Heterogeneous Distribution of Functional Glutamate Receptor Subtypes in Organotypic Slice Culture of Rat Hippocampus Revealed by Calcium Fluorometry

Abstract

The heterogeneous distribution of functional glutamate receptor subtypes was visualized by calcium fluorometry in the fura-2-loaded organotypic slice culture of the rat hippocampus. The organotypic culture prepared according to the method described by Gähwiler (1984) was loaded with fura-2, a fluorescent Ca²⁺ indicator, and subjected to image analysis. In the culture the cells grew in the mono- or bi-layer manner on the glass coverslip preserving their original arrangement. Monitoring the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), we detected the synaptic activities as spontaneous and electrically evoked [Ca²⁺]_i changes. Each of the agonists for glutamate receptor subtypes (at a concentration of 10 μM) showed characteristic two-dimensional patterns of [Ca²⁺]_i change. The responses to N-methyl-D-aspartic acid (NMDA) and DL-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) were always the largest in the CA1 region, but that to kainic acid was the largest in the CA3 region. Trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (t-ACPD) produced a clear elevation of [Ca²⁺]_i in the cells that migrated out of the margin of the slice, which were negative in the immunohistochemical staining with a neuron-specific marker, MAP-2 antibody. Present results confirmed the heterogeneous distribution of functional glutamate receptor subtypes in the hippocampus which we previously described in a fresh slice preparation.