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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0317fje. |
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* Research Institute Neurosciences Vrije Universiteit, Drug Abuse Program, Department of Medical Pharmacology, VU Medical Center, 1081 BT Amsterdam, The Netherlands;
Research Institute Neurosciences Vrije Universiteit, Drug Abuse Program, Department of Molecular and Cellular Neurobiology, Faculty of 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{at}vumc.nl
SPECIFIC AIM
In view of the differential role of the nucleus accumbens (NAc) shell and core in motivational behavior and the functional relationship between these subregions, the present study was designed to examine the long-term effects of heroin exposure on the expression in the NAc core of a preselected set of transcripts previously shown to be down-regulated in the NAc shell specifically by self-administration of heroin and cocaine. We also aimed to study the role of response contingency in mediating heroin-induced gene expression.
PRINCIPAL FINDINGS
1. The expression of transcripts down-regulated in the NAc shell long after self-administration of heroin is up-regulated in the core
Rats were trained to self-administer heroin during daily sessions, then subjected to an extinction protocol of 3 wk. After 18 extinction sessions, the number of responses for the heroin treatment group was strongly reduced relative to the first extinction session and reached the level of that for the saline treatment group. However, there was no statistically significant difference between the extinction and maintenance phases. To investigate whether the observed down-regulation of a recently identified set of transcripts in the NAc shell long after heroin self-administration displays regional specificity within the NAc, we determined expression levels in the NAc core of the same animals by means of transcript-specific, real-time quantitative PCR (qPCR) to detect subtle changes in the expression of even low abundant transcripts. Values were normalized to the transcript for HPRT. As shown before, the expression of additional endogenous controls (glyceraldehyde-3-phosphate dehydrogenase, neuron-specific enolase, 
-tubulin, ß-actin, and ubiquitin) was not differentially regulated. In contrast to down-regulation in the NAc shell, most transcripts were up-regulated in the core long after heroin self-administration. Of the 25 transcripts down-regulated in the NAc shell, 18 were significantly up-regulated and only 2 were also down-regulated in the NAc core (Fig. 1
). The differential regulation of five transcripts did not reach statistical significance.
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2. Possible role of response contingency in heroin-induced gene expression
To examine the possible role of response contingency in the expression of these 25 transcripts in the NAc core, we determined their expression long after noncontingent (passive/forced) administration of heroin in so-called yoked controls. As observed after heroin self-administration, the majority of the transcripts (20 of 25) was significantly up-regulated in the NAc core upon noncontingent administration of the opiate (Fig. 1
). In this case, the regulation of five transcripts did not reach statistical significance. Finally, from the 18 transcripts up-regulated in the NAc core upon heroin self-administration, no fewer than 16 transcripts were up-regulated by noncontingent administration of heroin.
CONCLUSIONS AND SIGNIFICANCE
Unraveling the molecular basis of long-term neuroplasticity underlying relapse behavior may ultimately lead to an adequate pharmacotherapy for drug addicts. In an initial study to identify the long-term genomic consequences of drug exposure, we discovered a set of gene transcripts that is down-regulated in rat NAc shell long (3 wk) after active, but not noncontingent, heroin administration, thereby revealing key cellular processes that may underlie reinstatement of drug-seeking behavior during abstinence. This set of transcripts was obtained from a well-controlled subtraction screening (validated by qPCR) between the NAc shell of rats that previously self-administered heroin and that of rats responding for saline. The transcripts are of interest because they encode for proteins involved in a wide variety of cellular processes such as neuronal growth, apoptosis, synaptic transmission, neuropeptide synthesis and release, transcription, translation, and cell metabolism. Moreover, they may be involved in the response to addictive drugs in general, as 17 of the 25 transcripts were shown to be down-regulated upon cocaine self-administration. Differential expression of the transcripts was observed long after drug exposure, suggesting they may be responsible for long-term reinstatement of the extinguished drug-seeking behavior upon reexposure to drugs or drug-associated cues as observed using this animal model for relapse behavior. To study the region specificity of drug-induced genomic responses when considering the functional compartmentation of the NAc, we focused on this set of 25 transcripts. We revealed striking differences in gene expression between the NAc core vs. the shell, and showed that most of these transcripts are up-regulated in the NAc core long after repeated heroin exposure independent of response contingency.
But how can the dichotomous response of the NAc subregions be explained? It is now widely accepted that the NAc cannot be regarded as a single unit, but that its subregions can be distinguished anatomically and functionally. Histochemical studies have revealed that the NAc shell comprises the medial, ventral, and rostral parts of the NAc, whereas the NAc core is located in the dorsal and central parts. Each subregion is connected to other brain areas, with the NAc shell embedded in the limbic emotional and motivational circuit and the NAc core in the circuits mediating motor expression in response to motivational stimuli. The embedding of the subregions in different brain circuits is also reflected in their differential function in several kinds of behavior. For instance, the NAc shell and core are differentially involved in latent inhibition, with the NAc shell modulating the activity of the core. In relation to self-administration of addictive drugs, two processes are of particular importance: reinforcement learning and the conversion from motivation to action. The NAc core seemed to mediate reinforcement learning whereas the NAc shell was shown to integrate reward-associated (Pavlovian) cues to trigger increased instrumental performance for obtaining drug reward. Differences in the release of dopamine in the NAc shell and core of drug-exposed rats have been reported. For example, noncontingent exposure to morphine, cocaine, or amphetamine acutely increased extracellular dopamine levels preferentially in the NAc shell compared with the core. Behavioral sensitization to morphine, cocaine, or nicotine was associated with enhanced dopamine release in the NAc core and with decreased dopamine release in the shell. In addition, preexposure to conditional appetitive stimuli seemed to inhibit the consummatory dopamine response in the NAc shell whereas this response was potentiated in the core.
The present study lends further support to the functional compartmentation of the NAc, since both subregions adapted differentially at the level of gene expression long after contingent or noncontingent exposure to heroin. Long after contingent administration of heroin there seems to be a reciprocal relationship between the opiate-induced genomic responses in these NAc subregions, in agreement with the aforementioned neurochemical observations. The activity of the NAc core has been suggested to be under control of the shell; as such, the latter subserves a neuronal mechanism to activate instrumental actions. Moreover, the NAc core is modulated by means of projections from the NAc shell to the ventral tegmental area and substantia nigra pars compacta, terminating upon dopaminergic projections to the NAc core. An important finding in the present study is that the genomic responses in the NAc core long after contingent and noncontingent heroin administration are qualitatively similar, whereas in the NAc shell these responses differ profoundly. Thus, unlike long-term genomic responses in the NAc shell, both contingent and noncontingent heroin exposure enhanced the expression of the studied set of transcripts (down-regulated in the NAc shell by heroin self-administration only). Therefore, the cognitive processes involved in drug self-administration may only direct the genomic response observed in the NAc shell, whereas the similar long-term genomic responses in the NAc core upon contingent and noncontingent heroin administration of heroin may reflect the persistent pharmacological effects of heroin (including Pavlovian conditioning) (Fig. 2
).
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Much work lies ahead in order to obtain a genome-wide overview of differentially expressed genes in both subregions of the NAc. For instance, future studies of gene expression should reveal transcripts that are up-regulated in the NAc shell long after self-administration of heroin and other addictive substances, as well as transcripts differentially regulated in the core, before addressing the question of which genes are responsible for the persistence of drug addiction.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0317fje; 
2 Present address: Department of Cell Biology and Genetics, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
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  L. Yao, K. McFarland, P. Fan, Z. Jiang, Y. Inoue, and I. Diamond Activator of G protein signaling 3 regulates opiate activation of protein kinase A signaling and relapse of heroin-seeking behavior PNAS, June 14, 2005; 102(24): 8746 - 8751. [Abstract] [Full Text] [PDF]
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0.05).