The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry

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Abstract
VD1 and RPD2 are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD1/RPD2 gene. To this end, in situ hybridization with the cDNA probe of the VD1/RPD2 gene and immunocytochemistry with antisera specific to VD1 and RPD2 (the α1-antiserum, Mab4H5 and ALMA 6) and to R15 (the α1 and 16-mer antisera) were performed on alternate tissue sections. A VD1/RPD2 neuronal system comprising three classes of neurons (A1–A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD1 and RPD2) immunoreact with the α1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1–5 small and 1–5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the α1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3–4 medium-sized neurons and a cluster of 4–5 small neurons located in the pedal ganglion) immunoreact with the α1-antiserum only. All neurons of the system are immunonegative to the R15 antisera. The observations suggest that the neurons of the VD1/RPD2 system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.

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