Potentiation by Kainate of Excitatory Amino Acid Release in Striatum: Complementary In Vivo and In Vitro Experiments

The effect of kainate on extracellular levels of amino acids in corpus striatum was investigated in vitro and in vivo, to elucidate the mechanism underlying its neurotoxicity. Kainate increased extracellular glutamate and aspartate in both striatal slices in vitro and intact striatum in vivo, as previously reported. Both in vitro and in vivo, DL-threo-3-hydroxyaspartate increased extracellular glutamate and aspartate levels (to between 150 and 200% of basal), and also enhanced their kainate-evoked release. The action of kainate in vivo was reduced by prior frontal decortication, whereas in vitro the kainate-evoked responses were only slightly reduced by tetrodotoxin, and remained above control values. These results confirm that kainate increases extracellular glutamate and aspartate, and provide evidence that this is due to synaptic release evoked by an action on receptors on glutamatergic neurone terminals. These findings may be relevant to the understanding of epilepsy.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

Substrates of Energy Metabolism Attenuate Methamphetamine-Induced Neurotoxicity in Striatum

Abstract: High doses of methamphetamine (METH) produce a long-term depletion in striatal tissue dopamine content. The mechanism mediating this toxicity has been associated with increased concentrations of dopamine and glutamate and altered energy metabolism. In vivo microdialysis was used to assess and alter the metabolic environment of the brain during high doses of METH. METH significantly increased extracellular concentrations of lactate in striatum and prefrontal cortex. This increase was significantly greater in striatum and coincided with the greater vulnerability of this brain region to the toxic effects of METH. To examine the effect of supplementing energy metabolism on METH-induced dopamine content depletions, the striatum was perfused directly with decylubiquinone or nicotinamide to enhance the energetic capacity of the tissue during or after a neurotoxic dosing regimen of METH. When decylubiquinone or nicotinamide was perfused into striatum during the administration of METH, there was no significant effect on METH-induced striatal dopamine efflux, glutamate efflux, or the long-term dopamine depletions measured 7 days later. However, a delayed perfusion with decylubiquinone or nicotinamide for 6 h beginning immediately after the last METH injection attenuated the METH-induced striatal dopamine depletions measured 1 week later. These results support the hypothesis that the compromised metabolic state produced by METH administration predisposes dopamine terminals to the neurotoxic effects of glutamate, dopamine, and/or free radicals.     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

L-Glutamate and Insulin Enhance Glycogen Synthesis in Cultured Astrocytes from the Rat Brain Through Different Intracellular Mechanisms

The effects of L-glutamate and insulin on glycogen synthesis in astrocytes were examined. L-Glutamate and insulin both stimulated glycogen synthesis in primary cultures of rat astrocytes in a dose-dependent manner, as measured by the incorporation of 14C from [14C]glucose into glycogen. D-Aspartate also increased the incorporation of 14C into glycogen. When insulin and L-glutamate were added together, the glycogen synthesis as well as glycogen content of the cells was additively increased. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had little effect on glycogen synthesis induced by L-glutamate, whereas it suppressed the insulin-induced glycogen synthesis. These results suggest that the insulin- and L-glutamate-induced glycogen syntheses are mediated by different intracellular mechanisms. In fact, insulin stimulated the conversion of glycogen synthase b to glycogen synthase a, which was suppressed by wortmannin. L-Glutamate and D-aspartate, however, did not increase the level of glycogen synthase a activity. By contrast, L-glutamate increased 2-deoxy-D-[3H]glucose uptake by the astrocytes, whereas insulin did not affect the uptake. These results suggest that insulin stimulates glycogen synthesis in astrocytes by activating glycogen synthase, which is dependent on a wortmannin-sensitive signaling pathway. L-Glutamate, however, enhances the glucose uptake, which contributes to the increase in glycogen synthesis in the cells.     

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.     

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.     

Regional Calcium Accumulation and Cation Shifts in Rat Brain by Kainate

Abstract: Following local application of kainic acid, changes in the contents of Na⁺, K⁺, Ca²⁺, and Mg²⁺ of the striatum, cerebellum, and hippocampus of the rat were observed at various times after surgery. Within 1 h the levels of K⁺ decreased 20% whereas the levels of Na⁺ and Ca²⁺ increased at least 50% and 20%, respectively. These changes persisted for more than 8 weeks. Ca²⁺ levels rose further, to more than 10-fold during 8 weeks. The Mg²⁺ levels were slightly and only transiently decreased. Unilateral injections of kainate into the striatum affected the contents of these cations not only in this area, but also in the overlying cerebral cortex, the olfactory tubercle, and the ipsilateral substantia nigra. The Ca²⁺ increases were less when rats were kept on a diet deficient in Ca²⁺ and vitamin D. ⁴⁵Ca²⁺, intravenously administered, accumulated significantly more in the kainate-lesioned striatum and substantia nigra than in the homotopic contralateral areas. Electron microscopic examination of the localization of Ca²⁺ with the oxalate-pyroantimonate technique showed the appearance of extracellularly located deposits and the accumulation of Ca²⁺ in (possibly degenerating) myelinated axons in kainate-lesioned striata. This study provides evidence that calcification of cerebral tissue is closely associated with neurodegenerative processes and shows that kainate may serve as a tool to elucidate the mechanism of brain calcification. The results are discussed in relation to idiopathic calcinosis (striopallidodentate calcinosis, Fahr's disease).     

Ontogeny of Dopamine D1 Receptors in Rat Striatum

The development of dopamine D1 receptors in rat striatum during the early postnatal period is described, using [3H]piflutixol as ligand. Dopamine D1 receptors increase in number from day of birth until about 21 days of age, when they reach adult levels. This increase in number parallels the increase in several other dopamine markers in striatum during the same time period. The increase is reflected in an increase in Bmax of ligand binding to D1 receptors. All other properties of D1 receptors that were examined do not change throughout this developmental period and are essentially the same as those found in adult tissue. These include association and dissociation rates, affinity for piflutixol as determined by kinetic and saturation studies, and pharmacology. These studies provide a biochemical and pharmacological basis for further studies on the ontogeny of dopamine receptors and of striatum and on factors regulating development of this region.     

Synaptosomal Uptake and Release of Dopamine in Rat Striatum After Hypoxia

Hypoxia induces alterations of central monoaminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long-lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short-term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+-evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2-11) caused long-lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+-stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.     

Induction of c-fos mRNA Expression in Rat Striatum by Neuroleptic Drugs

The effect of selective dopamine D2 receptor-acting drugs on striatal c-fos mRNA expression in the rat has been investigated by Northern hybridization and autoradiography to determine a possible role for c-fos in the initiation of adaptive changes in D2 receptor number by neuroleptic drugs. The neuroleptic drug haloperidol, a D2 receptor antagonist, was found to produce a rapid and transient induction of c-fos mRNA expression as compared with the expression in animals treated with saline. This induction by haloperidol was found to be dose dependent and D2 receptor mediated, inasmuch as a D2 agonist completely reversed the induction and the inactive isomer of the neuroleptic butaclamol, which does not produce an increase in D2 receptors, had no effect on c-fos mRNA expression. From these data, it can be concluded that c-fos expression in striatum is under dopamine D2 receptor-mediated inhibitory control. It is suggested that c-fos may play a role in the initiation of the increase in D2 receptor number produced by chronic neuroleptic drug treatment.     

Development of Haloperidol-Induced Dopamine Release in the Rat Striatum Using Intracerebral Dialysis

Haloperidol-induced dopamine (DA) release and metabolism were studied in the rat striatum at 10-11, 21-22, and 35-36 days of age using intracerebral dialysis and HPLC with electrochemical detection. There was an age-related increase in basal DA release and extracellular levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), with the greatest increases occurring between 10-11 and 21-22 days of age. Haloperidol (0.1 mg/kg, i.p.) significantly increased DA release at each age compared to control. Also, haloperidol produced a significantly greater increase in DA release at 10-11 days than at 21-22 or 35-36 days of age when expressed as percentage of predrug release. Haloperidol increased DA release over 60 min to 235%, 138%, and 158% above baseline at 10-11, 21-22, and 35-36 days of age, respectively, after which time the levels remained relatively constant. Haloperidol significantly increased extracellular DOPAC and HVA levels at each age compared to controls, but there were no significant differences in DOPAC or HVA levels between ages in response to haloperidol. The results indicate that, at 10 days of age, DA release in the striatum is physiologically functional and that the regulatory feedback control of DA release and metabolism in the striatum develops prior to 10 days of age.     

Effect of the Cerebral Tryptaminergic System on the Turnover of Dopamine in the Striatum of the Rat

The effect of the cerebral 5-hydroxytryptamine system on the turnover of striatal 3,4-dihydroxyphenyl-ethylamine (dopamine) was investigated by measuring the level of dopamine and one of its metabolites in rats depleted of cerebral 5-hydroxytryptamine or treated with a 5-hydroxytryptamine receptor blocker. Treatment with p-chlorophenylalanine induced, in addition to a reduction in striatal 5-hydroxytryptamine and 5-hydroxyindol-3-ylacetic acid, an increase in the striatal concentration of dopamine, a diminution in the concentration of homovanillic acid in the same cerebral area, and a reduction in the rise of this acid after the administration of a butyrophenone derivative or tetrabenazine. Treatment with methysergide also reduced the increase of homovanillic acid induced by the butyrophenone. When probenecid was given to rats treated with p-chlorophenylalanine, homovanillic acid failed to accumulate, whereas the accumulation of 5-hydroxyindol-3-ylacetic acid was unaffected. The decay of dopamine after alpha-methyl-p-tyrosine administration was normal for the first 6 h but was later reduced in rats given p-chlorophenylalanine or methysergide. The results suggest that the lack of activation of 5-hydroxytryptamine receptors leads to a reduction in the turnover of dopamine in the nigrostriatal pathway.     

N-Methyl-D-Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo

There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Rapid ATP Loss Caused by Methamphetamine in the Mouse Striatum: Relationship Between Energy Impairment and Dopaminergic Neurotoxicity

Abstract: To study the relationship between energy impairment and the effects of α-methamphetamine (METH) on dopaminergic neurons, ATP and dopamine levels were measured in the brain of C57BL/6 mice treated with either a single or four injections of METH (10 mg/kg, i.p.) at 2-h intervals. Neither striatal ATP nor dopamine concentrations changed after a single injection of METH, but both were significantly decreased 1.5 h after the multiple-dose regimen. The effects of METH on ATP levels appear to be selective for the striatum, as ATP concentrations were not affected in the cerebellar cortex and hippocampus after either a single or multiple injections of METH. In a second set of experiments, an intraperitoneal injection of 2-deoxyglucose (2-DG; 1 g/kg), an inhibitor of glucose uptake and utilization, was given 30 min before the third and fourth injections of METH. 2-DG significantly potentiated METH-induced striatal ATP loss at 1.5 h and dopamine depletions at 1.5 h and 1 week. These results indicate that a toxic regimen of METH selectively causes striatal energy impairment and raise the possibility that perturbations of energy metabolism play a role in METH-induced dopaminergic neurotoxicity.     

Selective Release of Spermine and Spermidine from the Rat Striatum by N-Methyl-d-Aspartate Receptor Activation In Vivo

The intrastriatal infusion of N-methyl-D-aspartate (NMDA; 250-1,000 microM) via a dialysis cannula in anesthetized rats resulted in a marked and rapid increase in the concentrations of spermine and spermidine recovered in the dialysate. Extracellular concentrations of NMDA-released spermine and spermidine were calculated to be in the low micromolar range. Putrescine levels were not significantly affected by NMDA. The effects of NMDA (500 microM) were blocked by the previous systemic injection of MK-801 (3 mg/kg, i.p.) but were insensitive to the intrastriatal infusion of tetrodotoxin (1 microM). Intrastriatally infused kainate or quisqualate (1,000 microM) did not increase polyamine levels in the dialysate. Spermine and spermidine dialysate levels were also significantly increased by the infusion of high concentrations of K+ (greater than 100 mM), although the effects of K+ were considerably less marked than those of NMDA. Striatal polyamines are released into the extracellular space specifically by NMDA receptor activation. Because of their multiple effects on receptor- and voltage-operated cation channels, polyamines that are released by NMDA receptor activation may play an important role in phenomena already attributed to NMDA receptor stimulation, such as long-term potentiation, synaptic plasticity, and neurotoxicity.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.