Effects of Antiepileptic Drugs on K + ‐Induced Glutamate Release and Spreading Depression in the Hippocampus Determined with an In Vivo Microdialysis Glutamate Biosensor (MGB)

Purpose : Glutamate is the principal excitatory neurotransmitter in the central nervous system, and excessive release of glutamate may produce seizures in patients with epilepsy and in various animal models of epilepsy. Glutamatergic transmission also plays an important role in the development of neuronal damage after a variety of insults to the brain, including those caused by status epilepticus. Thus, to clarify the effects of the antiepileptic drugs (AEDs) carbamazepine (CBZ) and zonisamide (ZNS) on the multiple components of hippocampal glutamate release, this study was carried out to determine the Ca 2+ ‐ and K + ‐evoked glutamate release in rat hippocampus with an in vivo microdialysis glutamate biosensor (MGB). Methods : The MGB was composed of a dialysis electrode (DE), a microdialysis probe, and a stainless steel recording electrode. The DE was filled with 100 U/ml of glutamate oxidase dissolved in a phosphate‐modified Ringer's solution (PMRS) containing (in m M ): 145 Na + , 2.7 K + , 1.0 Mg 2+ , 154.4 C1 − and adjusted with 4.3 m M phosphate buffer to pH 7.4. The MGB was implanted into the rat hippocampus (A =−5.8 L = 4.8, and V =−4.0 mm from Bregma). The perfusion rate was maintained at 1.0 μl/min, by using a modified Ringer's solution (MRS) composed of (in m M ): 14.5 Na + , 2.7 K + , 1.2 Ca 2+ , 1.0 Mg 2+ , 154.4 Cl − , 0.2 ascorbate, and adjusted to pH 7.4 with 2 m M phosphate buffer and 1.1 m M Tris buffer. To study the effects of an increase in extracellular Ca + or K + levels (Ca 2+ ‐ or K + ‐evoked) on hippocampal glutamate release, MRS including either 3.4 m M Ca 2+ , 50 m M K + , 100 m M K + , 3.4 m M Ca 2+ , and 50 m M K + , Ca 2+ ‐free, and 50 mM K + , or Ca 2+ ‐free and 40 m M Mg 2+ was infused for 60 min. To study the effects of CBZ and ZNS on basaMlippocampa1 extracellular glutamate levels, and on Ca 2+ ‐ or K + ‐evoked hippocampal glutamate release, 100 m M CBZ or 1 m M ZNS was dissolved in each perfusate. The neuronal firing frequencies were recorded with a telemeter set at a bandpass of 0.1–3 kHz and were fed into the computer as discharge rates. Results : An increase in the extracellular K + levels produced concentration‐dependent increases in the extracellular glutamate levels in the hippocampus. This K + ‐evoked glutamate release consisted of three components: an initial transient rise (ITR), a late gentle rise (LGR), and multiple phasic transient rises (MPTR). An increase in the extracellular Ca 2+ levels did not affect hippocampal extracellular glutamate levels; however, it did enhance the K + ‐evoked hippocampal glutamate release. Therapeutic concentrations of either CBZ and ZNS had no effect on basal extracellular glutamate levels in the hippocampus, whereas they did inhibit the K + ‐evoked hippocampal glutamate release. Therapeutic concentrations of CBZ or ZNS also inhibited the stimulatory effects of Ca 2+ on K + ‐evoked hippocampal glutamate release. Conclusions : This study demonstrated that K + ‐evoked hippocampal glutamate release consists of three phases: Ca 2+ ‐dependent ITR, which was dependent on neuronal activity, and followed by the LGR and MPTR, which were Ca 2+ dependent and neuronal activity independent (spreading depression). The inhibitory effect of ZNS on ITR was stronger than that of CBZ, whereas the inhibitory effects of CBZ on the series of LGR and MPTR of K + ‐evoked hippocampal glutamate release were more effective than those of ZNS. The new method used in this study, the MGB, should be useful for studying the mechanisms of action of AEDs.