Using FACS to investigate the opposing neuronal pathways in Cocaine addiction

Cocaine is a powerfully addictive stimulant drug. Behavioral abnormalities associated with addiction coincide with changes in gene transcription. Striatal Spiny Neurons implicated in addiction form two pathways: Direct & Indirect. Our approach used Fluorescent Automated Cell Sorting to separate the functionally different neurons from the pathways and probe them for changes in gene expression. Rats were trained to self-administer cocaine by lever press for either 1 hour each day (short access) or 6 hours each day (long access). In 9 days, long access rats showed a marked escalation of the total dose taken during each session reflecting compulsive drug use. Fluorescently labeled spheres were injected in substantia nigra and globus pallidus on day 10 to mark the direct and indirect pathways respectively. These were retrogradely transported into the striatum by day 17 when the brains were harvested. Striatal tissue was enzymatically digested and processed to produce a single cell suspension. Cells were stained with antibodies to neuronal cell bodies (NeuN) and DNA marker (DAPI). Cells were then successfully separated on size, presence of cell bodies and DNA, and the presence of retrograde labels. Sufficient quantities of RNA was extracted from the fluorescently sorted cells and quantitative RTPCR on candidate genes was run. Next we propose to use a Gene array and look at global changes in gene expression. Our study is the first of its kind in rats to use FACS sorting to separate heterogeneous neuronal populations for expression studies in cocaine addiction.