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

    817—Amphetamine and Related Drugs: Neural Mechanisms of Addiction

    Wednesday, November 13, 2013, 1:00 pm - 5:00 pm

    817.12: Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS)

    Location: Halls B-H

    *Q.-R. LIU, F. J. RUBIO, J. M. BOSSERT, N. J. MARCHANT, X. HOU, Y. SHAHAM, B. T. HOPE;
    Behavior Neurosci. Res. Br., Intramural Res. Program, NIDA/ NIH, BALTIMORE, MD

    Abstract Body: Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a FACS-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 6-7% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p) while less than 1% of neurons were activated by vehicle. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as 100 Fos-positive neurons. Methamphetamine induced 3-20-fold increases of the immediate early genes arc, homer-2, fosB and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not in Fos-negative neurons. Apparent IEG mRNA induction was 10-fold lower or absent when assessed in unsorted samples from homogenates of single dorsal striatum. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.

    Lay Language Summary: We developed an improved method for assessing gene expression in strongly activated neurons obtained from a single adult rat striatum following methamphetamine injections.
    Stimuli-directed behaviours are thought to be mediated by stimuli-selective activation of specific patterns of sparsely distributed neurons called ‘neuronal ensembles’. We previously developed methods for assessing gene expression induced uniquely within these activated neuronal ensembles. Activated neurons were labelled with antibodies against the neural activity marker Fos and purified using fluorescence-activated cell sorting (FACS). However, this method required overly large brain areas from many rats to obtain enough tissue for analysis of gene expression, which made it less useful for use in more complex animal models of addiction.
    The current paper used gene-targeted pre-amplification (PreAmp) with PCR to assess expression of up to 30 genes from as few as 5-100 Fos-positive neurons from a single rat dorsal striatum. Using our improved method, we found that acute methamphetamine induced 3-20-fold increases of the immediate early genes arc, homer-2, fosB and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not in Fos-negative neurons. In contrast, induction of the same immediate early genes was 10-fold lower or absent when assessed in homogenates obtained from bulk dorsal striatum.
    The sensitivity of the new FACS-PreAmp method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in more complex addiction models.