GABAB Receptor-Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Abstract: Previously, we have shown a significant increase in number of GABA_B receptor binding sites in neocortex and thalamus of lethargic ( lh/lh ) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABA_B receptors would inhibit [³H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [³H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [³H]GABA release evoked by 12 m M KCl, an effect mediated by GABA_B receptors. The maximal inhibition ( I _max) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC₅₀ (3 µ M ) was unchanged. In the thalamic preparation, the effect of baclofen (50 µ M ) was 58% less robust in lh/lh mice. Other effects mediated by GABA_B receptors (inhibitions in Ca²⁺ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABA_B receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABA_B receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.     

Repeated Administration of γ-VinylGABA Reduces Rat Brain Glutamine Synthetase Activity

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ-vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ-vinylGABA administration. Amino acid analysis of the cortex from γ-vinylGABA-treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ-vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.     

Uptake and Release of d-Aspartate, GABA, and Glycine in Guinea Pig Brainstem Auditory Nuclei

Abstract: This study attempts to determine if the medial (MSO) and lateral superior olive (LSO), medial nucleus of the trapezoid body (MNTB), ventral nucleus of the lateral lemniscus (VNLL), and central nucleus of the inferior colliculus (ICc) contain glutamatergic synaptic endings. Micropunch and microdissection procedures provided fresh samples of these auditory nuclei for the measurement of the high-affinity uptake and electrically evoked release of exogenous d -[³H]ASP. The study also determined if the LSO and MSO contain glycinergic synaptic endings by measuring uptake and release of [¹⁴C]-Gly in these nuclei, and whether the MNTB, VNLL, and ICc contain GABAergic endings by assessing the uptake and release of [¹⁴C]GABA in these structures. Several strategies optimized the evoked Ca²⁺-dependent release of the labeled amino acids. These included the enhancement of high-affinity uptake during loading of the markers into the tissues, inhibition of uptake during the subsequent measurement of release, and use of an electrical stimulus current that evoked maximal Ca²⁺-dependent release. Each of these nuclei manifested the high-affinity uptake and the evoked Ca²⁺-dependent release of d -[³H]Asp, suggesting the presence of synaptic endings that may use Glu or Asp as a transmitter. Similar findings suggest the presence of glycinergic synaptic endings in the LSO and MSO, and of GABAergic synaptic endings in the MNTB, VNLL, and ICc.     

Evidence for Glutamatergic Projections from the Cochlear Nucleus to the Superior Olive and the Ventral Nucleus of the Lateral Lemniscus

Abstract: This study attempts to determine if projections ascending from the guinea pig cochlear nucleus (CN) could be glutamatergic and/or aspartatergic. Multiple radio frequency lesions were made to ablate the right CN. The ablation was verified histologically. To identify the principal targets of CN efferents, silver impregnation methods were used to localize the preterminal degeneration of fibers in transverse sections of the brainstem 5 and 7 days after CN ablation. CN efferents projected heavily to the lateral superior olive (LSO) ipsilaterally, the medial superior olive (MSO) bilaterally, and contralaterally to the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body, the ventral (VNLL) and intermediate nuclei of the lateral lemniscus and the central nucleus of the inferior colliculus (ICc). There were smaller projections to the lateral nucleus of the trapezoid body ipsilaterally, the dorsal and dorsomedial periolivary nuclei bilaterally, and the dorsal nucleus of the lateral lemniscus contralaterally. There were sparse projections to the VNLL and ICc ipsilaterally and the CN contralaterally, and a very sparse projection to the contralateral LSO. To determine if CN efferents were glutamatergic and/or aspartatergic, the fresh brainstem was sectioned transversely and samples of the LSO, MSO, MNTB, VNLL, and ICc were taken to measure the electrically evoked release and the uptake of d -[³H]Asp and [¹⁴C]Gly or [¹⁴C]GABA 3–5 days after the CN ablation. The release studies suggest that only certain of the histologically identified projections ascending from the CN may be glutamatergic and/or aspartatergic. CN ablation depressed d -[³H]Asp release in the MSO bilaterally and in the contralateral MNTB and VNLL, suggesting that the CN efferents to these nuclei may use glutamate or aspartate as a transmitter. It was unclear whether a marginal depression of d -[³H]Asp release in the ipsilateral LSO reflected the presence of glutamatergic CN projections to this nucleus. d -[³H]Asp release in the ICc was unaffected, suggesting that CN efferents to this nucleus may not be glutamatergic. There were no deficits in d -[³H]Asp uptake. [¹⁴C]Gly release from the LSO and MSO was unchanged. [¹⁴C]Gly uptake was unchanged in the MSO and depressed only in the contralateral LSO, possibly reflecting subnormal uptake activity in endings contributed by contralateral MNTB cells that had lost their CN efferents. [¹⁴C]GABA uptake in the MNTB, VNLL, and ICc was unchanged. [¹⁴C]GABA release was unchanged in the VNLL and ICc. [¹⁴C]GABA release was depressed only in the contralateral MNTB, possibly reflecting the loss of a small complement of GABAergic CN efferents and the reaction of GABAergic projections from the contralateral VNTB to their loss of CN efferents.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Selective Inhibition of the Tricarboxylic Acid Cycle of GABAergic Neurons with 3-Nitropropionic Acid In Vivo

Abstract: The effects of 3-nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase, on cerebral metabolism were investigated in mice by NMR spectroscopy. 3-NPA, 180 mg/kg, caused a dramatic buildup of succinate. Succinate was labeled 5.5 times better from [1-¹³C]glucose than from [2-¹³C]acetate, showing a predominantly neuronal accumulation. [1-¹³C]Glucose labeled GABA in the C-2 position only, compatible with inhibition of the tricarboxylic acid (TCA) cycle associated with GABA formation, at the level of succinate dehydrogenase. Aspartate was not labeled by [1-¹³C]glucose in 3-NPA-intoxicated animals. In contrast, [1-¹³C]glucose labeled glutamate in the C-2, C-3, and C-4 positions showing uninhibited cycling of label in the TCA cycle associated with the large, neuronal pool of glutamate. The labeling of glutamine, and hence GABA, from [2-¹³C]acetate showed that the TCA cycle of glial cells was unaffected by 3-NPA and that transfer of glutamine from glia to neurons took place during 3-NPA intoxication. The high ¹³C enrichment of the C-2 position of glutamine from [1-¹³C]glucose showed that pyruvate carboxylation was active in glia during 3-NPA intoxication. These findings suggest that 3-NPA in the initial phase of intoxication fairly selectively inhibited the TCA cycle of GABAergic neurons; whereas the TCA cycle of glia remained uninhibited as did the TCA cycle associated with the large neuronal pool of glutamate, which includes glutamatergic neurons. This may help explain why the caudoputamen, which is especially rich in GABAergic neurons, selectively undergoes degeneration both in humans and animals intoxicated with 3-NPA. Further, the present results may be of relevance for the study of basal ganglia disorders such as Huntington's disease.     

Neurotoxic Effect of Intranigral Injection of 1-Methyl-4-Phenylpyridinium on GABA-Containing Neurons and Its Relation to Circling Behavior

Abstract: The ionic species 1-methyl-4-phenylpyridinium (MPP⁺) seems to be the metabolite responsible for the damage to dopaminergic neurons occurring after administration of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In the present study we show that the unilateral stereotaxic microinjection of MPP⁺ into the substantia nigra pars reticulata in rats produces immediately intense and long-lasting (up to 96 h) contralateral turning behavior in a dose-dependent manner. This behavioral effect was correlated with a dose- and time-dependent decrease (up to 90%) of glutamate decarboxylase activity and with a notable loss of neurons in the injected nigra reticulata. GABA levels in the injected nigra were also decreased, whereas the dopamine concentration in the ipsilateral striatum was not affected at 24 h, when maximal behavioral effects were observed. The circling behavior was prevented by the dopamine carrier blocker nomifensine only during the first 2 h, whereas the dopamine receptor antagonist haloperidol was ineffective. The results indicate that MPP⁺ is toxic for inhibitory GABAergic neurons in the nigra pars reticulata and, furthermore, suggest that disruption of the function of these GABAergic neurons may be involved in the abnormal motor behavior produced by the injection of MPP⁺ in the substantia nigra.     

The effect of kainate on protein kinase C,GABA, and the uptake of serotonin in the rabbit retina in vivo

The purpose of this study was to investigate the effect of kainate on protein kinase C (PKC), -aminobutyrate (GABA) and serotonin uptake in the rabbit retina. Kainate when injected into the vitreous humour produces a change in the GABA immunoreactivity within 6 hours. After 3 days, remnants of the normal GABA immunoreactivity still persist and additionally astrocyte and microglia-like elements stain positively for GABA. After 7 days exposure to kainate none of the normal GABA immunoreactivity is apparent, instead a number of round-shaped elements which may be reactive astrocytes and/or microglia stain positively for GABA. During these stages kainate does not affect the PKC immunoreactivity associated with the on-bipolar cells. Six hours following kainate treatment the ability of certain GABA amacrine cells to take up exogenous serotonin is unaffected. After three days only a few of these cells can still take up exogenous serotonin and then not avidly. After seven days the GABA/serotonin amacrine cells cannot take up exogenous serotonin suggesting that all of these neurons are irreversibly damaged. One hour after treatment with kainate both calcium-dependent and-independent PKC species are translocated from the cytosolic to membrane compartments. After 5 hours and 7 days there was also evidence from the enzyme assay experiments that kainate caused the calcium-dependent and-independent PKC enzymes to be translocated but because the total enzyme activity was reduced due perhaps to down-regulation of the enzyme this was difficult to assess precisely. However, the electrophoresis/blotting experiments of tissues exposed for 5 hours (but not one hour) to kainate established clearly that , , and PKC are translocated from cytosolic and membrane compartments.     

GABA concentrations in the striatum following repetitive cerebral ischemia

GABAergic neurons in the striatum are very sensitive to the effects of ischemia. The progressive decline in striatal GABA following transient forebrain ischemia in gerbils may be secondary to either a decreased production or an increase in reuptake mechanisms or both. The current experiment was designed to evaluate release of GABA by stimulation with K+ or inhibition of its uptake with nipecotic acid or their combination (K+ nipecotic) after repetitive forebrain ischemia in gerbils by in-vivo microdialysis on Days 1, 3, 5, and 14 following the insult. Infusion of nipecotic acid or potassium chloride, resulted in a significant increase in extracellular GABA. This response was significantly decreased in the post-ischemic animals. The synergistic effect of increased GABA concentrations by the infusion of nipecotic acid+potassium chloride seen in the controls was not evident in the post-ischemic animals. In conclusion, though there is a reduction in the extracellular GABA concentrations in the first week following an ischemic insult, restorative mechanisms are operative in the second week as seen by the increasing GABA concentrations.