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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0272fje. |
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Research Institute Neurosciences Vrije Universiteit, Drug Abuse Program, Department of Medical Pharmacology, VU Medical Center, 1081 BT Amsterdam, The Netherlands; and
* Department of Molecular and Cellular Neurobiology, Faculty Earth and Life Sciences, 1081 HV Amsterdam, The Netherlands
3Correspondence: Research Institute Neurosciences Vrije Universiteit, Department of Medical Pharmacology, VU Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail: anm.schoffelmeer.pharm{at}med.vu.nl
SPECIFIC AIM
In view of possible context-dependent factors in drug-induced neuroplasticity, the objective of this study was to compare the genomic response in the rat nucleus accumbens (NAc) shell after long-term withdrawal of active and passive heroin administration. To identify differentially regulated transcripts in the NAc shell after long-term extinction of heroin self-administration (SA), we constructed a subtraction library and preselected a set of transcripts of which expression was assessed in different cohorts of rats with a history of heroin or cocaine exposure (SA and forced administration; FA) by means of real-time quantitative PCR. We found that active drug consumption during SA (instrumental learning) is a crucial psychological factor directing long-term genomic consequences in the brain.
PRINCIPAL FINDINGS
1. Construction of the subtraction library
Rats were trained using a widely employed heroin self-administration paradigm. After 18 daily sessions of extinction of SA behavior, we constructed a subtraction library of the NAc shell. To preselect a set of transcripts coherently regulated in the NAc shell long after heroin SA vs. saline SA, we chose a unidirectional subtractive hybridization method. During subtraction, both cDNA populations were hybridized to enrich for differentially expressed transcripts. Suppression PCR was performed to obtain a set of cDNA clones representing genes expressed at a lower level in the NAc shell after heroin SA. Subtraction was successful, as the abundance of GAPDH in the subtracted and unsubtracted pools of cDNA differed 
10 times. After subtraction, amplified cDNA was cloned into an M13 vector. From a library of 7964 primary clones, 1060 plaques were picked and analyzed. Size determination by PCR and sequencing revealed that 317 clones contained inserts. Different cDNA fragments were found to correspond to the same transcript due to initial RsaI digestion. The 317 clones yielded 152 individual transcripts, with 40 transcripts encoding for known proteins, 79 known as ESTs, and 33 so far unidentified.
2. Validation of the library
The group of 40 known transcripts was assessed for its regulation by reverse Northern blot analysis to independently validate the subtractive hybridization. After normalization to HPRT, the average regulation of the transcripts was -2.7 ± 0.3-fold, in agreement with the construction of a library representing down-regulated genes. Transcript-specific real-time quantitative PCR (qPCR) was used to measure the long-term genomic responses in the NAc shell after active heroin administration for the set of 40 transcripts in pooled RNA from an independent cohort of rats. These experiments showed that 25 transcripts were significantly down-regulated (P<0.05) (Fig. 1
). Fifteen transcripts did not reach statistical significant differential regulation, suggesting that they represent false positives from the subtraction procedure.
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3. Active and passive heroin administration induce different gene expression profiles
We found that only 8 of the 25 transcripts were significantly down-regulated in the NAc shell of rats that received doses of heroin passively (FA) (Fig. 1)
. These data indicate that SA of heroin in rats primarily causes long-term regulation of the expression of transcripts that is specific for active drug intake (i.e., not apparent after a passive form of drug administration).
4. Active and passive cocaine administration induce different gene expression profiles
To test this novel concept further, we measured the validated transcripts long after SA and FA of cocaine in the NAc shell of another cohort of rats. Rats were trained to self-administer cocaine essentially as described for heroin. QPCR experiments revealed that 17 of the 25 transcripts of the library also appeared to be down-regulated long after cocaine SA (common denominators). In contrast, and in line with the results obtained for heroin, after FA of cocaine only 4 of these 17 transcripts were significantly down-regulated. Thus, the striking difference between the genomic responses after SA and FA of heroin was also apparent in animals with a history of active and passive cocaine administration.
CONCLUSIONS AND SIGNIFICANCE
Several genes have been reported to be differentially expressed in rat NAc on administration of drugs of abuse, usually soon after cessation of drug exposure. These acutely regulated genes may cause immediate effects and trigger the long-term expression of genes that contribute to enduring neuroplasticity associated with the persistence of addiction behavior, including persistent changes in the altered responsiveness and morphology of NAc neurons. We investigated long-term changes in gene expression in rat NAc with a heroin SA paradigm used extensively to investigate relapse behavior induced by drugs of abuse, drug-associated cues and stressors. Since the NAc shell may be an important subregion regarding the effectiveness of stimuli in mediating retrieval of learned associations triggering goal-directed behavior, we focused on this subregion. To study the context dependence of genomic responses, we created a set of transcripts that is down-regulated in the NAc shell after long-term extinction of heroin SA.
Open subtraction screening was used that does not discriminate between types of transcripts and includes low abundant transcripts. The library is the result of a successful subtraction, as a parallel control experiment in which skeletal muscle cDNA was artificially enriched with cDNA and subsequently subtracted yielded the expected subtraction products. Second, several sequences are present multiple times in the library, indicating the preferential cloning of certain types of transcripts (317 clones represent 152 different transcripts). Third, the occurrence of transcripts encoding certain known proteins shows that their representation in the library is different from a nonsubtracted population of transcripts and hence indicates that they were selectively cloned. For example, transcription- and growth-related factors are highly abundant in the library but are low abundant in a normal population of transcripts. Ribosomal proteins are normally highly abundant, but in the subtracted library only one transcript (ribosomal protein L4) belongs to this class. Fourth, analysis of the subtraction efficiency showed a profound difference in the abundance of GAPDH between the subtracted and unsubtracted pools of cDNA. Most important, two independent methods—reverse Northern blot analysis and qPCR— performed on separate cohorts of rats confirmed down-regulation of most of a selected group of transcripts after heroin SA.
The validated set of down-regulated transcripts in the subtracted library comprises a large functional diversity in proteins involved in neuronal growth, apoptosis, signal transduction, neuropeptide synthesis and release, transcription, translation, and cell metabolism. The down-regulation of transcripts involved in the modulation of neuronal growth is consistent with the drug-induced alterations in neuronal morphology observed recently. Even more drastic rearrangements in the limbic system might result from apoptotic processes. Accordingly, several transcripts encoding for apoptotic proteins appeared to be regulated in the NAc shell. Our results further suggest that signal transduction is still affected long after cessation of heroin SA, as indicated by regulation of transcripts for receptors, phosphatases, kinases, and molecules involved in neuropeptide synthesis and release. Down-regulation of modulators of transcription, translation, and cell metabolism might suggest that heroin SA persistently alters cellular activity in the NAc shell. Elucidating whether and how these proteins are involved in adaptive changes related to addiction behavior is beyond the scope of the present study. The functional diversity observed in the proteins encoded by the long-term regulated genes indicates that the NAc shell is a target of regulation at various levels of cellular physiology. Because of the integrative role of the NAc shell within the limbic system, long-lasting alterations in cellular functioning may be expected to change synaptic transmission in related brain structures. The persistence of addiction behavior may therefore be a direct result of altered genetic and cellular networks.
The major finding of our study is that psychological processes involved in active, but not in passive administration of addictive drugs, appear to be crucial to the drug-induced long-term gene expression profiles in the NAc shell. Two types of drug-induced neuroadaptations can be distinguished: those caused by forced exposure to a drug and those caused by SA. Adaptations of the first type might result from pharmacological effects of the drug; those of the second type may be derived from psychological processes (instrumental learning) superimposed on these drug effects. Our findings indicate that long-term genomic consequences in the NAc shell resulting from previous drug SA are profoundly distinct from those caused by the pharmacological effects of drugs. Obviously, the unique neuroadaptations due to drug SA may be crucial for the ability of stimuli (drugs, stressors and drug-associated cues) to elicit relapse to compulsive drug-seeking behavior (Fig. 2
). Therefore, these findings have important ramifications for the design of genome-wide gene expression studies of the neurobiological basis of addiction behavior.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0272fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0272fje 
2 Present address: Centre for Neuroscience Research, Guy’s, King’s and St. Thomas’ Schools of Biomedical Sciences, King’s College London, St. Thomas St., London SE1 1UL, UK.
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0.05).