A fixation procedure for ultrastructural investigation of synaptic connections in resected human cortex.

ABSTRACT

Electron microscopic investigations of the fine circuitry of human central nervous system require a well-preserved tissue ultrastructure. Because the deterioration of subcellular structures occurs rapidly in postmortem human brain, the use of a fixation by immersion of surgically resected human nervous tissue would be advantageous to investigate directly its synaptic circuitry. To obtain an optimal preservation of subcellular elements in immersion-fixed brain tissue, different conditions of fixation were first tested on 400 microns-thick sections of rat neocortex. Parameters tested were temperature of the fixative solution, concentrations of glutaraldehyde and of cacodylate buffer with or without microwave irradiation, and finally, the presence of dimethyl sulfoxide. The best ultrastructural preservation was obtained by immersing the tissue in 0.1 M cacodylate buffer, 3.0 mM CaCl2, 2% paraformaldehyde, 2.5% glutaraldehyde, and 2.5% dimethyl sulfoxide at 37 degrees C for 5 min and then at 4 degrees C for 4 h. This procedure of fixation was then applied to human neocortical tissue resected to alleviate temporal lobe epilepsy. This method led to good tissue preservation in addition to retaining the antigenicity to the inhibitory amino acid neurotransmitter, gamma-aminobutyric acid (GABA). Therefore, the tissue preservation obtained would permit these chemically defined connections to be investigated quantitatively at the electron microscopic level in resected human cortex.