Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.     

Molecular characterization of the human VIP prohormone

Two distinct cell groups contain α-melanocyte-stimulating hormone-like immunoreactivity in rat hypothalamus. Only one group, located in the arcuate nucleus, contains other opiocortin peptide immunoreactivity. Combined immunocytochemistry, radioimmunoassay and high performance liquid chromatography, using two different antisera, were used in an attempt to characterise the immunoreactive material present in each cell group. The results thus obtained from normal rats, using an antiserum against α-melanocyte-stimulating hormone and one against COOH-terminal adrenocorticotropin, were compared with those obtained from rats treated neonatally with monosodium glutamate, which destroys the arcuate nucleus.In animals treated with monosodium glutamate, cells of the arcuate nucleus, staining with both antisera, were reduced in number. Cells containing only α-melanocyte-stimulating hormone-like immunoreactivity in the lateral hypothalamus were unaffected. Peptide levels detected by radioimmunoassay with both antisera were reduced in parallel. Chromatographed extracts showed parallel reductions in α-melanocyte-stimulating hormone-like and COOH-terminal adrenocorticotropin-like immunoreactivities.These results suggest that if the immunostained cells of the lateral hypothalamus contain conventional α-melanocyte-stimulating hormone, it constitutes only a very small proportion of the total hypothalamic concentration. However, the possibilities that the antiserum is crossreacting with a different molecular species, or with a similar compound synthesised by a different pathway cannot be excluded.     

Amino acids and the synaptic pharmacology of granule cells in the dentate gyrus of the rat

Granule cells in the dentate gyrus in the hippocampi of anaesthetized rats were excited by stimulation of the contralateral hippocampus (the commissural input) and the ipsilateral entorhinal cortex (the perforant path). The cells were also activated by the electrophoretic administration of various amino acids. A selective antagonism of glutamate and perforant path excitations was obtained with glutamic acid diethylester, and of aspartate and other amino acid induced and commissural excitations with D- or DL-alpha-aminoadipate. An excitatory effect of alpha-aminoadipate which was sometimes observed was prevented by the gamma-aminobutyric acid antagonist bicuculline, and may be a disinhibitory phenomenon. The results lend support to the proposition that the transmitter of the perforant path is glutamate while that of the commissural fibres is aspartate.     

Release by Electrical Stimulation of Endogenous Glutamate, γ-Aminobutyric Acid, and Other Amino Acids from Slices of the Rat Medulla Oblongata

Evidence was obtained for the release of amino acids by electrical stimulation of slices of regions of the rat medulla oblongata: rostral ventrolateral, caudal ventrolateral and caudal dorsomedial. There was a Ca2+-dependent, tetrodotoxin-sensitive increase in the efflux of aspartate, glutamate, gamma-aminobutyric acid (GABA), glycine, and beta-alanine in all regions examined. There were distinct regional differences in the relative amounts of amino acids released. These results provide evidence for the possible neurotransmitter role of aspartate, glutamate, GABA, glycine, and beta-alanine in these regions of the rat medulla oblongata.     

Immunohistochemistry of acid phosphatase in the human prostate: normal and pathologic. Cytochemistry and biochemistry of acid phosphatases II

Three different antisera against human prostatic acid phosphatase were used for direct and indirect immunohistochemical demonstration of acid phosphatase in paraffin sections of infantile and adult normal, hyperplastic and carcinomatous prostatic tissue. All antisera were prepared in rabbits. Antiserum A was prepared from highly purified acid phosphatase extracted from autopsy specimens. Antiserum B was a concentrate of a commercial antiserum used in radioimmunoassay and was prepared from purified extracts of human seminal fluid. Antiserum C was a peroxidase-conjugated antiserum prepared from purified extracts of human seminal fluid. The specificity of the three antisera was compared using different immunohistochemical methods and tissues. It was comparably high in all three antisera which gave only slightly different staining results in prostatic tissue. The staining results in prostatic carcinoma were only dependent on the titer of the respective antiserum. Carcinomas with a cribriform growth pattern showed variable staining, but always had a positive immunoreactions, provided the titer of the antiserum was sufficiently high. Striking differences were observed in metaplastic, atrophic and hyperplastic prostatic epithelium. The most intense reaction was observed in atrophic glands: it was much less intense in hyperplastic and normal epithelium and negative or slightly positive in metaplastic epithelium.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Ultrastructural immunocytochemical localization of excitatory amino acids in the somatosensory system.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.     

Excitatory Amino Acid-Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.     

A study of the variable heavy chain (VH) region of membrane-bound Ig on human chronic leukemic lymphocytes.

Lymphocytes from 20 patients with chronic lymphocytic leukemia (CLL) were studied for membrane staining by direct immunofluorescence by employing anti-F(ab')2, anti-VHI, anti-VHII, anti-VHIII subgroup-specific antisera, as well as light chain-specific antisera. Some lymphocyte preparations were also studied in indirect immunofluorescence with an antiserum raised against a fragment (VH) corresponding to the variable region of the heavy chain of a human IgG3 myeloma protein (Kup). Lymphocytes from each CLL patient demonstrated a restriction of VH subgroups expressed on the cell membrane; six were restricted to the VHI subgroup, seven to VHII, and seven to the VHIII subgroup. This restriction gave further evidence for monoclonality of the membrane-bound Ig and the leukemic cell proliferation. Antiserum to the VH fragment stained closely similar percentages of CLL lymphocytes to that obtained with anti-F(ab')2 antiserum. Furthermore, double staining revealed that the same cells were stained with anti-VH antiserum as were stained with anti-F(ab')2 antiserum, i.e., only the B lymphocytes.     

Dynamic release of amino acid transmitters induced by valproate in PTZ-kindled epileptic rat hippocampus

In the present communication, the dynamic release of amino acid (AA) transmitters induced by valproate (VPA) in pentylenetetrazol (PTZ)-kindled freely moving rats hippocampus has been determined. The results showed that glutamate and aspartate release were significantly increased during the seizure/interical periods, and markedly decreased after the application of 200mg/kg valproate. In contrast, gamma-aminobutyric acid and taurine release were markedly decreased during interical period, and significantly increased during the seizure period. Glycine release was similar to the case of glutamate and aspartate release. The increase of either gamma-aminobutyric acid/taurine or glycine releases during the seizure period could be inhibited by the application of valproate likewise. The results indicate that: (a) the imbalance between excitatory and inhibitory neurotransmitters is really involved in epilepsy; (b) the modulation of valproate on the major amino acid neurotransmitters certainly plays one of important roles on antiepilepsy efficacy; (c) the pentylenetetrazol-kindled epileptogenesis model is a fit one for approaching the mechanisms of valproate modulating amino acid neurotransmitters.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin

In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gamma-amino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H2O2 (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H2O2 (brown reaction product in target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices

The effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices have been examined. Methylmercury induced a concentration-dependent increase in the spontaneous release of glutamate, aspartate, gamma-aminobutyric acid, and taurine from mouse cerebellar slices. Glycine release was slightly increased, but not in a concentration-dependent manner. The spontaneous release of glutamine from mouse cerebellar slices was not altered by any concentration of methylmercury examined (10, 20, and 50 microM). The tissue content of glutamate, gamma-aminobutyric acid, glutamine, and taurine decreased after exposure to methylmercury. Exposure of cerebellar slices to 20 microM methylmercury resulted in a significant enhancement in glutamate release during stimulation with 35 mM K+. This increase could be accounted for by the methylmercury-induced increase in spontaneous glutamate release. The increase in spontaneous release of glutamate and gamma-aminobutyric acid was independent of the availability of extracellular calcium. These results suggest that methylmercury increases the release of neurotransmitter amino acids, particularly gamma-aminobutyric acid and glutamate, by acting at intracellular sites to increase release from a neurotransmitter pool. The increase in the potassium-stimulated release of glutamate may reflect an increased sensitivity of the cerebellar granule cell to the effects of methylmercury. It is suggested that alterations in amino acid neurotransmitter function in the cerebellum may contribute to some of the neurological symptoms of methylmercury intoxication.     

The novel anticonvulsant BIA 2-093 inhibits transmitter release during opening of voltage-gated sodium channels: a comparison with carbamazepine and oxcarbazepine

(S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz/b,f/azepine-5-carboxamide (BIA 2-093) is endowed with high anticonvulsant activity and shares with carbamazepine (CBZ) and oxcarbazepine (OXC) the capability to inhibit voltage-gated sodium channels (VGSC). The present study was aimed to compare the effects of BIA 2-093, CBZ and OXC on the release of glutamate, aspartate, gamma-aminobutyric acid (GABA) and dopamine from striatal slices induced by the VGSC opener veratrine. The release of glutamate, aspartate, GABA and aspartate by veratrine from rat striatal slices was a concentration and time dependent process. All the three dibenzazepine carboxamide derivatives, BIA 2-093, CBZ and OXC inhibited in a concentration dependent manner (from 30 to 300 microM) the veratrine-induced release of glutamate, aspartate, GABA and dopamine. CBZ, OXC and BIA 2-093 were endowed with similar potencies in inhibiting veratrine-induced transmitter release. It is concluded that BIA 2-093, CBZ and OXC inhibit veratrine-induced transmitter release, which is in agreement with their capability to block VGSC. This property may be of importance for the anticonvulsant effects of BIA 2-093.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid,choline acetyltransferase or serotonin

Summary In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gammaamino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of primary antisera, the sections are incubated in a cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H₂O₂ (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H₂O₂ (brown reaction product in the target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices

Effects of diazepam and gamma-aminobutyric acid-related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., gamma-aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 microM, respectively. gamma-Aminobutyric acid (1 to 10 microM) and muscimol (100 microM) significantly reduced high K+-stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 microM by itself, antagonized the inhibitor effects of diazepam and gamma-aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 microM) of gamma-aminobutyric acid was required for the inhibition. These results indicate the modulation by gamma-aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Distribution of gamma-aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat

The distributions of gamma-aminobutyric acid (GABA), glycine, glutamate and aspartate were measured in cochlear nuclei of two rats by quantitative histochemical mapping procedures. The levels and distributions in the two rats were comparable, and resembled those previously reported for cat cochlear nucleus. The results are consistent with a concept that these putative transmitter amino acids have similar levels and distributions in the cochlear nucleus among mammals.     

Abnormalities in Amino Acid Neurotransmitters in Discrete Brain Regions of Genetically Dystonic Hamsters

The concentrations of 11 amino acids, including the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, glycine, and taurine, were determined by HPLC in 12 brain regions of genetically dystonic (dtSZ) hamsters and age-matched nondystonic controls. Since dystonia in mutant dtSZ hamsters is transient and disappears after about 70 days of age, amino acids were determined at the age of maximum severity of dystonia (30-40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In dtSZ hamsters with the maximum severity of dystonia, significant changes in concentrations of the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, and taurine were found in several regions involved in motor functions, e.g., cerebellum, thalamus, and corpus striatum. Most of these changes were not permanent but disappeared in parallel with dystonia, implicating a causal relationship between altered aminoacidergic neurotransmission and dystonia in mutant dtSZ hamsters.     

Levels and Synthesis of Glutamate and Aspartate in the Olfactory Cortex Following Bulbectomy

Guinea pigs were unilaterally bulbectomised and the contents of aspartate, glutamate and GABA measured in slices of olfactory cortex taken from the lesioned and intact hemispheres. Two days after the operation there was a fall in the aspartate and glutamate levels, which persisted for over 120 days, whereas gamma-aminobutyric acid (GABA) showed a transient fall followed by a small rise. The fall in glutamate and aspartate was much greater in small, thin slices containing a high density of nerve terminals. The synthesis of 13C aminoacids from [13C]glucose during electrical stimulation was greater in the slices taken from the normal side than in those from the operated side. The GABA synthesis, however, was four times greater on the lesioned side. This time-course for the fall in acidic amino acids correlates with the fall in electrical responses, and this lends weight to the idea that aspartate and/or glutamate mediate synaptic transmission in the area.