This study represents the first microarray analysis of mesolimbic gene expression following long-term enforced abstinence from cocaine self-administration. Transcriptomic studies of cocaine-induced gene expression changes have been conducted, but these have focused on non-contingent cocaine administration and no or limited (~ 1 day) abstinence. The work conducted in the present study used a model with well-characterized behavioral changes during periods of abstinence, and used animals not subjected to behavioral testing during abstinence (e.g. progressive ratio or extinction responding) so that the gene expression changes observed are free from the effects elicited by behavioral testing conducted before sacrifice. Additionally, it is important to note that all groups (1, 10, and 100 days of abstinence) self-administered equivalent amounts of cocaine over the 10 days of discrete trial cocaine self-administration. This time-course analysis of gene expression allows for discrimination of gene expression changes associated with increased drug seeking (10 and 100 days of abstinence) from those that occur with cocaine self-administration, but do not persist for as long as increased drug seeking and taking (1 day of abstinence).
The literature describes a number of neurobiological changes (e.g. altered gene/protein expression, neurotransmitter levels, epigenetic events) with different models of cocaine abuse, (for a review see [11, 28]). Whether these changes persist into periods of abstinence, however, has generally not been determined. In this study, gene expression changes that occurred as a result of cocaine self-administration and abstinence segregated into three categories of expression patterns. Category 1 changes were defined as those that occur with cocaine use, but do not persist into periods of abstinence. These were observed as changes only between naïve animals and 1-day abstinent animals. After only 1 day of abstinence, an increase in drug seeking and drug taking is not observed , so the changes in gene expression observed at this point may be necessary, but are not sufficient, to cause the incubated phenotype and are primarily due to exposure to cocaine. Category 2 changes are those that occur with cocaine use that persist with periods of abstinence. These were observed to be altered in the comparisons between naïve and 1-day abstinent animals and between naïve and 10- or 100-day abstinent animals. These alterations may result from cocaine exposure, but do not return to naïve levels with cessation of the cocaine stimulus. The persistence of these changes may indicate their potential role in the development (10 days) or maintenance (100 days) of abstinence-persistent increases in drug seeking and drug taking behaviors. Category 3 changes consist of genes that were unchanged with cocaine use, but are altered during the abstinence period. While not immediately affected by cocaine exposure, this set of changes may result from initiation or continuation of abstinence. These may function synergistically with other (category 2) changes to contribute to the development of increased drug-seeking and -taking.
The mPFC mediates executive function and decision making processes and is therefore a key neuroanatomical region in addictive behaviors [29, 30]. In response to cocaine administration, changes in metabolic activity, neurotransmitter systems, and gene or protein expression occur in the mPFC (for a review see ). In this study, a large number of gene expression changes were observed in the mPFC both as a result of cocaine self-administration and with subsequent enforced abstinence. Most of these changes occurred as a direct result of the cocaine self-administration (981) and a majority (793) returned to cocaine-naïve levels with cessation of cocaine self-administration. As expected many changes (category 1) in gene expression require continued cocaine stimulus to remain altered, and return to normal levels after the stimulus is removed. A subset of genes (188) remained changed after 100-days of enforced abstinence. Persistence of gene expression changes with abstinence (category 2) requires maintenance via other mechanisms. Epigenetic changes occur in response to cocaine, and may constitute a regulatory mechanism for persistent changes in gene expression [12, 31]. Changes (480) that do not occur during cocaine self-administration, but are induced with abstinence (category 3) may reflect the withdrawal of the cocaine stimulus and development of the incubated phenotype.
Altered gene expression of Adora2b, Arc, CART, Cd47, Drd5, Egr1, Fos, Nefl, NPY, and Nr4a1 were confirmed by qPCR. We have previously described altered expression of Arc, CART, Egr1, Fos, NPY, and Nr4a1 in these samples in a directed study of genes with known relevance to drug abuse . A number of the qPCR confirmation analyses that did not reach statistical significance demonstrated expression profiles similar to those observed in the microarray. This may reflect the effects of neuroanatomical complexity on quantitation of gene expression endpoints and the inclusion of larger numbers of animals in the confirmatory experiments.
This work identified altered expression of two G-protein coupled receptors (GPCRs; Adora2b, Drd5), a cell-surface signaling molecule (CD47), and a component of the neuronal cytoskeleton (Nefl). Increased Drd5, and signaling through this receptor, have been reported to decrease responsiveness to cocaine [32, 33]. Similarly, adenosine signaling has been implicated in drug addiction. Specifically, activation of Adora2b receptors attenuates cocaine-conditioned place preference . Although the mechanisms underlying these effects are unclear, Drd5 signaling is implicated in neuronal activities including long-term potentiation (LTP; ), and reinforcement learning , while both Drd5  and Adora2b  appear to affect Ca2+ dynamics.
The decrease in CD47 expression in this model is a novel observation and is of interest due to its function in neuronal development [39, 40]. Nefl functions in cytoskeletal organization and cell-surface receptor remodeling [41, 42], which may be impaired with the observed decrease in expression at 1-day of abstinence. Previously, changes in protein levels and post-translational modifications of Nefl, and other neurofilament isoforms, have been reported with cocaine, morphine, alcohol, and nicotine administration [43–45].
We have previously reported a directed analysis of immediate early genes (IEGs; Arc, Egr1, Fos, and Nr4a1) and neuropeptides (NPY and CART) in this animal model . These genes were also identified in the current discovery microarray analysis, providing increased confidence in the microarray findings. These genes play important roles in a number of neuronal processes including learning and memory [46, 47], synaptic plasticity [48–50], Ca2+ signaling [51, 52], and MAPK signaling .
Network analysis was conducted using the set of confirmed mPFC gene expression changes, and revealed that Cart, NPY, Nr4a1, Fos, Egr1, Adora2b and Drd5 all interact (directly or indirectly) with the MAPK/ERK pathway. While altered expression of MAPK/ERK pathway elements was not detected in this study, changes in expression and activity levels of MAPK/ERK genes have been reported (for a review see ) and this pathway is thought to play an important role in drug-induced changes in the brain [53, 54]. Regulators of Ca2+ dynamics were also identified in the network analysis. The changes in Drd5, Adora2b, CD47 and CART expression may indicate a decrease in intracellular Ca2+ signaling that occurs with cocaine self-administration and persists into periods of abstinence [40, 52, 55, 56].
Additionally, the gene expression changes identified indicate that synaptic plasticity may be affected by cocaine self-administration and abstinence. Persistent reductions in levels of CD47 and Arc, and inductions in levels of Drd5 and NPY suggest altered synaptic plasticity process involved in memory formation and removal of old memory traces, respectively [50, 57, 58]. A potential reduction in synaptic plasticity in the mPFC with cocaine self-administration/abstinence is hypothesized based on levels of CD47, Nefl, Arc, Egr1, and NPY [39, 42, 48–50]. These data are in agreement with studies of the direct role of psychostimulants on mechanisms of synaptic plasticity, including LTP and LTD, in the mesolimbic system [59, 60]. In total, these gene expression changes may contribute to persistently altered synaptic plasticity in the mPFC.
The central role of the NAc in psychostimulant reward is well documented . While cocaine exerts common actions on the NAc and mPFC , we observed little overlap (50 of the 1875 total gene expression changes (mPFC + NAc)) between these brain regions. The regulated genes common to both brain regions include IEGs reported previously , various signaling molecules, and genes involved in cellular metabolism. When the microarray datasets were examined by ontological analysis distinct molecular functions were observed in each brain region. This indicates that the functional changes occurring in the mPFC and NAc may differ and may ultimately play different roles in abstinence-dependent behaviors.
Unlike the mPFC, fewer category 1 and 2 changes were observed in the NAc (100 of 414 total), than category 3 changes (those changed specifically during abstinence) (314). Of the cocaine-induced changes, only 32 persisted into periods of abstinence (category 2), while the remainder returned to pre-cocaine levels. Arc, Beta-catenin, Cap2, Crip2, Dnm2, Egr2, Fos, Fut8, GFAP, Gpr88, Htr1d, and Nr4a1 were all confirmed by qPCR to be differentially expressed. We have previously demonstrated the responsiveness of Arc, Fos and Nr4a1 in this animal model .
Published data regarding Cap2, Crip2, Fut8, and Gpr88 in the brain are limited, with no previous reports of cocaine-responsiveness. Crip2 (a LIM-domain protein), Cap2 (an adenylate cyclase-associated protein) and Dnm2 are cytoskeletal function and organization genes [63, 64]. Interestingly, Dnm2 is regulated by the transcription factor Arc, also altered in the NAc with cocaine [12, 65]. Among the remaining changes, Egr2 and GFAP have been previously demonstrated to be cocaine-responsive [66–68]. Htr1d has been linked with a number of psychiatric disorders [69, 70]. Changes in the expression of these genes may also indicate cocaine induced alterations in receptor signaling, glial cell function, and synaptic plasticity.
Beta-catenin, which was increased at 10-days of abstinence in this study, is a well-characterized protein that regulates cell growth as a part of the Wnt signaling pathway. As a part of Wnt signaling, beta-catenin also plays a role in synaptic plasticity [71, 72]. In response to chronic cocaine, beta-catenin has been shown to increase in a number of brain regions [73–75]. Fut8, a fucosyltransferase protein, also increased at 10-days of abstinence in this study, has been shown to increase upon Wnt/beta-catenin activation , indicating that there may be a coordinated activation of Wnt signaling during periods of abstinence from cocaine.
Network analysis of the confirmed genes in the NAc identified a TNF-centered network. Generally involved in inflammatory processes, TNF has not been historically associated with behavioral responses to cocaine. Studies performed on the effects of cocaine on macrophages have reported that cocaine suppresses LPS-stimulated TNF expression [77, 78]. TNF induction was recently demonstrated to reduce conditioned place preference and locomotor sensitization caused by methamphetamine and morphine administration . If TNF does play a role in the behavioral responses to cocaine, these additional genes may represent regulatory and effector elements of a TNF network.
While these reported changes represent new insights into abstinence-induced changes in the brain, localization of these changes to specific cell types is still to be determined. As with other functional genomic and proteomic approaches looking at dissected brain regions, even these specific dissections contain a heterogeneous cellular population. Future molecular neurobiology studies that seek to extend these, and other findings, will need to utilize techniques (e.g. laser capture microdissection and fluorescent in-situ hybridization) to localize changes to specific cell types and neuronal networks .