Carbocyanine postmortem neuronal tracing: Influence of different parameters on tracing distance and combination with immunocytochemistry

Carbocyanines (Dil, DiA, DiO) are able to travel along membranes by diffusion and therefore have been used as postmortem neuronal tracers in aldehyde-fixed tissues. Surprisingly, detailed data on the influence of different parameters on tracing distances are still missing. This study was carried out to optimize tracing procedures and to reveal the validity of the combination of postmortem tracing with immunocytochemistry. Carbocyanine crystals were applied to the cervical spinal cord, sciatic nerves, and brachial plexuses of humans and guinea pigs. Incubation in the dark at 37C for 12-15 weeks proved optimal to achieve longest tracing distances (28.9 +/- 2.2 mm) in human and animal tissues. Longer incubation times and incubation temperatures higher than 37C did not result in longer tracing distances. No differences were evident between adult acid newborn animals and between central and peripheral nervous system. The diffusion coefficient for Dil was calculated to be 2.5 x 10(-7) cm(2) sec(-1). After application of Dil to nerves of guinea pig extraocular muscles, Dil-positive afferent perikarya were observed iri the anteromedial part of the trigeminal ganglion. These perikarya were identified by calcitonin gene-related peptide immunoreactivity (CG RP-IR). The percentage of CGRP-IR neurons after tracing was concordant with the percentage of CGRP-IR in trigeminal ganglia exclusively processed for CGRP-IR without previous postmortem tracing. These results demonstrate carbocyanines to be specific tracers for exact neuronal mapping studies.