Yuko Kawata

Determination of the effects of caffeine and carbamazepine on striatal dopamine release by in vivo microdialysis

The effects of carbamazepine and caffeine on adenosine receptor subtypes were determined using in vivo microdialysis in an attempt to elucidate their different psychotropic mechanisms of action. Adenosine and a selective adenosine A1 receptor agonist decreased the striatal extracellular dopamine level, whereas caffeine, carbamazepine and a selective adenosine A1 receptor antagonist increased it, but neither an adenosine A2 receptor agonist nor an antagonist affected it. Under conditions of adenosine A1 receptor blockade, adenosine, carbamazepine and a selective adenosine A2 receptor agonist increased the striatal extracellular dopamine level, whereas caffeine and a selective adenosine A2 receptor antagonist decreased it. These results suggest that adenosine A1 receptor stimulation reduces the striatal extracellular dopamine level, and that adenosine A2 receptor stimulation under conditions of adenosine A1 receptor blockade increases it. Therefore, caffeine is an antagonist of both adenosine A1 and A2 receptor subtypes, and carbamazepine is an adenosine A1 receptor antagonist as well as an adenosine A2 receptor agonist. These properties support the hypothesis that the central actions of both carbamazepine and caffeine result from effects on both adenosine A1 and A2 receptors.

Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode

1 Multiple components of hippocampal glutamate release were examined by study of Ca2+‐ and K+‐evoked hippocampal extracellular glutamate release using an in vivo microdialysis glutamate biosensor in urethane‐anaesthetized rats. In addition, the effects of the antiepileptic drugs, carbamazepine (CBZ) and zonisamide (ZNS) perfused through the probe on glutamate release were assessed. 2 Basal glutamate levels were below detection limits (∼0.1 μm). An increase in extracellular KCl (from 2.7 to 50 and 100 mm) increased extracellular hippocampal glutamate levels to 9.2±1.4 and 20.0±2.6 μm, respectively, calculated from the area under curve (AUC) for 60 min. 3 This KCl‐evoked glutamate release consisted of three components: an initial transient rise, a late gentle rise, and late multiple phasic transient rises. 4 An increase in or removal of extracellular CaCl2 levels respectively enhanced and reduced the 50 mm KCl‐evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 12.4±2.1 and 5.8±0.9 μm. 5 Perfusion with 100 μm CBZ or 1 mm ZNS inhibited both the 50 mm KCl‐evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 5.5±1.1 and to 5.8±1.3 μm, respectively, as well as the stimulatory effects of Ca2+ on KCl‐evoked hippocampal glutamate release. 6 These results suggest that both CBZ and ZNS may reduce epileptiform events by inhibiting excitatory glutamatergic transmission.

Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5‐HT) release and 5‐HT reuptake activity, hippocampal extracellular 5‐HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5‐HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5‐HT levels. Adenosine and A1 receptor agonist, 2‐chloro‐N6‐cyclopentyl‐adenosine (CCPA), decreased extracellular 5‐HT levels, whereas non‐selective antagonist, caffeine, and A1 antagonist, 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT) increased them. When 5‐HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5‐HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX), and A3 receptor agonist, N6–2‐(4‐aminophenyl)ethyladenosine (APNEA) did not affect 5‐HT levels; however, when A1 receptor was blocked by CPT, 5‐HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5‐HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5‐HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5‐HT reuptake activity might be modulated by hippocampal A3 receptor.

Biphasic effects of zonisamide on serotonergic system in rat hippocampus

To study the mechanisms of antiepileptic action of zonisamide (ZNS), we determined the effects of ZNS on extracellular, total levels and re-uptake activity of serotonin (5-HT) in rat striatum and hippocampus. After acute administrations, plasma ZNS concentrations associated with anticonvulsive action (effective concentrations) increased the total levels of 5-HT, its metabolise (5-hydroxyindoleacetic acid: 5-HIAA) and precursor (5-hydroxytryptophan: 5-HTP). After chronic administration of ZNS, effective plasma concentrations also increased the extracellular and total levels of 5-HT, 5-HIAA, and 5-HTP. On the other hand, after both acute and chronic administrations of ZNS, a supra-effective ZNS concentration either decreased or did not affect the total levels of these substances. Therefore, the stimulatory effects of ZNS on the 5-HT system were reduced by an increase in ZNS concentration to supra-effective concentrations. ZNS concentrations of 30-1000 microM did not affect hippocampal 5HT re uptake activity in vitro. These results suggest that ZNS has biphasic effects on the 5-HT system, in that effective concentrations of ZNS enhance and supra-effective concentrations of ZNS reduce the function of the 5-HT system. These biphasic effects of ZNS on the 5-HT system may be involved in the mechanisms of action of the antiepileptic and psychotropic effects, and side effects of ZNS.

Determination of effects of antiepileptic drugs on SNAREs‐mediated hippocampal monoamine release using in vivo microdialysis

To elucidate possible mechanisms underlying the effects of carbamazepine (CBZ), valproate (VPA) and zonisamide (ZNS) on neurotransmitter exocytosis, the interaction between these three antiepileptic drugs (AEDs) and botulinum toxins (BoNTs) on basal, Ca2+‐ and K+‐evoked release of dopamine (DA) and serotonin (5‐HT) were determined by microdialysis in the hippocampus of freely moving rats. Basal release of monoamine was decreased by pre‐microinjection of the syntaxin inhibitor, BoNT/C, but only weakly affected by the synaptobrevin inhibitor, BoNT/B. Ca2+‐evoked release was inhibited by BoNT/C selectively. K+‐evoked release was reduced by BoNT/B predominantly and BoNT/C weakly. Perfusion with low and high concentrations of CBZ and ZNS increased and decreased basal monoamine release, respectively. Perfusion with VPA increased basal 5‐HT release concentration‐dependently, whereas basal DA release was affected by VPA biphasic concentration‐dependently, similar to CBZ and ZNS. This stimulatory action of AEDs on basal release was inhibited by BoNT/C predominantly. Ca2+‐evoked monoamine release was increased by low concentrations of CBZ, ZNS and VPA, but decreased by high concentrations. These effects of the AEDs on Ca2+‐evoked release were inhibited by BoNT/C, but not by BoNT/B. K+‐evoked monoamine release was reduced by AEDs concentration‐dependently. The inhibitory effect of these three AEDs on K+‐evoked release was inhibited by BoNT/B, but not by BoNT/C, These findings suggest that the therapeutic‐relevant concentration of CBZ, VPA and ZNS affects exocytosis of DA and 5‐HT, the enhancement of syntaxin‐mediated monoamine release during resting stage, and the inhibition of synaptobrevin‐mediated release during depolarizing stage.

Biphasic effects of carbamazepine on the dopaminergic system in rat striatum and hippocampus

To clarify the effects of carbamazepine (CBZ) on dopamine (DA) release and their metabolism, the extracellular and total levels of DA, its metabolites (DOPAC and HVA) and precursor, 3,4-dihydroxyphenylalanine (DOPA) in the striatum and hippocampus were studied. DA re-uptake and DOPA accumulation in the striatum and hippocampus, and monoamine oxidase (MAO) activities were also determined. After acute and chronic administrations of CBZ, the plasma concentration of CBZ associated with therapeutic activity increased the extracellular and total levels of all substances determined, whereas supratherapeutic concentration of CBZ decreased extracellular and total levels of all substances. Neither therapeutic nor supratherapeutic concentrations of CBZ affected MAO-A nor -B activities, nor DA re-uptake. DOPA accumulation caused by NSD1015 was inhibited by therapeutic and supratherapeutic concentrations of CBZ. These results suggest that a therapeutic concentration of CBZ enhances DA turnover, whereas a supratherapeutic concentration of CBZ inhibits DA turnover. These effects of CBZ on dopaminergic systems may be, at least partially, involved in the mechanisms of action of CBZ.

Effects of carbamazepine on hippocampal serotonergic system

To establish the mechanism of action of the antiepileptic and psychotropic effects of carbamazepine (CBZ), its effects on serotonin (5-HT) transmission, metabolism and re-uptake activity in the rat hippocampus were studied. After acute and chronic administrations of 25 mg/kg CBZ, the plasma concentration of CBZ was found to be within the therapeutic range, whereas both acute and chronic administrations of 50 and 100 mg/kg CBZ resulted in a supratherapeutic plasma concentration. Acute administration of the therapeutic dose of CBZ resulted in an increase in the hippocampal extracellular and total level of 5-HT, its metabolite, 5-hydroxydoleacetic acid (5-HIAA) and its precursor, 5-hydroxytryptophan (5-HTP). The acute administration of 50 mg/kg CBZ resulted in an increase in the hippocampal levels of extracellular 5-HT and 5-HIAA as well as in the total levels of 5-HTP, whereas hippocampal levels of extracellular 5-HTP, total 5-HT and 5-HIAA remained unaffected. CBZ at a dose of 100 mg/kg decreased the levels of all of these substances. After chronic administration, 25 mg/kg/day CBZ increased hippocampal total levels of 5-HT, 5-HTP and 5-HIAA, whereas 100 mg/kg/day CBZ decreased all of these total levels. CBZ at a dose of 50 mg/kg/day decreased total levels of 5-HT, however neither total levels of 5-HIAA nor 5-HTP were affected. Both therapeutic and supratherapeutic plasma concentrations of CBZ inhibited 5-HTP accumulation, and did not affect 5-HT re-uptake activity in vitro. These results suggest that a therapeutic concentration of CBZ enhances 5-HT turnover and transmission, whereas a supratherapeutic concentration of CBZ inhibits 5-HT turnover and transmission without affecting 5-HT re-uptake activity. These effects of CBZ on serotonergic systems may be, at least partially, involved in the mechanisms of action of CBZ.

Effects of Ca2+ channel antagonists on striatal dopamine and DOPA release, studied by in vivo microdialysis

1 To elucidate the mechanisms regulating the release of striatal dopamine and its precursor, 3,4‐dihydroxyphenylalanine (DOPA), we determined the effects of various Ca2+ channel antagonists, an N‐type Ca2+ channel antagonist, ω‐conotoxin GVIA, a P‐type Ca2+ channel antagonist, ω‐agatoxin IVA, and a Q‐type Ca2+ channel antagonist, ω‐conotoxin MVIIC, on the basal and Ca2+‐ and K+‐evoked release of striatal dopamine and DOPA, by use of in vivo microdialysis. 2 ω‐Conotoxin GVIA strongly inhibited striatal basal dopamine release (IC50=0.48 nM), whereas this toxin only weakly modulated basal striatal DOPA release (IC50=9.55 nM). Neither ω‐agatoxin IVA nor ω‐conotoxin MVIIC affected the basal striatal release of dopamine and DOPA. 3 ω‐Conotoxin GVIA strongly inhibited Ca2+‐evoked striatal dopamine release (IC50=0.40 nM), whereas Ca2+‐evoked striatal DOPA release only was weakly modulated (IC50=10.51 nM). Neither ω‐agatoxin IVA nor ω‐conotoxin MVIIC affected the Ca2+‐evoked release of striatal dopamine and DOPA. 4 Both ω‐agatoxin IVA and ω‐conotoxin MVIIC inhibited the K+‐evoked release of striatal dopamine (IC50 of ω‐agatoxin IVA=2.65 nM; IC50 of ω‐conotoxin MVIIC=12.54 nM) and DOPA (IC50 of ω‐agatoxin IVA=0.15 nM; IC50 of ω‐conotoxin MVIIC=3.05 nM), whereas ω‐conotoxin GVIA had no effect on the K+‐evoked release of striatal dopamine and DOPA. 5 An increase in the extracellular Ca2+ and K+ concentrations (Ca2+‐ and K+‐evoked stimulation) did not affect tyrosine hydroxylase activity in vivo. 6 These findings suggest that striatal DOPA release is neurotransmitter‐like and that, unlike the mechanisms of striatal dopaminergic transmission, this striatal DOPA transmission is at least partly regulated by voltage‐sensitive Ca2+ channels.

Pharmacological discrimination between effects of carbamazepine on hippocampal basal, Ca2+‐ and K+‐evoked serotonin release

To elucidate mechanisms of hippocampal serotonin release and possible mechanisms of clinical action of carbamazepine (CBZ), we determined interaction between antagonists of N‐type (ω‐conotoxin GVIA:GVIA), P‐type (ω‐agatoxin IVA:IVA) Ca2+ channels, Na+ channel (tetrodotoxin: TTX) and CBZ on hippocampal basal, Ca2+‐ and K+‐evoked serotonin releases, using microdialysis in freely moving rats. Basal release was reduced by TTX, GVIA and IVA (GVIA>IVA). Ca2+‐evoked release was reduced by GVIA but unaffected by TTX and IVA. K+‐evoked release was reduced by TTX, GVIA and IVA (GVIA

Effects of adenosine receptor subtypes on hippocampal extracellular serotonin level and serotonin reuptake activity.

Abstract: To clarify the effects of adenosine receptor subtypes (A1, A2, and A3) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5‐HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A1 receptor agonist, 2‐chloro‐N6‐cyclopentyladenosine, decreased extracellular 5‐HT levels, whereas an adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT), and caffeine increased these levels. A selective A2A receptor agonist (CGS‐21680), an adenosine A2 receptor agonist (PD‐125944), an adenosine A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX), and an adenosine A3 receptor agonist, N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5‐HT levels. When the adenosine A1 receptor was blocked by CPT, the hippocampal extracellular 5‐HT level was increased by adenosine, CGS‐21680, and PD‐125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A1 and A2 receptors were blocked by CPT and DMPX, the extracellular 5‐HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5‐HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A1 receptor was blocked, irrespective of whether the adenosine A2 receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5‐HT levels, during both adenosine A1 and A2 receptor blockade, were inhibited by selective 5‐HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A2 receptor and the inhibitory effects of the A3 receptor on hippocampal extracellular 5‐HT levels are masked by the inhibitory effects of the adenosine A1 receptor.