Summary Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries we discovered an unexpectedly abundant expression of a 28 nucleotide-sized class of piRNAs in brain, which had been thought to be germ-line specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter important for memory. We find that the piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small-RNA mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory.

Using a simplified preparation of the Aplysia siphon-withdrawal reflex, we previously found that associative plasticity at synapses between sensory neurons and motor neurons contributes importantly to classical conditioning of the reflex. We have now tested the roles in that plasticity of two associative cellular mechanisms: activity-dependent enhancement of presynaptic facilitation and postsynaptically induced long-term potentiation. By perturbing molecular signaling pathways in individual neurons, we have provided the most direct evidence to date that each of these mechanisms contributes to behavioral learning. In addition, our results suggest that the two mechanisms are not independent but rather interact through retrograde signaling.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.

Plasticity at central synapses has long been thought to be the most likely mechanism for learning and memory, but testing that idea experimentally has proven to be difficult. For this reason, we have developed a simplified preparation of the Aplysia siphon withdrawal reflex that allows one to examine behavioral learning and memory while simultaneously monitoring synaptic connections between individual identified neurons in the CNS. We previously found that monosynaptic connections from LE siphon sensory neurons to LFS siphon motor neurons make a substantial contribution to the reflex in the siphon withdrawal preparation (Antonov et al., 1999a). We have now used that preparation to assess the contribution of various cellular mechanisms to classical conditioning of the reflex with a siphon tap conditioned stimulus (CS) and tail shock unconditioned stimulus (US). We find that, compared with unpaired training, paired training with the CS and US produces greater enhancement of siphon withdrawal and evoked firing of LFS neurons, greater facilitation of the complex PSP elicited in an LFS neuron by the siphon tap, and greater facilitation of the monosynaptic PSP elicited by stimulation of a single LE neuron. Moreover, the enhanced facilitation of monosynaptic LE-LFS PSPs is greater for LE neurons that fire during the siphon tap and correlates significantly with the enhancement of siphon withdrawal and evoked firing of the LFS neurons. These results provide the most direct evidence to date that activitydependent plasticity at specific central synapses contributes to behavioral conditioning and support the idea that synaptic plasticity is a mechanism of learning and memory more generally.

Synaptic plasticity and learning involve different mechanisms depending on the following: (1) the stage of plasticity and (2) the history of plasticity, or metaplasticity. However, little is known about how these two factors are related. We have addressed that question by examining mechanisms of synaptic plasticity during short-term and intermediate-term behavioral sensitization and dishabituation in a semi-intact preparation of the Aplysia siphon-withdrawal reflex. Dishabituation differs from sensitization in that it is preceded by habituation, and is thus a paradigm for metaplasticity. We find that whereas facilitation during short-term sensitization by one tail shock involves presynaptic covalent modifications by protein kinase A (PKA) and CamKII, facilitation during intermediate-term sensitization by four shocks involves both presynaptic (PKA, CaMKII) and postsynaptic (Ca 2ϩ , CaMKII) covalent modifications, as well as both presynaptic and postsynaptic protein synthesis. The facilitation also involves presynaptic spike broadening 2.5 min after either one or four shocks, but not at later times. Dishabituation by four shocks differs from sensitization in several ways. First, it does not involve PKA or CaMKII, but rather involves presynaptic PKC. In addition, unlike sensitization with the same shock, dishabituation by four shocks does not involve protein synthesis or presynaptic spike broadening, and it also does not involve postsynaptic Ca 2ϩ. These results demonstrate that not only the mechanisms but also the site of plasticity depend on both the stage of plasticity and metaplasticity during memory formation.

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.long-term memory storage | RNA transport | local protein synthesis | neurogenomics | cystoskeleton D espite decades of research on the importance of local translation in learning-related synaptic plasticity and long-term memory storage, we know relatively little about the identity of the "synaptic transcriptome," the various components of the total RNA population that is transported from the cell body, and how this transcriptome becomes localized to and translated at specific synapses (1-12). To develop a general strategy for isolating and characterizing all of the RNAs transported from the cell body to the synapse, we have focused on the RNA transport complexes that interact with the molecular motor kinesin that mediate transport of gene products from the cell body to synapses (13,14).Our approach offers four distinct advantages over previously described methods: (i) it allows the identification of RNAs based on their association with transport machinery that is destined for synapses; (ii) it reflects dynamically regulated RNAs; (iii) it allows for identification of the targeted RNAs; and (iv) it can be used in different regions of the central nervous system (CNS), thereby facilitating genomic characterization of synaptic transcriptome of the entire CNS or region of the CNS. These advantages should aid the study of stoichiometric changes in localized RNAs and their role of local translation, not only in memory storage, but also in a variety of other physiological conditions, such as development. Using this strategy, we have succeeded in obtaining a comprehensive collection of RNAs targeted to Aplysia CNS synapses. We further show that myosin heavy chain mRNA, a cargo of kinesin, is localized to sensory neuron processes and is required specifically for the induction of long-term facilitation (LTF) at sensory and motor neuron synapses. Results Strategy for Identifying Synaptically Targeted RNAs: Isolation andCharacterization of RNA Transport Complexes. We assumed that successful isolation of the RNA-protein complexes associated with the kinesin transport machinery would help identify the full repertoire of RNAs that are actively transported to synapses. Because the CNS contains both neuronal and nonneuronal cells, this approach will also yield RNAs found in the kinesin complex in nonneuronal cells, such as glia. We first optimized conditions for isolating RNA transport complexes from the CNS of Aplysia based on a previous...

Nitric oxide (NO) is thought to be involved in several forms of learning in vivo and synaptic plasticity in vitro, but very little is known about the role of NO during physiological forms of plasticity that occur during learning. We addressed that question in a simplified preparation of the Aplysia siphon-withdrawal reflex. We first used in situ hybridization to show that the identified L29 facilitator neurons express NO synthase. Furthermore, exogenous NO produced facilitation of sensory-motor neuron EPSPs, and an inhibitor of NO synthase or an NO scavenger blocked behavioral conditioning. Application of the scavenger to the ganglion or injection into a sensory neuron blocked facilitation of the EPSP and changes in the sensory-neuron membrane properties during conditioning. Injection of the scavenger into the motor neuron reduced facilitation without affecting sensory neuron membrane properties, and injection of an inhibitor of NO synthase had no effect. Postsynaptic injection of an inhibitor of exocytosis had effects similar to injection of the scavenger. However, changes in the shape of the EPSP during conditioning were not consistent with postsynaptic AMPA-like receptor insertion but were mimicked by presynaptic spike broadening. These results suggest that NO makes an important contribution during conditioning and acts directly in both the sensory and motor neurons to affect different processes of facilitation at the synapses between them. In addition, they suggest that NO does not come from either the sensory or motor neurons but rather comes from another source, perhaps the L29 interneurons.

The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.