Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.     

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.     

Excitatory Amino Acid Receptor Potency and Subclass Specificity of Sulfur-Containing Amino Acids

The sulfur-containing amino acids, L- and D-cysteate, L-cysteine, L- and D-cysteine sulfinate, L- and D-cysteine-S-sulfate, L-cystine, L- and D-homocysteate, L- and D-homocysteine sulfinate, L-homocysteine, L-serine-O-sulfate, and taurine were tested in two excitatory amino acid receptor functional assays and in receptor binding assays designed to label specifically the AA1/N-methyl-D-aspartate (NMDA), AA2/quisqualate, and AA3/kainate receptor recognition sites, as well as a CaCl2-dependent L-2-amino-4-phosphonobutanoate site, and a putative glutamate uptake site. Agonist efficacies were determined by chick retinal excitotoxicity and stimulated sodium efflux from rat brain slices. D-Homocysteine sulfinate, L-homocysteate, and L-serine-O-sulfate had affinities most selective for the NMDA binding site, whereas the binding affinities of D-cysteate, D-cysteine sulfinate, D-homocysteate, and L-homocysteine sulfinate were less selective. However, the correlation of agonist activity sensitive to blockade by D-2-amino-7-phosphonoheptanoate or D-2-amino-5-phosphonopentanoate in the functional assays with affinity in the NMDA binding assay (r = 0.87, p less than 0.005 and r = 0.98, p less than 0.005 for excitotoxicity and sodium efflux, respectively) allows characterization of these sulfur-containing amino acids as acting at NMDA subclass receptors. L-Homocysteate, which has been found in the brain, and L-serine-O-sulfate are selective agonists and could serve as endogenous neurotransmitters at the NMDA receptor.     

Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Cellular Origins of Cyclic GMP Responses to Excitatory Amino Acid Receptor Agonists in Rat Cerebellum In Vitro

Incubated slices and freshly dissociated cells from 8-day-old rat cerebellum were used to try to identify the cells that participate in the large increases in cyclic GMP levels that follow activation of excitatory amino acid receptors in this tissue. In the slices, cyclic GMP responses to L-glutamate and related excitants were unaffected by tetrodotoxin and could be replicated by the guanylate cyclase activator nitroprusside. Nitroprusside and the receptor agonists appeared to activate the same pool of the enzyme. Prior destruction of neuroblasts, deep nuclei, or Golgi neurones did not cause loss of responses to L-glutamate. If granule cells were rendered necrotic, however, the cyclic GMP responses to all excitants tested were reduced by greater than or equal to 90%. Substantial losses of responses to veratridine and high K+ levels also occurred, but the nitroprusside-induced elevations were unaffected. In dissociated cell suspensions, the magnitude of responses to receptor agonists, but not those to nitroprusside, was markedly dependent on cell concentration. Responses to L-glutamate were the same in cell suspensions that were Purkinje cell depleted and Purkinje cell enriched. It is concluded that granule cells are primarily involved in the cyclic GMP responses to excitatory amino acids but that the cyclic GMP accumulations occur elsewhere, probably in glial cells.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel Glutamate Receptor Antagonists Selectively Protect Against Kainic Acid Neurotoxicity in Cultured Cerebral Cortex Neurons

The effect on excitatory amino acid (EAA)-induced toxicity of two novel non-N-methyl-D-aspartate (non-NMDA) antagonists 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA) and 2-amino-3-[2-(3-hydroxy-5-methyl-isoxazol-4-yl)methyl-5-methyl-3- oxoisoxazolin-4-yl]propionic acid (AMNH) was tested in primary cultures of cerebral cortex neurons. Such cultures provide a useful model for the investigation of the toxicity of EAAs and a convenient screening system for potential neuroprotective activity of pharmacological agents. It was demonstrated that AMNH and AMOA abolished neurotoxicity induced by kainic acid with IC50 values of 62 +/- 10 and 120 +/- 19 microM, respectively. No effect on neuronal damage induced by NMDA or AMPA could be detected.     

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)     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Administration of Vigabatrin (γ-Vinyl-γ-Aminobutyric Acid) Affects the Levels of Both Inhibitory and Excitatory Amino Acids in Rat Cerebrospinal Fluid

The effect of vigabatrin (gamma-vinyl-gamma-aminobutyric acid), a new anticonvulsant drug, on the transmitter amino acids in rat cisternal CSF was studied. CSF was collected through a permanently implanted polyethylene cannula from freely moving rats at 5, 24, 48, and 96 h after administration of 1,000 mg/kg of vigabatrin. The free gamma-aminobutyric acid (GABA) level was elevated maximally (13.5-fold; p less than 0.01) at 24 h after injection. The homocarnosine (GABA-histidine) level also was increased (123%; p less than 0.01) at 24 h after injection, and its concentration remained at the same level for the next 3 days. Glycine and taurine concentrations had increased [31% (p less than 0.05) and 63% (p less than 0.01), respectively] at 5 h after injection. It is interesting that the levels of glutamate and aspartate increased [330% (p less than 0.05) and 421% (p less than 0.01), respectively] at 96 h after injection, the time when the free GABA level had returned to the baseline concentration and the vigabatrin level was 3% of the maximal concentration. The present study indicates that a single dose of vigabatrin in rats elevates levels of both the inhibitory and excitatory amino acids in CSF. However, the temporal profile of observed changes in relation to vigabatrin injection shows that neither the long-lasting elevation of GABA content nor the increase in glutamate and aspartate levels correlates with the level of vigabatrin in CSF. These findings suggest that the excitatory mechanisms are also augmented following acute administration of vigabatrin, especially when the content of GABA had decreased to the baseline level and the level of vigabatrin was low.     

Magnesium-Dependent Inhibition of Agonist-Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids

The excitatory amino acid agonists kainate, N-methyl-D-aspartate (NMDA), and quisqualate inhibited ligand-stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK-801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol-stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol-stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol-stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol-stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol-stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol-stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.     

Ammonia-Induced Extracellular Accumulation of Taurine in the Rat Striatum In Vivo: Role of Ionotropic Glutamate Receptors

Accumulation of taurine (Tau), glutamate (Glu) and glutamine (Gln) was measured in vivo in microdialysates of the rat striatum following a direct application to the microdialysis tube of 60 mM ammonium chloride which renders the final ammonia concentration in the extracellular space to 5 mM. The following compounds were coadministered with ammonia to distinguish between the different mechanisms that may underlie the accumulation of amino acids: ion transport inhibitors, diisothiocyanostilbene-2,28-disulfonate (DIDS) and furosemide, a Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an NMDA receptor antagonist dizocilpine (MK-801) and an 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Ammonia stimulated Tau accumulation in the microdialysates to 250% of the basal value. Furosemide did not significantly affect the stimulation by ammonia and DIDS only moderately depressed the effect. The ammonia-dependent Tau accumulation was increased by 50% in the presence of PDC and reduced by 35% in the presence dizocilpine and DNQX. In the microdialysates ammonia stimulated Glu and Gln accumulation somewhat less than Tau accumulation. Except for stimulation of Gln accumulation by DNQX, the effects were not modified by any of the cotreatments. The results are consistent with the assumption that ammonia stimulates Tau efflux mainly via activation of ionotropic Glu receptors.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Changes in Extracellular Concentrations of Glutamate, Aspartate, Glycine, Dopamine, Serotonin, and Dopamine Metabolites After Transient Global Ischemia in the Rabbit Brain

Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmitters (e.g., glycine and dopamine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1-, 5-, and 13-fold and by 4-, 9-, and 31-fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700-fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4-fold, but remained elevated throughout the 80-min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann-Whitney test). That glutamate and aspartate concentrations in the hippocampus co-vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Synaptosomal Transport of Radiolabel from N-Acetyl-Aspartyl-[3H]Glutamate Suggests a Mechanism of Inactivation of an Excitatory Neuropeptide

This study was undertaken to explore in synaptosomal preparations the disposition of N-acetyl-aspartyl-glutamate (NAAG), an endogenous acidic dipeptide neurotransmitter candidate. Radiolabel from N-acetyl-aspartyl[3H]glutamate was taken up rapidly into an osmotically sensitive compartment by rat brain synaptosomal preparations in a sodium-, temperature-, and time-dependent manner. HPLC analysis of the accumulated radiolabel indicated that the bulk of the tritium cochromatographed with glutamic acid and not with NAAG. In contrast, [14C]NAAG, labeled on the N-terminal acetate, was not taken up by the synaptosomal preparation. All effective inhibitors of synaptosomal, Na+-dependent [3H]glutamate uptake were found to exhibit similar potency in inhibiting uptake of tritium derived from [3H]NAAG. However, certain alpha-linked acidic dipeptides, structurally similar to NAAG, as well as the potent convulsant quisqualic acid inhibited synaptosomal transport of [3H]NAAG but were ineffective as inhibitors of [3H]glutamate transport. Together with a demonstration of disparities between the regional accumulation of radiolabel from [3H]NAAG and high-affinity [3H]glutamate uptake, these data suggest the presence in brain of a specific peptidase targeting carboxy-terminal glutamate-containing dipeptides that may be coupled to the Na+-dependent glutamate transporter. These findings provide a possible mechanism for NAAG inactivation subsequent to its release from nerve endings.     

Increase in the Extracellular Histamine Concentration in the Rat Striatum by μ-Opioid Receptor Activation

Abstract: The effects of morphine and selective ligands for μ-, κ-, and δ-opioid receptors on the extracellular histamine (HA) concentration in the striatum of freely moving rats were examined by in vivo microdialysis. On the day after implantation of the dialysis probe, the HA output per 30-min period was measured using HPLC-fluorometry. Morphine (3.8 mg/kg, s.c.) significantly increased the HA output by ∼200% 1–3 h after treatment. This effect was completely antagonized by naltrexone (1.6 mg/kg, s.c.). The HA output decreased to a level below 10% of the basal value by 4 h after treatment with ( S )-α-fluoromethylhistidine (77 mg/kg, s.c.). In such animals, morphine (3.8 mg/kg, s.c.) had no influence on the HA output. [ d -Ala²,MePhe⁴,Gly(ol)⁵]Enkephalin (DAGO; 0.2 µg, i.c.v.), a selective μ-agonist, significantly increased the HA output by ∼150% 0.5–1.5 h after treatment, and this effect was also completely blocked by naltrexone. A selective κ-agonist, U-50,488 (3.8 and 7.6 mg/kg, s.c.), and a selective δ-agonist, [ d -Pen², d -Pen⁵]enkephalin (0.5 and 2 µg, i.c.v.), had no effect on the HA output. These findings suggest that the stimulation of μ-opioid receptors by morphine and DAGO increases the extracellular HA concentration by accelerating HA release from nerve endings.     

Differential Effects of δ- and μ-Opioid Receptor Antagonists on the Amphetamine-Induced Increase in Extracellular Dopamine in Striatum and Nucleus Accumbens

Abstract: The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine-induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective δ-opioid receptor antagonist, but not by the irreversible μ-opioid receptor antagonist β-funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 µg) or vehicle was given 15 min before and β-funaltrexamine (10 µg) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10-µl volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneous d-amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30-min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose-dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 µg) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 µg of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, β-funaltrexamine (10 µg) had no effect in the striatum but significantly attenuated the amphetamine-induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that δ-opioid receptors play a relatively larger role than μ-opioid receptors in mediating the amphetamine-induced increase in extracellular dopamine content in the striatum, whereas μ-opioid receptors play a larger role in mediating these effects in the nucleus accumbens.