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.

Induction of long-term synaptic changes at one synapse can facilitate the induction of long-term plasticity at another synapse. Evidence is presented here that if Aplysia sensory neuron somata and their remote motor neuron synapses are simultaneously exposed to serotonin pulses insufficient to induce long-term facilitation (LTF) at either site alone, processes activated at these sites interact to induce LTF. This coincident induction of LTF requires that (i) the synaptic pulse occur within a brief temporal window of the somatic pulse, and (ii) local protein synthesis occur immediately at the synapse, followed by delayed protein synthesis at the soma.

The motor pattern that drives each crayfish swimmeret consists of alternating bursts of impulses in power-stroke (PS) and return-stroke (RS) motor neurons. A model of the neural circuit that generates this pattern focused on connections between motor neurons themselves (Heitler, 1978, 1981). The model predicts that synergist motor neurons are electrically coupled, whereas antagonists make mostly inhibitory synapses. We tested this model by observing the responses of motor neurons to pressure ejection of GABA and glutamate, transmitters that crayfish motor neurons release at neuromuscular junctions, and by measuring the strengths and delays of synapses between pairs of motor neurons. Both GABA and glutamate inhibited motor neurons. This inhibition persisted when synaptic transmitter release was blocked by high Mg2+. The effects of GABA were mimicked by muscimol, but not by baclofen or the GABAc receptor agonist cis-4-aminocrotonic acid, and they were not blocked by bicuculline. The effects of glutamate were mimicked by ibotenic acid. Picrotoxin partially blocked glutamate's inhibition of the motor pattern, but did not affect GABA responses. Most (87%) pairs of synergist motor neurons tested made weak, noninverting connections. Approximately half of these had synaptic delays of <2 msec, consistent with direct electrical or chemical synapses. Individual motor neurons were dye-coupled to between one and three other motor neurons, and to interneurons. Less than half (44%) of the pairs of antagonist motor neurons tested made synaptic connections. These connections were weak, had long latencies (>4 msec), and therefore were probably polysynaptic. We conclude that direct synapses between swimmeret motor neurons cannot account for alternation of PS and RS bursts.

The induction of different phases of memory depends on the amount and patterning of training, raising the question of whether specific training patterns engage different cellular mechanisms and whether these mechanisms operate in series or in parallel. We examined these questions by using a cellular model of memory formation: facilitation of the tail sensory neuron-motor neuron synapses by serotonin (5-hydroxytryptamine, 5-HT) in the CNS of Aplysia. We studied facilitation in two temporal domains: intermediate-term facilitation (1.5-3 h) and long-term facilitation (LTF, >24 h). Both forms can be induced by using several different temporal and spatial patterns of 5-HT, including (i) repeated, temporally spaced pulses of 5-HT to both the sensory neuron soma and the sensory neuron-motor neuron synapse, and (ii) temporally asymmetric exposure of 5-HT to the soma and synapse under conditions in which neither exposure alone induces LTF. We first examined the protein and RNA synthesis requirements for LTF induced by these two patterns and found that asymmetric (but not repeated) 5-HT application induced LTF that required postsynaptic protein and RNA synthesis. We next focused on the patterning and protein synthesis requirements for intermediate-term facilitation. We found that intermediate-term facilitation (i) is induced locally at the synapse, (ii) requires multiple pulses of 5-HT, and (iii) requires synaptic protein synthesis. Our findings show that different temporal and spatial patterns of 5-HT induce specific temporal phases of long-lasting facilitation in parallel by engaging different cellular and molecular mechanisms. I t has long been appreciated that memories can persist in dramatically different time frames, from seconds and minutes up to a lifetime (1, 2). A major question now concerns the relationship between memories that exist in different temporal domains. One possibility, for which there is considerable empirical support (see ref. 1), is that memories are processed in series. However, there is a growing body of evidence showing that, at least under certain circumstances, memories can also be processed in parallel (3-7).Like memory, neuronal plasticity can exist in several temporal domains. This feature of plasticity has been revealed in many experimental contexts, including three model systems that have been especially useful for studying the synaptic basis for learning and memory: the mammalian hippocampus (8-11), the photoreceptor neurons in Hermissenda (12, 13), and the sensory neuron-motor neuron (SN-MN) synapses of Aplysia. In Aplysia, the tail-elicited siphon-withdrawal reflex has been useful for examining the temporal relationships between synaptic plasticity and memory. Memory for sensitization induced by repeated tail shocks can exist in at least three temporal domains: short-term memory (Ͻ25 min), intermediate-term memory (Ϸ90 min), and long-term memory (Ͼ24 h) (14-16). Because tail shock releases serotonin (5-hydroxytryptamine, 5-HT) systemically (17) On the synaptic level, ITF exists in m...

BDNF, which acts through tropomyosin-related kinase B (TrkB) receptors during mammalian development, also enhances longterm synaptic facilitation (LTF) in adult Aplysia. Because LTF is a substrate for long-term memory (LTM) in Aplysia, we examined the requirement of a secreted TrkB ligand in LTM formation at molecular, synaptic, and behavioral levels. Using an extracellular fusion protein that sequesters secreted TrkB ligands, we show that TrkB function is required for serotonin-induced activation of extracellular signal-regulated kinase, tail nerve shock-induced LTF in the CNS, and tail shock-induced LTM but is not necessary for short-term synaptic facilitation or short-term memory. These results show that a secreted growth factor, acting through a TrkB signaling cascade, is critical for the induction of long-lasting plasticity and memory formation in Aplysia.growth factor ͉ molecular signaling ͉ sensitization T he assembly of the nervous system during development and the encoding of information during memory formation in the adult appear to share common mechanistic features (1, 2). For example, neurotrophins are known to play a critical role in differentiation and survival of developing neurons, and at least one neurotrophin, BDNF, can also play important roles in synaptic plasticity and memory formation in adult animals (for review, see ref.3). Thus a functional analysis of BDNF and related growth factors provides a powerful vantage point to address the theoretically important issue of whether development and memory formation are mechanistically related.BDNF is a ligand for the tropomyosin-related kinase B (TrkB) receptor, a member of the receptor tyrosine kinase family. The binding of BDNF to the TrkB receptor initiates a wide range of downstream signaling events, including ERK activation (4, 5). An important role for BDNF has been established in studies examining long-term potentiation in mammalian systems (4,(6)(7)(8)(9)(10)(11)(12)(13)(14). In addition, a role for BDNF in memory formation has also been established (15-18). Thus, there is now strong evidence that TrkB signaling can play important roles in synaptic plasticity on the one hand and memory formation on the other. What is currently missing is the ability to forge direct mechanistic links between these different levels of analyses. The marine mollusk Aplysia is well suited to bridge this gap, because it has proven to be a useful model system for the investigation of the molecular mechanisms underlying both synaptic facilitation and memory formation (see refs. 19-21).We recently showed that human recombinant BDNF significantly facilitates the induction of long-term synaptic facilitation (LTF) at identified tail sensory neuron-motor neuron (SN-MN) synapses in Aplysia, and that this enhancing effect of BDNF in LTF induction requires ERK signaling (Fig. 1A and ref. 22). Because activation of TrkB receptors is known to initiate downstream signaling events including ERK activation (4, 5), we here examine the role of TrkB ligand signaling in memory for ...

The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation.

Four different functional types of motor neurons innervate each swimmeret: return-stroke excitors (RSEs), power-stroke excitors (PSEs), return-stroke inhibitors (RSIs), and power-stroke inhibitors (PSIs). We studied the structures and passive electrical properties of these neurons, and tested the hypothesis that different types of motor neurons would have different passive properties that influenced generation of the swimmeret motor pattern. Cell bodies of neurons innervating one swimmeret were clustered in two anatomic groups in the same ganglion. The shapes of motor neurons in both groups were similar, despite the differences in locations of their cell bodies and in their functions. Diameters of their axons in the swimmeret nerve ranged from <2 to approximately 35 microm. Resting membrane potentials, input resistances, and membrane time constants were recorded with microelectrodes in the processes of swimmeret motor neurons in isolated abdominal nerve cord preparations. Membrane potentials had a median of -59 mV, with 25th and 75th percentiles of -66.0 and -53 mV. The median input resistance was 6.4 M omega, with 25th and 75th percentiles of 3.4 and 13.7 M omega. Membrane time constants had a median of 9.3 ms, with 25th and 75th percentiles of 5.7 and 15.0 ms. Excitatory and inhibitory motor neurons had similar passive properties. RSE motor neurons were typically more depolarized than the other types, but the passive properties of RSE, PSE, RSI, and PSI neurons were not significantly different. Membrane time constants measured from cell bodies were briefer than those measured from neuropil processes, but membrane potentials and input resistances were not significantly different. The relative sizes of different motor neurons were measured from the sizes of their impulses recorded extracellularly from the swimmeret nerve. Smaller motor neurons had lower membrane potentials and were more likely to be active in the motor pattern than were large motor neurons. Motor neurons of different sizes had similar input resistances and membrane time constants. Motor neurons that were either oscillating or oscillating and firing in phase with the swimmeret motor pattern had lower average membrane potentials and longer time constants than those that were not oscillating. When the state of the swimmeret system changed from quiescence to continuous production of the motor pattern, the resting potentials, input resistances, and membrane time constants of individual swimmeret motor neurons changed only slightly. On average, both input resistance and membrane time constant increased. These similarities are considered in light of the functional task each motor neuron performs, and a hypothesis is developed that links the brief time constants of these neurons and graded synaptic transmission by premotor interneurons to control of the swimmeret muscles and the performance of the swimmeret system.

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