In vitro odor-aversion conditioning in a terrestrial mollusk.
We developed an in vitro odor-aversion conditioning system in the terrestrial mollusk, Limax, and found a behavioral correlate of network oscillation in the olfactory CNS. We first examined the odor-induced behavior of Limax, after odor-aversion conditioning in vivo. Shortening of mantle muscles was specifically observed in response to aversively conditioned odors. We previously identified that parietal nerves, which project to the mantle muscle in Limax, regulate shortening of the mantle muscle. We therefore isolated whole brains containing noses (sensory organs) and parietal nerves (motor output), and applied an odor-aversion conditioning paradigm to these in vitro preparations. Before the in vitro conditioning, application of attractive odors to the noses did not elicit any discharge in the parietal nerves. However, after odor-aversion conditioning, discharges in the parietal nerves were observed in response to the natively attractive but aversively conditioned odors. We also found that network oscillation frequency in the procerebrum (PC), the olfactory CNS of Limax, increased specifically in response to the aversively conditioned odors that elicited avoidance behavior. In naive (nonconditioned) preparations, increases in the PC oscillation frequency were observed specifically in response to innately aversive odors. These results indicate that the isolated brains have an ability of odor learning. They also suggest that changes in PC network oscillation are associated with aversively conditioned and innately aversive odors, both of which elicit avoidance behavior. This in vitro conditioning system would be an effective approach for exploring the neural mechanism to determine the aversion to odors.