Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

Synaptic Chemistry Associated with Aberrant Neuronal Development in the Reeler Mouse

Abstract: Pre- and postsynaptic neurochemical markers for several afferent and intrinsic neuronal systems were measured in the mouse mutant, reeler. In the neocortex of the reeler, the relative positions of the polymorphic and pyramidal cells were inverted but this was not associated with alterations in the content/mg protein of synaptic markers for noradrenergic [tyrosine hydroxylase (TH), norepinephrine (NE), NE uptake], cholinergic [choline acetyltransferase (ChAT), quinuclidinyl benzilate (QNB) binding], γ-aminobutyric acid (GABA)ergic (glutamate decarboxylase, GABA uptake, GABA receptors, GABA) or glutamatergic (glutamate uptake, receptors, glutamate) neurons. The laminar distributions of the hippocampal neurons were disrupted and associated with mild hypoplasia; consistent with this alteration, the content/mg protein of some GABAergic (GABA uptake) and glutamatergic (glutamate receptors) markers were slightly increased. The reeler cerebellum was characterized not only by misalignment of neurons but also by a marked loss of granule cells. Commensurate with the degree of cerebellar hypoplasia, the total amount of glutamate content, [³H]l-glutamate uptake activity, [³H]muscimol, and [³H]QNB ligand binding were reduced in the reeler cerebellum. In contrast, presynaptic markers for the noradrenergic (TH, NE) climbing fibers and the cholinergic (ChAT) mossy fibers were significantly increased/mg protein but their total content/cerebellum was near normal. Our data support suggestions that cerebellar granule cells use glutamate as their neurotransmitter and contain GABA and cholinergic receptors. The findings also suggest that misplaced cortical and cerebellar neurons retain normal neurochemical characteristics and that the morphologic alterations do not markedly affect the quantitative development of aminergic afferent systems.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.     

Collapse of extracellular glutamate regulation during epileptogenesis: down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid

We used northern and western blotting to measure the quantity of glutamate and GABA transporters mRNA and their proteins within the hippocampal tissue of rats with epileptogenesis. Chronic seizures were induced by amygdalar injection of kainic acid 60 days before death. We found that expression of the mRNA and protein of the glial glutamate transporters GLAST and GLT-1 were down-regulated in the kainic acid-administered group. In contrast, EAAC-1 and GAT-3 mRNA and their proteins were increased, while GAT-1 mRNA and protein were not changed. We performed in vivo microdialysis in the freely moving state. During the interictal state, the extracellular glutamate concentration was increased, whereas the GABA level was decreased in the kainic acid group. Following potassium-induced depolarization, glutamate overflow was higher and the recovery time to the basal release was prolonged in the kainic acid group relative to controls. Our data suggest that epileptogenesis in rats with kainic acid-induced chronic seizures is associated with the collapse of extracellular glutamate regulation caused by both molecular down-regulation and functional failure of glutamate transport.     

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

2-Amino-4-Phosphonobutyric Acid Exerts a Light-Dependent Effect on Post-Gabaculine Levels of Retinal γ-Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission

Abstract: The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal γ-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.     

Effects of x-irradiation on the levels of glutamate,aspartate and GABA in different regions of the cerebellum of the rat

The effect of the x-irradiation-induced loss of cerebellar granule and stellate cells on the levels of glutamate, aspartate and GABA in regions of the rat cerebellum was determined. The level of glutamate was significantly lower in the neuron-depleted cerebellar cortex while GABA levels were higher than control values in the cerebellar cortex and white matter of the x-irradiated rats. Aspartate levels were not changed by x-irradiation in any cerebellar region. The data is discussed in terms of the proposed role of glutamate as the excitatory neurotransmitter released from granule cells.     

Cortical modulation of cholinergic neurons in the striatum

Previous reports suggest the existence of a cortico-striatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (1 hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striatal cholinergic neurons can be influenced by this cortico-striatal pathway.     

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%.     

High-affinity transport of choline and amino acid neurotransmitters in synaptosomes from brain regions after lesioning the nucleus basalis magnocellularis of young and aged rats

Bilateral lesion of the nucleus basalis with ibotenic acid infusions in young and aged rats results in the degeneration of cholinergic neurons which innervate the cortex. As expected, high-affinity uptake of choline was decreased in the frontal cortex subsequent to the lesion. Twenty one days after surgery there was a significantly decrease of the transport rate of GABA, glutamate and glycine in the frontal cortex of young rats, but those activities showed a recovery six months after lesion. On the contrary, 12-month old rats lesioned with the same experimental protocol showed no recovery of the transport rates in the frontal cortex. Uptake of choline, GABA, glutamate and glycine has also been studied in other areas of the brain, namely, hippocampus, olfactory bulb and cerebellum. The present results suggest that lesioning the nucleus basalis of rats led to a more effective and permanent impairment of some biochemical functions of the brain, when compared to young lesioned animals, and also suggest a functional relationship between the nucleus basalis and other areas of the brain.     

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing -γ-aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K⁺-stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium-dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (K_m) was increased and maximal velocity (V_max) was reduced. Sodium-independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.     

Pentylenetetrazole decreases metabolic glutamate turnover in rat brain

Seizures were induced in rats by intraperitoneal injection of pentylenetetrazole (PTZ, 70 mg/kg), followed, 30 min later, by injection of [1-13C]glucose and [1,2-13C]acetate. Analyses of extracts from cortex, subcortex and cerebellum were performed using 13C magnetic resonance spectroscopy and HPLC. It could be shown that PTZ affected different brain regions differently. The total amounts of glutamate, glutamine, GABA, aspartate and taurine were decreased in the cerebellum and unchanged in the other brain regions. GABAergic neurones in the cortex and subcortex were not affected, whereas those in the cerebellum showed a pronounced decrease of GABA synthesis. However, glutamatergic neurones in all brain regions showed a decrease in glutamate labelling and in addition a decreased turnover in cerebellum. It could be shown that this decrease was in the metabolic pool of glutamate whereas release of glutamate was unaffected since glutamine labelling from glutamate was unchanged. Aspartate turnover was also decreased in all brain regions. Changes in astrocyte metabolism were not detected, indicating that PTZ had no effect on astrocyte metabolism in the early postictal stage.     

Effects of 3-acetylpyridine on the levels of several amino acids in different CNS regions of the rat

The effects of one intraperitoneal injection of 60–65 mg/kg of 3-acetylpyridine (3-AP) on the levels of aspartate, glutamate, GABA, taurine, glycine, and alanine in the cerebellum, medulla, telencephalon, and diencephalon-mesencephalon of the rat were studied at various times (4–28 days) after injection. In the first 4–7 days, the levels of glutamate, GABA, glycine, and alanine in the cerebellum were 10–30% higher in the 3-AP-treated rats than in the control animals. By day 14, the levels of these four amino acids were normal (in the case of glutamate and glycine) or below normal (for GABA and alanine). By day 21, the values for GABA and alanine returned to normal. In the first 7 days, the level of aspartate in the cerebellum was the same in both the 3-AP- and saline-injected groups. From days 14 to 28, the level of aspartate in the cerebellum was 10–20% lower in the 3-AP-injected group than in the saline-treated animals. The level of taurine in the cerebellum was 15–30% lower in the 3-AP group than in the control group from days 7 to 28. The pattern of changes observed in the medulla in the first 7 days was similar to that found in the cerebellum for this period. However, unlike the data for the cerebellum, the level of aspartate in the medulla was unchanged by the 3-AP injection from day 14 to day 28, and the level of glutamate in the medulla remained higher (10–15%) from days 14 to 28 in the 3-AP-injected animals with respect to control values. The levels of taurine in the medulla were lower (10–15%) from day 7 to day 28 in the 3-AP injected group with respect to control values. The injection of 3-AP did not alter the levels of aspartate, glutamate, GABA, taurine, glycine, or alanine in the telencephalon on days 7, 14, 21, or 28 and in the diencephalon-mesencephalon on day 21 with respect to control levels.     

Effects of selective doses of x-irradiation on the levels of several amino acids in the cerebellum of the rat

The cerebella of rats were exposed to selective doses of low levels of x-irradiation beginning on day 4, 8, or 12 following birth. The doses of x-irradiation given on days 12, 13, and 15 (12–15X group) resulted in a 24% reduction in the wet weight of the cerebella; the doses given on days 8, 9, 11, 13, and 15 (8–15X group) resulted in a 57% weight reduction; the doses given on days 4, 5, 7, 9, 11, 13, and 15 (4–15X group) resulted in a 67% weight reduction. The schedule of x-irradiation begun on day 12, which prevented the acquisition of the late-forming granule cells, reduced the levels (nmole/mg dry tissue weight) of alanine (22%) and glutamate (10%), and increased the levels of glycine (15%), GABA (13%), and taurine (71%), with respect to control values. The schedule begun on day 8, which prevented the acquisition of stellate and granule cells, reduced the levels of alanine (15%), glutamate (12%), and taurine (21%), and increased the levels of glycine (102%) and GABA (56%). The schedule begun on day 4, which prevented the acquisition of basket, stellate, and granule cells, reduced the level of glutamate (15%) and increased the levels of glycine (186%) and GABA (78%). The levels of alanine and taurine in the cerebella of the 4–15X group were the same as control values. The level of aspartate in the cerebella of the 3 groups of x-irradiated animals was not significantly different from control values. The consistent reduction in the level of glutamate as a function of the number of doses of x-irradiation is suggestive that glutamate may have a higher level in the granule cells than in other cells in the cerebellum, and that the higher level may be a reflection of a possible excitatory transmitter role for glutamate. In addition, the data are interpreted in terms of taurine being associated with the stellate cells and possibly serving as a transmitter for these inhibitory interneurons.     

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.     

Alterations of Amino Acid Transmitter Systems in Spinal Cords of Chronic Paraplegic Dogs

The high-affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid [( 3H]GABA) and the Na+-independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosacral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity uptake and for preparing synaptic plasma membranes for Na+-independent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie-Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high-affinity uptakes of 1 microM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60-70% higher for the spastic group than for the control animals. For both amino acids, Eadie-Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+-independent binding of 5 nM [3H]glutamate was 40-85% higher and the binding of 10 nM [3H]GABA was 40-60% lower for the paraplegic groups relative to the values for the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depolarization elicits,while hyperpolarization blocks uptake of endogenous glutamate by retinal horizontal cells of the turtle

We have employed an immunoreaction against glutamate to qualitatively demonstrate varying levels of glutamate in retinal horizontal cells of the turtle. Glutamate-like immunoreactivity (GLI) in horizontal cells could be demonstrated after glutamate decarboxylase was inhibited by aminooxy acetic acid (AOAA) and its degradation to GABA was blocked. Depolarization of horizontal cells by kainic acid (KA) induces strong glutamate immunoreactivity in these cells, whereas hyperpolarization by 2,3-cis piperidine dicarboxylate (PDA) abolishes glutamate-like immunoreactivity in horizontal cells. When glutamate release from cones and bipolar cells is blocked in the absence of calcium, or when glutamate uptake is blocked by DL-threo -hydroxy aspartate, KA/AOAA treatment of the retina does not induce GLI in horizontal cells. Our data show that horizontal cells are capable of taking up glutamate from the endogenous retinal pool in an activity dependent way. Our interpretation of these findings is that retinal horizontal cells are capable of regulating glutamate levels in the extracellular space of the cone pedicle complex by an activity-dependent uptake system. We suggest that inhibition of glutamate uptake upon hyperpolarization rather than inhibition of GABA release may evoke the antagonistic surround response of retinal bipolar cells.