Operant conditioning is a form of associative learning through which an animal learns about the consequences of its behavior. Here, we report an appetitive operant conditioning procedure in Aplysia that induces long-term memory. Biophysical changes that accompanied the memory were found in an identified neuron (cell B51) that is considered critical for the expression of behavior that was rewarded. Similar cellular changes in B51 were produced by contingent reinforcement of B51 with dopamine in a single-cell analog of the operant procedure. These findings allow for the detailed analysis of the cellular and molecular processes underlying operant conditioning.

SUMMARY Operant conditioning is a ubiquitous but mechanistically poorly understood form of associative learning in which an animal learns the consequences of its behavior. Using a single-cell analogue of operant conditioning in neuron B51 of Aplysia, we examined second-messenger pathways engaged by activity and reward and how they may provide a biochemical association underlying operant learning. Conditioning was blocked by Rp-cAMP, a peptide inhibitor of PKA, a PKC inhibitor and by expressing a dominant negative isoform of Ca2+-dependent PKC (apl-I). Thus, both PKA and PKC were necessary for operant conditioning. Injection of cAMP into B51 mimicked the effects of operant conditioning. Activation of PKC also mimicked conditioning, but was dependent on both cAMP and PKA, suggesting that PKC acted at some point upstream of PKA activation. Our results demonstrate how these molecules can interact to mediate operant conditioning in an individual neuron important for the expression of the conditioned behavior.

Learning can lead to changes in the intrinsic excitability of neurons. However, it is unclear to what extent these changes persist and what role they play in the expression of memory. Here, we report that in vitro analogues of operant conditioning produce a long-term (24 h) increase in the excitability of an identified neuron (B51) critical for the expression of feeding in Aplysia. This increase in excitability, which is cAMP dependent, contributes to the associative modification of the feeding circuitry, providing a mechanism for long-term memory storage.

A long-standing debate in neuroscience is whether classical and operant conditioning are mechanistically similar or distinct. The feeding behavior of Aplysia provides a model system suitable for addressing this question. Here we report that classical and operant conditioning of feeding behavior differentially modify the intrinsic excitability of neuron B51, a critical element for the expression of the feeding response, thus revealing that these two forms of associative learning differ at the cellular level.

Although classical and operant conditioning are operationally distinct, it is unclear whether these two forms of learning are mechanistically distinct or similar. Feeding behavior of Aplysia provides a useful model system for addressing this issue. Both classical and operant appetitive behavioral training enhance feeding and neuronal correlates have been identified. Behavioral training was replicated by in vitro analogs that use isolated ganglia. Moreover, a single-cell analog of operant conditioning was developed using neuron B51, a cell important for the expression of the conditioned behavior. Here, a single-cell analog of classical conditioning was developed. Acetylcholine (ACh) mediated the CS-elicited excitation of B51 in ganglia and mimicked the CS in the single-cell analog of classical conditioning. Pairing ACh with dopamine (DA), which mediates the US in ganglia, decreased the excitability of B51 and increased the CS-elicited excitation of B51, similar to results following both in vivo and in vitro classical training. Finally, a D1R agonist failed to support classical conditioning in the cellular analog, whereas D1R mediates reinforcement in operant conditioning.