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

Abstract

1. 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, Ca(2+)- and K(+)-evoked release of dopamine (DA) and serotonin (5-HT) were determined by microdialysis in the hippocampus of freely moving rats. 2. 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. Ca(2+)-evoked release was inhibited by BoNT/C selectively. K(+)-evoked release was reduced by BoNT/B predominantly and BoNT/C weakly. 3. 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. 4. Ca(2+)-evoked monoamine release was increased by low concentrations of CBZ, ZNS and VPA, but decreased by high concentrations. These effects of the AEDs on Ca(2+)-evoked release were inhibited by BoNT/C, but not by BoNT/B. 5. 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. 6. 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.

Figure 1

Typical chromatograms of ECD-HPLC. (a,b) Typical chromatograms obtained from 20 μl of a standard solution containing DA and 5-HT, and hippocampal perfusate, respectively. (a) The four lines express chromatograms of different concentrations of DA and 5-HT (1, 2, 5 and 10 fmol). The quantification limits for DA and 5-HT were both 100 amol 20 μl⁻¹. (b) The three lines express chromatograms of basal, Ca²⁺- and K⁺-evoked releases.

Figure 2

Effects of AEDs on hippocampal basal releases of DA and 5-HT. The effects of CBZ, VPA and ZNS on the hippocampal basal 5-HT release are shown in a, c and e, respectively. The effects of CBZ, VPA and ZNS on the hippocampal basal DA release are shown in b, d and f, respectively. The ordinates indicate the mean±s.d. (n=6) of extracellular levels of 5-HT or DA (fmol sample⁻¹), and abscissas show the time in minutes (min). The open bars indicate perfusion with AEDs. The effects of CBZ, VPA and ZNS on hippocampal basal releases of DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 3

Concentration-dependent effects of AEDs on basal releases of hippocampal DA and 5-HT. The concentration-dependent effects of CBZ, VPA and ZNS on basal releases of hippocampal 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of basal releases of DA and 5-HT (fmol sample⁻¹), and abscissas indicate the concentration of AEDs (μM). The concentration-dependent effects of CBZ, VPA and ZNS on basal releases of hippocampal DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 4

Effects of AEDs on hippocampal Ca²⁺-evoked releases of DA and 5-HT. The effects of CBZ, VPA and ZNS on the hippocampal Ca²⁺-evoked 5-HT release are shown in a, c and e, respectively. The effects of CBZ, VPA and ZNS on the hippocampal Ca²⁺-evoked DA release are shown in b, d and f, respectively. The ordinates indicate the mean±s.d. (n=6) of extracellular levels of 5-HT or DA (fmol sample⁻¹), and abscissas show the time in minutes (min). The open bars indicate perfusion with AEDs, and stripped bars indicate the Ca²⁺-evoked stimulation for 20 min. The effects of CBZ, VPA and ZNS on hippocampal Ca²⁺-evoked releases of DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 5

Concentration-dependent effects of AEDs on Ca²⁺-evoked releases of hippocampal DA and 5-HT. The concentration-dependent effects of CBZ, VPA and ZNS on Ca²⁺-evoked releases of hippocampal 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of Ca²⁺-evoked releases of DA and 5-HT (fmol sample⁻¹), and the abscissas indicate the AEDs concentration (μM). The concentration-dependent effects of CBZ, VPA and ZNS on Ca²⁺-evoked releases of hippocampal DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 6

Effects of AEDs on hippocampal K⁺-evoked releases of DA and 5-HT. The effects of CBZ, VPA and ZNS on hippocampal K⁺-evoked 5-HT release are shown in a, c and e, respectively. The effects of CBZ, VPA and ZNS on the hippocampal K⁺-evoked DA release are shown in b, d and f, respectively. The ordinates indicate the mean±s.d. (n=6) of extracellular levels of 5-HT or DA (fmol sample⁻¹), and abscissas show the time in minutes (min). The open bars indicate perfusion with AEDs, and stripped bars indicate the K⁺-evoked stimulation for 20 min. The concentration-dependent effects of CBZ, VPA and ZNS on hippocampal K⁺-evoked releases of DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 7

Concentration-dependent effects of AEDs on K⁺-evoked releases of hippocampal DA and 5-HT. The concentration-dependent effects of CBZ, VPA and ZNS on K⁺-evoked releases of hippocampal 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of K⁺-evoked releases of DA and 5-HT (fmol sample⁻¹), and the abscissas indicate the concentration of AEDs (μM). The concentration-dependent effects of CBZ, VPA and ZNS on hippocampal K⁺-evoked releases of DA and 5-HT were compared using one-way ANOVA and Tukey's multiple comparison (*P<0.05; **P<0.01).

Figure 8

Interaction between AEDs and BoNTs on hippocampal basal releases of DA and 5-HT. The interactions between AEDs and BoNTs on hippocampal basal release of 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of basal releases of DA and 5-HT (fmol sample⁻¹). The interaction between AEDs and BoNTs on hippocampal basal releases of DA and 5-HT were compared using two-way ANOVA and Tukey's multiple comparison (control vs *P<0.05; **P<0.01, no AEDs vs #P<0.05; ##P<0.01).

Figure 9

Interaction between AEDs and BoNTs on hippocampal Ca²⁺-evoked releases of DA and 5-HT. The interactions between AEDs and BoNTs on hippocampal Ca²⁺-evoked release of 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of Ca²⁺-evoked releases of DA and 5-HT (fmol sample⁻¹). The interaction between AEDs and BoNTs on hippocampal Ca²⁺-evoked releases of DA and 5-HT were compared using two-way ANOVA and Tukey's multiple comparison (control vs *P<0.05; **P<0.01, no AEDs vs #P<0.05; ##P<0.01).

Figure 10

Interaction between AEDs and BoNTs on hippocampal K⁺-evoked releases of DA and 5-HT. The interactions between AEDs and BoNTs on hippocampal K⁺-evoked release of 5-HT and DA are shown in a and b, respectively. The ordinates indicate the mean±s.d. (n=6) of levels of K⁺-evoked releases of DA and 5-HT (fmol sample⁻¹). The interaction between AEDs and BoNTs on hippocampal K⁺ (control vs *P<0.05; **P<0.01, no AEDs vs #P<0.05; ##P<0.01).