Identification and characterization of neuronal ensembles activated during drug-induced behaviors 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 changes in sparsely distributed neurons, called neuronal ensembles, that are activated by drug-associated stimuli. Until now there has been no method to identify these neuronal ensembles for analysis. All methods to date examine 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 has developed novel methods for identifying and characterizing neuronal ensembles that are activated during locomotor sensitization or self-administration of cocaine and heroin. We have found that the ability of cocaine or amphetamine to activate neurons and induce Fos expression in nucleus accumbens is enhanced for up to 6 months after repeated drug administration in rats that developed context-specific sensitization to these drugs. We also examined the role environmental stimuli play in selectively activating specific neurons during cocaine administration. We used FosB immunohistochemistry to identify neurons that were repeatedly activated during each repeated drug administration. We then used c-fos in situ hybridization to identify the neurons that were activated only on challenge day. Double-labeling for FosB and c-fos revealed that while the drug administration environment modulates the degree of neuronal activation in the striatum, the same set of sparsely distributed neurons is activated during each administration of cocaine. This suggests that only a small number of neurons in the striatum are repeatedly activated and undergo neuroplastic changes during repeated cocaine administration. Overall, our finding of enhanced cocaine-induced Fos induction in nucleus accumbens is the only brain alteration shown to persist as long as the altered behavior and modulated by environmental stimuli similar to that of the altered behavior. Identifying these neurons will help us to characterize the molecular and cellular alterations that mediate behavioral responses to cocaine and other drugs of abuse. Unfortunately, no technique has been available for identifying these neurons in live tissue for electrophysiological analysis or for selectively manipulating these neurons in behaving animals. To address this problem in the past year, we breed a strain of cfos-lacZ transgenic rats in order to identify and selectively manipulate these neurons in live tissue. The transgene in these rats contains a c-fos promoter that regulates transcription of the bacterial gene lacZ, which encodes the enzyme beta-galactosidase (Kasof et al. 1995, J. Neurosci. 15:4238-4249). We have observed induction of beta-galactosidase in fixed striatal tissue following acute and repeated cocaine administration. Beta-galactosidase is induced with a similar time course, dose-response relationship, as Fos protein and is co-induced in all Fos-labeled neurons indicating its use as a marker of activated neurons. Beta-galactosidase labeling has enabled us to identify and manipulate the enzyme in live tissue. For the first time in live tissue, we can study neuronal ensembles activated during drug-induced behavior. We developed a novel technique to selectively inactivate nucleus accumbens neuronal ensembles activated during cocaine-induced locomotor activity following context-specific sensitization. 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). Wehave shown that after cocaine has induced beta-galactosidase, subsequent injection of Daun02 into the nucleus accumbens will inactivate only those neurons activated during cocaine administration and attenuate sensitized cocaine-induced locomotor activity. Daun02 does not inactivate neurons after saline injections to cfos-lacZ rats or in wild-type rats injected with cocaine. Daun02 appears selective for only activated neuronal ensembles because when neurons are activated in one environment and Daun02 is injected into the accumbens, it does not alter locomotor activity in a distinct environment. Selective lesions of activated neuronal ensembles during behavior has never been done before. It allows us to determine causal roles for neuronal ensembles in behavior. We are now testing whether the same neurons are involved in other behaviors, particularly following heroin self-administration and relapse. The technique has immense potential for understanding many different forms of learning processes. We have bred cfos-GFP mice that express green fluorescent protein in activated neurons. In collaboration with Dr. Carl Lupica, we have detected the fluorescent signal in sparsely distributed neurons in striatal slices obtained from cfos-GFP mice that had previously received cocaine. Identification of live neurons in striatal preparations that were active during drug-induced behavior has also never been done before. Following sensitization to cocaine, we have found unique electrophysiological alterations that occur only in neurons that contain GFP versus those that do not. We have found altered AMPA/NMDA ratios and spontaneous ESPCs following activation of glutamatergic afferents. We are now examining We are now using our newly developed cfos-GFP rats to examine similar electrophysiological alterations in behaviorally-activated neurons during reinstatement of cocaine and heroin-seeking in rats. We have developed a method to dissociate striatal neurons from cfos-lacZ rats and from wild-type rats following cocaine-induced behavior and sort the activated neurons that contain beta-galactosidase or 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 messages that are different for activated versus non-activated neurons using microarrays and real-time PCR. We have used FACS to identify the unique molecular alterations in activated neurons during context-specific sensitization to cocaine. We will now use our newly developed cfos-GFP rats to examine similar molecular alterations in behaviorally-activated neurons during reinstatement of cocaine and heroin-seeking in rats. The methods we have developed in the past several years have enabled us to identify and characterize a unique class of neurons that are selectively activated during drug administration. These neurons 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. We have ended our studies with the locomotor sensitization model and are now focusing our newly developed methods on cocaine and heroin self-administration models. To further advance our studies, we are developing new transgenic rats and viruses to manipulate neuronal ensembles in these self-administration models. A particular emphasis is on utilizing recently developed optogenetic methods.
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