The mesolimbic reward circuit is comprised of dopaminergic neurons in the ventral tegmental area and their targets in the nucleus accumbens (NAc) and other associated limbic brain regions. This circuit is the major site of action for addictive drugs such as psychostimulant amphetamine (AMPH). The reinforcing properties of AMPH are mediated by changes in the physiology and synaptic conectivity of neurons in the NAc. Considerable evidence suggests that AMPH-induced changes in striatal gene expression are essential for these cellular adaptations, and chromatin regulation has been implicated as a mechanism that may contribute to the persistence of these changes in neuronal physiology. However the striatum is comprised of multiple kinds of neurons that are synaptically interconnected into functional microcircuits, and very little is known about whether or how cell-type specific differences in AMPH-regulated transcription impact striatal function. We propose to take a novel approach to this question by using a protocol we have developed for fluorescence- activated cell sorting (FACS) to characterize AMPH-induced changes in gene transcription in striatal fast- spiking GABAergic interneurons (FSIs). FSIs play a crucial role in gating striatal output, however it remains unknown whether these neurons experience AMPH-dependent adaptations. The diffuse distribution of FSIs has presented a significant barrier to biochemical analysis of their gene transcription and chromatin regulation. However we discovered that AMPH administration drives rapid and robust phosphorylation of the methyl-DNA binding protein MeCP2 at Ser421 (pMeCP2) in the NAc, and that this AMPH-induced phosphorylation occurs selectively in FSIs. On the basis of these findings we have developed FACS protocols to use the pMeCP2 antibody as a label to purify AMPH-activated FSIs from the mouse striatum. Here we propose to use this technique in order to determine whether FSIs show plasticity of transcriptional regulation in response to repeated AMPH exposure.
In Aim 1 we will FACS purify FSIs from the striatum of mice that received either acute or repeated AMPH injections and profile changes in gene expression by RNA-Seq.
In Aim 2 we will FACS purify FSI nuclei from the striatum of mice that received either acute or repeated AMPH injections and perform ChIP-Seq with antibodies against acetylated histone H3 as an indication of chromatin regulation. If we observe differences in gene expression or chromatin regulation, these data would provide the first evidence that this interneuron population experiences molecular adaptations in response to repeated AMPH exposure. This outcome would be exciting because it would raise the posibility that transcriptional plasticity is accompanied by AMPH-induced adaptations in FSI function. We anticipate that identifying FSI genes regulated by repeated AMPH will suggest new hypotheses of how plasticity of this important interneuron population may contribute to AMPH-induced changes in mesolimbic circuit functions.
Chronic drug abuse can be attributed to the ability of addictive substances to induce persistent adaptations in the reward circuits of the brain. This study will identify cell-type specific amphetamine-induced changes in neuronal gene expression that may contribute to this process. Because reward circuit adaptations are part of the pathology that leads to addiction, these genes represent important potential targets for therapeutic intervention.
|Gadalla, Kamal K E; Bailey, Mark E S; Spike, Rosemary C et al. (2013) Improved survival and reduced phenotypic severity following AAV9/MECP2 gene transfer to neonatal and juvenile male Mecp2 knockout mice. Mol Ther 21:18-30|