MEASUREMENT OF γ-AMINOBUTYRIC ACID IN ISOLATED NERVE CELLS OF CAT CENTRAL NERVOUS SYSTEM

Abstract— A sensitive method for measuring γ-aminobutyric acid (GABA) has been developed. This method consists of a combination of the enzymic GABA assay of J akoby and S cott (1959) with the enzymic cycling technique of L owry , P assonneau , S chulz and R ock (1961) and permits the measurement of as little as 2 × 10⁻¹⁴ mol of GABA. Using this method, GABA analyses were made on single isolated nerve cell bodies of different types from the CNS of the cat. Average GABA concentrations in these cell bodies were: spinal ganglion cells, 0.2 m m ; spinal mononeurons, 0.9 m m ; large cells of the ventral part of Deiters' nucleus, 2.7 m m ; large cells of the dorsal part of Deiters' nucleus, 6.3 m m ; cerebellar nuclei cells, 6.0 m m ; cerebellar Purkinje cells, 6.6mM; cerebral Betz cells, 2.5 m m .
The GABA concentrations in the isolated dorsal Deiters' cells were greatly reduced (1.7 m m ) after the removal of the cerebellar vermis while those of the ventral Deiters' cells were unaffected by the denervation. These results suggest that GABA is concentrated within axon terminals, probably of Purkinje neurons, synapsing with the dorsal Deiters' cells. The results of GABA analyses on isolated nerve cells are discussed in relation to the relevant neuronal functions and the possible role of GABA as an inhibitory transmitter.     

SOME EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE METABOLISM OF γ-AMINOBUTYRIC ACID IN RAT BRAIN

(1) The inhibitor of γ-aminobutyrate transaminase (GABA-T), amino-oxyacetic acid (AOAA), drastically reduced the activity of GABA-T to 30 per cent of the control value, with a corresponding increase of brain GABA, but had no effect on the activity of glutamate decarboxylase (GAD). (2) The monoamine oxidase (MAO) inhibitors phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine, lowered GABA-T activity to 58, 49 and 48 per cent, respectively; this was associated with a marked elevation of brain GABA. (3) The action of phenelzine and phenylpropylhydrazine in vivo and in vitro could be abolished by pre-treatment of the tissue with the structurally related MAO inhibitors phenylisopropylhydrazine and trans-2-phenylcyclopropylamine. These had no action on the GABA system in vivo, either on the GABA content or on the GABA-T activity. These latter drugs, however, were unable to influence the effects of AOAA either on GABA or on GABA-T. (4) The possible mechanism of action on GABA and the enzyme activities of the GABA system is discussed.     

THE COMPARATIVE EFFECTS OF VARIOUS HYDRA-ZIDES ON γ-AMINOBUTYRIC ACID AND ITS METABOLISM

Abstract— The administration of different hydrazides to chicks (20-23 days post-hatching) in amounts giving similar latent periods before the onset of seizures produced (i) similar rates of decrease in content of cerebral GABA, (ii) considerable but dissimilar inhibitions of cerebral GAD activity, (iii) slight inhibitions of cerebral GABA-T activity. The results support the view that low GABA levels were involved in. the etiology of the seizures but seemed to rule out the possibility that a reduced turnover of GABA was responsible for the occurrence of the convulsions.     

NEURONAL AND GLIAL SYSTEMS FOR γ-AMINOBUTYRIC ACID METABOLISM

—Bulk prepared neuronal perikarya, nerve endings and glial cells have been used to study amino acid concentrations and GABA metabolism in vitro. All amino acids were more concentrated in synaptosomes and glial cells than in neuronal perikarya. Cell specificity was found with respect to the relative distribution of some amino acids. Glutamate decarboxylase activity was considerably higher in synaptosomes than in glial cells. The inhibitory effect of amino-oxyacetic acid on glutamate decarboxylase activity differed between synaptosomes and glial cells. γ-Aminobutyric acid-α-ketoglutarate transaminase had the highest activity in the glial cell fraction; the inhibition of amino-oxyacetic acid differed between glial and neuronal material. The metabolism of exogenous GABA just accumulated by a cell showed similar time characteristics in neuronal and glial material.     

LOCALIZATION OF THE SITES OF γ-AMINOBUTYRIC ACID (GABA) UPTAKE IN LOBSTER NERVE-MUSCLE PREPARATIONS

The principal sites of γ-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.     

THE EFFECT OF 4-AMINO HEX-5-YNOIC ACID (γ-ACETYLENIC GABA, γ-ETHYNYL GABA) A CATALYTIC INHIBITOR OF GABA TRANSAMINASE, ON BRAIN GABA METABOLISM IN VIVO

Abstract— 4-Amino hex-5-ynoic acid (γ-acetylenic GABA, γ-ethynyl GABA, RM171.645), a catalytic inhibitor of GABA transaminase in vitro , induces a rapid, long-lasting dose-dependent decrease of GABA transaminase activity and, to a lesser extent, of glutamate decarboxylase activity in the brains of rats and mice when given by a peripheral route. The GABA concentration in whole brain increases up to 6-fold over control values. The action of γ-acetylenic GABA is relatively specific, as no in vivo inhibition of brain aspartate and alanine transaminase activities could be detected. Furthermore, the amount of radioactive drug bound to the protein fraction of brain homogenate is of the same order of magnitude as that of the GABA transaminase present, as calculated from total GABA transaminase activity, molecular weight and specific activity of the pure enzyme. γ-Acetylenic GABA illustrates the usefulness of a catalytic irreversible enzyme inhibitor in altering neurotransmitter metabolism in vivo .     

DEVELOPMENTAL CHANGES IN THE KINETICS OF γ-AMINOBUTYRIC ACID TRANSPORT BY CHICK RETINA

—Isolated retinas from chick embryos and mature animals were incubated in [³H]GABA at 25°C for 10 min in order to investigate kinetic properties of the amino acid uptake system. Embryo retina accumulated [³H]GABA by two distinct kinetic systems with K_m values of the order 10⁻⁴m and 10⁻⁵m for the low- and high-affinity mechanisms respectively. However, as the retina matured, the high-affinity process disappeared and only the low-affinity system was detectable. No obvious explanation can be offered for this phenomenon although a similar observation has previously been made in chick brain by other workers.     

REDUCTION OF γ-AMINOBUTYRIC ACID LEVEL IN THE CAT CEREBRAL CORTEX BY AFFERENT ELECTRICAL STIMULATION

—Electrical stimulation for 30 s of one brachial plexus in cat (afferent electrical stimulation = AES) produced a 20% decrease in GABA level of the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex in the same animal. This change in GABA was reversed within a few seconds after cessation of stimulation. Inhibition of GABA catabolism by aminooxyacetic acid elevated considerably the cortical level of GABA but failed to prevent lowering GABA by AES. When AES was performed in preconvulsive condition induced by administration of picrotoxin, the decrease in GABA was negligible, while similar treatment with pentylenetetrazol had no influence on the decrease in GABA produced by AES. The observed lowering cortical GABA by AES is interpreted as being associated with some mechanism of the inhibitory transmitter inactivation.