Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. Here, we identify a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrate that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory.

The cerebellum funnels its entire output through a small number of presumed glutamatergic premotor projection neurons in the deep cerebellar nuclei and GABAergic neurons that feed back to the inferior olive. Here we use transgenic mice selectively expressing green fluorescent protein (GFP) in glycinergic neurons to demonstrate that many premotor output neurons in the medial cerebellar (fastigial) nuclei are in fact glycinergic, not glutamatergic as previously thought. These neurons exhibit similar firing properties as neighboring glutamatergic neurons and receive direct input from both Purkinje cells and excitatory fibers. Glycinergic fastigial neurons make functional projections to vestibular and reticular neurons in the ipsilateral brainstem, while their glutamatergic counterparts project contralaterally. Together these data suggest that the cerebellum can influence motor outputs via two distinct and complementary pathways.

The mitogen-activated protein kinase (MAPK) pathway has been implicated recently in synaptic plasticity and memory. Here we used tail shock-induced sensitization of the tail-elicited siphon withdrawal reflex in Aplysia to examine the role of MAPK in three different phases of memory. We show that a specific pattern of serotonin (5-HT) application that produces intermediate-term and long-term synaptic facilitation (ITF and LTF, respectively) of the sensory-motor (SN-MN) synapses in Aplysia leads to sustained activation of extracellular signal-regulated kinase in the ventrocaudal cluster sensory neurons (SNs), which include the tail SNs. Furthermore, repeated tail shocks that induce intermediate-term and long-term memory (ITM and LTM, respectively) for sensitization also lead to sustained MAPK activation in the SNs. Given these results, we next examined the requirement of MAPK activity in (1) SN-MN synaptic facilitation and (2) memory for sensitization in Aplysia, by inhibiting MEK, the upstream kinase that phosphorylates and activates MAPK. In cellular experiments, we show that MAPK activity is required for ITF of tail SN-tail MN synapses, and, in parallel behavioral experiments, we show that ITM requires MAPK activity for its induction but not its expression. In contrast, short-term memory for sensitization does not require MAPK activity. Finally, 5-HT-induced LTF has been shown previously to require MAPK activity. Here we show that LTM for sensitization also requires MAPK activity. These results provide evidence that MAPK plays important roles specifically in long-lasting phases of synaptic plasticity and memory.

Summary Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, while long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.