The role of neuronal ensembles in context-induced reinstatement of ethanol seeking: pharmacogenetic, optogenetic and molecular investigation

Abstract

Learned associations play an important role in addiction. With repeated drug use, addicts learn to associate drug effects with stimuli or cues in the drug environment, such as drug paraphernalia, context, and people. Over time, these cues can promote drug craving and relapse. These drug-related cues are complex combinations of different stimuli that are recognized with a high degree of resolution. Thus, any neural mechanism capable of encoding these learned associations must have a comparably high degree of resolution. In this way, 1949, Donald Hebb hypothesized that learned associations are encoded by sparsely distributed patterns of synchronously activated neurons now called neuronal ensembles. We hypothesize that these strongly activated patterns of neurons, in accumbens and prefrontal cortex (PFC), form neuronal ensembles that encode learned associations between ethanol effects and environmental cues associated with these effects. One of the main problems for treating ethanol addiction is high rates of relapse to drug use. In human addicts, environmental stimuli associated with previous drug use can provoke relapse to drug use after prolonged abstinence. Many laboratories model drug relapse in rodents using a context-induced reinstatement procedure. In this procedure, rodents learn to associate particular environments or contexts with drug-taking. At a later time, investigators can re-expose rodents to the drug-associated context and examine its effects on relapse to drug seeking. We will investigate whether context-induced reinstatement of ethanol seeking is mediated by projections from dorsal medial PFC (dmPFC) to accumbens core and we will look for unique molecular alterations in these synapses, which could be involved with this behavior. For that we will train rats to self-administer ethanol in context A and extinguished lever pressing in a distinct context B. On test day, the context-induced reinstatement of ethanol seeking will be tested putting the rats back in the ethanol context (A). To assess a causal role for neural ensembles from dmPFC and accumbens core in context-induced reinstatement, we will use c-fos-lacZ transgenic rats and a pro-drug called Daun02 to inactivate only those ensembles. Next, to investigate whether context-induced reinstatement of ethanol seeking could be mediated by activation of synapses between neuronal ensembles of the accumbens core and dmPFC, we will use an optogenetic approach, in which male rats received bilateral dmPFC microinjections of a virus vector, coding for either light-sensitive eNpHR3.0-eYFP or control eYFP protein with optical fibers implanted into accumbens core. Then, will be performed two counterbalanced tests in which either light or no light will be delivered bilaterally into accumbens core. To complement our optogenetic studies that identify, we will develop a procedure that uses flow citometry to analyze behaviorally activated synaptoneurosomes (preparation which contain the presynaptic terminal attached to post-synaptic density) in order to identify unique molecular alterations that are distinct from those in non-activated synapses. We hope that our preclinical procedure for context-induced reinstatement could find new targets for ethanol relapse treatment. (AU)