The idea that an excessive release of glutamate mediates neuronal cell death was proposed, as the excitotoxicity hypothesis, by Olney and de-Gubareff.1) Glutamate activates two types of its receptor, N-methyl-D-aspartate (NMDA) type and non-NMDA one.2) Excessive activation of glutamate receptors, resulting in increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) and generation of reactive oxygen species (ROS), is involved in neuronal disorder and degeneration.
3)Both phenomena induced by glutamate are related to cell injury and death. 4,5) In fact, antagonists for NMDA and non-NMDA glutamate receptors are proven to possess neuroprotective action. [6][7][8] Extract of Ginkgo biloba leaves (EGBL) is expected to treat patients with some neuronal disorders such as dementia and Alzheimer's disease. 9,10) The extract exerts protective action (or antioxidant action) on neurons suffering from oxidative stress induced by hydrogen peroxide, one of ROS, and by overload of intracellular Ca 2ϩ . 7,11,12) EGBL and its constituents inhibit the response mediated by activation of NMDA receptor. 13,14) However, there is limited information concerning the effect of EGBL on response induced by activation of non-NMDA glutamate receptor. Kainate, an agonist for non-NMDA glutamate receptor, increases [Ca 2ϩ ]i in neurons via activations of non-NMDA glutamate receptor and voltage-dependent Ca 2ϩ channel. 15) Therefore, in order to elucidate one of mechanisms for neuroprotective action of EGBL, we examined the effect of EGBL on kainate-induced increase in [Ca 2ϩ ]i in rat brain neurons using a laser microscope with fluorescent Ca 2ϩ -indicator.
MATERIALS AND METHODS
MaterialsExtract of Ginkgo biloba leaves (EGBL) was obtained from Icho-Ha Sangyo Co., Ltd., Ibaraki, Japan. EGBL was initially dissolved in 50% ethanol. EGBL solution (1-30 mg/ml) was added to bathing solution to achieve appropriate concentrations (1-30 mg/ml). Ethanol as a solvent at 0.05% did not affect any fluorescence measurements from cells. Other chemical reagents except for fluorescent probes were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) unless otherwise mentioned.Cell Preparation Experimental method for dissociation of rat cerebellar neurons was similar to those described in previous studies. 16,17) In brief, the slices of cerebellum dissected from 10-to 16-d-old Wistar strain rats were treated with 1000 protease units/ml dispase (Godo Shusei, Tokyo, Japan) in Tyrode's solution for 50-60 min at a temperature of 32-34°C. After enzymatic treatment, cerebellar neurons were dispersed by triturating in Tyrode's solution. Since the majority of neurons were characterized by small size and spherical shape under microscopic observation, they were ascertained as cerebellar granule cells. These cells responded to kainate, resulting in an increase in [Ca 2ϩ ]i.15) The cells were placed on culture dishes and incubated at 35-36°C in Tyrode's solution for 50-60 min before use. The application of chemicals to neurons was performed by a Y-tube...