Identification and characterization of neuronal ensembles activated during drug-induced behaviors? ? Bruce T. Hope? ? Repeated cocaine 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 to cocaine and amphetamine. ? ? 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 ?-galactosidase (Kasof et al. 1995, J. Neurosci. 15:4238-4249). We have observed induction of ?-galactosidase in fixed striatal tissue following acute and repeated cocaine administration. ?-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 is enabling 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 lesion nucleus accumbens neuronal ensembles activated during cocaine-induced locomotor activity following context-specific sensitization. Daun02 is a suicide substrate for ?-galactosidase that, in culture following bath application of Daun02, kills only cells that contain the enzyme (Farquhar et al. 2002, Cancer Chemother. Pharmacol. 50:65-70). We have shown that after cocaine has induced ?-galactosidase, subsequent injection of Daun02 into the nucleus accumbens will kill only those neurons activated during cocaine administration and attenuate sensitized cocaine-induced locomotor activity. Daun02 does not kill neurons after saline injections to cfos-lacZ rats or in wild-type rats injected with cocaine. Selective lesions of activated neuronal ensembles during behavior has never been done before. We will begin testing whether the same neurons are involved in other behaviors, such as cocaine self-administration and relapse. The technique has immense potential for understanding many different forms of learning processes. ? ? We have already labeled neurons that contain ?-galactosidase in live slice preparations for electrophysiological analysis. We bath applied a cell permeable substrate of beta-galactosidase (Imagene Green, Molecular Probes) that is catalyzed by the enzyme to form a fluorescent product. In collaboration with Dr. Carl Lupica, we have detected the fluorescent signal in sparsely distributed neurons in striatal slices obtained from cfos-lacZ transgenic rats that had previously received cocaine. Identification of live neurons in striatal preparations that were active during a behavior has also never been done before. Following sensitization to cocaine, we will isolate the electrophysiological alterations that occur in neurons that contain beta-galactosidase versus those that do not. Our primary objective is to examine alterations in sensitivity to glutamatergic input. We are examining altered responsiveness following bath application of NMDA or AMPA and following activation of glutamatergic afferents, including alterations in the ratio of NMDA to AMPA responsiveness. We have already obtained recordings from ?-galactosidase-positive neurons.? ? We have recently developed a method to dissociate striatal neurons from cfos-lacZ rats following cocaine-induced behavior and sort the activated neurons that contain ?-galactosidase from the majority of non-activated neurons that do not contain ?-galactosidase. We use fluorescence-activated cell sorting (FACS) to sort these neurons. We are present analyzing mRNA message levels in these neurons using real-time PCR techniques. This technique will allow us to identify the unique molecular alterations in these neurons during context-specific sensitization to cocaine. ? ? The methods we developed in the past two years will enable 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.

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National Institute on Drug Abuse (NIDA)
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Klanecky, Alicia K; McChargue, Dennis E (2009) Emotional reactivity across individuals with varying trauma and substance dependence histories. Ment Health Subst Use 2:191-202
McChargue, Dennis E; Doran, Neal (2009) Cue-evoked positive affect, depression vulnerability and smoking years. Am J Health Behav 33:49-57
Onaivi, Emmanuel S; Ishiguro, Hiroki; Gong, Jian-Ping et al. (2008) Brain neuronal CB2 cannabinoid receptors in drug abuse and depression: from mice to human subjects. PLoS ONE 3:e1640
Hope, Bruce T; Nagarkar, Deepti; Leonard, Sherry et al. (2007) Long-term upregulation of protein kinase A and adenylate cyclase levels in human smokers. J Neurosci 27:1964-72
Carriba, Paulina; Ortiz, Oskar; Patkar, Kshitij et al. (2007) Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids. Neuropsychopharmacology 32:2249-59
Harvey, Brandon K; Hope, Bruce T; Shaham, Yavin (2007) Tolerance to opiate reward: role of midbrain IRS2-Akt pathway. Nat Neurosci 10:9-10
Mattson, Brandi J; Crombag, Hans S; Mitchell, Tim et al. (2007) Repeated amphetamine administration outside the home cage enhances drug-induced Fos expression in rat nucleus accumbens. Behav Brain Res 185:88-98
Quarta, Davide; Ciruela, Francisco; Patkar, Kshitij et al. (2007) Heteromeric nicotinic acetylcholine-dopamine autoreceptor complexes modulate striatal dopamine release. Neuropsychopharmacology 32:35-42
Hope, Bruce T; Simmons, Danielle E; Mitchell, Tim B et al. (2006) Cocaine-induced locomotor activity and Fos expression in nucleus accumbens are sensitized for 6 months after repeated cocaine administration outside the home cage. Eur J Neurosci 24:867-75
Hope, B T; Crombag, H S; Jedynak, J P et al. (2005) Neuroadaptations of total levels of adenylate cyclase, protein kinase A, tyrosine hydroxylase, cdk5 and neurofilaments in the nucleus accumbens and ventral tegmental area do not correlate with expression of sensitized or tolerant locomotor responses to co J Neurochem 92:536-45

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