Behavioral and cellular analysis of classical conditioning of feeding behavior in Aplysia

In classical conditioning, an associative form of learning, animals learn to associate two stimuli. Cellular and molecular mechanisms for the induction and consolidation of associative learning and memory at the level of single cells and synaptic connections have been studied in both vertebrate and invertebrate animals. The majority of studies, however, relied on aversive stimuli to induce learning. This bias may limit the extent to which identified mechanisms generalize to other forms of associative learning and memory, such as appetitive forms. The goal of the present study was to develop a classical conditioning procedure for the marine mollusk Aplysia californica using appetitive reinforcement, and to analyze associative learning using behavioral and electrophysiological techniques. Using tactile stimulation of the lips as the conditional stimulus (CS) and food as the unconditional stimulus (US) a training protocol was developed that reliably induced classical conditioning of feeding behavior. Memory persisted for at least 24 hours. The gross organization of reinforcement-mediating pathways was analyzed in additional behavioral experiments. Moreover, neurophysiological correlates of classical conditioning were identified and characterized in an in vitro preparation containing the circuitry for feeding behavior. In vitro stimulation of a nerve (AT4) that may mediate the CS during training, resulted in a greater number of buccal motor patterns (BMPs) in brains from conditioned animals, as compared to control animals. The majority of these BMPs were ingestion-like, consistent with the increased number of bites in response to the CS after classical conditioning. Moreover, classical conditioning correlated with increased excitatory synaptic input to BMP-initiating neuron B31/32, in response to stimulation of AT 4, as compared to controls. The expression of the correlates of classical conditioning identified in this study was specific to stimulation of AT 4, which is consistent the stimulus specificity that is characteristic for classical conditioning. The identification of cellular correlates of classical conditioning documented here provides the basis for future, more detailed analyses of an appetitive form of associative learning and memory, that may extend the working knowledge of the cellular and molecular mechanisms for associative plasticity in general