Two-color in situ hybridization in the CNS of Aplysia californica

Aplysia californica is an attractive model organism for cellular and systems neuroscience. Currently, there is a growing body of sequence data from Aplysia that includes many interesting genes. To fully exploit this molecular data it must be integrated with the large body of physiological data that are already available for identified neurons in Aplysia networks. In situ hybridization is a powerful technique that enables this to be done. Expression patterns of selected mRNA transcripts can be mapped to individual cells in the central nervous system (CNS). Here, we describe a detailed non-radioactive in situ hybridization protocol optimized for whole-mount preparations of Aplysia ganglia. The indirect alkaline phosphatase-based chromogenic detection method we employ may be used with one or two colors in order to detect one or two different transcripts in the same preparation. The procedure is also compatible with intracellular dye labeling, making it possible to couple localization of transcripts with electrophysiological studies in positively identified neurons. Double labeling was done for transcripts encoding the neuropeptides FMRFamide and sensorin. The sensitive detection of mRNA and great preservation of CNS morphology makes this method a useful tool for analyzing expression patterns of neuron specific genes in Aplysia.