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.     

3-Nitropropionic Acid Toxicity in the Striatum

Abstract: We examined the effects of chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) in doses ranging from 12 to 16 mg/kg/day for 30 days on striatal cytoarchitecture in rats. Administration of 3-NP at a dose of 16 mg/kg/day resulted in large lesions with a central necrotic core that was depleted of both neurons and glia. Glial fibrillary acidic protein (GFAP) gene expression was decreased in the lesion core, whereas the tissue surrounding this area showed a massive increase in signal intensity. Enkephalin and substance P mRNA expression in the striatum showed dose-dependent decreases following administration of 3-NP. A substantial decrease occurred even in animals treated with 3-NP at a dose of 12 mg/kg/day, in which there was little discernible neuronal loss and no increase in GFAP gene expression. In contrast to the decrease in enkephalin and substance P mRNA expression, somatostatin mRNA-expressing neurons were largely preserved. There was no preferential loss of [³H]naloxone patches in the rat striatum following chronic administration of 3-NP. In animals treated with 12–15 mg/kg/day neither the area nor binding density of the patches was changed. To study the effect of 3-NP on N -methyl- d -aspartate (NMDA)-gated Ca²⁺ channels we used in vivo administration of [³H]MK-801. Three hours after a single injection of 3-NP at a dose of 30 mg/kg there was a three- to fivefold increase in [³H]MK-801 binding in cortex and striatum as compared with saline-treated animals, consistent with an activation of NMDA receptors.     

Partial Inhibition of Brain Succinate Dehydrogenase by 3-Nitropropionic Acid Is Sufficient to Initiate Striatal Degeneration in Rat

Abstract: Chronic inhibition of succinate dehydrogenase (SDH) by systemic injection of the selective inhibitor 3-nitropropionic acid (3NP) has been used as an animal model for Huntington's disease (HD). However, the mechanisms by which 3NP produces lesions in the striatum are not fully characterized. A quantitative histochemical method was developed to study the level of regional SDH inhibition resulting from intraperitoneal injection of 3NP or chronic intoxication using osmotic pumps. The results showed that (a) 3NP was an irreversible SDH inhibitor in vivo, (b) the level of SDH inhibition in the striatum (the brain region most vulnerable to 3NP) was similar to that observed in other brain regions not affected by the toxin, such as the cerebral cortex, and (c) the neurotoxic threshold of SDH inhibition in the brain was 50–60% of control levels. The present study demonstrates that the selective degeneration in the striatum observed after chronic 3NP administration cannot be ascribed to a preferential inhibition of SDH in this particular brain region. This work also suggests that the partial decrease in the activity of the respiratory chain complex II–III reported in HD patients may be sufficient to induce the selective striatal degeneration observed in this disorder.     

Exacerbation of NMDA, AMPA, and l-Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-d -aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and l -glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm³), as did coinjection of malonate with S-AMPA (11.0 ± 1.0 mm³) or glutamate (12.8 ± 0.7 mm³). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm³). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 ± 0.7 mm³), but it attenuated the toxicity of malonate plus glutamate by ～40% (7.5 ± 0.9 mm³). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm³, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 ± 0.9 mm³). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.     

Increased Vulnerability to 3-Nitropropionic Acid in an Animal Model of Huntington's Disease

Abstract: There is substantial evidence for both metabolic dysfunction and oxidative damage in Huntington's disease (HD). In the present study, we used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of hydroxyl radical production in a transgenic mouse model of HD, as well as in littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was unchanged in the striatum of transgenic HD mice at baseline. Following administration of the mitochondrial toxin 3-nitropropionic acid (3-NP), there were significant increases in 3,4-DHBA generation in both control and transgenic HD mice, and the increases in the transgenic HD mice were significantly greater than those in controls. Furthermore, administration of 3-NP produced significantly larger striatal lesions in transgenic HD mice than in littermate controls. The present results show increased sensitivity to the mitochondrial toxin 3-NP in transgenic HD mice, which suggests metabolic dysfunction in this mouse model of HD.     

3-Nitropropionic Acid Neurotoxicity Is Attenuated in Copper/Zinc Superoxide Dismutase Transgenic Mice

Abstract: The mitochondrial toxin 3-nitropropionic acid (3-NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3-NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non-Tg littermate controls. The Tg-SOD mice showed a pronounced attenuation of Nissl-stained striatal lesions compared with non-Tg mice. Systemic administration of 3-NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid. This production was attenuated significantly in Tg-SOD mice. In a similar way, 3-NP produced significant increases in 3-nitrotyrosine/tyrosine, a marker for peroxynitrite-mediated damage, which were significantly attenuated in Tg-SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3-NP neurotoxicity.     

Modulation of In Vivo Dopamine Release by D2 but Not D1 Receptor Agonists and Antagonists

The capacity of D1 and D2 agonists and antagonists to regulate the in vivo release and metabolism of dopamine (DA) in mesolimbic and nigrostriatal DA neurons of the mouse was determined using gas chromatographic and mass fragmentographic (GC-MF) analysis. DA release was inferred from levels of 3-methoxytyramine (3-MT) and DA metabolism was inferred from levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). DA release was increased by the D2 antagonists haloperidol and metoclopramide but not by the D1 antagonists SCH 23390 and SKF 83566. DA metabolism was increased by each of the four antagonists but to a greater extent with the D2 antagonists. The D2 agonists CGS 15855A and LY 171555 decreased DA release whereas the D1 agonist SKF 38393, at relatively high doses, only slightly affected DA release. Each of the three agonists decreased DA metabolism but again metabolism was more affected by the D2-selective drugs. The in vivo release of DA from mesolimbic and neostriatal DA neurons appears to be modulated by D2 but not by D1 receptors, whereas both receptor types can modulate DA metabolism.     

Eating-Induced Dopamine Release from Mesolimbic Neurons Is Mediated by NMDA Receptors in the Ventral Tegmental Area: A Dual-Probe Microdialysis Study

Abstract: This study was aimed at identifying the neuronal pathways that mediate the eating-induced increase in the release of dopamine in the nucleus accumbens of the rat brain. For that purpose, a microdialysis probe was implanted in the ventral tegmental area and a second probe was placed in the ipsilateral nucleus accumbens. Receptor-specific compounds acting on GABA_A (40 µ M muscimol; 50 µ M bicuculline), GABA_B (50 µ M baclofen), acetylcholine (50 µ M carbachol), NMDA [30 µ M (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)], and non-NMDA [300 µ M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] receptors were infused into the ventral tegmental area by retrograde dialysis, whereas extracellular dopamine was recorded in the ipsilateral nucleus accumbens. Intrategmental infusion of muscimol or baclofen decreased extracellular dopamine in the ipsilateral nucleus accumbens; CPP and CNQX were without effect, and bicuculline and carbachol increased dopamine release. During infusion of the various compounds, food-deprived rats were allowed to eat for 10 min. The infusions of muscimol, bicuculline, baclofen, carbachol, and CNQX did not prevent the eating-induced increase in extracellular dopamine in the nucleus accumbens. However, during intrategmental infusion of CPP, the eating-induced increase in extracellular dopamine in the nucleus accumbens was suppressed. These results indicate that a glutamatergic projection to the ventral tegmental area mediates, via an NMDA receptor, the eating-induced increase in dopamine release from mesolimbic dopamine neurons.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of NMDA and Non-NMDA Receptors to In vivo Glutamate-Induced Calpain Activation in the Rat Striatum. Relation to Neuronal Damage

Glutamate, the major excitatory neurotransmitter, can cause the death of neurons by a mechanism known as excitotoxicity. This is a calcium-dependent process and activation of the NMDA receptor subtype contributes mainly to neuronal damage, due to its high permeability to calcium. Activation of calpain, a calcium-dependent cysteine protease, has been implicated in necrotic excitotoxic neuronal death. We have investigated the contribution of NMDA and non-NMDA ionotropic receptors to calpain activation and neuronal death induced by the acute administration of glutamate into the rat striatum. Calpain activity was assessed by the cleavage of the cytoskeletal protein, α-spectrin. Caspase-3 activity was also studied because glutamate can also lead to apoptosis. Results show no caspase-3 activity, but a strong calpain activation involving both NMDA and non-NMDA receptors. Although neuronal damage is mediated mainly by the NMDA receptor subtype, it can not be attributed solely to calpain activity. Special issue article in honor of Dr. Ricardo Tapia.     

Effects of Dibutyryl Cyclic AMP and Retinoic Acid on the Differentiation of Dopamine Neurons: Prevention of Cell Death by Dibutyryl Cyclic AMP

Abstract: Immature neurons, including fetal and tumoral cells, are used for investigating neuronal differentiation in vitro. The human neuroblastoma cell line NB69 could be induced to differentiate to dopamine or acetylcholine neurons by different compounds, including neurotrophins and activators of the protein kinases. In these NB69 cells dibutyryl cyclic AMP (dbcAMP) at 2 m M reduced the division rate and increased the levels of catecholamines, tyrosine hydroxylase (TH) activity, and monoamine oxidase activity. The dbcAMP also increased cell size, dendritic arborization, density of the sites for high-affinity dopamine uptake, and activity of choline acetyltransferase. In fetal rat midbrain neurons treatment with dbcAMP increased the levels of dopamine and the number of TH-immunoreactive neurons in the culture. When embryonic day 14 fetal midbrain neurons, previously exposed to 1 µ M retinoic acid (a compound that severely reduces the number of fetal midbrain dopamine neurons), were treated with dbcAMP, the levels of dopamine and the number of TH-immunoreactive cells returned to normal levels. This suggests that dbcAMP induces the differentiation to dopamine neurons of quiescent progenitor or facilitates expression of the dopamine phenotype in immature neurons. Therefore, dbcAMP not only differentiates uncommitted immature dopamine neurons, but also reverses the antidopaminergic effects of retinoic acid. These properties of dbcAMP could be of therapeutic value in Parkinson's disease.     

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3-hydroxyanthranilic acid oxygenase (3HAO), catalyzing the conversion of 3-hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3-hydroxyanthranilic acid caused less than 25% inhibition of activity at 500 microM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10-fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at -80 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Dopamine D3 (Auto)receptors Inhibit Dopamine Release in the Frontal Cortex of Freely Moving Rats In Vivo

Abstract: In freely moving rats, the novel, selective dopamine (DA) D₃ receptor agonist PD 128,907 dose-dependently [effective dose (ED₂₅) = 0.07 mg/kg, s.c.] reduced dialysate levels of DA in the frontal cortex, a structure innervated by the ventral tegmental area (VTA). This action of PD 128,907 (0.16 mg/kg, s.c.) was abolished by a selective DA D₃ receptor antagonist S 14297 (1.25 mg/kg, s.c.), which alone did not modify levels of DA. In contrast to S 14297, its inactive distomer, S 17777, did not modify the actions of PD 128,907. In addition, PD 128,907 dose-dependently and potently inhibited the firing rate of VTA-localized neurons in anesthetized rats (ED₅₀ = 0.001 mg/kg, i.v.). S 14297, but not S 17777, completely reversed the actions of PD 128,907 (0.005 mg/kg, i.v.) with a 50% inhibitory dose of 0.03 mg/kg, i.v. and did not itself significantly modify the firing rate. In conclusion, these data provide the first direct evidence that DA D₃ (auto)receptors modulate (inhibit) the release of DA in the frontal cortex.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).     

Domoic Acid Neurotoxicity in Cultured Cerebellar Granule Neurons Is Mediated Predominantly by NMDA Receptors that Are Activated as a Consequence of Excitatory Amino Acid Release

Abstract: The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 µMl -glutamate or 10 µM domoate for 2 h in physiologic buffer at 22°C followed by a 22-h incubation in 37°C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist d (?)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate-induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65–77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate-stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors. Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux.     

Stimulation of γ-[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur-Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release

In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.     

<Emphasis Type="Italic">N</Emphasis>-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate Receptor Antagonists Have Variable Affect in 3-Nitropropionic Acid Toxicity

There is accumulating evidence that excitotoxicity and oxidative stress resulting from excessive activation of glutamate (N-methyl-d-aspartate) NMDA receptors are major participants in striatal degeneration associated with 3-nitropropionic acid (3NP) administration. Although excitotoxic and oxidative mechanisms are implicated in 3NP toxicity, there are conflicting reports as to whether NMDA receptor antagonists attenuate or exacerbate the 3NP-induced neurodegeneration. In the present study, we investigated the involvement of NMDA receptors in striatal degeneration, protein oxidation and motor impairment following systemic 3NP administration. We examined whether NMDA receptor antagonists, memantine and ifenprodil, influence the neurotoxicity of 3NP. The development of striatal lesion and protein oxidation following 3NP administration is delayed by memantine but not affected by ifenprodil. However, in behavioral experiments, memantine failed to improve and ifenprodil exacerbated the motor deficits associated with 3NP toxicity. Together, these findings suggest caution in the application of NMDA receptor antagonists as a neuroprotective agent in neurodegenerative disorders associated with metabolic impairment.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory Effects of Ascorbic Acid on the Binding of [3H]Dopamine Antagonists to Neostriatal Membrane Preparations : Relationship to Lipid Peroxidation

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo-isomer of ascorbic acid) inhibited the stereospecific binding of [³H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 m M ) or lower (0.006 m M ) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α-naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [³H]spiroperidol binding. These data strongly suggest that an iron-catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 m M ) and subsequently washed still had greatly diminished capacity to bind [³H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [³H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.