Ricardo Tapia

Demonstration of central γ-aminobutyrate-containing nerve terminals by means of antibodies against glutamate decar☐ylase

Abstract A new procedure is described to obtain specific antibodies against mouse glutamate decar☐ylase using a purified glutamate decar☐ylase preparation obtained by affinity chromatography. Antibodies prepared against the Sepharose-bound glutamate decar☐ylase caused a concentration-dependent inhibition of glutamate decar☐ylase activity. Furthermore, the glutamate decar☐ylase antibodies gave a single precipitation band in double immunodiffusion, immunoelectrophoresis and in counterimmunoelectrophoresis analysis when reacted against crude extracts from mouse or rat brain. Immunohistochemical analysis in the rat brain demonstrated specific glutamate decar☐ylase-related immunofluorescence, presumably in γ-aminobutyrate-containing nerve terminals, but in no case in cell bodies in any part of the brain and the spinal cord. Two principal types of presumed γ-aminobutyrate-containing nerve terminals could be demonstrated. One type is strongly glutamate decar☐ylase immunoreactive, appears to have fairly large varicosities, may possibly make axosomatic and axodendritic contacts, and is confined mainly to the deep cerebellar nuclei, the nucleus vestibularis lateralis, the substantia nigra, the globus pallidus and the substantia innominata together with adjacent parts of the medial forebrain bundle and the dorsal part of the olfactory tubercle. In all these areas they are found in high densities. It is suggested, partly in agreement with previous findings that this type of γ-aminobutyrate-containing nerve terminal belongs to Golgi type I nerve cells (with long projections), contains high amounts of glutamate decar☐ylase enzyme protein and may mediate postsynaptic inhibition. The other type of γ-aminobutyrate-containing nerve terminal is weakly to moderately glutamate decar☐ylase immunoreactive, appears to have fine varicosities, may possibly make both axosomatic, axodendritic and axoaxonic contacts and is found all over the brain and the spinal cord in low to high densities. The distribution of this type correlates fairly well with the known distribution of glutamate decar☐ylase activity in the rat brain. It is suggested that this type of γ-aminobutyrate-containing nerve terminal mainly belongs to Golgi type II neurons (interneurons), contains relatively low amounts of glutamate decar☐ylase enzyme protein, and mediates both pre- and postsynaptic inhibition. The findings underline the existence of a new type of γ-aminobutyrate-containing Golgi type I neuron, controlling activity in the substantia innominata and in the outflow from Callejas islands of the olfactory tubercle and the view that γ-aminobutyrate may play a role in synaptic processes in all parts of the brain and the spinal cord.

Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GABA-mediated neurotransmission and of ion channels

Infusion of the K(+) channel blocker 4-aminopyridine in the hippocampus induces the release of glutamate, as well as seizures and neurodegeneration. Since an imbalance between excitation and inhibition, as well as alterations of ion channels, may be involved in these effects of 4-aminopyridine, we have studied whether they are modified by drugs that block glutamatergic transmission or ion channels, or drugs that potentiate GABA-mediated transmission. The drugs were administered to anesthetized rats subjected to intrahippocampal infusion of 4-aminopyridine through microdialysis probes, with simultaneous collection of dialysis perfusates and recording of the electroencephalogram, and subsequent histological analysis. Ionotropic glutamate receptor antagonists clearly diminished the intensity of seizures and prevented the neuronal damage, but did not alter substantially the enhancement of extracellular glutamate induced by 4-aminopyridine. None of the drugs facilitating GABA-mediated transmission, including uptake blockers, GABA-transaminase inhibitors and agonists of the A-type receptor, was able to reduce the glutamate release, seizures or neuronal damage produced by 4-aminopyridine. In contrast, nipecotate, which notably increased extracellular levels of the amino acid, potentiated the intensity of seizures and the neurodegeneration. GABA(A) receptor antagonists partially reduced the extracellular accumulation of glutamate induced by 4-aminopyridine, but did not exert any protective action. Tetrodotoxin largely prevented the increase of extracellular glutamate, the electroencephalographic epileptic discharges and the neuronal death in the CA1 and CA3 hippocampal regions. Valproate and carbamazepine, also Na(+) channel blockers that possess general anticonvulsant action, failed to modify the three effects of 4-aminopyridine studied. The N-type Ca(2+) channel blocker omega-conotoxin, the K(+) channel opener diazoxide, and the non-specific ion channel blocker riluzole diminished the enhancement of extracellular glutamate and slightly protected against the neurodegeneration. However, the two former compounds did not antagonize the 4-aminopyridine-induced epileptiform discharges, and riluzole instead markedly increased the intensity and duration of the disharges. Moreover, at the highest dose tested (8mg/kg, i.p.), riluzole caused a 75% mortality of the rats. We conclude that 4-aminopyridine stimulates the release of glutamate from nerve endings and that the resultant augmented extracellular glutamate is directly related to the neurodegeneration and is involved in the generation of epileptiform discharges through the concomitant overactivation of glutamate receptors. Under these conditions, a facilitated GABA-mediated transmission may paradoxically boost neuronal hyperexcitation. Riluzole, a drug used to treat amyotrophic lateral sclerosis, seems to be toxic when combined with neuronal hyperexcitation.

Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high‐affinity sodium‐dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl‐threo‐β‐hydroxyaspartate and l‐aspartate‐β‐hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate or by intrastriatal coinjection of 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.

Effect of 4-aminopyridine on transmitter release in synaptosomes.

The effect of 4-aminopyridine (4-AP) on the release of labeled transmitters in mouse brain synaptosomes was studied in a superfusion system. 4-AP at microM concentrations notably stimulated the spontaneous release of labeled GABA and glutamate, and of acetylcholine (ACh) derived from tritiated choline. No effects on the release of labeled alpha-aminoisobutyric acid were observed. The stimulation of GABA and ACh release was dependent on the presence of Ca2+ in the superfusion media, whereas the effect on glutamate release was more variable and no clear Ca2+-dependence was observed. In contrast to these results, 4-AP did not have any effect on the release of the above transmitters stimulated by K+-depolarization in the presence of Ca2+. These results are discussed in terms of the possible participation of Ca2+ in the action of 4-AP on spontaneous transmitter release in isolated nerve endings.

Glutamate uptake impairment and neuronal damage in young and aged rats in vivo

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3‐month‐old and 22–24‐month‐old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.

Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes

The mechanism of the Ca2+-dependent stimulation of neurotransmitter release by 4-aminopyridine in synaptosomes was studied. The stimulation of gamma-[3H]aminobutyric acid and [3H]acetylcholine release by 4-aminopyridine was not significantly affected either by tetrodotoxin or by the absence of Na+ in the medium, whereas the toxin notably inhibited the release of both transmitters induced by veratridine. On the other hand, the release of labeled gamma-aminobutyric acid induced by 4-aminopyridine was inhibited by both La3+ and ruthenium red, two blockers of Ca2+ transport in synaptosomes. In other experiments, 4-aminopyridine had only a slight stimulatory effect, if any, on the influx of 45Ca2+ into synaptosomes, under both resting and K+-depolarizing conditions. Ruthenium red inhibited the stimulation by K+ of the 45Ca2+ uptake, and 4-aminopyridine did not antagonize this inhibition. We conclude that the transmitter-releasing action of 4-aminopyridine in synaptosomes does not involve an excitatory effect on the membrane which may result in the opening of voltage-sensitive Na+ channels. 4-Aminopyridine does not seem to act either by enhancing Ca2+ entry into the synaptosomes. It is proposed that 4-aminopyridine facilitates the coupling between Ca2+ binding and transmitter secretion at the presynaptic membrane.

Preferential stimulation of glutamate release by 4-aminopyridine in rat striatum in vivo

The potassium channel blocker 4-aminopyridine (4-AP) is a potent convulsant drug which, in vitro, stimulates the release of neurotransmitter amino acids. We have studied the effect of 4-AP in vivo on the extracellular concentration of amino acids in rat striatum, by means of microdialysis and HPLC. Perfusion with 4-AP in the awake animal produced intense motor alterations, including barrel turning and running fits. Therefore, most microdialysis experiments were carried out in anesthetized rats. Perfusion with 20-75 mM 4-AP for 12.5 min resulted in a massive increase in extracellular glutamate (up to 20-fold), smaller increases in aspartate and taurine (up to 10-fold) and slight increments in glutamine, alanine, glycine and GABA. In contrast, perfusion with 100 mM K+ produced, mainly, an increment in taurine (7-fold) and modest increases in glutamate and aspartate (100-300%), as well as a notable decrease in glutamine. Tetraethylammonium (TEA, 120 mM) perfusion induced taurine and glutamate elevations similar to those after high K+, but glutamine was not affected. In unanesthetized rats, perfusion with 40 mM 4-AP induced changes in extracellular amino acids similar to those observed under anesthesia. In these animals neither high K+ nor TEA affected significantly the motor behavior. The results suggest that an enhancement of glutamatergic synaptic transmission, rather than a general depolarizing action, is an important factor in the neuronal hyperexcitability induced by 4-AP, which is consistent with the previously demonstrated inhibition of its convulsant effect by glutamate receptor antagonists.

Evidence for a role of glutamate decarboxylase activity as a regulatory mechanism of cerebral excitability

AMPLE experimental evidence indicates that GABA is an inhibitory synaptic transmitter in the CNS of mammals. This evidence can be summarized as follows: when GABA is applied to neurons it produces electrical and ionic changes similar to those observed with the natural transmitter (I(RNJEV1C & SCHWARTZ, 1967; CURTIS & JOHNSTON, 1970; CURTIS et al., 1971), glutamate decarboxylase (Lglutamate-I-carboxylyase, EC 4.1.1.15) is present in the nerve endings (SALGANICOFF & DE ROBERTIS, 1965; FONNUM, 1968) and concentrated in regions where GABA seems to act as a transmitter (FONNUM & WALBERG, 1973; SAITO r t a/.. 1974). GABA is released from brain tissue tipon stim~ilation by a Ca”-dependent mechanism (SRINIVASAN et al., 1969; OBATA & TAKEDA, 1969; IVERSEN et al., 1971; ROBERTS, 1974), and it is eliminated from extracellular spaces by a Naf-dependent uptake into nerve cells, glial cells or nerve endings (IVERSEN & NEAL, 1968; HENN & HAMBERGER, 1971; BLOOM & IVERSEN, 1971; SCHRIER & THOMPSON, 1974). Pharmacologically, the physiological action of GABA may theoretically be blocked by at least four mechanisms: (1) inhibition of the synthesis of GABA; (2) blocking the postsynaptic receptor; (3) blocking the release of GABA; and (4) stimulating the rate of uptake of GABA. At present, no pharmacological way of stimulating the uptake of GABA or of blocking its release in uivo is available, but at least two drugs which seem to act by blocking the receptor of GABA, namely bicuculline (CURTIS et al., 1970, 1971) and picrotoxin (HILL et al., 1972), are known. As it might be expected, these substances are powerful convulsants when injected into experimental animals (CURTIS et al., 1970; MELDRUM & HORTON, 1971; TEWS et al., 1963). With regard to the inhibition of GABA synthesis, the decrease of glutamate decarboxylase activity observed after the administration of a number of drugs has been related also to the occurrence of convulsions (HOLTZ & PALM, 1964; LOVELL. 1971). In many of these cases an unexpected finding has been consistently reported: independently of the total brain GABA levels, which may be markedly elevated, convulsions are observed immediately when the activity of glutamate decarboxylase, and thus the rate of synthesis of GABA, is inhibited to a certain extent (TAPIA et a/., 1967a, 1969; TAPIA & AWAPARA, 1967; WOOD & PEESKER, 1972, 1973; TAPIA, 1974). These results suggest that under normal conditions GABA is not being released from a storage pool specifically related to i ts synaptic function (no lag period exists between the inhibition of glutamate decarboxylase and the appearance of seizures), but, instead, that the newly synthesized GABA is being liberated into the synaptic cleft (TAPIA, 1974). In the present paper we give further evidence supporting this suggestion. We reported previously that the administration of Lglutamic acid y-hydrazide (GAH) results in an increase

Glutamate excitotoxicity and therapeutic targets for amyotrophic lateral sclerosis

Two forms of amyotrophic lateral sclerosis (ALS) are known, the familial (FALS), due in part to mutations in superoxide dismutase 1 (SOD1), and the sporadic (SALS), which accounts for > 90% of all cases. The cause of SALS is not known, but excitotoxicity due to overactivation of glutamate receptors may mediate the motor neuron degeneration in the spinal cord, which is the hallmark of this disease. Overactivation of calcium-permeable α-amino-3-hydroxy-5-isoxazole propionate receptors lacking the subunit glutamate receptor 2, leading to an increase in calcium cytoplasmic concentration, seems to play an important role in the mechanism of neuronal death. The knowledge of this mechanism, in addition to other factors, provides several possible targets for therapeutic strategies that are reviewed in this article. Some of these strategies have proven to be partially effective in both human mutant superoxide dismutase 1 transgenic rodents (FALS model) and the few existing in vivo models of spinal motor neurodegeneration induced by excitotoxicity (SALS models), although observable benefits are still to be shown in clinical trials.

Glutamine and glutamate as precursors of the releasable pool of gaba in brain cortex slices

The release of labeled GABA, synthesized from [(14)C]glutamine and from [(14)C]glutamate, was studied in mouse brain cortex slices. The spontaneous release of radioactive GABA derived from glutamate was higher than that of GABA synthesized from glutamine. In contrast, depolarization by 48 mM K(+) notably stimulated the release of GABA synthesized from glutamine but did not affect significantly the release of GABA formed from glutamate. It is concluded that glutamate is a better precursor than glutamine of the GABA pool spontaneously released, whereas the reverse is true for the pool releasable by K(+)-depolarization. The release of [(14)C]glutamate was stimulated by K(+) depolarization both when it was exogenously supplied and when it was derived from [(14)C]glutamine.