Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Effect ofd- andl-α-aminoadipate on the efflux ofl-aspartate,l-glutamate and γ-aminobutyrate from superfused rat brain slices

d-α-Aminoadipate (d-AA) andl-α-aminoadipate (l-AA) were found to significantly reduce spontaneous efflux of [¹⁴C]l-aspartate from preloaded rat brain slices. Onlyd-AA significantly reduced spontaneous efflux of [¹⁴C]l-glutamate and [³H]γ-aminobutyric acid (GABA);l-AA reduced but not significantly the efflux of these 2 labeled amino acids.d-AA reduced K⁺-stimulated release of [¹⁴C]l-aspartate and [^14]C]l-glutamate significantly, andl-AA that of [³H]GABA significantly. Since bothd-AA andl-AA inhibit the uptake ofl-aspartate,l-glutamate and GABA, their effects on the efflux of these amino acids are more specific. These results also suggest that it is unlikely that the depressant effect ofd-AA, and the excitant effect ofl-AA on neurons when applied locally by iontophoresis are secondary to the accelerated or decelerated release of more specific transmitter amino acids from neighboring cells.     

In vivo release of endogenously synthesized [H]GABA from the cat substantia nigra and the pallido-entopeduncular nuclei

Halothane anesthetized cats were implanted with push-pull cannulae to study the release of [³H]GABA continuously formed from [³H]glutamine in the substantia nigra (SN) and in the pallido-entopeduncular nuclei (PEP). A spontaneous release of [³H]GABA was observed from both structures and it reached a steady state level 1 h after the beginning of the superfusion with [³H]glutamine. In cats implanted with two push-pull cannulae, the local application of potassium (47 mM) in the PEP stimulated the release of [³H]GABA from the ipsilateral SN.In cats implanted with 4 push-pull cannulae, the unilateral 10 min electrical stimulation of the caudate nucleus evoked the release of [³H]GABA not only from the ipsilateral SN, but also in most cases from the contralateral structure. This stimulus also enhanced the release of [³H]GABA from PEP but the effects were mainly observed in the medio-caudal part of the ipsilateral PEP and in the latero-rostral part of the contralateral structure. In all cases, the changes in [³H]GABA release were observed during and after the electrical stimulation. The ipsilateral effects can be attributed to the direct activation of the caudato-PEP or caudato-SN GABAergic neurons. A polysynaptic neuronal loop must be involved in the symmetric contralateral effects.     

Storage and release of endogenous and labelled GABA formed from [H]Glutamine and [C]Glucose in hippocampal slices: Effect of depolarization

To study the effect of depolarization on the synthesis, storage and release of GABA, hippocampal slices were incubated in 0.25 mM [³H]glutamine and 2.5 mM [¹⁴C]glucose in the presence of 3 or 50 mM K⁺. Total and labelled glutamine, glutamate and GABA contents were measured by high-performance liquid chromatography. Depolarization in the presence of Ca²⁺ led to a two-fold increase of labelled glutamate and a 3-fold increase of labelled GABA content originating from both labelled precursors. In the absence of Ca²⁺ and in the presence of 10 mM Mg²⁺, depolarization failed to increase labelled glutamate content and labelled GABA formation was increased by only 30%. Following superfusion with unlabelled 0.25 mM glutamine and 2.5 mM glucose a second depolarization with 50 mM K⁺ released twice as much labelled GABA from slices that had been incubated in the presence of 50 mM K⁺, than from those incubated in 3 mM K⁺. This difference remained unchanged in slices that were superfused with 1 mM aminooxyacetic acid, an inhibitor of GABA synthesis. The contribution of labelled GABA, especially of GABA derived from [³H]glutamine, to released GABA was significantly higher than to GABA stored in the slices. Results suggest that depolarization in the presence of Ca²⁺ results in increased glutamate and GABA synthesis from both glutamine and glucose and that part of GABA released by high K⁺ originates from preformed GABA stores.     

The entry of [1-C]glucose into biochemical pathways reveals a complex compartmentation and metabolite trafficking between glia and neurons: a study by C-NMR spectroscopy

Glial–neuronal interactions were investigated in rats injected intraperitoneally with [1-¹³C]glucose and killed after 15, 30, 45, or 60 min. Brain extracts were analyzed by ¹³C-NMR spectroscopy and the fractional ¹³C-enrichment at individual carbon positions was measured for amino acids, lactate, and N-acetyl-aspartate. [1-¹³C]Glucose was shown to be metabolized by both neurons and glia, with the anaplerotic pathway through pyruvate carboxylase (PC) accounting for 10% of total cerebral glucose metabolism. The PC-mediated pathway accounted for 39% of the glutamine synthesis, and for 8, 6, 14% of glutamate, GABA, and aspartate synthesis, respectively. These results reflect a compartmentation of the cerebral amino acids synthesis within glial and neuronal cells. The appearance of the ¹³C-label in C5 of glutamate and glutamine, C1 of GABA and C2 of lactate, is suggestive of pyruvate, formation from TCA cycle intermediates and provides evidence of metabolite trafficking between astrocytes and neurons.     

Sites ofd-[H]aspartate accumulation in mouse cerebellar slices

Slices of mouse cerebellar vermis, cut in the parasagittal plane, were incubated for various times (up to 3 h) in the presence of 1μMd-[³H]aspartate, a non-metabolized substrate for the glutamate/aspartate carrier in brain tissue. Light microscopic autoradiography indicated that in regions away from the cut edges of the slices the amino acid accumulated in glia and granule cells. Relatively few grains were seen over Purkinje, Golgi, stellate and basket cells or over white matter. Grain counts over the granule cell layers in the middle parts of the sliced indicated that after short (15 min) exposures to the labelled substrate, non-granule cell areas (which included glia) contained, on average, slightly more grains than granule cells but wit longer exposures (1.5 and 3 h) the relative grain density over granule cells became much higher, possibly because glial uptake preventsd-[³H]aspartate gaining access to neuronal sites in adequate amounts during short incubations and/or because the longer incubations allow time for retrograde migration of the label from parallel fibre terminals to occur. The demonstration of selective uptake ofd-[³H]aspartate into granule cells contrasts with previous autoradiographic results (possible reasons for which are discussed) and supports the notion thatl-glutamate is the transmitter of granule cells. The results also have a bearing on the importance of the metabolic compartmentation of glutamate in relation to its proposed transmitter role.     

In vivo release of [H]GABA in cat caudate nucleus and substantia nigra. I. Bilateral changes induced by a unilateral nigral application of muscimol

Push-pull cannulae were implanted in both caudate nuclei and both substantiae nigrae (SN) of halothane-anesthetized cats and the release of [³H]GABA, continuously synthesized from [³H]glutamine, was measured in these structures during 4 h of superfusion. In some experiments, multi-unit neuronal activity was recorded at the tip of the nigral push-pull cannulae, using a bipolar electrode. Two hours after the onset of superfusion with [³H]glutamine, 10⁻⁶M of muscimol was added (for 1 h) in the superfusion medium delivered to one SN. This treatment increased locally the release of [³H]GABA and enhanced the neuronal activity of the nigral cells in the zona reticulata. An increased release of [³H]GABA was also observed in the contralateral SN, in association with an inhibition of the activity of the zona reticulata cells. The unilateral nigral application of muscimol also induced asymmetric changes in the release of [³H]GABA in both caudate nuclei, since [³H]GABA release was increased ipsilaterally and reduced on the contralateral side. The present findings are considered in relation to possible GABAergic neuronal populations affected by this local pharmacological treatment.     

Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo C NMR spectroscopic study of rats after administering [2-C] glucose with or without ammonium acetate

The ¹³C-label incorporation into glutamate, glutamine, aspartate and γ-aminobutyric acid (GABA) from [2-¹³C] glucose was measured by ¹³C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [2-¹³C] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of ¹³C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The ¹³C entered the TCA cycle via pyruvate carboxylase from [2-¹³C] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate>GABA>glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the ¹³C-skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.     

Sodium ion movements and the spontaneous and electrically stimulated release of [H]GABA and [C]glutamic acid from rat cortical slices

The effect on spontaneous and electrically stimulated release of [³H]GABA and [¹⁴C]glutamic acid from rat cortical slices of agents and ionic conditions that affect sodium ion movements, was studied in anin vitro superfusion system. Those conditions that elevated non-inulin Na⁺ content of the slice accelerated spontaneous efflux and inhibited electrically stimulated release of the labeled amino acids. Agents which blocked the entry of Na⁺ with electrical field stimulation failed to diminish release of the amino acids. Therefore, the entry of Na⁺ into the slice with membrane depolarization is not a requirement for amino acid neurotransmitter release from cortical slices in response to electrical field stimulation. The acceleration of spontaneous efflux and inhibition of electrically stimulated release in the presence of an elevated non-inulin Na⁺ content was discussed in relation to sodium coupled transport of amino acids.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

The distribution and function of DL-[H]2-Amino-4-Phosphonobutyrate binding sites in the rat striatum

The binding of the glutate-like radioligand,DL-[³H]2-amino-4-phosphhonobutyrate (DL-[³H]APB), to L-glutamate-sensitive sites in the rat striatum was investigated. A single, saturable population of binding sites, indistinguishable from that characterized previously on rat whole brain synaptic membranes, was identified. The effects of specific lesions of the striatum: (a) decortication; (b) striatal injection of kainic acid; and (c) 6-hydroxydopamine injections into the substantia nigra, were also examined. SpecificDL-[³H]APB binding in the striatum was elevated significantly following decortication. An increase in the number of binding sites was found to be responsible for this enhancement in binding. Lesions of the postsynaptic tartets of corticostriatal fibres reduced the number of DL-[³H]APB binding sites in the striatum without affecting binding site affinity. This finding suggests thatL-APB sensitive excitatory amino acid receptors are located predominantly on membranes derived from structures postsynaptic with regard to the glutamatergic innervation. The possible physiological role of these receptors was examined using an in vitro release technique. BothL-glutamate and L-APB were found to facilitate potassium evoked [³H]dopamine release from striatal slices. This finding supports the proposed existence of functional acidic amino acid receptors on dopaminergic terminals in the striatum. These receptors may play an important role in the control of motor function.     

Regulation of NMDA-stimulated [14C]GABA and [3H]acetylcholine release by striatal glutamate and dopamine receptors.

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [14C]GABA and [3H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [14C]GABA release was more sensitive to NMDA and glycine antagonists than was [3H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [3H]ACh release was inhibited by a D2 receptor mechanism whereas NMDA-stimulated [14C]GABA release was enhanced by D1 receptor activation. NMDA and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [3H]ACh release, and synergistically to evoke [14C]GABA release. An additive effect of NMDA and kainate (KA) was found on [14C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [3H]ACh release. KA-stimulated [3H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [14C]GABA and [3H]ACh release. On the other hand, NMDA-stimulated [14C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

GABA and benzodiazepine-induced modification of [C]l-glutamic acid release from rat spinal cord slices

The spontaneous and potassium-evoked release of [¹⁴C]-label from rat spinal cord slices preloaded with [¹⁴C]l-glutamic acid and its modification by GABA and related drugs, such as flurazepam, was studied as a possible indirect measure of presynaptic inhibition and of the ability of benzodiazepines to augment it. GABA (100 μM) reduced the spontaneous release of [¹⁴C]-label (glutamate) provided that GABA metabolism was blocked by amino-oxyacetic acid (AOAA), but failed to reduce the potassium-evoked release of glutamate, although muscimol (10 μM) had some effect. In contrast, flurazepam (1–100 μM) did not affect spontaneous release but produced some inhibition of the evoked release (through a system insensitive to 10 μM bicuculline). This inhibition became more marked in the presence of both GABA and AOAA, and was then overcome by bicuculline. It is concluded that either some benzodiazeophine receptors must be occupied for GABA to produce an effect on evoked release and/or, that the benzodiazepines can only augment GABA function once a certain amount has been released. Studies of the rapid distribution of [¹⁴C]-label from glutamate, to GABA, glutamine and other amino acids, using high voltage electrophoresis, showed the importance of blocking metabolic pathways in studies of this kind.     

Excitatory/inhibitory neuronal metabolic balance in mouse hippocampus upon infusion of [U-13C6]glucose

Hippocampus plays a critical role in linking brain energetics and behavior typically associated to stress exposure. In this study, we aimed to simultaneously assess excitatory and inhibitory neuronal metabolism in mouse hippocampus in vivo by applying ¹⁸FDG-PET and indirect ¹³C magnetic resonance spectroscopy (¹H-[¹³C]-MRS) at 14.1 T upon infusion of uniformly ¹³C-labeled glucose ([U-¹³C₆]Glc). Improving the spectral fitting by taking into account variable decoupling efficiencies of [U-¹³C₆]Glc and refining the compartmentalized model by including two γ-aminobutyric acid (GABA) pools permit us to evaluate the relative contributions of glutamatergic and GABAergic metabolism to total hippocampal neuroenergetics. We report that GABAergic activity accounts for ∼13% of total neurotransmission (V_NT) and ∼27% of total neuronal TCA cycle (V_TCA) in mouse hippocampus suggesting a higher V_TCA/V_NT ratio for inhibitory neurons compared to excitatory neurons. Finally, our results provide new strategies and tools for bringing forward the developments and applications of ¹³C-MRS in specific brain regions of small animals.     

Stimulus-dependent labeling of cultured ganglionic cell with [C]2-deoxyglucose

We prepared cultures of dissociated cells from the ciliary (CG) and dorsal root ganglion (DRG) of 10–12-day-old chick embryos, and applied [¹⁴C]2-deoxyglucose ([¹⁴C]2-DG) to the cultured cells to examine the effects of stimulation on the labeling with [¹⁴C]2-DG at the single cell level. Electrical current stimulation increased [¹⁴C]2-DG uptake in CG and DRG neurons. The increase depended on frequency of the stimulation. These effects were potentiated by the application of tetraethylammonium, but suppressed by tetrodotoxin. Externally applied potassium ions increased the [¹⁴C]2-DG uptake in the CG cell, depending logarithmically on the concentration of applied KC1. The concentration-dependent increase agreed with potassium effect on the equilibrium potential. For CG cells, acetylcholine (ACh), glutamate, γ-aminobutyric acid (GABA) and glycine induced remarkable increases of the [¹⁴C]2-DG uptake, while dopamine did not induce any change. For DRG cells, GABA and glycine facilitated the [¹⁴C]2-DG uptake, while ACh, glutamate and dopamine did not have any significant effects on it. These facilitatory actions of neurotransmitters on the [¹⁴C]2-DG uptake are mostly consistent with the excitatory effects of the substrates on both CG and DRG cells in culture. The results suggest that the [¹⁴C]2-DG uptake in single cells is intimately correlated with action potential generation and change in the resting potential.     

Bilateral asymmetrical changes in the nigral release of [H]GABA induced by unilateral application of acetylcholine in the cat caudate nucleus

In halothane anaesthetized cats, a push-pull cannula was implanted into the right caudate nucleus (CN) and in each substantia nigra (SN). The release of [³H]GABA continuously formed from [³H]glutamine was estimated in each structure. Acetylcholine (ACh, 5 × 10⁻⁵M) added in presence of eserine (5 × 10⁻⁵M) for 50 min in the right caudate nucleus 2 h after the onset of superfusion with [³H]glutamine, stimulated the [³H]GABA release locally. The effect was biphasic when ACh application was made in the median two-thirds of the structure and it was monophasic and transient when the ACh application was restricted to the lateral part. ACH application in the right caudate nucleus also induced changes in [³H]GABA release in the anterior (pars reticulata) and posterior (pars compacta) parts of both SN. While [³H]GABA release was enhanced in the ipsilateral anterior SN, it was reduced in the contralateral anterior SN. Respective opposite effects were observed in the posterior parts of the ipsi- and contralateral SN. These bilateral asymmetrical changes in [³H]GABA release were not dependent on the site of ACh application in the right caudate nucleus. These results indicate that the facilitation of cholinergic transmission in one caudate nucleus influences in an opposite way the striato-nigral GABA neurones on both sides of the brain.     

Age-related changes of sodium-dependentd-[3H]aspartate and [3H]FK506 binding in rat brain

Summary We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependentd-[³H]aspartate and [³H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependentd-[³H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [³H]FK506 binding was significantly lower only in the hippocampus, thalamus and cerebellum. [³H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependentd-[³H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependentd-[³H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10–25% reduction in sodium-dependentd-[³H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Neuronal uptake of [H]gaba and [H]glycine in laminae I–III (substantia gelatinosa rolandi) of the rat spinal cord. An autoradiographic study

[³H]GABA or [³H]glycine were injected into the subarachnoidal space of adult rats at C₄–C₅ level. After 10–60 min, the animals were perfused with 4% paraformaldehydee-0.5% glutaraldehyde and thick sections of the cervical spinal cord were postosmicated and Epon embedded. Light microscope autoradiographs of transverse cord sections showed numerous silver grains over the dorsal column and laminae I–III, higher grain densities occurring over lamina I for GABA and laminae III for glycine. In [³H]GABA-injected animals nerve cell bodies in lamina I or at the transition to lamina II appeared strongly labeled in light and electron microscope autoradiographs. These cells were smaller and less rich in RER than marginal cells and poor in axosomatic synaptic contacts. High grain densities appeared over axon terminals synapsing with dendrites in laminae I–II and over the light peripheral axon endings of synaptic glomeruli of laminae II–III. After [³H]glycine treatment, a number of nerve cell bodies were labeled in lamina III.It is suggested that two types of inhibitory interneurons occur in the rat gelatinosa, one GABAergic with cell body in lamina I, and another glycinergic in lamina III.     

Depolarization-induced release of [H] -aspartate from GABAergic neurons caused by reversal of glutamate transporters

Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to γ-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [³H] -aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [³H] -aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [³H] -aspartate was decreased by about 40%. These results indicated that some of the released [³H] -aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [³H] -aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [³H] -aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [³H] -aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [³H] -aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [³H] -aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [³H] -aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.     

In vivo release of newly synthesized [H]GABA in the substantia nigra of the rat: relative contribution of GABA striato-pallido-nigral afferents and nigral GABA neurons

The release of [3H]gamma-aminobutyric acid ([3H]GABA) continuously formed from [3H]glutamine has been measured with a push-pull cannula implanted in the substantia nigra of the rat anesthetized with ketamine. Consistent with the high density of GABA terminals coming from both the striato-pallido-nigral afferents, and from GABA nigrofugal neurons, our results showed that a large amount of [3H]GABA was spontaneously released in the reticulata, about 4 times higher than in the compacta. In the absence of calcium the spontaneous [3H]GABA release was reduced (-30%), as well as the K(+)-induced release of [3H]GABA (-66%). Bicuculline (10(-4) M) did not affect the K(+)-evoked release of [3H]GABA, suggesting that autoreceptors on GABA afferent fibers are distinct from the GABAA subtype. Partial lesions of striato- and pallido-nigral GABA neurons with kainic acid (1.2 micrograms) decrease by 40% the glutamic acid decarboxylase (GAD) activity in the ipsilateral SN without decreasing the spontaneous release of [3H]GABA; even following extensive lesions with kainic acid (2.5 micrograms), GAD activity (-72%) and spontaneous [3H]GABA release (-83%) were not completely abolished. These results suggest that a non-negligible contribution of GABA nigral neurons accounts for the spontaneous GABA release measured in the substantia nigra. This is further supported by the decrease (-20%), and the increase (+40%) of [3H]GABA release produced by the local application of glycine (10(-6) M), and bicuculline (10(-4) M), which respectively, inhibits and activates the nigral neuron activity. The contribution of nigral GABA neurons to the amount of [3H]GABA release from the substantia nigra, is likely linked to their high spontaneous firing rate.