Localization of opioid binding sites in rat brain by electron microscopic radioautography

Abstract

AbstractTwo met‐enkephalin analogs (FK 33–824 and FW 34–569, Sandoz) were utilized for in vitro labeling of opioid binding sites in the rat central nervous system. Binding kinetics determined in 20‐μm‐thick frozen tissue sections of the striatum revealed that both pentapeptides bind to a single population of sites at 20°C with an apparent dissociation constant (KD) of approximately 1–2 nM and a maximum capacity (B max) of 65–170 fmoles/mg protein. Radioautographic data suggest that this population is the same for iodinated and tritiated forms of the FK compound and the iodinated FW analog. Fixation of labeled sections with high concentrations of glutaraldehyde allowed proportional retention of more than 50% of specifically bound 125I‐FK molecules in all brain regions after histological processing for high‐resolution radioautography. In contrast, glutaraldehyde fixation did not prevent the loss of bound 125I‐FW molecules. These differences are attributed to the presence in FK, but not in FW molecules, of a free primary amino group considered essential for cross‐link formation between aldehydes and proteins, and imply that a majority of FK‐receptor complexes may be stabilized by glutaraldehyde. Consistent with this observation is the fact that the radioautographic distribution of specifically bound 125I‐FK was unchanged after fixation and dehydration. In electron microscopic radioautographs prepared from prefixed, vibratome‐cut striatal sections that were incubated with 125I‐FK and fixed with glutaraldehyde, silver grains were found to be mostly associated with neuronal plasma membrane interfaces. The present methodological approach thus appears to be compatible with electron microscopic localization of opioid binding sites in the central nervous system and might be applicable to the localization of other types of binding sites using radioligand molecules that contain a free primary amino group.