Identification and characterization of striatal neurons 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 that are activated by drug-associated stimuli. Until now there has been no method to identify these specific neurons 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 specific neurons that are activated during locomotor sensitization to cocaine and amphetamine. In the past year, we have found that the capacity 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 behavioral sensitization to the drugs in a novel environment. 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 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. [We are now performing final analysis of the data for a manuscript] 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 have bred a strain of cfos-lacZ transgenic rats in order to identify and selectively manipulate these neurons in live tissue. The rats' inserted transgene contains a c-fos promoter that controls transcription of the bacterial gene lacZ, which encodes the enzyme beta-galactosidase. In the cfos-lacZ transgenic rats, we have observed induction of beta-galactosidase in fixed striatal tissue following acute and repeated cocaine administration. This induction follows a similar time course, dose-response relationship, and regional distribution to those previously observed using Fos immunohistochemistry as a marker of activated neurons. We are presently confirming colocalization of Fos and beta-galactosidase using double-labeling techniques. Beta-galactosidase labeling is enabling us to identify and manipulate the enzyme in live tissue. For the first time, we can study, in live tissue, the specific neurons that are activated during drug-induced behavior. We are selectively lesioning neurons activated during drug-induced locomotor activity to analyze their role in mediating this behavior. Daun02 is a suicide substrate for beta-galactoosidase that, in culture following bath application of Daun02, kills only cells that contain the enzyme. When cocaine administration induces beta-galactosidase in the striatum cfos-lacZ transgenic rats, subsequent injection of Daun02 into the striatum will kill only those neurons activated during cocaine administration. We are then testing for cocaine-induced locomotor activity to determine the lesioned neurons' role in behavior. Selective lesions of only those neurons that are active during a behavior has never been done before. We are also testing whether the same neurons are involved in other behaviors, such as cocaine self-administration and relapse. We have already labeled neurons that contain beta-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. The methods we developed this year will enable us to identify and characterize a unique class of neurons that are selectively activated during drug administration. Understanding these neurons' function 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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