Effects of Valproate on K+‐Evoked Serotonin Release and Intracellular Ca2+ Elevation in Rat Hippocampus.
Authors: Murakarni Takuya , Okada Motohiro , Kawata Yuko , Mizuno Kazuhisa , Wada Kazumaru , Kancko Sunao 
Published: August 2005

Purpose: Several investigations have suggested that voltagescnsitive Ca+ channcl (VSCC) subtypes activities modulate seizure threshold. In addition, it is well established that presynaptic VSCC activity regulates the releases of neurotransmitters. We have already demonstrated that several types of inonoamine (MA) releases are regulated by N‐, P‐, and Q‐type VSCC subtypes. Specifically, our findings have shown that basal and Ca2‐evoked MA rclease arc rcgulated by N‐type VSCC, whereas K+‐evoked MA rclcase is regulated by P/Q‐ type VSCC. Therefore, to clarify the mechanisms of anticpileptic action of valproate (VPA), the present study determined the effect of VPA on K+‐evoked hippocampal neurotransmitter rclease and intracellular Ca2+ mobilization.

Methods: To study thc elfects of VPA (700pM or 3.5inM) on K+‐evoked (SO and 100 mM) elevation of intraccllular Ca2+ levels, we prepared the rat hippocampal brain sliccs (350 pm thick) loaded with 100 pM Fura‐2/AM. lntracellular Ca2+ levels were determined using fluorescence microscopy with digital fluorescencc analyzer system. The microscope was equipped with a computer‐driven interfcrence filter cxchange system, and 340 or 380 nm filters were usc for measurement. Then, 3401380 nm fluorescence ratio was calculated by tligital fluorescence analyzer (Argus‐50). Fura‐2 loaded hippocampal slices were pcrfused continuously with 37°C oxygenated A‐CSF at 2ml/min flow rate. Slices were alternately excited with 340 and 380 nm light. The ratio or 2 signals was used to determine the change in intracellular free Ca2+. The effects of 3 mM VPA (estimated eflective concentration in the brain tissue: 693 pM) on 50 and 100 mM K+‐cvoked hippocam pal 5‐HT relcasc were deterinincd by in vivo microdialysis with ECD‐HPLC system in freely moving rats. A concentric dialysis probe was implanted into the hippocampus. The perfusion rate was always llp, 1/min, using modified Ringer's solution (MRS). MRS containing 50 or 100 mM K+ was perrused for 20 minutes.

Results: Fifty and I00 mM K+‐evoked stimulation produced the elevation of intracellular Ca2+ levels. Prc‐superfusion with therapeutic (700/AM) and supratherapcutic (3.5 mM) levels of VPA inhibited both 50 and I00 mM K+ ‐evoked elcvation of hippocampal intraccllular Ca2+ levels in a concentration‐dependent manner (p<O.OS). An increase in the extraccllular level of K+ (from 2.7 lo SO or 100 mM) produced the elevation of hippocampal extraccllular levels of 5‐HT. Pre‐perfusion with a therapeutic concentriition of VPA inhibited the S O and 100 mM K+‐evoked 5‐HT release (p<O.OS).

Conclusions: Elevation of the intracellular Ca2+ level is produced by 2 major pathways. The first is an increasing the inflow of Ca2+ from extracellulu space by passing through several types of VSCC, ionotropic glutamate receptor and nicotinic acetylcholine receptor linked to ion channels. The other is the enhancement of intracellular Ca2+ mo‐ biliation. lectrophysiological experiments have demonstrated the existence 0 1 at least 7 subtypes of VSCC (L‐,N‐,0‐,P‐,Q‐,R‐and T‐types). I n our previous study, we have clarified the effects of N‐, P‐, and Q‐type VSCCs on various types of MA relcase. Both basal and Ca2+‐ evoked MA releases were regulated by N‐type VSCC, whereas K+‐ evoked MA release was regulated by P/Q‐type VSCC. These results suggest that a combination between inhibition of P/Q‐type VSCC, which regulate release of excitatory neurotransmitters, with an activation of N‐type VSCC, which regulates release of inhibitory neurotransmitters, might elevate seizure threshold, yielding the anticonvulsive activity. I n the present study, VPA reduced the K+‐evoked elevation of hippocampal intraccllular Ca2+ level in a concentration‐dependent manner. The therapeutic concentration of VPA inhibited K+‐evoked hippocampal MA release, which might he regulated by P/Q‐type VSCC activity. These rcsults suggest that the inhibitory effects of VPA on K+‐cvokcd hippocampal excitation may involve in the mechanisms of action of the anticpileptic action of VPA.