Mapping of interneurons that contribute to food aversive conditioning in the slug brain

To determine the distribution of neurons that contribute to memory formation induced by odor-taste associative conditioning in the slug's brain, we examined neuronal activity of the central nervous system of the slug Limax marginatus using a fluorescent activity marker [Lucifer yellow (LY)]. When LY was injected into the body cavity just after the conditioning, many of the procerebral (PC) interneurons were labeled. The PC lobe was considered to play important roles in the olfaction of the slug, because the olfactory afferent fibers from both the inferior and the superior tentacular noses innervate it. Such strong dye-uptake activity of PC interneurons was not observed when LY was injected just after unpaired control treatment. Thus, it was suggested that enhancement of dye-uptake activity upon conditioning was caused by the association of a conditioning stimulus (CS) with an unconditioned stimulus (UCS). The distribution patterns of PC interneurons that were labeled by LY after conditioning showed a characteristic feature: They usually formed a belt-shaped cluster parallel to the dorsoventral axis. This feature of the distribution was maintained when different odors were used as a CS. Furthermore, the number of the clusters reflected the number of CS odors but not the number of conditioning sessions. From these observations, we considered that enhancement of the neural activity involving dye uptake in each belt-shaped cluster contributed to formation of each odor memory.