Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.     

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).     

Retinoic acid and development of the central nervous system.

We consider the evidence that RA, the vitamin A metabolite, is involved in three fundamental aspects of the development of the CNS: 1) the stimulation of axon outgrowth in particular neuronal sub-types; 2) the migration of the neural crest; and 3) the specification of rostrocaudal position in the developing CNS (forebrain, midbrain, hindbrain, spinal cord). The evidence we discuss involves RA-induction of neurites in cell cultures and explants of neural tissue; the teratological effects of RA on the embryo's nervous system; the observation that RA can be detected endogenously in the spinal cord; and the fact that the receptors and binding proteins for RA are expressed in precise domains and neuronal cell types within the nervous system.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Immunocytochemical demonstration of met-enkephalin in the central nervous system of the domestic fowl

Summary In the central nervous system of the male domestic fowl, met-enkephalin (ENK) immunoreactive perikarya and fiber tracts as well as extensive but sharply delimited fiber networks were visualized by means of the PAP technique. The most striking results were: (1) The demonstration of an association of ENK-containing structures with branchial nerves; (2) the spatial relationship of ENK-containing perikarya and fibers to somatostatin (SOM) and arginine-vasotocin (AVT)-immunoreactive systems; (3) the presence of dense and extensive ENK fiber networks within (a) the caudo-basal wall of the third ventricle and (b) the septal-preoptic area; in both regions mainly ENK fibers, but also SOM and AVT fibers, may cross to the contralateral side.     

Insect mucosubstances. II. The mucosubstances of the central nervous system

Synopsis The glycosaminoglycans of the glial lacunar system and neural lamella of cockroach and locust ganglia have been characterized histochemically, using primarily Alcian Blue binding methods at various pH levels and salt concentrations, the periodic acid-Schiff test together with recent modifications, the high iron diamine test, and enzymatic digestions. The results suggest the presence of hyaluronic acid in the glial lacunar system and of a mixture of chondroitin and dermatan sulphates, together with keratan sulphate in the neural lamella. The significance of the presence of these substances in the central nervous system of insects is discussed.     

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.     

Immunocytochemical localization of the precursor protein for beta-amyloid in the rat central nervous system

Two rabbit polyclonal antibodies generated against different portions of the amyloid precursor protein were used to localize this protein in normal rat brain. Light and electron microscopic immunohistochemical localizations demonstrate that the protein is widely distributed throughout the neuraxis, with the highest concentrations of immunoreactive neurons occurring in the olfactory bulb, cerebral cortex, septum-diagonal band, globus pallidus, cerebellum, and hippocampus. Immunoreactive astrocytes are also present in the cerebral cortex in relation to both neurons and capillaries. However, immunoreactivity was not observed within the endothelium of the cerebral vasculature. These data demonstrate that the beta-amyloid precursor is widely distributed in the CNS and provide further insight into the cellular elements that may be involved in the neuropathological changes associated with Alzheimer's disease.     

Ghrelin acts in the central nervous system to stimulate gastric acid secretion

Ghrelin is a novel acylated peptide that functions in the regulation of growth hormone release and energy metabolism. It was isolated from rat stomach as an endogenous ligand for growth hormone secretagogue receptor. Ghrelin is also localized in the arcuate nucleus of rat hypothalamus. Intracerebroventricular (ICV) administration increases food intake and body weight. We examined the effect of ghrelin on gastric acid secretion in urethane-anesthetized rats and found that ICV administration of ghrelin increased gastric acid output in a dose-dependent manner. Vagotomy and administration of atropine abolished the gastric acid secretion induced by ghrelin. ICV administration of ghrelin also induced c-fos expression in the neurons of the nucleus of the solitary tract and the dorsomotor nucleus of the vagus, which are key sites in the central nervous system for regulation of gastric acid secretion. Our results suggest that ghrelin participates in the central regulation of gastric acid secretion by activating the vagus system.     

The renin-angiotensin system and the central nervous system.

One of several factors affecting the secretion of renin by the kidneys is the sympathetic nervous system. The sympathetic input is excitatory and is mediated by beta-adrenergic receptors, which are probably located on the membranes of the juxtaglomerular cells. Stimulation of sympathetic areas in the medulla, midbrain and hypothalamus raises blood pressure and increases renin secretion, whereas stimulation of other parts of the hypothalamus decreases blood pressure and renin output. The centrally active alpha-adrenergic agonist clonidine decreases renin secretion, lowers blood pressure, inhibits ACTH and vasopressin secretion, and increases growth hormone secretion in dogs. The effects on ACTH and growth hormone are abolished by administration of phenoxybenzamine into the third ventricle, whereas the effect on blood pressure is abolished by administration of phenoxybenzamine in the fourth ventricle without any effect on the ACTH and growth hormone responses. Fourth ventricular phenoxybenzamine decreases but does not abolish the inhibitory effect of clonidine on renin secretion. Circulating angiotensin II acts on the brain via the area postrema to raise blood pressure and via the subfornical organ to increase water intake. Its effect on vasopressin secretion is debated. The brain contains a renin-like enzyme, converting enzyme, renin substrate, and angiotensin. There is debate about the nature and physiological significance of the angiotensin II-generating enzyme in the brain, and about the nature of the angiotensin I and angiotensin II that have been reported to be present in the central nervous system. However, injection of angiotensin II into the cerebral ventricles produces drinking, increased secretion of vasopressin and ACTH, and increased blood pressure. The same responses are produced by intraventricular renin. Angiotensin II also facilitates sympathetic discharge in the periphery, and the possibility that it exerts a similar action on the adrenergic neurons in the brain merits investigation.