K. Stöcklin

Effect of carbamazepine, oxcarbazepine and lamotrigine on the increase in extracellular glutamate elicited by veratridine in rat cortex and striatum

Lamotrigine, carbamazepine and oxcarbazepine inhibit veratrine-induced neurotransmitter release from rat brain slices in concentrations corresponding to those reached in plasma or brain in experimental animals or humans after anticonvulsant doses, presumably due to their sodium channel blocking properties. Microdialysis measurements of extracellular glutamate and aspartate were carried out in conscious rats in order to investigate whether corresponding effects occur in vivo. Veratridine (10 μM) was applied via the perfusion medium to the cortex and the corpus striatum in the presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM in perfusion medium). Maximally effective anticonvulsant doses of carbamazepine (30 mg/kg), oxycarbazepine ( 60 mg/kg) and lamotrigine (15 mg/kg) were given orally.The uptake inhibitor increased extracellular glutamate and aspartate about 2-fold in striatum and about 7-fold and 3-fold, respectively, in cortex. Veratridine caused a further 2–3-fold increase in extracellular glutamate in striatum and cortex, respectively, but its effect on extracellular aspartate was less marked in both areas. None of the anticonvulsant compounds affected the veratridine-induced increases in extracellular glutamate or aspartate in the striatum which were, however, markedly inhibited by tetrodotoxin (1 μM) and thus are sensitive to sodium channel blockade. In the cortex, the same drugs at the same doses did cause about 50% inhibition of the veratridine-induced increase in extracellular glutamate. Carbamazepine and to a lesser extent lamotrigine, but not oxcarbazepine, also inhibited the veratridine-induced increase in extracellular aspartate in the cortex.Although our results might seem to support the view that inhibition of glutamate and aspartate release is responsible for the anticonvulsant effects of lamotrigine, carbamazepine and oxcarbazepine, two complementary findings argue against this interpretation. First, as previously shown, inhibition of electrically induced release of glutamate requires 5 to 7 times higher concentrations of these compounds than release elicited by veratrine. Second, the present study indicates that doses totally suppressing convulsions caused no inhibition in the striatum and at best a 50% inhibition in the brain cortex. From this we conclude that the doses used here, although to some extent effective against veratridine, did not suppress the release of GLU and ASP elicited by the normal ongoing electrical activity of the glutamatergic and aspartatergic neurons and that the mechanism of the suppression of convulsions must be sought elsewhere.

The reversible MAO inhibitor, brofaromine, inhibits serotonin uptake in vivo

The inhibition of the binding of [3H]cyanoimipramine in the rat brain in vivo, with and without pretreatment with proadifen (SK & F 525 A) to prevent its metabolism, has recently been shown to reflect inhibition of 5-HT uptake. This method has been suggested to be more sensitive than other more indirect methods. We have used this method to study the reversible MAO-A inhibitor, brofaromine, which has been shown previously to inhibit 5-HT uptake into synaptosomal preparations in vitro and ex vivo at doses about 30 times higher than those that inhibited MAO-A, and a number of 5-HT uptake inhibitors. The effects of orally administered clomipramine, imipramine, citalopram, CGP 6085 A, fluoxetine and brofaromine on [3H]cyanoimipramine binding were similar to those obtained in the synaptosome ex vivo model in the absence of proadifen; ifoxetine seemed to be more potent in inhibiting [3H]cyanoimipramine binding. The effects of clomipramine, imipramine, citalopram and ifoxetine were moderately to markedly enhanced by proadifen, indicating a first-pass effect. The effect of brofaromine on [3H]cyanoimipramine binding showed a time course similar to that on MAO-A, and was not altered upon repeated treatment. The inactivity of another MAO-A inhibitor, moclobemide, suggest that the effect of brofaromine was due to true inhibition of 5-HT uptake and not to displacement of [3H]cyanoimipramine by elevated synaptic 5-HT. The possible role of inhibition of 5-HT uptake in the antidepressant effect of brofaromine is discussed.

Weak effects of local and systemic administration of the GABA uptake inhibitor, SK&F 89976, on extracellular GABA in the rat striatum

SummaryThe effects of local and systemic administration of the potent GABA uptake inhibitor, SK&F 89976, on GABA overflow from the striatum of conscious rats were investigated in brain dialysis experiments. Administration of the compound via the dialysis probe at concentrations of 25 or 100 μgmol/l significantly increased basal GABA overflow about 2-fold. Overflow evoked by 104 mmol/l K+ remained unaltered at the lower and was almost doubled at the higher concentration; this increase did, however, not reach statistical significance.Given systemically at 50 mg/kg i.p., a dose which is severalfold higher than those which exhibit anticonvulsant effects, SK&F 89976 caused a significant enhancement of K+-stimulated GABA overflow by about a factor of 2; the lower dose of 20 mg/kg i.p. was not effective. Basal GABA overflow was not significantly increased by either dose. These results suggest that the marked effects of nipecotic acid on basal GABA overflow reported by several authors seem to be related to GABA displacement rather than uptake inhibition, and that uptake inhibition does not improve the interpretability of measurements of GABA release by brain dialysis. They neither support the idea that the relative insensitivity of extracellular GABA to low Ca2+ and tetrodotoxin is indirectly due to very efficient removal of GABA by neuronal and/or glial uptake, leaving only residual amounts to be measured.

Systemic administration of baclofen and the GABAB antagonist, CGP 35348, does not affect GABA, glutamate or aspartate in microdialysates of the striatum of conscious rats

SummaryPrevious in vitro experiments have shown that the GABAB agonist, baclofen, and the antagonist, CGP 35348, respectively, decrease and increase the autoreceptor-mediated release of GABA in brain slices and synaptosomes. Since it is not clear whether these autoreceptors are operative in vivo, an attempt was made to reproduce these results in brain dialysis experiments, knowing that only positive results would permit a conclusion in view of the doubts expressed in the literature with respect to the origin of extracellular GABA. Because of older reports of an inhibitory action of baclofen on the in vitro release of glutamate, which might be ascribed to the action of presynaptic GABAB heteroreceptors, extracellular glutamate and aspartate were also measured.Neither (−)-baclofen, administered systemically at a dose of 20 mg/kg i.p., nor the GABAB antagonist, CGP 35348 (300 mg/kg i.p.) had significant effects on basal overflow of GABA, glutamate, or aspartate nor on that evoked by 100 mmol/l K+ in the striatum of the conscious, freely moving rat. To ascertain this result, (−)-baclofen was also administered between two K+ stimulations, so that the first stimulation could serve as an intraindividual control of the second. The compound did not significantly affect K+ evoked overflow of any of the three transmitter amino acids under these conditions.It must be emphasized that these data do not exclude the operativity of presynaptic GABAB auto- and hetero-receptors in vivo. They only suggest that this question must, in all probability, be addressed by other techniques than brain dialysis.

Binding of [3H]brofaromine to monoamine oxidase A in vivo: displacement by clorgyline and moclobemide

Short duration of action, displaceability by endogenously released monoamines, and absence of cumulation of effect of the selective inhibitor of monoamine oxidase type A (MAO-A), brofaromine (CGP 11305 A), indicate reversibility of its interaction with the enzyme in vivo. However, its in vitro interaction with the enzyme showed features commonly associated with irreversible inhibition. To clarify this issue, the in vivo binding of [3H]brofaromine to MAO-A in areas of the rat brain, and in rat heart and liver was investigated. Specific binding, defined by pretreatment with the irreversible inhibitor, clorgyline, was between 15 and 75% of total binding depending ont eh tissue and the time elapsed after injection of radioactivity. In brain and heart tissue, unlabelled brofaromine, another reversible inhibitor of MAO-A, moclobemide and clorgyline were able to displace [3H]brofaromine when administered after the labelled compound with ED50s of 1-3 mg/kg p.o., 3 mg/kg p.o. and 0.3-1 mg/kg s.c., respectively. In the liver, brofaromine and moclobemide and the inhibitor of drug-metabolizing enzymes, proadifen (SK&F 525 A), were able to significantly inhibit [3H]brofaromine binding. Clorgyline was only marginally effective, suggesting that, in this organ, [3H]brofaromine binds predominantly to such enzymes. In conclusion, the binding of [3H]brofaromine to MAO-A in rat brain and heart in vivo was found to be displaceable by other MAO inhibitors and is therefore reversible. In the liver, the compound bound predominantly to other sites, probably microsomal drug-metabolizing enzymes.

Determination of rat brain and plasma levels of the orally active GABAB antagonist 3-amino-propyl-n-butyl-phosphinic acid (CGP 36742) by a new GC/MS method

An involvement of GABAergic neurons has been suggested in the process of memory consolidation based on anatomical evidence and increasing physiological and biochemical data. With the advent of orally active GABAB antagonists, such as CGP 36742, the question of their therapeutic value, for example in Alzheimers disease, becomes relevant. Therefore, a new GC/MS method was developed to determine the concentration of CGP 36742 (3-amino-propyl-n-butyl phosphinic acid) in various intra- and extracerebral tissues after different routes of application. The compound was chemically derivatised in a two-step process (acylation of the amino group and esterification of the phosphinic acid). The limit of detection of the method was 0.01 microgram/g tissue and 0.0005 microgram/mL plasma. The time-course after i.p. treatment showed peak levels of CGP 36742 between 30 min and 1 hr after injection. After a dose of 100 mg/kg, the concentration in the brain ranged from 1 to 1.4 microgram/g or 6 to 8 microM, assuming that 1 mg tissue equals 1 microL (i.e., below the IC50 of the interaction with GABAB receptors as measured by [3-3H]-aminopropyl-phosphinic acid binding [35 microM]). These results are discussed in light of the psychopharmacological effects (improvement of cognitive performance of rats) of CGP 36742 observed at very low oral doses.

Effects of CGP 28014 on the in vivo release and metabolism of dopamine in the rat striatum assessed by brain microdialysis

The effects on rat striatal dopamine (DA) metabolism of systemic and local administration of CGP 28014, an inhibitor of catechol-O-methyl-transferase (COMT), were studied by in vivo microdialysis. CGP 28014 (30 mg/kg i.p.) significantly reduced the levels of homovanillic acid (HVA), but did not modify DA and 3,4-dihydroxyphenylacetic acid (DOPAC). The intrastriatal administration (via the microdialysis probe) of 5, 7.5, 10, and 20 mM of CGP 28014 elicited a concentration-dependent, several-fold increase in extracellular DA but did not alter the levels of HVA and DOPAC. Thus, the effects of CGP 28014 observed after i.p. injection (decrease in HVA levels) are different from those measured after intrastriatal administration (increase in DA release). Therefore, the inhibition of COMT is likely to be due to the action of a metabolite of CGP 28014 formed in the periphery and not in the brain.

Effects of baclofen on striatal 5-HT mediated by the nigrostriatal dopamine system.

In rats, baclofen injected unilaterally into the substantia nigra increased dopamine (DA) and serotonin (5-HT) levels in the ipsilateral striata. Homovanillic acid levels first decreased and then increased to a maximum after 4 h. The content of 3,4-dihydroxyphenylacetic acid also increased after a latency of about 1 h. The effects of baclofen on striatal DA metabolism are consistent with the assumption that impulse flow in these DA neurones was diminshed by the drug. DA metabolites were also found in greater quantities in the contralateral striata, probably owing to compensatory activation of these DA neurones. Baclofen injected intranigrally did not increase the striatal 5-HT content in rats with a 6-hydroxydopamine-induced lesion of the nigrostriatal DA pathway. Our results suggest the presence of DA receptors on striatal 5-HT terminals or on interneurones synapsing axo-axonally with them.

Displacement of in vivo binding of [3H]brofaromine to rat intestinal monoamine oxidase A by orally administered tyramine.

The reversibility of the interaction of inhibitors with monoamine oxidase (MAO) is thought to provide a safety valve with respect to tyramine potentiation. We sought experimental evidence for this concept by studying the binding of orally administered [3H]brofaromine to the A-form of MAO in the rat ileum. Specific binding, defined by pretreatment with 10 mg/kg clorgyline p.o., amounted to 70-90% of total binding between 30 min and 6 h after administration of the radioligand. Brofaromine and clorgyline dose dependently displaced [3H]brofaromine with ED50 values of about 0.2 mg/kg when administered orally after the radioligand; so did orally administered tyramine in doses relevant for tyramine potentiation in the rat. It was also found that tyramine was relatively more effective in partially MAO-inhibited rats. The data suggest that the concept of reversibility functioning as a safety valve with respect to the potentially hazardous effects of tyramine ingestion is realistic.

Differential effects of competitive and noncompetitive NMDA receptor antagonists on GABA turnover in the mouse brain

The modulation of GABA-mediated neurotransmission by excitatory amino acid inputs was examined by testing the effects of competitive NMDA receptor antagonists (CPP, CGS 19755, CGP 37849 and CGP 39551) and noncompetitive antagonists (dizocilpine, PCP, ketamine, dextromethorphan and SKF 10047) on the rate of GABA synthesis and depletion, two indices of GABAergic neuronal activity. The rate of GABA synthesis in the mouse was assessed in four brain regions by the elevation in GABA levels after inhibition of GABA-T with gabaculine. The rate of GABA depletion was estimated from the decrease in GABA concentrations observed after inhibition of GAD with isoniazid. The administration of anticonvulsant doses of all competitive NMDA antagonists dose-dependently decreased the rate of GABA synthesis. With the exception of CGP 39551, the time course of effects of competitive antagonists on the rate of GABA synthesis corresponded with the time course of their anticonvulsant potencies. Unlike the competitive antagonists, the noncompetitive blockers, dizocilpine, PCP, dextromethorphan and SKF 10047 had no effect on the rate of GABA synthesis.