe anemia. We have used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the cellular distribution of hepcidin mRNA and protein in brain, spinal cord, and dorsal root ganglia. Our results show a wide-spread distribution of hepcidin in different brain areas, including the olfactory bulb, cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon, cerebellum, pons, spinal cord, as well as in dorsal root ganglia of the peripheral nervous system. Hepcidin immunoreactivity is not restricted to neurons, but can be detected in both neurons and GFAP-positive glia cells. Because hepcidin action in organs outside the CNS is linked to iron homeostasis, we speculate that it is also involved in such processes in the CNS, putatively together with other iron regulating proteins. Cellular mechanisms and functions of hepcidin in the CNS remain to be elucidated.     

Expression of trk receptors in the developing and adult human central and peripheral nervous system

A family of tyrosine receptor kinases known collectively as trk receptors plays an essential role in signal transduction mediated by nerve growth factor and related neurotrophins. To localize the major trk receptors (trkA, B and C) in the developing and adult central (CNS) and peripheral (PNS) nervous system, we generated monoclonal antibodies (MAbs) to extracellular (MAbs E7, E13, E16, E21, E29) and intracellular (MAb I2) domains of human trkA fused to glutathione S-transferase. Several MAbs (E7, E13, E16) recognized glycosylated trkA (gp 140^trk and gp110^trk) in Western blots, one MAb (E7) recognized non-glycosylated (p80^trk) and glycosylated trkA in immunoprecipitation assays, and two MAbs (E13, E29) detected trkA on the cell surface of NIH3T3 cells transfected with a trkA cDNA. Although generated to trkA fusion proteins, this panel of MAbs also recognized trkB and trkC in flow cytometric studies of NIH3T3 cells transfected with trkB or trkC cDNAs. Thus, we used these pan-trk MAbs to probe selected regions of the CNS and PNS including the hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and dorsal root ganglion (DRG) to localize trkA, B, and C receptors in the developing and adult human nervous system. These studies showed that trk receptors are expressed primarily by neurons and are detectable very early in the developing hippocampus, cerebellum, spinal cord, and DRG. Although the distribution and intensity of trk immunoreactivity changed with the progressive maturation of the CNS and PNS, immunoreactive trk receptors were detected in neurons of the adult human hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and DRG. This first study of trk receptor proteins in the developing and adult human CNS and PNS documents the expression of these receptors in subsets of neurons throughout the developing and adult nervous system. Thus, although the expression of trk receptor proteins is developmentally regulated, the constitutive expression of these neurotrophin receptors by neurons in many regions of the adult human CNS and PNS implies that mature trk receptor-bearing neurons retain the ability to respond to neurotrophins long after terminal neuronal differentiation is complete. © 1995 Wiley-Liss, Inc.     

Localization of Bcl-xbeta in the developing and adult rat central nervous system

bcl-xbeta is a novel apoptosis-regulating member of the bcl-x family that has recently been isolated from rats and mice. To explore the functional role of Bcl-xbeta, we raised a monoclonal antibody against rat Bcl-xbeta protein and investigated the cellular localization of the molecule in the rat CNS. Immunohistochemistry revealed that, in the fetal and neonatal stages, Bcl-xbeta was intensively and widely expressed in the CNS. Many neurons in the diencephalon and brain stem showed intense cytoplasmic labeling. The immunoreactivity decreased during the postnatal development and reached to the level of adulthood by P14. In the adult brain and spinal cord, labeling was restricted to specific types of neurons and distributed throughout their somata and dendrites. Weak immunoreactivity was present in many CNS regions such as the cerebral cortex, hippocampal dentate gyrus, caudate-putamen, globus pallidus, thalamus, locus ceruleus, pontine nuclei, inferior olive, reticular formation, cerebellar cortex and spinal anterior horn. Amygdaloid nuclei and hippocampal CA1 to CA3 sectors showed restricted expression of Bcl-xbeta in a subset of neurons. Neuronal labeling was almost undetectable in several regions, including the piriform cortex, hypothalamus, posterior column nuclei and spinal posterior horn. These results suggest that Bcl-xbeta plays an important role throughout the CNS in developing stage and may regulate the apoptosis of postnatal CNS neurons.     

Chondroitin sulfate proteoglycans in the developing central nervous system. II. Immunocytochemical localization of neurocan and phosphacan

Using immunocytochemistry, we have compared the distribution of neurocan and phosphacan in the developing central nervous system. At embryonic day 13 (E13), phosphacan surrounds the radially oriented neuroepithelial cells of the telencephalon, whereas neurocan staining of brain parenchyma is very weak. By E16–19, strong staining of both neurocan and phosphacan is seen in the marginal zone and subplate of the neocortex, and phosphacan is present in the ventricular zone and also has a diffuse distribution in other brain areas. Phosphacan is also widely distributed in embryonic spinal cord, where it is strongly expressed throughout the gray and white matter, in the dorsal and ventral nerve roots, and in the roof plate at E13, when neurocan immunoreactivity is seen only in the mesenchyme of the future spinal canal. Neurocan first begins to appear in the spinal cord at E16–19, in the region of ventral motor neurons. In early postnatal and adult cerebellum, neurocan immunoreactivity is seen in the prospective white matter and in the granule cell, Purkinje cell, and molecular layers, whereas phosphacan immunoreactivity is associated with Bergmann glial fibers in the molecular layer and their cell bodies (the Golgi epithelial cells) below the Purkinje cells. These immunocytochemical results demonstrate that the expression of neurocan and phosphacan follow different developmental time courses not only in postnatal brain (as previously demonstrated by radioimmunoassay) but also in the embryonic central nervous system. The specific localization and different temporal expression patterns of these two proteoglycans are consistent with other evidence indicating that they have overlapping or complementary roles in axon guidance, cell interactions, and neurite outgrowth during nervous tissue histogenesis. © 1996 Wiley-Liss, Inc.     

Changes of motor corticobulbar projections following different lesion types affecting the central nervous system in adult macaque monkeys

Functional recovery from central nervous system injury is likely to be partly due to a rearrangement of neural circuits. In this context, the corticobulbar (corticoreticular) motor projections onto different nuclei of the ponto‐medullary reticular formation (PMRF) were investigated in 13 adult macaque monkeys after either, primary motor cortex injury (MCI) in the hand area, or spinal cord injury (SCI) or Parkinson's disease‐like lesions of the nigro‐striatal dopaminergic system (PD). A subgroup of animals in both MCI and SCI groups was treated with neurite growth promoting anti‐Nogo‐A antibodies, whereas all PD animals were treated with autologous neural cell ecosystems (ANCE). The anterograde tracer BDA was injected either in the premotor cortex (PM) or in the primary motor cortex (M1) to label and quantify corticobulbar axonal boutons terminaux and en passant in PMRF. As compared to intact animals, after MCI the density of corticobulbar projections from PM was strongly reduced but maintained their laterality dominance (ipsilateral), both in the presence or absence of anti‐Nogo‐A antibody treatment. In contrast, the density of corticobulbar projections from M1 was increased following opposite hemi‐section of the cervical cord (at C7 level) and anti‐Nogo‐A antibody treatment, with maintenance of contralateral laterality bias. In PD monkeys, the density of corticobulbar projections from PM was strongly reduced, as well as that from M1, but to a lesser extent. In conclusion, the densities of corticobulbar projections from PM or M1 were affected in a variable manner, depending on the type of lesion/pathology and the treatment aimed to enhance functional recovery.     

Immunohistochemical localization of μ-opioid receptors in the central nervous system of the rat

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the ‘patch’ areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguus nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-L1. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the μ-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic μ-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic μ-opioid receptors. © 1996 Wiley-Liss, Inc.     

Topography and cytoarchitecture of the motor nuclei in the brainstem of salamanders

The organization of the motor nuclei of cranial nerves V (including mesencephalic nucleus), VI, VII, IX, and X is described from HRP-stained material (whole mounts and sections) for 25 species representing five families of salamanders, and the general topology of the brainstem is considered. Location and organization of the motor nuclei, cytoarchitecture of each nucleus, and target organs for nuclei and subnuclei are described. The trigeminal nucleus is separated distinctly from the facial and abducens nuclei and consists of two subnuclei. The abducens nucleus consists of two distinct subnuclei, one medial in location, the abducens proper, and the other lateral, the abducens accessorius. The facial nucleus has two subnuclei, and in all but one species it is posterior to the genu facialis. The facial nucleus completely overlaps the glossopharyngeal nucleus and partially overlaps that of the vagus. In bolitoglossine plethodontid salamanders, all of which have highly specialized projectile tongues, the glossopharyngeal and vagus nuclei have moved rostrally to overlap extensively and intermingle with the anterior and posterior subnuclei of the facial nerve. In the bolitoglossines there is less organization of the cells of the brainstem nuclei: dendritic trunks are less parallel and projection fields are wider than in other salamanders. Some aspects of function and development are discussed; comparisons are made to conditions in anurans; and phylogenetic implications are considered.