Unique gene alterations are induced in FACS‐purified Fos‐positive neurons activated during cue‐induced relapse to heroin seeking

Abstract

Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6-h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell-sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial co-expression of NPY- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons.

New technologies for examining the role of neuronal ensembles in drug addiction and fear

“…This finding, along with our immunohistochemical findings 29, 82 , supports the idea of sparse coding, in which only a small proportion of sparsely distributed neurons undergo the molecular and cellular alterations needed to encode conditioned drug effects, whereas the surrounding majority of neurons presumably play a much smaller role. As many of these IEGs are also transcription factors, it is likely that they can induce further alterations of gene expression within activated neurons that may play uniquely important roles in learned behaviours mediated by activated neuronal ensembles.…”

“…qPCR analyses indicated several unique alterations in gene expression levels for IEGs and other genes within activated neurons. Cue-induced heroin seeking increased the expression of the IEGs arc , fosB , egr1 and egr2 in activated neurons relative to levels in the non-activated neurons from the same ‘test’ rats or in all neurons from the ‘no test’ rats 82 .…”

“…In the second study, we used the method to assess unique alterations of heroin cue-induced gene expression in activated versus non-activated neurons following FACS purification of activated Fos-expressing neurons from the mPFC and OFC 82 . Rats were trained to self-administer heroin as above and then remained in their home cages for 14–30 days.…”

microRNA-206 in Rat Medial Prefrontal Cortex Regulates BDNF Expression and Alcohol Drinking

“…Arc is an inducible early gene involved in drug-related behaviors. For example, Arc expression is altered in the mPFC during a single session of cocaine self-administration (Fumagalli et al, 2009) and relapse to heroin seeking (Fanous et al, 2013). More importantly, Arc plays a prominent role in mediating the effects of acute as well as long-term alcohol exposure (Pandey et al, 2008).…”

Clustered c-Fos Activation in Rat Hippocampus at the Acquisition Stage of Appetitive Instrumental Learning

“…The sections were then washed in tap water, counterstained, dehydrated and coverslipped with the mounting medium. For Fos staining we used a conventional procedure as it is often used [33] [34] without fluorescent double-labe- ling for Fos and mature neuron marker NeuN because Fos is known as a marker of neuronal activation [35] [36], and it was shown in studies that all cells labeled for Fos also were labeled for NeuN, which supports that only neurons expressed Fos in brains during learning [37].…”