Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish.

A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.