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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    062—Amphetamine and Related Drugs: Reinforcement, Seeking, and Reinstatement

    Saturday, November 09, 2013, 1:00 pm - 5:00 pm

    62.02: Unique molecular alterations in Fos-expressing neurons from single rat striatum following context-induced reinstatement of methamphetamine seeking using fluorescence-activated cell sorting (FACS)

    Location: Halls B-H

    *F. J. RUBIO1, Q.-R. LIU1, S. KAMBHAMPATI1, F. C. CRUZ1, R. M. LEAO1, A. W. MAIONE1, R. CIMBRO2, J. M. BOSSERT1, Y. SHAHAM1, B. T. HOPE1;
    1Behavioral Neurosci. Res. Branch, NIDA IRP, NIH, Baltimore, MD; 2Div. of Rheumatology, Johns Hopkins Sch. of Med., Baltimore, MD

    Abstract Body: We recently developed a novel FACS-based method to isolate and characterize molecular alterations in acute methamphetamine-activated Fos-expressing neurons of a single rat dorsal striatum (Liu et al., JNC, in revision). Here, we used this method to assess gene expression in Fos-expressing striatal neurons that were activated during context-induced reinstatement of methamphetamine seeking. Rats were trained to self-administer methamphetamine for 12 days in a distinct context (A); each drug infusion was paired with a discrete tone-light cue. Lever-pressing in the presence of the discrete cue was subsequently extinguished in context (B). Rats were then tested for reinstatement of methamphetamine in the drug-associated (A) or extinction-associated (B) contexts for 90 min. In all experiments, we observed robust context-induced reinstatement of methamphetamine seeking, indicated by higher non-reinforced lever-presses in context A than in context B. In Exp. 1, we used Fos immunohistochemistry to quantify the number of activated striatal neurons. In Exp. 2, we used FACS to separate Fos-positive and Fos-negative neurons. For FACS, cells from single dorsal and ventral striatum were dissociated by enzymatic and mechanical dissociation and fluorescently labeled with NeuN and Fos antibodies. Immunohistochemistry and FACS both indicated that context-induced reinstatement increased Fos expression in neurons from dorsal striatum but not nucleus accumbens. We used Picopure RNA isolation, gene-targeted preamplification of cDNA, and quantitative PCR to assess mRNA expression in the FACS-isolated Fos-positive and Fos-negative neurons from dorsal striatum. Context A-induced reinstatement was associated with increased expression of several immediate early genes, as well as the dopamine D2 (but not D1) receptor, Darpp-32, cannabinoid receptor 1, and NMDA- and AMPA-receptor subunits in Fos-positive but not Fos-negative neurons.

    Lay Language Summary: Environmental cues or contexts associated with methamphetamine seeking behavior activate a specific pattern of neurons called neuronal ensembles in rat dorsal striatum and induce unique gene alterations in these selectively activated neurons.
    The set of environmental cues or context associated with drug use can provoke relapse in drug addicts long after the last drug use. We model relapse behavior in rats using a procedure called context-induced reinstatement. In the current study, rats were trained to press a lever for intravenous injections of methamphetamine for 12 days in a context A that the rats learn to associate with taking methamphetamine. Next, rats are allowed to press the lever in the presence of a different set of environmental cues that we call context B but no longer receive methamphetamine infusions. Rats gradually stop pressing the active lever and learn to associate context B with extinction of lever pressing. Rats were then tested for reinstatement of methamphetamine seeking by re-exposing them to either the drug-associated context A or the extinction-associated B contexts for 90 min. We observed robust context-induced reinstatement of methamphetamine seeking in the context A but not in context B.
    The dorsal striatum brain area is important for drug seeking and hypothesized to encode learned associations between drug taking and context. We hypothesized that sparsely distributed patterns of neurons called neuronal ensembles are selectively activated by context A during methamphetamine taking and are gradually altered during training to encode the learned association. Different ensembles are activated in context B to encode extinction. Following re-exposure of rats to context A and expression of reinstatement behavior, we used Fos immunohistochemistry to identify a sparsely distributed set of neurons. Fos is a commonly used protein marker of strongly activated neurons. We found two-fold higher number of Fos-positive cells in the dorsal, but not ventral, striatum compared to rats exposure to context B.
    Until recently, it was not possible to study molecular alterations selectively in activated Fos-expressing neurons of a given brain area. We recently developed an improved fluorescence-activated cell sorting (FACS)-based method for assessing gene expression in strongly activated neurons obtained from a single adult rat striatum following methamphetamine injections (Liu et al., JNC, accepted). Here, we used this method to assess gene expression in activated striatal neurons that were activated by the drug-associated context A that lead to reinstatement of methamphetamine seeking behavior. We were able to isolate the activated Fos-expressing neurons (2% and 1% of total neurons for context A and B, respectively) from a single rat dorsal striatum from the non-activated neurons using our FACS method. Up to 20 genes were assessed from 5-487 Fos-positive neurons using gene-targeted pre-amplification (PreAmp) with PCR. We found that context A-induced reinstatement increased expression of immediate early genes fosB, the trophic factor BDNF and the NMDA-type glutamate receptor subunit NR2 in Fos-positive but not in Fos-negative neurons.
    Unique gene alterations are induced in dorsal striatal neuronal ensembles thought to encode the learned association between context and drug taking behavior in rats.