Gamma-aminobutyric acid receptors in the central nervous system

Conclusions Recent research has raised a whole set of new and interesting points concerning the arrangement of GABA receptor sites. The most important of these is the separation of two distinct GABA receptor categories, namely bicuculline-sensitive and bicuculline-insensitive, which control the chloride and calcium conductance of the postsynaptic membrane. Information regarding the membrane and intracellular processes involved in activating GABA_B receptors remains particularly limited as yet. Accordingly, findings from the literature maintain that calcium ions are not the sole transmitter of transmembrane current during activation of this category of receptor, while data from biochemical research suggests that the intracellular activity of cAMP and cGMP is changed when bicuculline-insensitive receptors are activated [15, 38]. It should be added that the physiological role played by these receptors is not yet known.The study of bicuculline-sensitive GABA receptor complexes using benzodiazepines, as well as their antagonists and reversible agonists, also offers considerable interest. Such research is particularly apposite in view of the widely discussed possibility of related endogenous-type substances existing and consequently of hitherto unknown inherent mechanisms controlling inhibitory processes within the CNS.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 273–282, March–April, 1986.     

Acetaminophen distribution in the rat central nervous system

Based on the evidence that the antinociceptive effects of acetaminophen could be mediated centrally, tissue distribution of the drug after systemic administration was determined in rat anterior and posterior cortex, striatum, hippocampus, hypothalamus, brain stem, ventral and dorsal spinal cord. In a first study, rats were treated with acetaminophen at 100, 200 or 400 mg/kg per os (p.o.), and drug levels were determined at 15, 45, 120, 240 min by high performance liquid chromatography (HPLC) coupled with electrochemical detection (ED). In a second study, 45 min after i.v. administration of [3H]acetaminophen (43 microCi/rat; 0.65 microg/kg), radioactivity was counted in the same structures, plus the septum, the anterior raphe area and the cerebellum. Both methods showed a homogeneous distribution of acetaminophen in all structures studied. Using the HPLC-ED method, maximal distribution appeared at 45 min. Tissue concentrations of acetaminophen then decreased rapidly except at the dose of 400 mg/kg where levels were still high 240 min after administration, probably because of the saturation of clearance mechanisms. Tissue levels increased with the dose up to 200 mg/kg and then leveled off up to 400 mg/kg. Using the radioactive method, it was found that the tissue/blood ratio was remarkably constant throughout the CNS, ranking from 0.39 in the dorsal spinal cord to 0.46 in the cerebellum. These results, indicative of a massive impregnation of all brain regions, are consistent with a central antinociceptive action of acetaminophen.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.     

Systemic administration of kainic acid produces elevations in TRH in rat central nervous system

Several studies have suggested that the concentration of thyrotropin releasing hormone (TRH) in the central nervous system (CNS) is influenced by the level of CNS activation. Hibernation in the ground squirrel and estivation in the lungfish result in region-specific decreases in TRH concentrations. Repeated electroconvulsive shock (ECS) and amygdaloid kindling have been shown to result in elevations of TRH in limbic brain regions. In the present study, limbic seizures induced by systemic administration of kainic acid resulted in substantial increases in the TRH content of posterior cortex and of dorsal and ventral hippocampus, and in moderate elevations in anterior cortex, amygdala/piriform cortex and corpus striatum. Maximal elevations in TRH were observed 2-4 days after kainic acid administration, and by 14 days TRH levels were similar to control values, with the exception of the dorsal hippocampus, which exhibited more prolonged elevations in TRH levels. Prior exposure to limbic seizure activity attenuated the magnitude of TRH elevation in response to a second administration of kainic acid in the posterior cortex but in no other region. These results indicate that seizure-related processes or events influence TRH systems in the CNS. Neuronal populations involved in limbic seizure induced damage may be involved in the modulation of posterior cortical TRH levels.     

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.     

Binding sites forl-glutamate in the central nervous system of the rat

The regional distribution of 2-amino-4-phosphonobutyrate (APB)/chloride-insensitivel-[³H]glutamate binding sites in the rat central nervous system was compared with that of APB/chloride-sensitive and with sodium-dependent binding sites. The distribution of APB-sensitive and APB-insensitive sites was not corelated, but the latter was identical to that of the sodium-dependent sites. The pharmacological specificity of the APB-insensitive sites was not consistent with that of an N-methylaspartate-preferring receptor, and was also different from the specificity determined for the sodium-dependent sites. The APB-insensitive sites appear to be unrelated to any other previously described excitatory amino acid binding site.Dedicated to K. A. C. Elliott on his 80th birthday.     

Corticosteroid receptors in the central nervous system of the rat.

Corticosteroid receptors were demonstrated in the medial hypothalamus, the hippocampus and the parietal cortex of the rat while no such receptors were found in the hypophysis, the amygdala and the anterior hypothalamus. The findings suggest the role of extrahypothalamic regions in the perception of corticosteroid feedback as well as in the regulation of the hypothalamo-hypophysial-adrenal function and do not support the assumption that corticosteroids would inhibit corticotrophin secretion by acting directly on the hypophysis.     

Atrial natriuretic peptide in the central nervous system of the rat

1. Studies of the presence of atrial natriuretic peptide immunoreactivity and receptor binding sites in the central nervous system have revealed unusual sites of interest. 2. As a result, numerous studies have appeared that indicate that brain atrial natriuretic peptide is implicated in the regulation of blood pressure, fluid and sodium balance, cerebral blood flow, brain microcirculation, blood-brain barrier function, and cerebrospinal fluid production. 3. Alteration of the atrial natriuretic peptide system in the brain could have important implications in hypertensive disease and disorders of water balance in the central nervous system.     

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.     

Distribution of tripeptidyl-peptidase II in the central nervous system of rat

Tripeptidyl-peptidase II (TPP II) is a high molecular weight serine peptidase which removes tripeptides from a free N-terminus of longer peptides. Since it had previously been demonstrated that the enzyme can inactivate enkephalins and dynorphins in vitro by removing the N-terminal Tyr-Gly-Gly peptide, we wanted to see whether TPP II could be involved in this process also in vivo. Therefore, the localization of TPP II in different cerebral regions of rat was investigated by immunoblot analysis and activity measurements. It could be shown that TPP II is relatively evenly distributed in the central nervous system of rat. This indicates that the physiological role of the enzyme is probably not a specific degradation of enkephalins, but rather pertains to the general turnover of proteins.     

On the histochemistry of N-acetyl-beta-D-glucosaminidase in rat central nervous system.

The optimal conditions for histochemical demonstration of NAG activity in the cerebrum, diencephalon, midbrain, cerebellum, medulla oblangata, and spinal cord were studied in a series of 37 Wistar rats of either sex. The following more important results were obtained: Each CNS zone required a definite methodlogical approach. Optimal fixation for most structures was achieved after 2 h treatment with formol-calcium and subsequent immersion of tissue blocks in formol-calcium 0,88 M saccharose. By this fixation technique it was possible to preserve high enzyme activity and good tissue structure. Only for the large pyramidal cells of the cerebral cortex the method of Holt provided optimal fixation. Formol-calcium-saccharose mixture and pure 0,88 M saccharose produced the opposide osmotic effect on nervous tissue previously fixed with formol-calcium: the former induced tissue shrinkage, the latter edema. The use of hexazonium p-rosaniline coupler prompted preliminary alcohol treatment of sections and introduction of 0.1 M acetate buffer in the incubation solution. Acetate buffer concentrations lower than 0.2 M diminished the diffuse cytoplasmic coloration and permitted a clear-cut demonstration of the lysosomal reaction. Ample information on the distribution of NAG activity in the CNS was obtained by using fast garnet GBC coupler and 0.1 M citrate buffer. Manganese chloride in a 0.2 mM concentration activates the reaction. The distribution of NAG reaction product in the cells of the different sections of the CNS was studied. The distribution of NAG reaction product in the cells of the different sections of the CNS was studied. The neurons, glial cells, and blood vessels showed positive reaction. Strongest activity was reported for the neurons of the supraoptic and paraventricular necleus, the epithelial cells of the chorioid plexus, nucleus ruber of the mesencephalon, and the vascular wall pericytes.     

Comparison of opioid receptor distributions in the rat central nervous system

The opioid receptors, mu, delta and kappa, conduct the major pharmacological effects of opioid drugs, and exhibit intriguing functional relationships and interactions in the CNS. Previously established hypotheses regarding the mechanisms underlying these phenomena specify theoretical patterns of relative cellular localisation for the different receptor types. In this study, we have used double-label immunohistochemistry to compare the cellular distributions of delta and kappa receptors with those of mu receptors in the rat CNS. Regions of established significance in opioid addiction were examined. Extensive mu/delta co-localisation was observed in neuron-like cells in several regions. mu and kappa receptors were also often co-localised in neuron-like cell bodies in several regions. However, intense kappa immunoreactivity (ir) also appeared in a separate, morphologically distinct population of cells that did not express mu receptors. These small, ovoid cells were often closely apposed against the larger, mu-ir cell bodies. Such cellular appositions were seen in several regions, but were particularly common in the medial thalamus, the periaqueductal grey and brainstem regions. These findings support proposals that functional similarities, synergy and cooperativity between mu and delta receptors arise from widespread co-expression by cells and intracellular molecular interactions. Although co-expression of mu and kappa receptors was also detected, the appearance of a separate population of kappa-expressing cells supports proposals that the contrasting and functionally antagonistic properties of mu and kappa receptors are due to expression in physiologically distinct cell types. Greater understanding of opioid receptor interaction mechanisms may provide possibilities for therapeutic intervention in opioid addiction and other conditions.     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

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     

Distribution of glutamine synthetase in the rat central nervous system.

The results of a light microscopic immunohistochemical study of glutamine synthetase in rat nervous system are presented. In all sites studied the enzyme was confined to astrocytes. Except for trace amounts in ependymal cells, the enzyme was not observed in other cells of the nervous system including neurons, choroid plexus, third ventricular tanycytes, subependymal cells and mesodermally-derived elements. The intensity of astrocyte staining varied in different regions with the greatest degree noted in the hippocampus and cerebellar cortex while the least was noted in brain stem, deep cerebellar nuclei and spinal cord. The glutamine synthetase content correlated well with sites of suspected glutamergic activity in keeping with the view of a critical role of astrocytes in the regulation of the putative neurotransmitter glutamic acid.