Serotonergic modulation of sensory neurons in Aplysia and their synaptic connections with follower cells has been used extensively as a model system with which to study mechanisms underlying neuronal plasticity. Serotonin (5-HT)-induced facilitation of sensorimotor connections is due to at least two processes: a process related to the broadening of presynaptic action potentials and a spike-duration-independent (SDI) process that may involve mobilization of transmitter. We have examined the relationship between spike broadening and synaptic facilitation of relatively nondepressed sensorimotor connections in the intact pleural-pedal ganglia. Previously, 5-HT-induced spike broadening in the sensory neuron was shown to be primarily due to the modulation of a voltage-dependent K+ current (Ik.v). Low concentrations (20-30 microM) of 4-aminopyridine (4-AP) were used to rather selectively block Ik.v. 4-AP increased spike duration in the sensory neuron and the excitatory postsynaptic potential (EPSP) in the motor neuron. The temporal development of 4-AP-induced spike broadening closely parallel that of synaptic facilitation. Thus spike broadening via the reduction of Ik.v can directly contribute to synaptic facilitation. The relationship between spike broadening induced by 5-HT (10 microM) and enhancement of the EPSP was also analyzed. We found that components of 5-HT-induced synaptic facilitation preceded the development of 5-HT-induced spike broadening. The comparison between the results of 4-AP and 5-HT revealed that the SDI processes made an important contribution to the rapid development of 5-HT-induced synaptic facilitation and that spike broadening made an important contribution to its maintenance. The SDI process and a slowly developing component of 5-HT-induced spike broadening are mediated, at least in part, by the activation of protein kinase C (PKC). Application of phorbol 12,13-diacetate (PDAc), an activator of PKC, partially mimicked the effects of 5-HT on spike duration and the EPSP. PDAc-induced enhancement of the EPSP preceded the slower development of PDAc-induced spike broadening. Like 5-HT, PDAc enhanced the EPSP via both spike broadening and the SDI processes. In addition, a 15-min exposure to PDAc occluded 5-HT-induced enhancement of the EPSP, suggesting that PKC and 5-HT engage similar or overlapping mechanisms. On the basis of these results and others, we propose a time-dependent hypothesis for the 5-HT-induced synaptic facilitation of nondepressed synapses, in which multiple second-messenger/protein kinase systems mediate the actions of 5-HT via both spike-duration-dependent and SDI processes.