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Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting ( FACS )
Author(s) -
Liu QingRong,
Rubio Francisco J.,
Bossert Jennifer M.,
Marchant Nathan J.,
Fanous Sanya,
Hou Xingyu,
Shaham Yavin,
Hope Bruce T.
Publication year - 2014
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.12381
Subject(s) - fosb , neun , striatum , methamphetamine , c fos , neuroscience , biology , immediate early gene , cell sorting , microbiology and biotechnology , gene expression , immunohistochemistry , pharmacology , flow cytometry , dopamine , gene , biochemistry , immunology
Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence‐activated cell sorting ( 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 5–6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. 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 five Fos‐positive neurons. Methamphetamine induced 3–20‐fold increases of immediate early genes arc , homer‐2, c‐fos, fosB, and its isoforms ( ΔfosB and a novel isoform ΔfosB‐2 ) in Fos‐positive but not Fos‐negative neurons. Immediate early gene mRNA induction was 10‐fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug‐associated cues in complex addiction models.Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre‐amplification to assess acute methamphetamine‐induced gene expression from as few as 5 Fos‐expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3–20‐fold increases of immediate early genes (IEGs) in Fos‐positive but not Fos‐negative neurons. Targeted PCR pre‐amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug‐associated cues in complex addiction models.