Effects of Zonisamide on K+‐Evoked Intracellular Ca2’ Mobilization, Releases of Serotonin, and Glutamate in Rat Hippocampus.
Authors: Kawata Yuko , Okada Motohiro , Murakami Takuya , Zuno Kazuhisa , Wada Kazumaru , Kaneko Sumo 
Published: August 2005

Purpose: Recently, it has been suggested that the dysfunction of K+ mobilisation is involved in the mechanisms or epileptic seizures. Therefore, to investigatethe mechanism of a novel antiepileptic drug, mnisamide (ZNS), the present study examined the effccts of ZNS on K+‐evoked elevation of intracellular Ca2+ levcl (Cazeli, and releases of scrotonin (5‐HT) and glutamate (Glu) in rat hippocampus.

Methods: (I) To examine the effects of therapeutic concentration of ZNS (TZ:0.2 mM) or supratherapcutic concentration of ZNS (SZ: I inM) on K+‐evokcd (50 and 100 mM) elcvation of [Ca2+]i, rat hippo‐ cainpal brain slices (350 km thick) loaded with 100 M Fura‐2/AM wcre prepared. The [C2+]i in thc hippocampal CAI, CA2, and CA3 rcgions was monitored by a fluorescence microscopy with a digital fluorcscence analyzer system (Hamamatsu photonics INC., Hama‐matsu, Japan) i n vitro. Thc superfiision with artificial‐cerebrospinali'luid (ACSF), ZNS (0.2 or 1 mM) containing ACSF was commenccd. After [CO2]i had stabilized, the supcrfusion inedium was switchcd to 50 or 100 mM K+ containing ACSF, TZ‐ACSF or SZ‐ACSF for 30 inin. (2) The hippocampal extracellular S‐HT level was determined by i n vivo microdialysis with ECD‐HPLC system (Eicom INC., Kyoto, Japan), i n frccly moving rats. Thc perfusion medium was commenced with modificd Ringer's solution (MRS) or 1 mM ZNS (estimated effectivc concentration was I87pM) containing MRS (Z‐MRS) and perfusion rtite was maintained at 1 I/min. Aftcr stabilization of thc extraccllular 5‐HT levcl, the perfusion medium was switched to 50 or 100 in M K+ containing MRS or Z‐MRS for 20 inin. (3) The real time monitoring of the hippocampal extracellular Glu level was determined by in vivo microdialysis glutamate bioscnsor which comprised an elcctrode to detcrmine Glu levcl and dialysis probe, in urethane anacsthctizcd rat. The perfusion ratc was maintained at I I/min, using MRS. The perfusion medium was commenced with MRS or Z‐MRS. After stabilization of the cxtracellular Glu level, the perfusion medium was switchcd to SO or 100 mM K+ containing MRS or Z‐MRS for 60 min.

Results: (I) The 50 mM K+‐evoked stirnulation produced a gentle rise of [C2+]i, however, the 100 mM K+‐cvoked stimulation produced a initial transient rise followed by the late gentle rise of [C2+]i. Su‐ perfusion with 0.2 and I mM ZNS inhibited both SO and 100 mM K+‐cvoked elevations of [C2+]i, in a concentration‐depcndeiit manner (P<O.O5). (2) K+‐cvoked stimulation (50 and 100 mM) increased the hippocampal extraccllular 5‐HT levels, in a concentration‐dependent mimncr (P<0.05). ZNS inhibited both 50 and 100 mM K+‐evoked 5‐HT rclease (P<O.OS). (3) The SO mM K+‐evoked stimulation produced the gentle rise of Glu release, however, I00 mM K+‐evoked stimulation produced multiplc phasic rises of Glu releases consisting of initial transient, late gcntlc and late multiple transient rises. ZNS inhibited these 4 typcs of Glu rcleases (P<O.OS).

Conclusions: The present study demonstrated that K+‐evoked stimulation produced various types of mobilizations of [C2+]i (the SO mM K+‐evoked gcntle rise, the 100 mM K+evoked initial transient and late gentle rises), rcleases of 5‐HT and Glu (thc SO mM K+‐evoked gentle rise, the 100 mM K+‐evoked initial transient rise, late gentle rise and the late multiple transient riscs). These elevations of [C2+]i, releases of 5‐HT and Glu induced by K+‐evoked stimulation wcre inhibited by therapeutic conccntration of ZNS. Thc present result suggcsts that ZNS reduces thc hyperactivity of neurons induccd by the dysfunction of K+ mobilizations. Thus, these effects of ZNS may, at least partially, be involved in the mechanisms of antiepileptic actions of ZNS.