BACKGROUND
the challenge for drug abuse treatment is maintaining abstinence despite a high propensity for abstinent patients to relapse to drug use.
although heroin has been abused for centuries, effective long-term preventions for heroin-relapse are still needed.
physiological and gene expression changes that may increase an individual's likelihood to relapse are known to exist well into a period of abstinence  <cit> .
therefore, relapse to drug use is currently being investigated on both the molecular and behavioral levels  <cit> .
these studies have been aided by advances in systems biology tools, including large-scale discovery techniques such as microarrays and proteomics.
these approaches are useful for discovering novel targets affected by drug abuse and examining hypotheses concerning categories of genes that are affected by drug use  <cit> .
as data on gene expression following relapse accumulates, the existence of a single "relapse gene" is becoming increasingly unlikely.
therefore, macroscopic views of gene expression  will prove very useful for guiding research into behavioral phenomena.
neurobiological, environmental, cue, and stress mechanisms have all been implicated in relapse to drug use.
the intense craving and motivation to seek drug, reported by humans during withdrawal from drug use, is challenging to model in animals, but the need for a relapse model continues to motivate the design of new behavioral procedures.
incubation is a behavioral phenomenon that is characterized by increased drug seeking following increasing periods of abstinence after the last self-administration session  <cit> .
increased drug-seeking has been inferred to represent the craving that drives humans to relapse  <cit> .
the prefrontal cortex is important for decision making and guiding behavior  <cit> , and the medial prefrontal cortex  is known to be especially important for goal-directed behaviors  <cit> .
because of its role in guiding goal-directed, drug-seeking behaviors, understanding the gene expression changes in this region following a period of abstinence from drug self-administration will be useful for understanding the neurobiological basis to relapse following a period of drug self-administration.
additionally, the ventral mpfc is believed to play an essential role for expression of incubation of cocaine-seeking  <cit>  and reinstatement of cocaine-seeking  <cit> .
in this study, we performed a whole genome analysis of gene expression in the medial prefrontal cortex of rats that displayed incubation of goal-directed behavior following  <dig> weeks of heroin self-administration and  <dig> weeks of home-cage enforced abstinence.
after the abstinence period, rats  were reintroduced to the testing chambers for a 90-minute extinction session during which behavioral responses were recorded.
this extinction session served not only to provide an opportunity to observe behavioral incubation, but also mimicked a real-life situation in which environmental cues precipitate relapse behavior following an extended period of abstinence.
following this experience of re-exposure to drug-associated context and cues, rna was isolated from the mpfc for whole genome microarray and qpcr analyses.
ontological analyses revealed that many of the genes identified to be changed have the potential to be key components to neuroadaptations that exist at the time of relapse.
methods
heroin self-administration
the self-administration and extinction procedures have been previously described  <cit> .
briefly, rats that self-administered heroin displayed incubation of heroin-seeking when tested in a 90-minute extinction session that occurred after  <dig> weeks of enforced abstinence .
as described in previous work, all rats  initially underwent a habituation procedure during which they received water on a spout in the self-administration chambers.
following habituation, the rats self-administered heroin  by licking the previously water-associated spout during  <dig> hour sessions that occurred on  <dig> consecutive days.
spout-licking has been the operant behavior in previous drug self-administration studies  <cit> .
two empty spouts, termed "active" and "inactive" were present in the self-administration chambers .
a fixed ratio  <dig>  schedule of reinforcement existed, under which  <dig> consecutive responses on the active spout produced an automated injection of heroin.
self-administering rats were yoked to rats that received infusions of saline.
that is, the control group was a separate set of animals that were treated exactly the same, but never received heroin .
the  <dig> days of heroin self-administration were followed by a 90-minute extinction session  that occurred following either  <dig> day or  <dig> days of drug-free enforced abstinence after the final self-administration session.
during the extinction session, responses on active and inactive spouts were recorded, but no drug was administered.
immediately after the extinction sessions, the rats were sacrificed and brain regions were harvested.
all studies were conducted in accordance with the pennsylvania state university institutional animal care and use committee , strictly adhering to the guidelines for the care and use of mammals in neuroscience and behavioral research.
national research council,  <dig> national research council, guidelines for the care and use of mammals in neuroscience and behavioral research
dissection and rna isolation
sacrifice and tissue dissection
immediately following the 90-minute extinction session, all rats were sedated using propofol  and decapitated within  <dig> minutes.
brains were rapidly removed from skulls, placed in pre-chilled phosphate buffered saline  and then sectioned in an ice-chilled asi brain slicer .
the section from bregma + <dig>  to  <dig>  mm was cut along the forceps minor  and the cortex medial to this cut was collected.
this includes the cingulate , prelimbic cortex, infralimbic cortex, and medial orbital cortex.
following dissection, the tissue was placed in prechilled tubes, immediately frozen on dry ice, and then stored at -80°c.
rna isolation
total cellular rna was isolated using tri reagent   <cit> .
isolated rna was further purified using an rneasy mini kit for rna clean-up .
rna quantity and quality were assessed using the rna  <dig> nano assay with an agilent  <dig> bioanalyzer .
microarray analysis
microarray analyses were performed on samples from rats that experienced  <dig> d of abstinence prior to an extinction session.
studies were performed by the penn state college of medicine functional genomics core facility on  <dig> arrays  according to standard procedures  <cit> .
following the manufacturer's protocol of the low input fluorescent linear amplification kit ,  <dig> ng rna with the addition of one-color spike mix was denatured and incubated with t <dig> promoter primer.
synthesis of cdna followed with the addition of first-strand buffer, dtt, dntp mix, mmlv-rt and rnase out and incubation at 40°c for  <dig> hours.
transcription of the product incorporated the cyanine 3-ctp in the master mix which includes transcription buffer, dtt, ntps, peg, rnase out, pyrophosphatases and t <dig> rna polymerase, with incubation for  <dig> hours at 40°c.
the resulting crna was purified using rneasy columns  followed by assessment of purity, concentration and quality using a nanodrop nd- <dig>  through calculated yield and specific activity.
<dig>  μg from each sample was fragmented, denatured, and then hybridized to agilent  <dig> ×  <dig> rat whole genome microarray slides for  <dig> hours at 65°c.
slides were then washed according to protocol.
microarrays were scanned with an axon 4000b scanner with genepix <dig> v <dig>  software at a  <dig> μm resolution and  <dig> nm with laser power at 100%, pmt voltage at  <dig> v, focus position  <dig> μm, and lines to average =  <dig>  images were then imported into agilent feature extraction software.
initial quality control , exclusion of manufacturing defects , background subtraction was then performed and the results exported to genespring gx  <dig>  .
all primary array data have been deposited to gene expression omnibus .
microarray data analysis
microarray data were normalized following import into genespring gx  <dig>   by transforming signal values less than  <dig>  to an intensity of  <dig> .
normalization was done per chip to the 50th percentile, and per gene to the median.
values were then normalized on a per gene basis to the control group.
potential differential expression was determined with a one-way anova , p <  <dig>  and filtered for  <dig>  fold and greater differences in expression in accordance with standards for microarray analysis  <cit> .
the use of a combination of statistical and fold-change cutoffs as opposed to traditional multiple testing corrections  produce gene lists with the lowest rate of type i and type ii errors  <cit> .
<dig> -fold was chosen as the fold-change cutoff, as this magnitude change is at the lower range of changes we find to be confirmable by rt-qpcr.
array data for the complete list of  <dig> genes that were identified to have changed expression at the p <  <dig>  level of significance is included in additional file  <dig>  also included are fold-changes in expression, accession numbers, and probe identification information.
rt-qpcr analysis of gene expression
complimentary dna synthesis was performed on total rna  using superscript iii reverse transcriptase .
the rna used for rt-qpcr was an aliquot from the same samples used to generate microarray probes.
comparisons at both  <dig> d of abstinence and  <dig> d of abstinence were made to gain insights into whether the expression changes observed after  <dig> d of abstinence had existed immediately after drug use and persisted during extended abstinence or had emerged during extended abstinence.
one μg rna,  <dig> ng oligo , and  <dig> mm each dntp, were incubated for  <dig> minutes at 65°c and then chilled on ice for  <dig> minutes.
5× first strand buffer ,  <dig> mm kcl, and  <dig> mm mgcl2),  <dig> mm dtt ,  <dig> u rnaseout, and  <dig> u superscript iii rt were then added.
the  <dig> μl reaction was incubated for  <dig> minutes at 50°c followed by a final incubation at 70°c for  <dig> minutes for termination.
the resulting cdna product was quantified and  <dig> ng of product was used in each subsequent qpcr reaction.
quantitative pcr was carried out on a real-time detection instrument  in 384-well optical plates using taqman universal pcr master mix and assay on demand primers and probes  as described previously  <cit> .
primer/probe sets used are listed in table  <dig>  sds  <dig> .
<dig> software and the 2-ΔΔct analysis method  <cit>  were used to quantitate relative amounts using β-actin as an endogenous control.
ontological, pathway, and network analysis
ontological analysis used gene ontology  categories to determine processes or functional categories that were differentially expressed, as described previously  <cit>  using genespring gx software.
this analysis determined the number of genes in a category present on the array and the number of expression changes that would be part of that category by random chance given the number of differentially expressed genes.
ingenuity pathway analysis  was used to create a network from rt-qpcr confirmed gene expression results from the rats described in this study.
statistical analysis
behavioral data were analyzed by t-tests at each time point of abstinence .
goal-directed behavior was determined by subtracting inactive spout responses from active spout responses.
rt-qpcr gene expression values were evaluated using t-tests between self-administering and yoked saline rats at each time point of abstinence .
for both behavioral and gene expression data, levels of significance were determined with α set at  <dig> .
correlational analyses were performed to determine whether a correlation between the goal-directed behavior or active spout responses during the extinction session and gene expression existed.
RESULTS
behavior
rats were allowed to self-administer heroin during  <dig> days of daily drug access.
the average daily heroin intake for the rats that experienced only  <dig> day of abstinence prior to extinction increased from  <dig>  ±  <dig>  infusions on day  <dig> to  <dig>  ±  <dig>  infusions on day  <dig> of self-administration.
average daily heroin intake for the group of rats that experienced  <dig> days of abstinence prior to extinction increased from  <dig>  ±  <dig>  infusions on day  <dig> to  <dig>  ±  <dig>  on day  <dig> of self-administration.
when rats were reintroduced to the self-administration chambers following  <dig> d or  <dig> d of abstinence, incubation of active spout responses was observed .
goal-directed behavior after  <dig> days of abstinence was significantly higher in self-administering vs. yoked saline rats .
additionally,  <dig> day abstinent heroin self-administering rats had higher goal-directed behavior than self-administering rats with only  <dig> day of abstinence .
microarray analysis
signals from  <dig>  probes  were detected as being present on all of the arrays.
filtering the detected genes produced a list of  <dig> genes that were identified as being changed by at least  <dig>  fold  relative to saline controls after  <dig> days of abstinence and a 90-minute extinction session.
a full listing of differentially expressed genes is provided in additional file  <dig>
rt-qpcr confirmation and validation
rt-qpcr was performed to confirm expression levels of genes for which significant differences in expression were detected by microarrays.
genes were chosen for confirmation analyses based on their ontological classifications and probable involvement in drug use.
many genes examined belonged to ontological categories of nervous system development or behavior.
for each gene examined, samples from self-administering and yoked saline rats from each of the abstinent period treatment groups  were examined.
among the genes that were detected by the arrays to be significantly changed, and confirmed by rt-qpcr, were genes that are important for intracellular signaling .
dual specificity phosphatase  <dig>  and dual specificity phosphatase  <dig>  expression levels were both increased by 39% and 24%, respectively, after  <dig> days of abstinence, although only dusp <dig> expression levels were increased after only  <dig> day of abstinence.
regulator of g-protein signaling  <dig>  expression was increased by 20% following  <dig> days of abstinence.
genes for additional intracellular molecules that each can be linked to physiological changes that occur following drug use were also confirmed to be significantly changed .
brain-derived neurotrophic factor  and calbindin  <dig>  both displayed increased expression levels, by 32% and 21%, respectively, relative to saline controls.
neuropeptide y  gene expression  was decreased by 18% at  <dig> day of abstinence and 20% at  <dig> days of abstinence.
ontological, pathway, and network analysis
analysis of microarray gene expression data by gene ontology revealed  <dig> genes that are involved in developmental processes.
of these genes,  <dig> were confirmed to be changed by rt-qpcr: bdnf, calb <dig>  dusp <dig>  egr <dig> and one  has been previously reported by another group of researchers  <cit> .
a second ontological category of interest was behavior, and  <dig> genes from our list were included in this category.
among these genes were egr <dig> and crybb <dig>
the ingenuity network analysis revealed that many genes validated in this study, together with genes previously reported from these samples to be significantly changed, can be linked through a hypothetical network that is involved with behavior, nervous system development and function, and cellular development .
the pathway depicted in this figure is based on known intermolecular relationships , although whether this entire network of interactions occurs in vivo remains to be elucidated.
all of the genes in this pathway were determined to be upregulated.
correlational analyses
behavioral data collected during extinction  was used in correlational analyses with gene expression data.
both pearson's and spearman's correlations were performed because of the small sample sizes that were not always normally distributed.
when separate correlations were performed for data from each time point, pearson's analyses identified a positive correlation between the number of active spout responses and dusp <dig> gene expression in rats that experienced  <dig> day of abstinence .
a significant negative correlation between dusp <dig> gene expression and inactive spout responses  was detected for rats that experienced  <dig> days of abstinence by a spearman's analysis.
combining the data from both abstinent periods yielded  <dig> more significant correlations .
goal-directed response and active spout response data from extinction were correlated with rt-qpcr data.
eight significant correlations were detected .
the upper number in each block of the table is the correlation coefficient and the lower number in each block of the table is the p value.
discussion
this study provides the first report of whole genome analysis of mpfc gene expression in rats that have expressed behavioral incubation and expands our knowledge of gene expression changes that exist after abstinence and during the time of relapse liability.
the approach of using a preliminary screen of the entire genome, followed by rigorous rt-qpcr confirmation of expression changes for genes likely to be involved in behavior or neuronal changes has yielded information on genes, such as dusp <dig> and dusp <dig>  whose role in addiction is only now beginning to emerge.
the majority of genes that were significantly changed using the criteria reported in this paper  had increased expression levels in heroin self-administering rats when compared to yoked saline following  <dig> days of enforced abstinence.
previously, increased gene expression has been found for opiate receptors in humans that died from an opiate overdose  <cit> , as well as components of the cyclic amp signaling system following opiate use  <cit> .
the reasons for a disproportionate number of up-regulated versus down-regulated genes following abstinence from heroin self-administration remain to be determined.
however, when gene expression nears the level of detection of the microarray platform , it is becomes more difficult to detect down-regulations.
for the genes identified, and confirmed in this study, there are several potential impacts on behavior.
phosphatase genes, such as dusp <dig> and dusp <dig> , encode proteins that have a direct impact on intracellular signaling.
dusp <dig> preferentially dephosphorylates erk  <cit>  and is intranuclear, while dusp <dig> is cytoplasmic  <cit> .
mdma  has been shown to increase expression of dusp  <dig>  dusp  <dig>  and dusp  <dig> genes  <cit> , while methamphetamine treatment increases dusp <dig> gene expression in multiple brain regions  <cit> .
both dusp <dig>  <cit>  and dusp <dig>  <cit>  regulate mitogen-activated protein kinase .
numerous studies have suggested mapk involvement in neuroadaptations that occur following drug use  <cit> .
beyond being affected by mere drug exposure, mapk molecules have also been shown to play a role in morphine tolerance  <cit>  and to be activated following opiate withdrawal  <cit> .
interestingly, the mapk pathway plays a central role in the relationships between genes that were reported as changed in this study and our previous study of gene expression .
molecules that regulate g-protein coupled receptor  signaling have been shown to be essential for reinstatement of heroin-seeking behavior  <cit> .
the observed increase in rgs <dig> gene expression following  <dig> days of abstinence and contextual re-exposure may represent an intracellular signaling change that affects communication between receptors and transcription factors, ultimately affecting cellular and organismal physiology.
a gpcr-regulating molecule  was shown, through a gene knock-down approach, to be essential to activating protein kinase a  signaling and observing reinstatement of heroin-seeking  <cit> .
the microarray analysis detected an npy receptor, npy5r, to be significantly increased after  <dig> days of abstinence.
while rt-qpcr for npy5r failed to replicate this change, the gene encoding npy, the ligand for this receptor, was tested by rt-qpcr and found to be changed.
npy gene expression differed from most other genes examined because it was decreased in rats that had self-administered heroin, both after  <dig> and  <dig> days of abstinence.
although npy is a neurotransmitter most recognized for its role in regulating food intake  <cit> , it has been hypothesized that npy may contribute to the negative motivational state of withdrawal  <cit> .
intracerebroventricular administration of npy has been found to block increased ethanol intake in rats  <cit> , and overexpression of the npy gene, using intra-amygdalar infusions of a viral expression vector, diminished alcohol intake following longer periods of abstinence or repeated alcohol withdrawals  <cit> .
in the present context, intraventricular injections of npy have been found to induce heroin-seeking behavior following extinction sessions  <cit> , suggesting that the decrease in npy gene expression observed in the present study may be related to the extinction component of the behavioral testing.
the majority of genes on which rt-qpcr was performed displayed expression levels significantly different from saline controls only after  <dig> days of abstinence .
this suggests that these changes are attributable either solely to the extended drug abstinence or to the combination of extended abstinence and contextual re-exposure.
it is documented that exposure to environments previously paired with drug administration can affect gene expression  <cit> , so future studies will be required to discern whether the observed changes in gene expression after  <dig> days of abstinence resulted from contextual re-exposure, the pharmacological drug abstinence, or a combination.
correlational analyses detected that bdnf was significantly correlated with both goal-directed behavior and active spout responses .
this observation supports the proposed importance of bdnf to drug-seeking behavior  <cit> .
while the study was not originally powered to conduct individual correlational analyses, the findings with bdnf suggest that future work may focus on individual behavior and gene expression.
several genes whose expression was changed in this study have also displayed changed expression levels following several extinction sessions  <cit> .
in the mpfc, there was an increase in gene expression relative to controls for arc, homer1a, ania- <dig>  mkp- <dig>  c-fos, egr <dig>  egr <dig>  and nr4a <dig>  homer <dig>  egr <dig>  and nr4a <dig> each were detected by the arrays to have increased expression values of at least  <dig> -fold in the current study.
the repeated extinction sessions in the koya et al.
study  <cit>  are a major difference from our study that may contribute to the differences in gene expression observed between studies.
genes that were changed in both koya's study, which included  <dig> extinction sessions, and the present study, which included  <dig> extinction session, are genes that apparently exhibit changed expression following a prolonged abstinence and maintain that change in expression regardless of the amount of environmental re-exposures.
the identities of the confirmed genes include not only transcription factors, but also genes for molecules involved in intracellular signaling and protein binding .
this range of functions  provides a reminder that drug use elicits changes in entire intracellular networks.
the importance of changes in gene expression of certain proteins, such as calb <dig> or dusp <dig>  to eliciting changed expression of genes encoding transcription factors, such as egr <dig> or fos, is an area for future investigation.
while this microarray analysis has illuminated genes whose expression is changed immediately following an extinction session, the gene expression profile that exists prior to extinction testing has not been examined in this study.
a comparison between the gene expression profile following abstinence versus the gene expression profile following abstinence and extinction would address the question of what expression changes are specifically induced by the extinction behavior itself.
the increased activity expressed after  <dig> days of abstinence may, in part, have been the cause, rather than the result, of observed gene expression changes.
however, such changes would fall into the category of immediate early changes as the extinction testing was only  <dig> minutes in duration.
the value of the present study is that it focuses on a time point at which the subjects have experienced contextual re-exposure, a key component for eliciting relapse.
this study has produced data that are relevant not only for future studies involving heroin, but also for understanding the molecular underpinnings of incubation of drug-seeking.
CONCLUSIONS
we have identified a group of genes whose expression is significantly changed following abstinence from heroin self-administration and incubation of heroin-seeking behavior.
confirmed genes are not limited to genes encoding transcription factors, but also encompass genes encoding molecules that are important for regulation of intracellular signaling.
these regulatory molecules may be effective targets for drug interventions to prevent relapse.
authors' contributions
klk performed behavioral procedures, data analysis, and drafted the manuscript.
rmb performed the microarray experimental procedures.
psg contributed to the behavioral design.
wmf contributed to the design and interpretation of the microarray experiment.
kev participated in the design and coordination of the study.
all authors made contributions to the final manuscript.
supplementary material
additional file 1
changed genes on array.
the data provided are the complete list of  <dig> genes that were identified to have changed expression at the p <  <dig>  level of significance.
click here for file
acknowledgements
we thank georgina bixler for her assistance with the rt-qpcr.
this work was supported by da <dig> , da <dig> , and da <dig> .

 the challenge for drug abuse treatment is maintaining abstinence despite a high propensity for abstinent patients to relapse to drug use. although heroin has been abused for centuries, effective long-term preventions for heroin-relapse are still needed. physiological and gene expression changes that may increase an individual's likelihood to relapse are known to exist well into a period of abstinence. therefore, relapse to drug use is currently being investigated on both the molecular and behavioral levels. these studies have been aided by advances in systems biology tools, including large-scale discovery techniques such as microarrays and proteomics. these approaches are useful for discovering novel targets affected by drug abuse and examining hypotheses concerning categories of genes that are affected by drug use. as data on gene expression following relapse accumulates, the existence of a single "relapse gene" is becoming increasingly unlikely. therefore, macroscopic views of gene expression will prove very useful for guiding research into behavioral phenomena. neurobiological, environmental, cue, and stress mechanisms have all been implicated in relapse to drug use. the intense craving and motivation to seek drug, reported by humans during withdrawal from drug use, is challenging to model in animals, but the need for a relapse model continues to motivate the design of new behavioral procedures. incubation is a behavioral phenomenon that is characterized by increased drug seeking following increasing periods of abstinence after the last self-administration session. increased drug-seeking has been inferred to represent the craving that drives humans to relapse. the prefrontal cortex is important for decision making and guiding behavior, and the medial prefrontal cortex is known to be especially important for goal-directed behaviors. because of its role in guiding goal-directed, drug-seeking behaviors, understanding the gene expression changes in this region following a period of abstinence from drug self-administration will be useful for understanding the neurobiological basis to relapse following a period of drug self-administration. additionally, the ventral mpfc is believed to play an essential role for expression of incubation of cocaine-seeking and reinstatement of cocaine-seeking. in this study, we performed a whole genome analysis of gene expression in the medial prefrontal cortex of rats that displayed incubation of goal-directed behavior following weeks of heroin self-administration and weeks of home-cage enforced abstinence. after the abstinence period, rats were reintroduced to the testing chambers for a 90-minute extinction session during which behavioral responses were recorded. this extinction session served not only to provide an opportunity to observe behavioral incubation, but also mimicked a real-life situation in which environmental cues precipitate relapse behavior following an extended period of abstinence. following this experience of re-exposure to drug-associated context and cues, rna was isolated from the mpfc for whole genome microarray and qpcr analyses. ontological analyses revealed that many of the genes identified to be changed have the potential to be key components to neuroadaptations that exist at the time of relapse. methods heroin self-administration the self-administration and extinction procedures have been previously described. briefly, rats that self-administered heroin displayed incubation of heroin-seeking when tested in a 90-minute extinction session that occurred after weeks of enforced abstinence. as described in previous work, all rats initially underwent a habituation procedure during which they received water on a spout in the self-administration chambers. following habituation, the rats self-administered heroin by licking the previously water-associated spout during hour sessions that occurred on consecutive days. spout-licking has been the operant behavior in previous drug self-administration studies. two empty spouts, termed "active" and "inactive" were present in the self-administration chambers. a fixed ratio schedule of reinforcement existed, under which consecutive responses on the active spout produced an automated injection of heroin. self-administering rats were yoked to rats that received infusions of saline. that is, the control group was a separate set of animals that were treated exactly the same, but never received heroin. the days of heroin self-administration were followed by a 90-minute extinction session that occurred following either day or days of drug-free enforced abstinence after the final self-administration session. during the extinction session, responses on active and inactive spouts were recorded, but no drug was administered. immediately after the extinction sessions, the rats were sacrificed and brain regions were harvested. all studies were conducted in accordance with the pennsylvania state university institutional animal care and use committee, strictly adhering to the guidelines for the care and use of mammals in neuroscience and behavioral research. national research council, national research council, guidelines for the care and use of mammals in neuroscience and behavioral research dissection and rna isolation sacrifice and tissue dissection immediately following the 90-minute extinction session, all rats were sedated using propofol and decapitated within minutes. brains were rapidly removed from skulls, placed in pre-chilled phosphate buffered saline and then sectioned in an ice-chilled asi brain slicer. the section from bregma + to mm was cut along the forceps minor and the cortex medial to this cut was collected. this includes the cingulate, prelimbic cortex, infralimbic cortex, and medial orbital cortex. following dissection, the tissue was placed in prechilled tubes, immediately frozen on dry ice, and then stored at -80°c. rna isolation total cellular rna was isolated using tri reagent. isolated rna was further purified using an rneasy mini kit for rna clean-up. rna quantity and quality were assessed using the rna nano assay with an agilent bioanalyzer. microarray analysis microarray analyses were performed on samples from rats that experienced d of abstinence prior to an extinction session. studies were performed by the penn state college of medicine functional genomics core facility on arrays according to standard procedures. following the manufacturer's protocol of the low input fluorescent linear amplification kit, ng rna with the addition of one-color spike mix was denatured and incubated with t promoter primer. synthesis of cdna followed with the addition of first-strand buffer, dtt, dntp mix, mmlv-rt and rnase out and incubation at 40°c for hours. transcription of the product incorporated the cyanine 3-ctp in the master mix which includes transcription buffer, dtt, ntps, peg, rnase out, pyrophosphatases and t rna polymerase, with incubation for hours at 40°c. the resulting crna was purified using rneasy columns followed by assessment of purity, concentration and quality using a nanodrop nd- through calculated yield and specific activity. μg from each sample was fragmented, denatured, and then hybridized to agilent × rat whole genome microarray slides for hours at 65°c. slides were then washed according to protocol. microarrays were scanned with an axon 4000b scanner with genepix v software at a μm resolution and nm with laser power at 100%, pmt voltage at v, focus position μm, and lines to average = images were then imported into agilent feature extraction software. initial quality control, exclusion of manufacturing defects, background subtraction was then performed and the results exported to genespring gx. all primary array data have been deposited to gene expression omnibus. microarray data analysis microarray data were normalized following import into genespring gx by transforming signal values less than to an intensity of. normalization was done per chip to the 50th percentile, and per gene to the median. values were then normalized on a per gene basis to the control group. potential differential expression was determined with a one-way anova, p < and filtered for fold and greater differences in expression in accordance with standards for microarray analysis. the use of a combination of statistical and fold-change cutoffs as opposed to traditional multiple testing corrections produce gene lists with the lowest rate of type i and type ii errors. -fold was chosen as the fold-change cutoff, as this magnitude change is at the lower range of changes we find to be confirmable by rt-qpcr. array data for the complete list of genes that were identified to have changed expression at the p < level of significance is included in additional file also included are fold-changes in expression, accession numbers, and probe identification information. rt-qpcr analysis of gene expression complimentary dna synthesis was performed on total rna using superscript iii reverse transcriptase. the rna used for rt-qpcr was an aliquot from the same samples used to generate microarray probes. comparisons at both d of abstinence and d of abstinence were made to gain insights into whether the expression changes observed after d of abstinence had existed immediately after drug use and persisted during extended abstinence or had emerged during extended abstinence. one μg rna, ng oligo, and mm each dntp, were incubated for minutes at 65°c and then chilled on ice for minutes. 5× first strand buffer, mm kcl, and mm mgcl2), mm dtt, u rnaseout, and u superscript iii rt were then added. the μl reaction was incubated for minutes at 50°c followed by a final incubation at 70°c for minutes for termination. the resulting cdna product was quantified and ng of product was used in each subsequent qpcr reaction. quantitative pcr was carried out on a real-time detection instrument in 384-well optical plates using taqman universal pcr master mix and assay on demand primers and probes as described previously. primer/probe sets used are listed in table sds. software and the 2-ΔΔct analysis method were used to quantitate relative amounts using β-actin as an endogenous control. ontological, pathway, and network analysis ontological analysis used gene ontology categories to determine processes or functional categories that were differentially expressed, as described previously using genespring gx software. this analysis determined the number of genes in a category present on the array and the number of expression changes that would be part of that category by random chance given the number of differentially expressed genes. ingenuity pathway analysis was used to create a network from rt-qpcr confirmed gene expression results from the rats described in this study. statistical analysis behavioral data were analyzed by t-tests at each time point of abstinence. goal-directed behavior was determined by subtracting inactive spout responses from active spout responses. rt-qpcr gene expression values were evaluated using t-tests between self-administering and yoked saline rats at each time point of abstinence. for both behavioral and gene expression data, levels of significance were determined with α set at. correlational analyses were performed to determine whether a correlation between the goal-directed behavior or active spout responses during the extinction session and gene expression existed.  behavior rats were allowed to self-administer heroin during days of daily drug access. the average daily heroin intake for the rats that experienced only day of abstinence prior to extinction increased from ± infusions on day to ± infusions on day of self-administration. average daily heroin intake for the group of rats that experienced days of abstinence prior to extinction increased from ± infusions on day to ± on day of self-administration. when rats were reintroduced to the self-administration chambers following d or d of abstinence, incubation of active spout responses was observed. goal-directed behavior after days of abstinence was significantly higher in self-administering vs. yoked saline rats. additionally, day abstinent heroin self-administering rats had higher goal-directed behavior than self-administering rats with only day of abstinence. microarray analysis signals from probes were detected as being present on all of the arrays. filtering the detected genes produced a list of genes that were identified as being changed by at least fold relative to saline controls after days of abstinence and a 90-minute extinction session. a full listing of differentially expressed genes is provided in additional file rt-qpcr confirmation and validation rt-qpcr was performed to confirm expression levels of genes for which significant differences in expression were detected by microarrays. genes were chosen for confirmation analyses based on their ontological classifications and probable involvement in drug use. many genes examined belonged to ontological categories of nervous system development or behavior. for each gene examined, samples from self-administering and yoked saline rats from each of the abstinent period treatment groups were examined. among the genes that were detected by the arrays to be significantly changed, and confirmed by rt-qpcr, were genes that are important for intracellular signaling. dual specificity phosphatase and dual specificity phosphatase expression levels were both increased by 39% and 24%, respectively, after days of abstinence, although only dusp expression levels were increased after only day of abstinence. regulator of g-protein signaling expression was increased by 20% following days of abstinence. genes for additional intracellular molecules that each can be linked to physiological changes that occur following drug use were also confirmed to be significantly changed. brain-derived neurotrophic factor and calbindin both displayed increased expression levels, by 32% and 21%, respectively, relative to saline controls. neuropeptide y gene expression was decreased by 18% at day of abstinence and 20% at days of abstinence. ontological, pathway, and network analysis analysis of microarray gene expression data by gene ontology revealed genes that are involved in developmental processes. of these genes, were confirmed to be changed by rt-qpcr: bdnf, calb dusp egr and one has been previously reported by another group of researchers. a second ontological category of interest was behavior, and genes from our list were included in this category. among these genes were egr and crybb the ingenuity network analysis revealed that many genes validated in this study, together with genes previously reported from these samples to be significantly changed, can be linked through a hypothetical network that is involved with behavior, nervous system development and function, and cellular development. the pathway depicted in this figure is based on known intermolecular relationships, although whether this entire network of interactions occurs in vivo remains to be elucidated. all of the genes in this pathway were determined to be upregulated. correlational analyses behavioral data collected during extinction was used in correlational analyses with gene expression data. both pearson's and spearman's correlations were performed because of the small sample sizes that were not always normally distributed. when separate correlations were performed for data from each time point, pearson's analyses identified a positive correlation between the number of active spout responses and dusp gene expression in rats that experienced day of abstinence. a significant negative correlation between dusp gene expression and inactive spout responses was detected for rats that experienced days of abstinence by a spearman's analysis. combining the data from both abstinent periods yielded more significant correlations. goal-directed response and active spout response data from extinction were correlated with rt-qpcr data. eight significant correlations were detected. the upper number in each block of the table is the correlation coefficient and the lower number in each block of the table is the p value. discussion this study provides the first report of whole genome analysis of mpfc gene expression in rats that have expressed behavioral incubation and expands our knowledge of gene expression changes that exist after abstinence and during the time of relapse liability. the approach of using a preliminary screen of the entire genome, followed by rigorous rt-qpcr confirmation of expression changes for genes likely to be involved in behavior or neuronal changes has yielded information on genes, such as dusp and dusp whose role in addiction is only now beginning to emerge. the majority of genes that were significantly changed using the criteria reported in this paper had increased expression levels in heroin self-administering rats when compared to yoked saline following days of enforced abstinence. previously, increased gene expression has been found for opiate receptors in humans that died from an opiate overdose, as well as components of the cyclic amp signaling system following opiate use. the reasons for a disproportionate number of up-regulated versus down-regulated genes following abstinence from heroin self-administration remain to be determined. however, when gene expression nears the level of detection of the microarray platform, it is becomes more difficult to detect down-regulations. for the genes identified, and confirmed in this study, there are several potential impacts on behavior. phosphatase genes, such as dusp and dusp, encode proteins that have a direct impact on intracellular signaling. dusp preferentially dephosphorylates erk and is intranuclear, while dusp is cytoplasmic. mdma has been shown to increase expression of dusp dusp and dusp genes, while methamphetamine treatment increases dusp gene expression in multiple brain regions. both dusp and dusp regulate mitogen-activated protein kinase. numerous studies have suggested mapk involvement in neuroadaptations that occur following drug use. beyond being affected by mere drug exposure, mapk molecules have also been shown to play a role in morphine tolerance and to be activated following opiate withdrawal. interestingly, the mapk pathway plays a central role in the relationships between genes that were reported as changed in this study and our previous study of gene expression. molecules that regulate g-protein coupled receptor signaling have been shown to be essential for reinstatement of heroin-seeking behavior. the observed increase in rgs gene expression following days of abstinence and contextual re-exposure may represent an intracellular signaling change that affects communication between receptors and transcription factors, ultimately affecting cellular and organismal physiology. a gpcr-regulating molecule was shown, through a gene knock-down approach, to be essential to activating protein kinase a signaling and observing reinstatement of heroin-seeking. the microarray analysis detected an npy receptor, npy5r, to be significantly increased after days of abstinence. while rt-qpcr for npy5r failed to replicate this change, the gene encoding npy, the ligand for this receptor, was tested by rt-qpcr and found to be changed. npy gene expression differed from most other genes examined because it was decreased in rats that had self-administered heroin, both after and days of abstinence. although npy is a neurotransmitter most recognized for its role in regulating food intake, it has been hypothesized that npy may contribute to the negative motivational state of withdrawal. intracerebroventricular administration of npy has been found to block increased ethanol intake in rats, and overexpression of the npy gene, using intra-amygdalar infusions of a viral expression vector, diminished alcohol intake following longer periods of abstinence or repeated alcohol withdrawals. in the present context, intraventricular injections of npy have been found to induce heroin-seeking behavior following extinction sessions, suggesting that the decrease in npy gene expression observed in the present study may be related to the extinction component of the behavioral testing. the majority of genes on which rt-qpcr was performed displayed expression levels significantly different from saline controls only after days of abstinence. this suggests that these changes are attributable either solely to the extended drug abstinence or to the combination of extended abstinence and contextual re-exposure. it is documented that exposure to environments previously paired with drug administration can affect gene expression, so future studies will be required to discern whether the observed changes in gene expression after days of abstinence resulted from contextual re-exposure, the pharmacological drug abstinence, or a combination. correlational analyses detected that bdnf was significantly correlated with both goal-directed behavior and active spout responses. this observation supports the proposed importance of bdnf to drug-seeking behavior. while the study was not originally powered to conduct individual correlational analyses, the findings with bdnf suggest that future work may focus on individual behavior and gene expression. several genes whose expression was changed in this study have also displayed changed expression levels following several extinction sessions. in the mpfc, there was an increase in gene expression relative to controls for arc, homer1a, ania- mkp- c-fos, egr egr and nr4a homer egr and nr4a each were detected by the arrays to have increased expression values of at least -fold in the current study. the repeated extinction sessions in the koya et al. study are a major difference from our study that may contribute to the differences in gene expression observed between studies. genes that were changed in both koya's study, which included extinction sessions, and the present study, which included extinction session, are genes that apparently exhibit changed expression following a prolonged abstinence and maintain that change in expression regardless of the amount of environmental re-exposures. the identities of the confirmed genes include not only transcription factors, but also genes for molecules involved in intracellular signaling and protein binding. this range of functions provides a reminder that drug use elicits changes in entire intracellular networks. the importance of changes in gene expression of certain proteins, such as calb or dusp to eliciting changed expression of genes encoding transcription factors, such as egr or fos, is an area for future investigation. while this microarray analysis has illuminated genes whose expression is changed immediately following an extinction session, the gene expression profile that exists prior to extinction testing has not been examined in this study. a comparison between the gene expression profile following abstinence versus the gene expression profile following abstinence and extinction would address the question of what expression changes are specifically induced by the extinction behavior itself. the increased activity expressed after days of abstinence may, in part, have been the cause, rather than the result, of observed gene expression changes. however, such changes would fall into the category of immediate early changes as the extinction testing was only minutes in duration. the value of the present study is that it focuses on a time point at which the subjects have experienced contextual re-exposure, a key component for eliciting relapse. this study has produced data that are relevant not only for future studies involving heroin, but also for understanding the molecular underpinnings of incubation of drug-seeking.  we have identified a group of genes whose expression is significantly changed following abstinence from heroin self-administration and incubation of heroin-seeking behavior. confirmed genes are not limited to genes encoding transcription factors, but also encompass genes encoding molecules that are important for regulation of intracellular signaling. these regulatory molecules may be effective targets for drug interventions to prevent relapse. authors' contributions klk performed behavioral procedures, data analysis, and drafted the manuscript. rmb performed the microarray experimental procedures. psg contributed to the behavioral design. wmf contributed to the design and interpretation of the microarray experiment. kev participated in the design and coordination of the study. all authors made contributions to the final manuscript. supplementary material additional file 1 changed genes on array. the data provided are the complete list of genes that were identified to have changed expression at the p < level of significance. click here for file acknowledgements we thank georgina bixler for her assistance with the rt-qpcr. this work was supported by da, da, and da.
extinction sessions were conducted on groups after either d or d of drug-free enforced abstinence to demonstrate behavioral incubation. in this study, genome-wide analysis of gene expression was conducted following an extinction session in rats that expressed behavioral incubation of heroin-seeking and goal-directed behavior. as an important modulator of goal-directed behavior, the medial prefrontal cortex was the target of genomic analysis. whole genome gene expression analysis was performed and results were confirmed by quantitative real-time pcr. rats were trained to self-administer heroin during h daily sessions for d. following the self-administration period, rats were reintroduced to the self-administration chambers for a 90-minute extinction session in which they could seek heroin, but received none. microarrays identified genes whose expression was identified as changed by at least fold following d of abstinence and the 90-minute extinction session compared to the saline treated controls. this increased drug-seeking took place despite the fact that they received no drug during this extinction session. a number of gene expression studies have investigated changes induced by drug exposure, but few reports describe changes that persist following relapse. the global gene expression analysis adds to the knowledge of heroin-induced changes and further highlights similarities between heroin and other drugs of abuse.
 a number of gene expression studies have investigated changes induced by drug exposure, but few reports describe changes that persist following relapse. in this study, genome-wide analysis of gene expression was conducted following an extinction session in rats that expressed behavioral incubation of heroin-seeking and goal-directed behavior. as an important modulator of goal-directed behavior, the medial prefrontal cortex was the target of genomic analysis. rats were trained to self-administer heroin during h daily sessions for d. following the self-administration period, rats were reintroduced to the self-administration chambers for a 90-minute extinction session in which they could seek heroin, but received none. extinction sessions were conducted on groups after either d or d of drug-free enforced abstinence to demonstrate behavioral incubation.  behavioral data demonstrated incubation of heroin-seeking and goal-directed behavior after the d abstinent period. that is, following d of enforced abstinence, animals displayed heightened drug-seeking behavior when returned to the environment where they had previously received heroin. this increased drug-seeking took place despite the fact that they received no drug during this extinction session. whole genome gene expression analysis was performed and results were confirmed by quantitative real-time pcr. microarrays identified genes whose expression was identified as changed by at least fold following d of abstinence and the 90-minute extinction session compared to the saline treated controls. orthogonal confirmation by rt-qpcr demonstrated significant alterations in bdnf, calb dusp dusp egr npy, rgs  ontological analysis indicates that several of the genes confirmed to be changed are important for neuroplasticity, and through that role may impact learning and behavior. the importance of drug-seeking behavior and memory of previous drug-taking sessions suggest that such genes may be important for relapse. the global gene expression analysis adds to the knowledge of heroin-induced changes and further highlights similarities between heroin and other drugs of abuse. 