FACS Identifies Unique Cocaine-Induced Gene Regulation in Selectively Activated Adult Striatal Neurons

Abstract

Numerous studies with the neural activity marker Fos indicate that cocaine activates only a small proportion of sparsely distributed striatal neurons. Until now, efficient methods were not available to assess neuroadaptations induced specifically within these activated neurons. We used fluorescence-activated cell sorting (FACS) to purify striatal neurons activated during cocaine-induced locomotion in naïve and cocaine-sensitized cfos-lacZ transgenic rats. Activated neurons were labeled with an antibody against β-galactosidase, the protein product of the lacZ gene. Cocaine induced a unique gene expression profile selectively in the small proportion of activated neurons that was not observed in the non-activated majority of neurons. These genes included altered levels of the immediate early genes arc, fosB, and nr4a3, as well as genes involved in p38 MAPK signaling and cell-type specificity. We propose that this FACS method can be used to study molecular neuroadaptations in specific neurons encoding the behavioral effects of abused drugs and other learned behaviors.

Incubation of Methamphetamine Craving Is Associated with Selective Increases in Expression ofBdnfandTrkb, Glutamate Receptors, and Epigenetic Enzymes in Cue-Activated Fos-Expressing Dorsal Striatal Neurons

“…We subsequently developed antibody-based FACS methods to purify activated (Fos-positive) neurons from adult wild-type rats (Guez-Barber et al, 2011;Liu et al, 2014). We used these methods to determine gene-expression changes in Fos-positive neurons in striatum and cortex in studies on cocaine-induced locomotor sensitization (Guez-Barber et al, 2011), incubation of heroin craving , and contextinduced reinstatement of methamphetamine seeking (Rubio et al, 2015). The main finding from the first two studies was that the Fos-positive neurons primarily showed higher expression of other IEGs (Guez-Barber et al, 2011;.…”

“…We used these methods to determine gene-expression changes in Fos-positive neurons in striatum and cortex in studies on cocaine-induced locomotor sensitization (Guez-Barber et al, 2011), incubation of heroin craving , and contextinduced reinstatement of methamphetamine seeking (Rubio et al, 2015). The main finding from the first two studies was that the Fos-positive neurons primarily showed higher expression of other IEGs (Guez-Barber et al, 2011;. In the third study, we found higher expression of IEGs in Fos-positive neurons, as well as increased expression of Grin2a and Grin2b mRNA in these neurons; the expression of other glutamate receptors and gene targets was similar in Fos-positive and Fos-negative neurons (Rubio et al, 2015).…”

“…We could not use expression levels in Fos-positive neurons in the No test group to compare with Fos-positive neurons in the Extinction test group, because this would be comparing different sets of neurons. Based on our previous studies showing cue selectivity of Fos-expressing neuronal ensembles Fanous et al, 2012;Cruz et al, 2014b), the Fos-positive neurons in the No test group were unlikely to be the same neurons that were selectively activated by cues in the Extinction test group. In other words, the cue-activated Fos-positive neurons (as well as Fos-negative neurons) in the Extinction test group were most likely Fos-negative before extinction tests.…”

“…Additionally, we previously used immunohistochemistry for the neuronal activity marker Fos (Cruz et al, 2014a) and reversible inactivation of DS to demonstrate a role for this brain region in context-induced reinstatement of methamphetamine seeking (Rubio et al, 2015). We also used fluorescence-activated cell sorting (FACS; Guez-Barber et al, 2011;Liu et al, 2014) and RNAscope in situ hybridization (ISH; Wang et al, 2012) methods and found that context-induced reinstatement was associated with selective increases in mRNA expression of NMDA receptor subunits Grin2a and Grin2b in Fos-positive but not Fos-negative neurons.…”

Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons

“…Cells with few processes, such as those in embryonic tissues, and synaptosomes have been sorted with some success (30)(31)(32). More recently, adult neurons have been isolated by FACS, taking advantage of genetically encoded fluorescent probes, and used for gene expression profiling studies (33,34). However, the entangled nature of mature neurons makes it difficult to separate cells without causing cellular damage or death.…”