Addiction to drugs of abuse is characterized by persistent structural and functional changes in synaptic connectivity analogous to the long-lasting synaptic plasticity that underlies learning and memory ?7?. A hallmark of long-lasting forms of synaptic plasticity is the change in the number and size of dendritic spines, the major sites of excitatory synaptic transmission. microRNAs (miRNAs), an abundant class of non-coding RNAs, were recently identified as important regulators of dendritic spine development in hippocampal neurons ?18?, and miRNAs control synaptic protein synthesis and learning and memory in the fruit fly ?36?. However, whether miRNA-dependent control of synaptic protein synthesis is similarly involved in dendritic spine morphogenesis in brain areas related to addiction, such as the nucleus accumbens (NAcc) of the striatum, is completely unknown. To begin to address a functional involvement of miRNAs in structural plasticity of NAcc medium spiny neurons (MSN), we plan to pursue the following five aims. First, we will test whether dendrites of MSN are capable to synthesize proteins locally near synapses without the contribution of the MSN cell bodies. This will be tested both with biochemical preparations of purified MSN synaptic terminals and at the single cell level using the dendritic synthesis of a membrane-targeted, non-diffusible reporter protein as a readout. Second, we will isolate the entire pool of synaptic miRNAs of MSN using large-scale expression profiling by microarrays, comparing the relative abundance of MSN miRNAs in synaptic terminals vs. the entire neuron. Synaptically enriched miRNAs represent good candidates for molecules involved in the regulation of dendritic spine morphogenesis and will be put forward to the functional analysis. Third, we will systematically interfere with the activity of synaptic miRNAs in MSN using complementary oligonucleotides (""""""""antimirs""""""""), and determine the effect of miRNA inhibition on dendritic spine development. Fourth, in order to understand how specific synaptic miRNAs control spine development, we will perform a screen for physiological miRNA target mRNAs whose polysome-association, a correlate of ongoing translation, is increased in the presence of the respective antimirs. Fifth, we will examine how elevated dopamine levels, a scenario evoked by the majority of drugs of abuse, modulates the activity of functionally important synaptic miRNAs in MSN. In particular, we will test whether activation of dopamine signaling is capable to relieve miRNA inhibition of the local translation of dendritic mRNA in MSN and thereby elicits exuberant dendritic spine plasticity as observed during chronic drug abuse. In conclusion, we hope that the presented line of investigation might give fundamental insight into the role of miRNA-mediated control of dendritic protein synthesis and structural plasticity in the NAcc. The obtained results might provide a framework for the investigation of miRNA function in addictive behavior in vivo and a first milestone towards the design of therapeutic strategies that target the miRNA pathway. ? ? ?
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