Cholecystokinin-like immunoreactivity in the dorsal horn of the rat spinal cord: an attempt to analyse contradictory results between immunocytochemistry and radioimmunoassay.

This study investigated the nature of the cholecystokinin-like immunoreactivity (CCKLI) in the dorsal horn of the rat spinal cord. Indeed, using the same antiserum, no variation of radioimmunoassayable CCKLI in the dorsal horn of rats treated neonatally with capsaicin could be seen, whereas the density of immunoreactive CCKLI fibres was greatly reduced in immunocytochemistry (IC). By comparing the technical conditions used in IC and radioimmunoassay (RIA), it could be concluded that the contradictory results obtained by the two techniques were very unlikely due to the fixation of the tissues, but rather to the concentration of the antiserum. First, CCKLI levels in extracts from fresh or 4% paraformaldehyde fixed tissues were not significantly different. Second, glomerular terminals, which are typical terminals of the fine primary afferents were immunoreactive at the surface of the block but unlabelled in its depth, where the concentration of antiserum might gradually reach a value closer to that used in radioimmunoassay. High pressure liquid chromatography analyses of acidic extracts of the dorsal part of the lumbar spinal cord of control rats revealed the presence, besides two major peaks coeluting with sulphated and non-sulphated CCK8, of two minor peaks '1' and '2' accounting for 23% of total CCKLI. Capsaicin treatment resulted in the disappearance of peak 2 and a marked reduction in the proportion of CCKLI in peak 1. Reported data suggested that the CCKLI material present in capsaicin-sensitive neurons, accounting for only about 10% of radioimmunoassayable CCKLI but for a much higher proportion of that detected in IC, was distinct from both genuine CCK and CGRP.