GABAergic Nerve Terminals Research Articles

Cellular compartments of GABA in brain and their relationship to anticonvulsant activity.

The effects of GABA-elevating agents were examined with respect to the cellular compartments in which GABA increases occurred and the brain region(s) that mediate the anticonvulsant activity of these compounds. Changes in GABA occurring in the presence and absence of GABAergic nerve terminals were estimated in vivo using rats in which the GABA projection to the substantia nigra (SN) was destroyed on one side of the brain. One week post-operatively, the GABA concentration in the denervated SN was 10–20% of control. The net increase in GABA content of the denervated SN was compared to that of the intact SN after intraperitoneal injection of amino-oxacetic acid (AOAA), di-n-propylacetate (DPA) and γ-vinyl GABA (GVG). In the intact SN, all drugs produced significant increases in GABA. In the denervated SN, both AOAA and GVG produced marked increases in GABA (nearly equivalent to those obtained in the intact SN) whereas DPA was without effect. It therefore appears that the DPA-induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA and GVG primarily elevate GABA in non-nerve terminal compartments. An increase in GABA associated with nerve terminals was obtained with GVG only after a latency of more than 12 h following a single injection. The time course of elevation of nerve terminal-associated GABA coincided with the time course of anticonvulsant action of GVG; both effects were maximal at 60 h after a single injection. Taken together, our results indicate that the ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures is directly correlated with increases in nerve terminal GABA and not related to increases in other GABA compartments.

Postnatal changes of gabaergic and glutamatergic parameters

In the rat cortex and striatum, glutamate decarboxylase, a marker for GABAergic nerve terminals, increased almost linearly for the first postnatal month, in agreement with previous reports. High affinity GABA and glutamate transport appeared to develop earlier, reaching, respectively, 170–190% and 72–80% of adult rates by the fifteenth postnatal day. Glial and neuronal uptakes of GABA in infant and adult tissues were investigated using β-alanine and cis-3-aminocyclohexanecarboxylic acid. The relatively high striatal uptake of GABA observed in 2-day-old rats was found to be mainly due to early development of neuronal rather than glial transport in this region. Both neuronal and glial uptake, however, contributed equally to the enhanced uptake of GABA obtained in both regions at day 15. Neuron/glia ratios were estimated to increase by more than 12-fold in the cortex, and about 4-fold in the striatum from newborns to adults. The present results also indicate that there may exist in the immature striatum some glioblasts which might accumulate β-alanine but not GABA. The 2-fold developmental increase in ornithine aminotransferase activity is consistent with the hypothesis that this enzyme may be enriched in glutamatergic neurons.

Biochemical development of the human brain. II. Some parameters of the GABA-ergic system.

The development of the gamma-aminobutyrate (GABA)-ergic system in the human cerebral cortex and cerebellum was studied in post mortem specimens, by estimating the activity of glutamate decarboxylase (GAD) and the binding capacity for muscimol as markers of GABA-ergic nerve terminals and GABA receptors respectively. The age periods studied were as follows (number of specimens in parentheses): fetal period, 17--24 and 28 weeks, gestational age (GA) (15); perinatal period, 26--42 weeks GA (9); postnatal period, 43--56 and 74 weeks GA (11); adult life, 26, 47, 57--73 years (9). Total protein and DNA were estimated in all specimens. Differences between the cerebral cortex and the cerebellum in the ontogenesis of the GABA-ergic system were revealed. In the cerebral cortex, GAD-specific activity increased progressively during development, but at term had only reached approximately 20% of the adult value, and the trend in the postnatal specimens indicated that the adult level is not reached until some time after 60 weeks GA. The concentration of muscimol binding sites, on the other hand, rose more rapidly than GAD activity with age in the cerebral cortex, attaining adult values by 60 weeks GA and being already at term approximately 45% of the mean adult figure. In the cerebellum, the relative development of pre- and postsynaptic markers was the reverse of that in the cerebral cortex: GAD specific activity had reached the adult value by 60 weeks GA and approximately 40% of this adult level was attained at term, while the muscimol binding site concentration was only about 10% of the adult value at term and was still increasing at 60 weeks GA. The affinity of the receptor for [3H]-muscimol did not change during development, and was the same in cerebral cortex and cerebellum.

Gabaergic nerve terminals decrease in the substantia nigra following hemitransections of the striatonigral and pallidonigral pathways

Glutamic acid decar☐ylase (GAD), the enzyme that synthesizes the neurotransmitter, GABA, was immunocytochemically localized in axon terminals as well as in small and medium-sized neurons of the rat substantia nigra. The pattern formed by GAD-containing axon terminals with the dendrites and somata of neurons in the substantia nigra was altered following ipsilateral hemitransections of the striatonigral and pallidonigral pathways. A marked reduction of GAD-positive terminals occurred throughout this brain region, but the ventral fifth of the pars reticulata showed a nearly normal pattern of GAD-positive axon terminals. The results of this investigation are consistent with results from biochemical studies which have indicated that the striatonigral and/or pallidonigral pathways are GABAergic. In addition, these results suggest that the residual GABAergic terminals remaining after hemitransection are derived from intrinsic neurons of the substantia nigra.

Radiohistochemical localization of GABA receptors

One can localize GABA receptors by light microscopic autoradiographic techniques. The technique involves labeling high affinity, sodium indepenent GABA binding sites with 3H-Muscimol in slide mounted tissue sections. Autoradiograms are generated by the apposition of emulsions-coated cover slips. We observed large variations in the distribution of GABA receptors throughout the brain. Some high density areas included the granule cell layer of the cerebellum, the dorsal lateral geniculate body, most thalamic nuclei, the external plexiform layer of the olfactory bulb and in the inner plexiform layer of the retina. The distribution of the high affinity receptors was not the same as that of suspected GABAergic nerve terminals or as that for benzodiazepine receptors in all areas. In general, this approach is valuable for obtaining information on GABA receptor densities in small regions of brain.

Evaluation of increases in nerve terminal-dependent vs nerve terminal-independent compartments of GABA in vivo

Evaluation of increases in nerve terminal-dependent and nerve terminal-independent compartments of GABA in vivo. Changes in GABA occurring in the absence and in the presence of GABAergic nerve terminals were estimated using rats in which the GABAergic projection to the substantia nigra (SN) was destroyed on one side of the brain. The net increase in GABA content of the GABA-denervated SN was compared with that of the intact SN after injection of amino-oxyacetic acid (AOAA), n-dipropylacetate (DPA) and γ-vinyl GABA (GVG). One week post-operative, GABA concentration in the SN on the transected hemisphere was 10–20% of control. AOAA (30 mg/kg) produced a two-fold increase in GABA content in the denervated SN, whereas DPA (300 mg/kg) was without effect. Since DPA caused an increase in GABA in the intact SN which was similar to that caused by AOAA (25–30%), it appears that the DPA induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA primarily elevates GABA in non-nerve-terminal compartments. GVG, an irreversible GABA-T inhibitor, increased GABA levels in both compartments. The predominant effect of GVG was on nerve terminal-independent GABA (4-fold increase after 900 mg/kg); the GABA increase associated with nerve terminals was smaller (30%) and did not appear until 60 hr after a single injection of GVG. The ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures was directly correlated with increases in nerve-terminal GABA, and not related to increases in other GABA pools.

Dissociation between drug-induced increases in nerve terminal and non-nerve terminal pools of GABA in vivo

To examine whether increased GABA levels produced by n-dipropylacetate (DPA) and amino-oxyacetic acid (AOAA) are associated with nerve terminals, we compared the effect of these drugs on the GABA content of substantia nigra (SN) in rats in which the GABAergic afferent projections to SN had been unilaterally destroyed. In the SN largely devoid of GABAergic nerve terminals, AOAA (30 mg/kg) produced a 2-fold increase in GABA, whereas DPA (300 mg/kg) was without effect. Since DPA and AOAA both increased GABA to a similar extent in the intact SN, it appears that the GABA increase produced by DPA is associated with GABAergic nerve terminals, while AOAA primarily elevates GABA in non-nerve terminal components (neural perikarya and glial cells) which are not destroyed by our lesions.

Presynaptic GABA autoreceptors on gabaergic nerve endings of the rat substantia nigra

The release of 3H-GABA evoked by exposure to 30 mM potassium during 1 min was found to be calcium independent in the rat occipital cortex and calcium dependent in the substantia nigra. Exposure to either muscimol 1 μM or GABA 1 μM inhibited the potassium-evoked release release of 3H-GABA from the substantia nigra but not from the occipital cortex. The inhibitory effect of muscimol 1 μM on the pitassium-evoked release of 3H-GABA from the substantia nigra was significantly antagonized by picrotoxin 10 μM. Exposure to either 10 or 100 μM pricotoxin alone did not affect the potassium-evoked release of 3H-GABA. These results are compatible with the presence of a negative feedback mechanism in gabaergic nerve terminals of the rat substantia nigra which is mediated by presynaptic GABA autoreceptors. In addition, these results emphasize the importance of the calcium-dependent dependent nature of the release of the neurotransmitter for demonstrating the modulation of transmitter release through presynaptic receptors.

Inhibitory, GABAergic Nerve Terminals Decrease at Sites of Focal Epilepsy

Using an immunocytochemical method for the localization of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamic acid decarboxylase (GAD), we have observed GABAergic nerve terminals distributed throughout all layers of normal monkey sensorimotor cortex. These terminals displayed ultrastructural characteristics that suggested that they arose from aspinous and sparsely spinous stellate neurons. In monkeys (Macaca mulatta and M. fascicularis) made epileptic by cortical application of alumina gel, a highly significant numerical decrease of GAD-positive nerve terminals occurred at sites of seizure foci indicating a functional loss of GABAergic inhibitory synapses. A loss of such inhibition at seizure foci could lead to epileptic activity of cortical pyramidal neurons.

Modulation of γ-aminobutyric acid transport in nerve endings: Role of extracellular γ-aminobutyric acid and of cationic fluxes

The aim of the present study was to elucidate the possible functional significance of gamma-aminobutyric acid (GABA) homoexchange at nerve endings. Using synaptosomes from adult rat cerebrum, we found that a number of conditions altering cationic fluxes produced a concomitant change in the stoichiometry of GABA homoexchange, In fact, exogenous GABA (10 muM), while not causing net release of intrasynaptosomal GABA in standard conditions, triggered a large net GABA release in the presence of veratridine, Na(+)-K(+)-ATPase inhibitors, or the ionophore A23187, superimposed on that due to the various agents tested alone. This extra release was mediated by the membrane carrier, being largely inhibited by the GABA carrier-blocker L-diaminobutyric acid. The altered stoichiometry of GABA homoexchange observed under these conditions (efflux > influx) appeared to be coupled to the influx of Na(+) (or of Ca(2+)), rather than determined by the establishment of a high intrasynaptosomal [Na(+)]. Under conditions of reversed Na(+) flux (Na(+) efflux), the GABA outward/inward flux ratio was also reversed, and the stoichiometry of GABA homoexchange was in favor of net influx. The possible contribution of K(+) to the effects observed is also discussed. It is concluded that the GABA transport system of nerve endings is susceptible to fine modulation by changes in cationic fluxes similar to those occurring in vivo during depolarization and repolarization. These fluxes may have a prominent role in determining the direction of net GABA transport in GABA-ergic nerve terminals of the living brain.

Neurochemical sequelae of kainate injections in corpus striatum and substantia nigra of the rat

Unilateral injection of 2 μg kainic acid into the substantia nigra of the rat results in a 45% decrease in tyrosine hydroxylase activity in the injected substantia nigra and in the ipsilateral corpus striatum. In contrast, the GABAergic nerve terminals in the substantia nigra are unaffected by this treatment. Injection of kainic acid into the striatum results in a 60% decrement in the activity of glutamate decarboxylase and of endogenous GABA levels in the ipsilateral substantia nigra whereas tyrosine hydroxylase activity remains unchanged; in addition, dopamine-sensitive adenylate cyclase activity in the ipsilateral substantia nigra decreases by 74%. These findings further support the hypothesis that intracerebral injections of kainic acid cause degeneration of neurons with cell bodies near the injection site while sparing axons passing through or terminating in the region.