Mechanisms of Cocaine-Induced Dendritic Spine Refinement and Plasticity

Anthony Koleske, Department of Molecular Biophysics and Biochemistry, Yale University

Significance

Neuronal refinement and stabilization in late adolescence are believed to confer resilience to poor decision making and addictive like behaviors. In previous work, we found that cocaine triggers dendritic spine destabilization on neurons in the orbital prefrontal cortex (oPFC). The Abl2/Arg nonreceptor tyrosine kinase acts downstream of integrin receptors to control cytoskeletal signaling pathways that stabilize dendrites and dendritic spines in the adolescent mouse brain. Loss of Arg function destabilizes dendritic spines in the oPFC, and this is accompanied by increased locomotor sensitization to cocaine and amphetamine, increased responding to reward-related cues, and decrements in instrumental reversal learning, all phenotypes implicated in addiction. Moreover, cocaine exposure led to spine enlargement in oPFC neurons in control animals, but this response is not observed following genetic or pharmacological inhibition of Arg.

Despite the clear implication of Arg-mediated signaling pathways in the refinement of oPFC neuron structure and determining the responsiveness to psychostimulants, we do not understand why loss of Arg function leads to reductions in oPFC stability or reduced structural plasticity in response to cocaine. We will use unbiased state-of-the-art proteomics approaches to identify cocaine- and Arg-regulated signaling processes that mediate oPFC stabilization and oPFC structural plasticity and assess their importance in regulating oPFC neuron structure and function.

Specific Aim 1

To identify candidate mediators of oPFC spine stabilization that are targeted by cocaine. We will use differential iTRAQ labeling and mass spectrometry of wild type and arg–/– mouse oPFC to identify novel proteins whose levels are altered upon loss of Arg function as candidate mediators of dendritic spine stabilization by Arg. We will also perform similar iTRAQ analysis on oPFC tissue from cocaine-treated wild type and arg–/– mice to identify potential pathways that mediate cocaine-induced dendritic spine plasticity. We will also conduct similar analyses on oPFC tissue from wild type mice treated chronically with corticosterone, which we have shown independently to destabilize oPFC spines. Comparison to the cocaine-treated mice will identify alterations that are specific to cocaine treatment. 
   

Specific Aim 2

To characterize the role of candidate mediators on oPFC neuron structure and function we will use a complementary set of biochemical, cell-based, and whole animal assays to assess the relative roles for the candidate molecules in oPFC spine stabilization and cocaine-induced plasticity. First, we will assess how cocaine affects the levels, subcellular localization, or activity of the candidate proteins in wild type (WT) or arg–/– mice. We will also how knockdown of the candidate proteins in long-term established cortical cultures affects dendritic spine and dendrite stability. We will monitor dendritic spine density, size, and shape as well as dendrite arbor size and branching patterns using methods we have described previously. In cases where we observe effects, we will test whether a GFP-tagged shRNA-resistant version of the candidate rescues defects in dendrite and dendritic spine stability. These studies will identify which of the novel candidates regulate dendritic spine and dendrite stability.