Neural changes after operant conditioning of the aerial respiratory behavior in Lymnaea stagnalis

In this study, we demonstrate neural changes that occurred during operant conditioning of the aerial respiratory behavior of Lymnaea stagnalis. Aerial respiration in Lymnaea occurs at the water interface and is achieved by opening and closing movements of its respiratory orifice, the pneumostome. This behavior is controlled by a central pattern generator (CPG), the neurons of which, as well as the motoneurons innervating the pneumostome, have previously been identified and their synaptic connections well characterized. The respiratory behavior was operantly conditioned by applying a mechanical stimulus to the open pneumostome whenever the animal attempted to breathe. This negative reinforcement to the open pneumostome resulted in its immediate closure and a significant reduction in the overall respiratory activity. Electrophysiological recordings from the isolated CNSs after operant conditioning showed that the spontaneous patterned respiratory activity of the CPG neurons was significantly reduced. This included reduced spontaneous activity of the CPG interneuron involved in pneumostome opening (input 3 interneuron) and a reduced frequency of spontaneous tonic activity of the CPG interneuron [right pedal dorsal 1 (RPeD1)]. The ability to trigger the patterned respiratory activity by electrical stimulation of RPeD1 was also significantly reduced after operant conditioning. This study therefore demonstrates significant changes within a CPG that are associated with changes in a rhythmic homeostatic behavior after operant conditioning.