A FIXATION PROCEDURE FOR ULTRASTRUCTURAL INVESTIGATION OF SYNAPTIC CONNECTIONS IN RESECTED HUMAN CORTEX

Electron microscopic investigations of the fine circuitry of human cen tral nervous system require a well-preserved tissue ultrastructure. Be cause the deterioration of subcellular structures occurs rapidly in po stmortem human brain, the use of a fixation by immersion of surgically resected human nervous tissue would be advantageous to investigate di rectly its synaptic circuitry. To obtain an optimal preservation of su bcellular elements in immersion-fixed brain tissue, different conditio ns of fixation were first tested on 400 mu m-thick sections of rat neo cortex. Parameters tested were temperature of the fixative solution, c oncentrations of glutaraldehyde and of cacodylate buffer with or witho ut microwave irradiation, and finally, the presence of dimethyl sulfox ide. The best ultrastructural preservation was obtained by immersing t he tissue in 0.1 M cacodylate buffer, 3.0 mM CaCl2, 2% paraformaldehyd e, 2.5% glutaraldehyde, and 2.5% dimethyl sulfoxide at 37 degrees C fo r 5 min and then at 4 degrees C for 4 h. This procedure of fixation wa s then applied to human neocortical tissue resected to alleviate tempo ral robe epilepsy. This method led to good tissue preservation in addi tion to retaining the antigenicity to the inhibitory amino acid neurot ransmitter, gamma-aminobutyric acid (GABA), Therefore, the tissue pres ervation obtained would permit these chemically defined connections to be investigated quantitatively at the electron microscopic level in r esected human cortex. (C) 1997 Elsevier Science Inc.