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This work reviews research on neural mechanisms of two types of associative learning in the marine mollusk Aplysia, classical conditioning of the gill- and siphon-withdrawal reflex and operant conditioning of feeding behavior. Basic classical conditioning is caused in part by activity-dependent facilitation at sensory neuron-motor neuron (SN-MN) synapses and involves a hybrid combination of activity-dependent presynaptic facilitation and Hebbian potentiation, which are coordinated by trans-synaptic signaling. Classical conditioning also shows several higher-order features, which might be explained by the known circuit connections in Aplysia. Operant conditioning is caused in part by a different type of mechanism, an intrinsic increase in excitability of an identified neuron in the central pattern generator (CPG) for feeding. However, for both classical and operant conditioning, adenylyl cyclase is a molecular site of convergence of the two signals that are associated. Learning in other invertebrate preparations also involves many of the same mechanisms, which may contribute to learning in vertebrates as well.

The simplicity and tractability of the neural circuits mediating behaviors in invertebrates have facilitated the cellular/molecular dissection of neural mechanisms underlying learning. The review has a particular focus on the general principles that have emerged from analyses of an example of nonassociative learning, sensitization in the marine mollusk Aplysia. Learning and memory rely on multiple mechanisms of plasticity at multiple sites of the neuronal circuits, with the relative contribution to memory of the different sites varying as a function of the extent of training and time after training. The same intracellular signaling cascades that induce short-term modifications in synaptic transmission can also be used to induce long-term changes. Although short-term memory relies on covalent modifications of preexisting proteins, long-term memory also requires regulated gene transcription and translation. Maintenance of long-term cellular memory involves both intracellular and extracellular feedback loops, which sustain the regulation of gene expression and the modification of targeted molecules.

Synaptic plasticity and memory storage have stages that involve different types of mechanisms at different sites (Bailey et al. 2008). Short-term plasticity involves covalent modifications that are generally thought to be restricted to either presynaptic or postsynaptic structures (Kandel 2001; Malinow and Malenka 2002). By contrast, long-term plasticity involves the protein and RNA synthesis-dependent growth of new synapses, which by its nature involves both pre- and postsynaptic alterations (Bailey and Chen 1988a,b; Glanzman et al. 1990; Bailey et al. 1992; Martin et al. 1997; Ma et al. 1999; Toni et al. 1999; Bozdagi et al. 2000; De Roo et al. 2008). However, it is not clear how these different stages of plasticity are related. On the one hand, under some circumstances short- and long-term plasticity can be produced independently, suggesting that they may be induced in parallel (Emptage and Carew 1993). On the other hand, in both Aplysia and hippocampus an intermediate-term stage of plasticity has been identified that usually involves protein but not RNA synthesis and structural alterations but not synaptic growth, and therefore might form a bridge between short- and long-term plasticity (Ghirardi et al. 1995; Winder et al. 1998; Sutton and Carew 2000; Sutton et al. 2001; Kim et al. 2003; Li et al. 2005, 2009; Villareal et al. 2007). That idea in turn suggests that aspects of the different stages of plasticity may be induced in series, similar to the states in artificial “cascade” models of memory storage (Fusi et al. 2005). Consistent with that idea, we have recently found that whereas short-term behavioral sensitization in Aplysia involves presynaptic mechanisms, intermediate-term sensitization involves both pre- and postsynaptic molecular mechanisms (Antonov et al. 2010), which may in turn be some of the initial steps in a program or cascade leading to synaptic growth during long-term plasticity. However, it has not been known whether that is also true of facilitation in vitro, where a more detailed analysis of the mechanisms involved in the different stages and their interrelations is feasible. We have therefore now examined both pre- and postsynaptic mechanisms of short- and intermediate-term facilitation in isolated cell culture, which has many technical advantages for such studies. Application of different concentrations or durations of the modulatory transmitter 5-HT to Aplysia sensory-motor neuron synapses in culture can produce short-, intermediate-, or long-term facilitation (Montarolo et al. 1986; Rayport and Schacher 1986; Ghirardi et al. 1995), making it possible to examine the mechanisms of each form of plasticity in the same preparation. In addition, because there are no other neurons in the culture dish, one can rule out indirect effects of plasticity at other sites. Furthermore, because both sides of the synapses are accessible to substances injected into the cell bodies, one can selectively manipulate pre- or postsynaptic mechanisms to investigate their contributions and possible interactions. We have examined facilitation induced by either a single brief 5-HT exposure (1 min) or a more prolonged 5-HT exposure (10 min) at rested or depressed sensory-motor neuron synapses. Previous studies have found that short-term facilitation by 1-min 5-HT involves covalent modifications but does not require protein synthesis (Montarolo et al. 1986; Martin et al. 1997). By contrast, facilitation by 10-min 5-HT involves both covalent modifications and protein synthesis (Li et al. 2005; Villareal et al. 2007), suggesting that it represents a form of intermediate-term facilitation. However, the relative contributions of pre- and postsynaptic mechanisms have not been fully explored with either protocol. Our results suggest that whereas short-term facilitation by 1-min 5-HT involves presynaptic PKA and CamKII, intermediate-term facilitation by 10-min 5-HT involves presynaptic PKC and postsynaptic Ca2+ and CamKII, as well as both pre- and postsynaptic protein synthesis. These results in culture are generally similar to those from experiments on short- and intermediate-term behavioral sensitization in a semi-intact preparation (Antonov et al. 2010), and suggest that not only the specific kinases involved but also their site of action depends on the stage of facilitation. In addition, they support the idea that the intermediate-term stage is the first to involve both pre- and postsynaptic molecular mechanisms.