The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system

Somatostatin-containing neurons in the rat central nervous system were localized by immunocytochemical methods. The detection of somatostatin-like immunoreactivity was facilitated by (1) the use of brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) a ‘double-bridge’ immunoperoxidase staining technique. In addition to the known distribution of somatostatin-like immunoreactivity we also observed immunoreactive perikarya in the following regions: the anterior olfactory nucleus, some areas of the preoptic and hypothalamic regions, the claustrum, the periaqueductal gray, the locus ceruleus, the central gray substance, the lateral parabrachial nucleus, the nucleus of the lateral lemniscus, the nucleus ambiguus, the spinal trigeminal nucleus, the nucleus of the solitary tract and various areas of the reticular formation. Immunoreactive neuronal processes were also observed in several major tracts of the brain, including the stria terminalis, the fornix and the medial forebrain bundle.Our results indicate that somatostatin-containing neurons may occur both as interneurons in some areas of the central nervous system and as projection neurons in others. The widespread but selective distribution of these neurons suggests that somatostatin is not only a hypothalamic regulator of neuroendocrine function, but may also function as a major neuromodulator mediating a variety of functions throughout the central nervous system.     

Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system

The acid-sensitive ion channel ASIC1 is a proton-gated ion channel from the mammalian nervous system. Its expression in sensory neurons and activation by low extracellular pH suggest that ASIC is involved in transmitting nociceptive impulses produced by the acidification caused by injury or inflammation. However, ASIC1 expression is not restricted to sensory neurons. To understand the functional role of ASIC1 in the CNS we investigated its expression and subcellular distribution therein. In particular, we examined the presence of ASIC1 in domains where the local pH may drop sufficiently to activate ASIC1 under physiological conditions. Immunostaining with specific antibodies revealed broad expression of ASIC1 in many areas of the adult rat brain including the cerebral cortex, hippocampus and cerebellum. Within cells, ASIC1 was found predominantly throughout the soma and along the branches of axons and dendrites. ASIC1 was not enriched in the microdomains where pH may reach low values, such as in synaptic vesicles or synaptic membranes. Pre- or postsynaptic ASIC1 was not gated by synaptic activity in cultured hippocampal neurons. Blockage or desensitization of ASIC1 with amiloride or pH 6.7, respectively, did not modify postsynaptic currents. Finally, the ontogeny of ASIC1 in mouse brain revealed constant levels of expression of ASIC1 protein from embryonic day 12 to the postnatal period, indicating an early and almost constant level of expression of ASIC1 during brain development.     

New data on the immunocytochemical localization of thyrotropin-releasing hormone in the rat central nervous system

An antiserum raised against the synthetic tripeptide pyroglutamyl-histidyl-proline (free acid) was used to localize thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) by immunocytochemistry. The distribution of TRH-immunoreactive structures was similar to that reported earlier; i.e., most of the TRH-containing perikarya were located in the parvicellular part of the hypothalamic paraventricular nucleus, the suprachiasmatic portion of the preoptic nucleus, the dorsomedial nucleus, the lateral basal hypothalamus, and the raphe nuclei. Several new locations for TRH-immunoreactive neurons were also observed, including the glomerular layer of the olfactory bulb, the anterior olfactory nuclei, the diagonal band of Broca, the septal nuclei, the sexually dimorphic nucleus of the preoptic area, the reticular thalamic nucleus, the lateral reticular nucleus of the medulla oblongata, and the central gray matter of the mesencephalon. Immunoreactive fibers were seen in the median eminence, the organum vasculosum of the lamina terminalis, the lateral septal nucleus, the medial habenula, the dorsal and ventral parabrachial nuclei, the nucleus of the solitary tract, around the motor nuclei of the cranial nerves, the dorsal vagal complex, and in the reticular formation of the brainstem. In the spinal cord, no immunoreactive perikarya were observed. Immunoreactive processes were present in the lateral funiculus of the white matter and in laminae V-X in the gray matter. Dense terminal-like structures were seen around spinal motor neurons. The distribution of TRH-immunoreactive structures in the CNS suggests that TRH functions both as a neuroendocrine regulator in the hypothalamus and as a neurotransmitter or neuromodulator throughout the CNS.     

Dystrophin in central nervous system: a developmental, regional distribution and subcellular localization study.

Dystrophin, the protein encoded by the Duchenne muscular dystrophy gene has been shown to be expressed in central nervous system. In the present study, polyclonal antibodies raised against 3 fusion proteins constructed from different structural domains of dystrophin were used to identify dystrophin in protein extracts from rat and mdx mouse brain. The developmental expression of the protein, its regional distribution in rat brain and its localization in rat brain subcellular fractions were also examined. We found that dystrophin or a 'dystrophin-related protein' is expressed in mdx mouse brain. Dystrophin is detectable at very early stages of rat brain development and is expressed in all adult brain regions examined, although quantitative regional differences were found. Subcellular distribution analysis indicates that dystrophin is absent in mitochondrial and synaptic vesicle-enriched fractions but is recovered in the synaptic plasma membrane fraction.     

Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody

The localization of glycine receptors was immunocytochemically examined in the rat brain using a monoclonal antibody against the affinity-purified glycine receptor. Glycine receptors were concentrated in the lower brainstem, whereas no immunoreactivity was observed in the diencephalon and forebrain except in a few diencephalic nuclei. The highest density of receptors was found in the cranial motor nuclei, reticular formation, parabrachial area, dorsal and ventral cochlear nuclei, and dorsal and ventral tegmental nuclei. Differences were observed in the distribution of immunoreactive elements in the various brain regions. In the cerebellar cortex, the immunoreactivity was exclusively seen along the dendrites of the Purkinje cells. On the other hand, glycine receptors were detected on the cellular membrane of the soma of the cochlear nuclei, trigeminal motor nucleus, parabrachial area, lateral reticular nucleus, dorsal nucleus of the lateral lemniscus, cerebellar nuclei, trigeminal spinal nucleus, anterior horn and reticular formation. In other regions, the receptors were evenly distributed throughout the neuropil.     

Quantitative autoradiographic evidence for axonal transport of imipramine receptors in the central nervous system of the rat

Interruption of the ascending serotonin axons of the medial forebrain bundle (MFB) in the rat brain produced a progressive time-dependent accumulation of imipramine receptors (labeled for autoradiography with [3H]imipramine). The largest accumulation of receptors occurred during the first 12 h at the caudal aspect of the lesion. An electrolytic lesion of the nucleus raphe dorsalis, administered 24 h prior to interruption of the medial forebrain bundle, markedly reduced the number of imipramine receptors on the caudal side of the lesion, while a significant accumulation was still evident on the rostral aspect. These results suggest that imipramine receptors are undergoing the process of orthograde axonal transport to terminals in the forebrain from the neuronal perikarya found in the nucleus raphe dorsalis. These receptors may also be undergoing retrograde transport back to their cell bodies of origin.     

Immunohistochemical localization of ryanodine receptors in mouse central nervous system.

The distribution of ryanodine receptor-like immunoreactivity in the mouse central nervous system was studied using two antibodies raised against synthetic peptides. These peptides represented a region conserved between the cardiac and skeletal muscle forms and a region specific to the cardiac form. Western blotting analysis and [3H]ryanodine binding analysis showed ryanodine receptors are expressed in all the brain regions. The activity was prominent in hippocampus and cerebral cortex. Immunohistochemical study demonstrated that the ryanodine receptors were localized unevenly in somata. Some apical and proximal dendrites in some cells were also labeled. In hippocampus pyramidal neurons in CA2-3 region were more labeled than CA1 region. Immunohistochemical distribution revealed by two antibodies was essentially the same but the fibers were more immunoreactive with the antibody raised against the cardiac muscle ryanodine form. The localization of ryanodine receptors was quite different from that of inositol 1,4,5-trisphosphate receptors.     

Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex

Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified anti-alpha-1 antibody was confirmed to react only with a single band of about 70-80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. Moreover, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons.     

Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda,Ascaroidea)

 The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining. Received: 3 April 1995 / Accepted: 16 June 1995     

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

Peroxisomal -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).     

Differential induction of c-Fos, c-Jun and Jun B in the rat central nervous system following unilateral entorhinal cortex lesion

In order to identify some of the molecular mechanisms that occur after a central nervous system trauma, the immediate early gene encoded proteins c-Fos, c-Jun and Jun B were analysed by immunocytochemistry following unilateral entorhinal cortex lesion (controls, 30 min, 2, 5, 12 and 24 h, two, six, 10 and 14 days, four weeks and six months postlesion). In the dentate gyrus, c-Fos was induced in some supragranular neurons (30 min), massively expressed in granule cells ipsilaterally to the lesion (2 h), expressed in hilar neurons (5 h and two days) and was absent at all later stages. A basal expression of c-Jun was found in dentate granule cells of controls, which was strongly increased on the lesion side (2 h) and on the side contralateral to the lesion (12 h). c-Jun expression returned to control levels by 24 h. Jun B was induced in granule cells ipsilateral to the lesion within 2 h and was back to control levels by 5 h. In the lateral septal area, c-Fos and c-Jun were induced 30 min postlesion and decreased rapidly thereafter. In the cerebral cortex, a widespread induction of c-Fos and c-Jun occurred within 30 min after entorhinal cortex lesion and this up-regulation lasted until two days postlesion. These data indicate that electrolytic lesion of the entorhinal cortex leads to a rapid and widespread induction of c-Fos, c-Jun and Jun B. Within the denervated fascia dentata, some of these changes may be linked to the reorganization processes following the lesion. Alternatively, the alterations in immediate early gene expression reported here may be due to changes in synaptic activity or postlesional seizures which occur in this lesioning paradigm.     

An electrophysiological study of 5-hydroxytryptamine receptors of neurones in the molluscan nervous system

1. 5-Hydroxytryptamine (5-HT) has been iontophoretically applied to the membrane of central neurones of Cryptomphallus aspersa; CILDA neurones (cells with inhibition of long duration) (Gerschenfeld & Tauc, 1964) are the only cells sensitive to 5-HT. The responses to 5-HT is always a depolarization. The CILDA cells studied were also depolarized by ACh.2. From experiments in which pulses of 5-HT and ACh were applied from a double-barrelled micropipette to the CILDA cell soma, it has been calculated that 5-HT and ACh receptors were located at different distances from the injecting micropipette tip. It has also been calculated from the diffusion equation that in the same CILDA cell a 5-HT concentration of 8.2 x 10(-9)M and a ACh concentration of 1.3 x 10(-8)M caused a similar peak depolarization.3. CILDA neurones show ;anomalous' rectification. 5-HT increases the membrane conductance of CILDA.4. 5-HT receptors of CILDA neurone are desensitized by repeated application of 5-HT. The desensitization lasts for ca. 40 sec.5. 5-HT receptors are blocked by lysergic acid diethylamide and its derivatives. Morphine chlorhydrate blocks them non-competitively.6. Some inhibitors of monoamine oxidase (trancylpromine, isocarboxazide, iproniazide and nialamide) have been tested. They do not prolong the action of 5-HT, but block the 5-HT receptors.7. No crossed desensitization between 5-HT and ACh has been observed. Atropine blocks both ACh-receptors and 5-HT receptors, 5-HT receptors appear to be blocked to a greater extent.8. The data presented support the assumption of a excitatory transmitter role of 5-HT to CILDA neurones, but further evidence is necessary to confirm this hypothesis.     

GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system

The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acidA (GABAA)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [3H]muscimol for high-affinity GABA agonist sites; [3H]bicuculline methochloride and [3H]SR-95531 for the low-affinity GABA sites; [3H]flunitrazepam for benzodiazepine sites, and [3H]2-oxo-quazepam for the 'BZ1'-type subpopulation; and [35S]t-butyl bicyclophosphorothionate (TBPS) and [3H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [35S]TBPS binding also were examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. In particular, the cerebellum, thalamus, hippocampus, substantia nigra and superior colliculus showed enrichment in the binding of some ligands compared to others, and other brain regions showed smaller discrepancies. In addition to the previously observed discrepancies between high-affinity GABA agonists binding and benzodiazepine receptor distribution, especially in the cerebellum, and the well-documented differences in 'BZ1'-selective versus non-selective ligands, significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. The major factor in regional variations within one ligand and between ligands involves differences in binding site densities, although other factors such as endogenous ligands and conformational flexibility may contribute to these findings. The lack of correspondence between components of the GABAA-receptor complex is most consistent with the existence of subtypes that vary in their binding affinities or even binding capabilities. At least four such subtypes are required to explain the regional dissimilarities between ligands. It is likely that these subtypes based on binding alone correspond to different gene products demonstrated recently by molecular cloning and protein chemistry, indicating a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.     

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function.Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.     

Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining.These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.     

Cellular and subcellular localization of an octadecaneuropeptide derived from diazepam binding inhibitor: immunohistochemical studies in the rat brain

Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI includes a newly discovered family of rat brain neuropeptides that are processing products of a common endogenous neuropeptide precursor, diazepam binding inhibitor (DBI). The members of this neuropeptide family have been shown to displace benzodiazepines and beta carbolines from their specific recognition sites located on the allosteric modulatory centers of GABAA receptors. We have previously examined the distribution of DBI-LI in rat brain. The anti-ODN antiserum used in this study does not cross-react with rat DBI, and thus allows a distinct analysis of ODN-LI as opposed to DBI-LI, in rat brain. Neuronal perikarya with ODN-LI were located in many brain nuclei, such as the pontine n., reticular thalamic n., subgeniculate n., supraoptic n. and suprachiasmatic n., and also in brain areas such as cerebral and cerebellar cortex, hippocampus, inferior colliculus, olfactory bulb and subiculum. In addition to perikaryal labelling, a punctate or diffuse immunostaining with ODN antibodies was detected in many brain regions such as cerebellum, hippocampus, amygdaloid area, olfactory tubercle, some of the deep cerebellar nuclei and some circumventricular organs. At the electron microscopic level ODN-LI was identified in neuronal perikarya, processes and terminals. In the axon terminals, ODN-LI appears to be associated with synaptic vesicles. Whenever ODN-LI was detected within neurons, DBI-LI was also found in identical cells. In addition to neurons, DBI-LI was found in glia or glial-like cells, while ODN-LI was not found in these cells. Our findings are consistent with the hypothesis that ODN may be a neuron-specific processing product of DBI and that ODN-like peptides may act as putative endogenous allosteric modulators of various GABAA receptor subtypes.