Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.     

Localization of urotensin I- and corticotropin-releasing factor-like immunoreactivity in the central nervous system of Catostomus commersoni

The distribution of urotensin I (UI) and corticotropin-releasing factor (CRF) immunoreactive (IR) structures was studied in the central nervous system (CNS) of the white sucker using the peroxidase-antiperoxidase immunocytochemical procedure. The close sequence homology between both peptides resulted in a high degree of crossreactivity. This was resolved by saturating the antisera solutions with heterologous antigens and specificity tests were done by adding excess of homologous peptides. UI immunoreactivity was seen in all of the identifiable caudal spinal cord neurosecretory cells, in their processes projecting to the urophysis, in thin beaded fibres coursing along the spinal cord, in brain stem, hypothalamus, proximal pars distalis and, especially, in the telencephalon. Some IR-UI specific and IR-CRF specific parvocellular neurons were also identified in the caudo-ventral tuberal region and ventral telencephalon. The IR-CRF was mainly present in parvocellular and magnocellular perikarya of the nucleus preopticus and in the preoptic-neurohypophysial pathway. Dense networks of IR-CRF reacting beaded fibres were also located in the lateral and posterior recessus nuclei. In the pituitary, IR-CRF fibre bundles were seen mainly in the neurointermediate lobe and in the rostral pars distalis. The cells of origin of the extraurophyseal system of IR-UI fibres in the sucker CNS have not been identified. The distribution of CRF immunostaining correlates well with the documented knowledge of CNS structures involved in the control of ACTH secretion in the goldfish. The probability of the occurrence of two UI-CRF related molecules, or of two different forms resulting from a common precursor molecule, forming two separate neuronal systems in the sucker CNS seems likely.     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

Localization of nerve growth factor-like immunoreactivity in rat nervous tissue

The use of immunofluorescence with affinity-purified antibodies enabled cytological localization of nerve growth factor-like material in the rat. Immunoreactivity was observed along various nerve tracts of the foetal rat brain and spinal cord at day 15 of gestation. Longitudinal pathways in ventral and dorsal spinal cord, ventral lower brain stem, posterior commissure, retroflex fascicle and in the olfactory bulb were all positive. A weaker and more widely spread immunostaining was visible in many areas in the central nervous system. Cranial nerves were strongly immunoreactive. Neuronal perikarya in the retina and the olfactory mucosa as well as filae olfactoriae and the olfactory nerve all the way to the olfactory bulb were also positive. In sensory ganglia and peripheral nerves most immunoreactivity was confined to supporting tissues, probably including Schwann cells. In irides, the pattern of immunoreactivity was similar to that of the sensory and autonomic innervation. More intensively fluorescent material was found in regrowing nerve fibres in iris transplants. Our histochemical results suggest that nerve growth factor and/or a related protein is present in large amounts along nerve pathways in supportive tissues of the peripheral nervous system as well as in the central nervous system during early development.     

Distribution and localization of neurokinin A-like immunoreactivity and neurokinin B-like immunoreactivity in rat peripheral tissue

Using specific radioimmunoassays and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of these peptides in rat peripheral tissues were studied. NKA-like immunoreactivity (NKA-LI) was present in highest levels of 15.7–23.9 pmol/g wet wt. and NKB-like immunoreactivity (NKB-LI) was in levels of 0.33–0.67 pmol/g wet wt., throughout the gastrointestinal tract involving stomach, duodenum, jejunum, ileum and colon. Immunocytochemical analysis of gastrointestinal tract revealed that NKA-LI and NKB-LI localized in ganglia of both the submucosal and myenteric plexuses as well as varicose neurons in the mucosa and the muscle layer of the small and large intestine. On the other hand, high levels of NKB-LI were observed in oesophagus (0.83 ± 0.08 pmol/g wet wt.), adrenal (1.02 ± 0.21), head of pancreas (0.73 ± 0.06) and kidney (0.98 ± 0.05).

The present study shows the difference of localization of NKA-LI and NKB-LI in peripheral tissues and suggests that NKB may have some physiological role differing from that of NKA in peripheral tissues.     

Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system

Using an antiserum directed against synthetic galanin (GAL) a sensitive radioimmunoassay was developed. The antiserum interaction with GAL was characterized by displacement curve characteristics and high performance liquid chromatography. Besides the main GAL-immunoreactive peak several small peaks with GAL-like immunoreactivity were observed. No cross-reactivity of the GAL-antiserum with several other peptides was observed. GAL-like immunoreactivity was measured in 37 microdissected areas of the rat central nervous system. High concentrations (greater than 2000 fmol/mg protein) were observed in the amygdaloid nuclei, the septum, globus pallidus, bed nuclei of the stria terminalis, all hypothalamic nuclei, the superior colliculus, locus coeruleus, the nucleus of the solitary tract and the neurointermediate lobe of the pituitary. Moderate concentrations (1000-2000 fmol/mg protein) were observed in the hippocampus, the nucleus accumbens and nucleus of the diagonal tract, the caudate-putamen, the central gray, the nucleus, tract and substantia gelatinosa of the spinal trigeminal nerve. The results generally correlate with those previously published by immunocytochemistry. The widespread distribution of GAL-like immunoreactivity in the rat central nervous system suggests an involvement of GAL in a variety of brain functions.     

Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system

FMRFamide immunoreactive material (irFMRFamide) was studied in rat brain and gastrointestinal tract. Highest irFMRFamide concentrations were found in tissues of the gastrointestinal tract and, in the brain, highest concentrations were found in the hippocampus, midbrain, brainstem and hypothalamus. High pressure liquid chromatographic characterization of irFMRFamide demonstrated that the immunoreactive material in brain, pancreas and duodenum was different from molluscan FMRFamide but it was also distinct from any known neuropeptide.     

Distribution and developmental expression of lamin-like immunoreactivity in the central nervous system.

Lamins are the major proteic constituents of the nuclear lamina, the innermost layer of the nuclear membrane. The immunolocalization of lamins in the rat central nervous system was studied using polyclonal antibodies. Besides an ubiquitarious localization in the nuclear membranes of neurons and glial cells, an intense lamin-like immunoreactivity was found in the soma and dendrites of cerebellar Purkinje cells. The same specific reaction was also observed in the human cerebellum. Experiments performed in newborn animals demonstrated that the cytoplasmic expression of lamins in Purkinje cells begins during postnatal development.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.     

Distribution of neurotensin-like immunoreactivity in the central nervous system of the Formosan monkey

The distribution of neurotensin-like immunoreactivity was investigated in the central nervous system of the Formosan monkey employing immunohistochemical techniques. Neurotensin-containing cells were found to be widely distributed in the forebrain. The principal densities of neurotensin-like neuronal perikarya were located in the limbic system, the basal ganglion and the cerebral cortex; particularly in the amygdala, the septum, the neostriatum, the claustrum and the insula. The stria terminalis and the preoptic area were also rich in immunostained neurotensin-like neurons. A large number of immunoreactive fibers were observed from the cerebral cortex to the spinal cord in locations such as the median eminence, the arcuate nucleus, the hippocampus, the central gray and the dorsal horn of the spinal cord. We analyzed in detail the distribution of neurotensin-like immunoreactivity in the brain of the Formosan monkey, and compared these results with those obtained in the brain of the rat, Japanese monkey and human. Some possible implications regarding differences in location of this peptide are also briefly discussed.     

Calcitonin gene-related peptide-like immunoreactivity in the central nervous system and peripheral organs of rats

The concentrations of rat calcitonin gene-related peptide-like immunoreactivity (rCGRP-LI) in various organs of male rats as well as the molecular heterogeneity of rCGRP-LI in tissue extracts was examined using a specific radioimmunoassay (RIA) for rCGRP and gel-filtration chromatography. rCGRP-LI was high in extracts of the spinal cord (202 +/- 22.6 pg/mg wet wt. of tissue; mean +/- S.E.M.) and of the thyroid (229 +/- 62.3 pg/mg). rCGRP-LI was detectable in the brainstem, hypothalamus, stomach, duedenum, pancreas and kidney. The elution pattern of the extracts on a Sephadex G-50 column showed 3 peaks of rCGRP-LI irrespective of organs and tissues. The first peak corresponded to authentic rCGRP-(1-37). The second and third rCGRP-LI peaks probably consisted of C-terminal fragments of rCGRP, because they had a lower molecular weight than rCGRP-(1-37) and because our antiserum cross-reacts with a synthetic C-terminal fragment. The ratio of 3 rCGRP-LI molecules, however, differed between neural tissue extracts and others. The main component of rCGRP-LI in neural tissue was authentic rCGRP-(1-37), while the smaller fragments of rCGRP were chiefly contained in other tissues like the stomach, pancreas and thyroid. The relative ratio of rCGRP-LI molecules with different size in respective tissue extracts was not changed after leaving the dissected tissues for 2 h at room temperature. These findings indicate that rCGRP-LI is abundantly present in the thyroid as well as the spinal cord and it is detected in lower amounts in the alimentary tract and central nervous system. rCGRP-LI in the extracts consists of 3 different components, the proportions of which vary from one tissue to another, probably reflecting tissue-specific differences in the processing of CGRP.     

Distribution of serotonin-, Gamma-Aminobutyric Acid- and substance P-like immunoreactivity in the central and peripheral nervous system of Mytilus galloprovincialis

The distribution of serotonin-, GABA- and substance P-like immunoreactivity has been studied in the cerebral and visceral ganglia and in some peripheral tissues of Mytilus galloprovincialis (Moleusca, Bivalvia). Cerebral ganglia contain a developed serotonin-immunoreactive neuronal subpopulation and numerous GABA-immunoreactive neurons, whereas neurons positive for substance P are sparse. In peripheral tissues innervated by the cerebral ganglia (labial palps and oesophagus) only serotonin-immunoreactive nerve fibers were found. In the visceral ganglia, serotonin- and GABA-immunoreactive neurons are far less numerous than in the cerebral ganglia, whereas several neurons positive for substance P are scattered in all cortical zones. Serotonin-immunoreactive plexuses innervate the posterior adductor muscle and the gill filaments which contain also a developed nerve network positive for substance P. The distribution pattern of the immunoreactive elements in the ganglia and in peripheral territories indicates that GABA should exert only a central action, whereas serotonin and a substance P-like peptide are involved both in central and peripheral neurotransmission.     

Immunohistochemical localization of Bis protein in the rat central nervous system

We studied the distribution of Bis (Bcl-2 interacting death suppressor) protein in the adult rat brain and spinal cord using immunohistochemistry. Immunoreactivity was observed in specific neuronal populations in distinct nuclei. The most intensely labeled cells were associated with the motor system, including most cranial nerve motor nuclei, Purkinje cells of the cerebellum, the red nucleus, and the ventral motor neurons of the spinal cord. Bis protein was also expressed in several structures associated with the ventricular system, including the subventricular zone of the lateral ventricle and its rostral extension, in the subcommissural organ, and in tanycytes, radial glial cells in the hypothalamus. Using double-labeling techniques, Bis-immunoreactive cells in the rostral migratory stream, coexpressing Bcl-2, were confirmed as glial fibrillary acidic protein-positive astrocytes comprising the glial tubes. The widespread distribution of Bis suggests that this protein has broader functions in the adult rat central nervous system than previously thought, and that it could be associated with a particular role in the rostral migratory system.J.-H. Lee and M.-Y. Lee contributed equally to this study. This work was supported by the KOSEF through the Cell Death Disease Research Center of MRC at the Catholic University of Korea (R13-2002-005-01001-0) and the Catholic Medical Center Research Foundation grant made in the program year of 2002     

Immunohistochemical localization of minor gangliosides in the rat central nervous system

We previously described the differential distribution of majorgangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) in adult rat braindetected by specific antibodies (Kotani,M., Kawashima,I., Ozawa,I.,Terashima,T. and Tai,T. Glycobiology, 3, 137–146, 1993).We report here the distribution of minor gangliosides in theadult rat brain by an immunofluorescence technique with mousemonoclonal antibodies (MAbs). Ten MAbs (GMR6, GMB28, GMR11,GMR19, GMR2, GMR7, GGR51, AMR10, NGR54 and NGR53) that specificallyrecognize GM3, GM2, GT1a, GD3, O-Acdisialoganglioside, GD2,GM1b, GM4, IV³NeuAc     

Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system

Peroxisomal β-oxidation, consisting of four steps catalysed by an acyl-CoA oxidase, a multifunctional protein and a thiolase, is responsible for the shortening of a variety of lipid compounds. The first reaction of this pathway is catalysed by a FAD-containing acyl-CoA oxidase, three isotypes of which have been so far recognised. Among these, straight-chain acyl-CoA oxidase (ACOX) acts on long and very long chain fatty acids, prostaglandins and some xenobiotics. We investigated ACOX localisation by means of a sensitive, tyramide based, immunocytochemical technique, thus obtaining a complete distribution atlas of the enzyme in adult rat CNS. Granular immunoreaction product was found in the cytoplasm of neuronal and glial cells, both in the perikarya and in the cell processes. ACOX immunoreactive neurons were present to variable extent, in either forebrain or hindbrain areas. Specifically, the strongest signal was detected in the pallidum, septum, red nucleus, reticular formation, nuclei of the cranial nerves, and motoneurons of the spinal cord. We then compared the ACOX immunoreactivity pattern with our previous distribution maps of other peroxisomal enzymes in the adult rat brain. While ACOX appeared to colocalise with catalase in the majority of cerebral regions, some differences with respect to d-amino acid oxidase were noted. These observations support the hypothesis of heterogeneous peroxisomal populations in the nervous tissue. The wide distribution of the enzyme in the brain is consistent with the severe and generalised neurological alterations characterising the peroxisomal disorder caused by ACOX deficiency (pseudo-neonatal adrenoleukodystrophy).     

Distribution of neuropeptide K-immunoreactivity in the rat central nervous system

The distribution of neuropeptide K (NPK), a 36-residue amidated peptide originally isolated from porcine brain, is described in the rat CNS by immunohistochemical methods. Antibodies were generated in rabbits to N-terminus and C-terminus regions of the peptide and the distribution of immunoreactive cell bodies and fibers was mapped in colchicine-treated and normal rat brains. Major areas of cell body staining included the medial habenular nucleus, the ventromedial nucleus of the hypothalamus, the interpeduncular nucleus, the lateral dorsal tegmental nucleus, the nucleus raphe pallidus, and the nucleus of the solitary tract. Some of the areas of dense NPK-fiber immunoreactivity included the ventral pallidum, the caudate-putamen, certain areas of the hypothalamus, the central and medial amygdaloid nuclei, the entopeduncular nucleus, the habenular nuclei, the substantia nigra pars reticulata, the caudal part of the spinal nucleus of the trigeminal nerve, the nucleus of the solitary tract and the dorsal horn of the spinal cord. A striking similarity exists between this pattern of immunoreactive staining and that described for substance P, suggesting that the tachykinin systems do not exist independently in the brain. The possible roles for multiple tachykinins in the brain are discussed.     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.