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A common feature of arousing stimuli used as reinforcement in animal models of learning is that they promote memory formation through widespread effects in the CNS. In the marine mollusk Aplysia, sensitization is typically induced by tail-shock, an aversive reinforcer that triggers a state of defensive arousal characterized by escape locomotion and increased heart rate. Serotonin (5-HT) contributes importantly to sensitization of defensive reflexes as well as to the regulation of locomotion and heart rate. Although specific serotonergic neurons increase their firing after tail-shock, it remains unclear whether this effect is restricted to these neurons or whether tail-shock recruits a more global serotonergic system. In this study, we recorded from serotonergic neurons throughout the CNS, which were prelabeled with 5,7-dihydroxytryptamine, during an in vitro analog of sensitization training, tail-nerve shock. We found that most of the serotonergic neurons that we recorded from (80%) increased their firing rate for several minutes after nerve shock. Most serotonergic neurons in the pedal and abdominal ganglion were also excited by 5-HT and by intracellular activation of the two serotonergic neurons CB1/CC3. This interconnectivity between serotonergic neurons might contribute to spread excitation within a large proportion of the serotonergic system during sensitization training. It is also possible that serotonergic neurons could be activated by 5-HT present in the hemolymph via a neuro-humoral positive feedback mechanism. Overall, these data indicate that sensitization training activates a large proportion of Aplysia serotonergic neurons and that this form of learning occurs in a context of increased serotonergic tone.

Serotonin (5-HT) plays an important role in sensitization of defensive reflexes in Aplysia and is also involved in several aspects of arousal, such as the control of locomotion and of cardiovascular tone. In the preceding paper, we showed that tail-nerve shock, a noxious stimulus that readily induces sensitization, increases the firing rate of a large number of serotonergic neurons throughout the CNS. However, the functional consequences of such an increase in serotonergic tone are still poorly understood. In this study, we examined this question by using the 5-HT precursor 5-hydroxytryptophan (5-HTP) to specifically increase 5-HT release in the CNS. Increased tonic 5-HT release after 5-HTP treatment was manifested by facilitation of sensorimotor (SN-MN) synapses, increased firing rate of serotonergic neurons in the pedal and abdominal ganglia, and enhanced 5-HT release evoked by tail-nerve shock. When 5-HTP was administered to freely moving animals, it produced a strong arousal response characterized by increased locomotion and heart rate, which was reminiscent of the defensive arousal reaction triggered by noxious stimulation such as tail-shock. In contrast, 5-HTP actually inhibited the tail-induced siphon-withdrawal reflex. It is possible that 5-HT-induced facilitation of SN-MN synapses was counteracted by inhibition of polysynaptic reflex pathways between SNs and MNs, resulting in transient behavioral inhibition of the reflex, which could favor escape locomotion and/or respiration shortly after an aversive stimulus. We conclude that a major function associated with the activation of the Aplysia serotonergic system evoked by noxious stimuli is the triggering of a defensive arousal response. It is known that tail-shock-induced serotonergic activation contributes to memory encoding at least in part by facilitating SN-MN synapses. However, this effect in isolation might not be sufficient for the behavioral expression of sensitization.