DiI tracing in combination with immunocytochemistry for analysis of connectivities and chemoarchitectonics of specific neural systems in a teleost, the Atlantic salmon.

An important goal in neuroanatomical research is to identify the neurotransmitters in specific neural pathways. One step towards this goal is to combine experimental neuronal tracing with immunocytochemistry. Unfortunately, optimal procedures for nerve tracing and immunocytochemistry are not always compatible. Carbocyanine compounds have recently been shown to be efficient tracers both in vivo and in paraformaldehyde-prefixed neural tissue. The possibility to apply them to prefixed tissue make them suitable for tracing of neural pathways that are not easily accessible in vivo. We have optimized the procedures for neural tracing with one carbocyanine compound, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in the CNS of a teleost fish, and evaluated its compatibility with different immunocytochemical protocols. We have compared several immunocytochemical protocols, taking into account cryostat and vibratome sectioning, glutaraldehyde post-fixation to stabilize DiI, antibodies with different capacity for tissue penetration and the use of detergents, and antibodies with different sensitivity to prolonged paraformaldehyde fixation. We have also evaluated the choice of marker for immunoreactivity and compared indirect immunofluorescence techniques using different fluorophores, and the peroxidase-antiperoxidase (PAP) technique with or without nickel enhancement of the diaminobenzidine reaction product. It appears that DiI tracing of neural connections in the teleost CNS yields very consistent results and that the combination with immunocytochemistry is very reliable. We present four different basic protocols for combined DiI tracing and immunocytochemistry, with notes on their specific applicability. Owing to their reliability, the protocols may prove useful in comparative neuroanatomical studies of other vertebrates, particularly fish and amphibians, as well as in studies of developmental changes and neural plasticity in fish and amphibians.