Although in vitro analyses of long-term changes in the sensorimotor connection of Aplysia have been used extensively to understand long-term sensitization, relatively little is known about the ways in which the connection is modified by learning in vivo. Moreover, sites other than the sensory neurons might be modified as well. In this paper, several different biophysical properties of sensory neurons, motor neurons, and LPl17, an identified interneuron, were examined. Membrane properties of sensory neurons, which were expressed as increased excitability and increased spike afterdepolarization, were affected by the training. The biophysical properties of motor neurons also were affected by training, resulting in hyperpolarization of the resting membrane potential and a decrease in spike threshold. These results suggest that motor neurons are potential loci for storage of the memory in sensitization. The strength of the connection between sensory and motor neurons was affected by the training, although the connection between LPl17 and the motor neuron was unaffected. Biophysical properties of LPl17 were unaffected by training. The results emphasize the importance of plasticity at sensory-motor synapses and are consistent with the idea that there are multiple sites of plasticity distributed throughout the nervous system.

The role of transforming growth factor-beta (TGF-beta) in long-term synaptic facilitation was examined in isolated Aplysia ganglia. Treatment with TGF-beta1 induced long-term facilitation (24 and 48 hours), but not short-term (5 to 15 minutes) or intermediate-term (2 to 4 hours) facilitation. The long-term effects of TGF-beta1 were not additive with those of serotonin. Moreover, serotonin-induced facilitation was blocked by an inhibitor of TGF-beta. Thus, activation of TGF-beta may be part of the cascade of events underlying long-term sensitization, consistent with the hypothesis that signaling molecules that participate in development also have roles in adult neuronal plasticity.

Learning and memory are influenced by the temporal pattern of training stimuli. The mechanisms that determine the effectiveness of a particular training protocol are not well understood, however. The hypothesis that the efficacy of a protocol is determined, in part, by interactions among biochemical cascades that underlie learning and memory was examined. Previous studies suggest that the PKA and ERK cascades are necessary to induce long-term synaptic facilitation (LTF) in Aplysia, a neuronal correlate of memory. A computational model of the PKA and ERK cascades was developed, and used the model to identify a novel training protocol that maximized PKA/ERK interactions. In vitro studies confirmed that the protocol enhanced LTF. Moreover, the protocol enhanced levels of phosphorylation of the transcription factor CREB1. Behavioral training confirmed that long-term memory also was enhanced by the protocol. These results illustrate the feasibility of using computational models to design training protocols that improve memory.

Biophysical, biochemical, and morphological studies have implicated sensory neurons as key sites of plasticity in the formation and retention of the memory of long-term sensitization in Aplysia californica. This study examined the effects of different sensitization training protocols on the structure of sensory neurons mediating the tail-siphon withdrawal reflex. A 4 d training period produced a robust localized outgrowth in these sensory neurons observed 24 hr after the end of training. These changes are consistent with previous results in siphon sensory neurons (Bailey and Chen, 1988a). In contrast, 1 d of sensitization training, which has been shown to effectively induce long-term behavioral sensitization and synaptic facilitation (Frost et al., 1985; Cleary et al., 1998), is not associated with morphological changes in tail sensory neurons at either 24 hr or 4 d after training. Similarly, a single treatment with the growth factor TGF-beta, which also induced facilitation, did not alter sensory neuron morphology. The different effectiveness of the two protocols was not simply a reflection of the number of stimuli presented, because a 1 d massed training protocol did not produce sensitization 24 hr after training, nor did it induce neuronal outgrowth. These results suggest that extensive sensitization training is required to induce neuronal outgrowth in tail sensory neurons, indicating that the memory of long-term sensitization induced by 1 d of training is mechanistically different from that induced by 4 d of training. Moreover, the induction of a form of long-term sensitization associated with neuronal outgrowth does not appear to be a function of the amount of stimulation but does appear to be dependent on the temporal spacing of the stimulation over multiple days.

Transforming growth factor beta1 (TGF-beta1) induces long-term synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has acute effects as well. Treatment of pleural-pedal ganglia with TGF-beta1 for 5 min activated mitogen-activated protein kinase (MAPK) and stimulated the phosphorylation of synapsin in a MAPK-dependent manner. This phosphorylation appeared to modulate synapsin distribution in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-beta1-treated sensory neurons showed a significant reduction in the number of these puncta, an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-beta1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-beta1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.