Characterization of neuronal ensembles activated during cue-induced reward seeking

National Institute On Drug Abuse

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Abstract

Identification and characterization of neuronal ensembles activated during cue-induced reward seeking Bruce T. Hope Repeated drug administration produces behavioral changes in rats as they learn to associate drug effects with stimuli present during drug administration. This form of learning is thought to involve neuroplastic alterations in sparsely distributed neurons, called neuronal ensembles, that are activated by drug-associated stimuli. Until recently, there no methods to identify these neuronal ensembles for analysis. Previous methods examined neurons in whole brain regions or a class of neurons identified by neurotransmitter or some other chemical characteristic. These methods miss or under-represent alterations in the specific neurons activated during drug-induced behavior. To address this problem, my lab develops novel methods for identifying and characterizing neuronal ensembles that are activated during self-administration and subsequent cue-induced seeking for cocaine, heroin, food, social interaction and other rewards. Identifying these neurons will help us to characterize the molecular and cellular alterations that mediate the learned responses to reward-related cues. We use our Daun02 inactivation procedure with transgenic Fos-LacZ rats to selectively inactivate Fos-expressing neuronal ensembles in nucleus accumbens, prefrontal cortex and other brain areas that were activated during various conditioned drug behaviors. Daun02 is a suicide substrate for beta-galactosidase that, in culture following bath application of Daun02, kills or inactivates cells that contain the enzyme (Farquhar et al. 2002, Cancer Chemother. Pharmacol.50:65-70). We have shown that after cocaine has induced beta-galactosidase, subsequent injection of Daun02 into the nucleus accumbens will inactivate only the small percentage (1% or less) neurons activated during cue-induced relapse to reward seeking, as well as separate ensembles for extinction of this behavior in separate experiments. Daun02 does not inactivate neurons that were not activated immediately prior to Daun02 injections. Selective inactivation of behaviorally activated neuronal ensembles allows us to determine causal roles for neuronal ensembles in behavior. We found specific neuronal ensembles are activated in prefrontal cortex, nucleus accumbens, and other brain areas following exposure to reward (and extinction) -related cues after prior operant training for a variety of rewards in rats. The Daun02 inactivation procedure has immense potential for understanding many different forms of learning processes. We breed Fos-GFP mice and rats that express green fluorescent protein in activated neurons. In collaboration with Dr. Carl Lupica, we previously detected the fluorescent signal in sparsely distributed neurons in striatal slices obtained from Fos-GFP mice and rats following Pavlovian and operant training and subsequent cue-induced reactivation of these neurons. Identification of live neurons in slice preparations that were active during drug- and cue-induced behaviors has allowed to identify a number of unique electrophysiological alterations that occur only in recently activated neurons that contain activated GFP versus neurons that do not. We found altered AMPA/NMDA ratios, spontaneous ESPCs, and particularly silent synapses following activation of glutamatergic afferents in mice and rats following a variety of drug- and cue-induced behaviors. We previously developed a method for dissociate neurons and synaptoneurosomes (synaptic terminals) from adult rat brains following conditioned drug behaviors and sort the activated neurons that Fos from the majority of non-activated neurons that do not contain these activation markers. We use fluorescence-activated cell sorting (FACS) to sort these neurons. Following FACS purification, we have found unique alterations in mRNA, protein and phosphorylation in the small percentage of behaviorally activated Fos-expressing neurons that are different from the alterations found in non-activated neurons using PCR and single cell and nuclei RNA-Seq. We have used FACS to identify these unique molecular alterations in behaviorally activated neurons from mice and rats following a variety of drug- and cue-induced behaviors. More recently we have been developing procedures for identifying behaviorally activated neuronal ensembles via the rapid increase of intracellular calcium levels in activated neurons. We have been using GCaMP and miniscopes in the brain to perform calcium imaging during both food and cocaine self-administration training and cue-induced relapse. We have also spent a great deal of effort towards using the irreversible fluorescent marker CaMPARI2 to identify, with one-minute resolution, neuronal ensembles that were activated during cue-induced relapse of cocaine seeking in rats. These methods, and more recently 3D volume imaging, have enabled us to identify and characterize a unique class of neurons and synapses that are selectively activated during drug administration. These neurons and synapses appear to be part of the neuronal ensembles that represent stimuli and learned associations between environment, interoceptive cues, and drug effects. Understanding the role of these neuronal ensembles in behavior and the ways that repeated drug administration alters them will help us to understand how drugs of abuse produce the learned behaviors associated with addiction that lead to relapse back to drug seeking. The long-term goal is to selectively attenuate or even erase addiction-related memories in human addicts without significant effects on their other memories.