Summary
Memory storage and memory-related synaptic plasticity relies on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain-enriched, and several of these were rapidly down-regulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present pre-synaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a novel role for miR-124 in long-term plasticity of synapses in the mature nervous system.
Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%-70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex-by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.
SUMMARY
The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that following repeated pulses of serotonin (5-HT) the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 hr or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF.
Summary
To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neurons to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have searched for anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.
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