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Keywords:

  • Addiction;
  • circadian rhythm;
  • drugs of abuse;
  • gene expression;
  • heroin;
  • methamphetamine;
  • microarray;
  • prodynorphin;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

The molecular alterations that underlie the long-lasting behavioural effects of drugs of abuse, such as psychomotor sensitization and physical dependence, are still not known. Moreover, it is not known which molecular effects are similar for addictive drugs from various pharmacological classes. In this study, we utilized whole-genome microarray profiling to evaluate the detailed time-course of transcriptional alterations in the mouse striatum during chronic treatment with heroin (HER) and methamphetamine (METH) and after period of spontaneous withdrawal. We identified 27 genes regulated by chronic drug administration. The overlap between lists of HER- and METH-induced genes was highly significant. The most substantial impact on the gene expression profile was observed for circadian genes (Per1, Per2 and Nr1d1). However, changing the treatment scheme from diurnal to nocturnal was sufficient to attenuate the drug-induced changes in circadian gene mRNA levels. Both of the drugs caused a dose-dependent induction in glucocorticoid-dependent genes with relatively long mRNA half-lives (Fkbp5, Sult1a1 and Plin4). The analysis also showed a drug-regulated group of transcripts enriched in the nucleus accumbens and includes well known (Pdyn, Cartpt and Rgs2) as well as new (Fam40b and Inmt) candidate genes. All identified alterations in the striatal transcriptome were transient and persisted up to 6 days after withdrawal. Behavioural sensitization, however, was maintained throughout the 12-day withdrawal period for both HER and METH. We suggest that transient gene expression alterations during drug treatment and in the early period of withdrawal are involved in the establishment of persistent neuroplastic alterations responsible for the development of drug addiction.

Methamphetamine (METH) and heroin (HER) are among the most harmful drugs of abuse and are associated with severe consequences, including mortality and incarceration (Hser et al. 2008). Although subsequent use of both of these drugs leads to addiction, the subjective feelings that occur because of these substances are significantly different. Administration of HER in humans produces a feeling of pleasure, satisfaction and relaxation (Seecof & Tennant 1986), whereas the administration of METH induces euphoria, increased attention and a feeling of power (Winslow et al. 2007). The effects of withdrawal from these substances are also radically different in human and mice (McGregor et al. 2005; Wesson & Ling 2003). Despite these differences, there are common effects of these two drugs. The most important of these are mood disorders and anhedonia following long-term use of HER or METH (Koob 1992; Newton et al. 2004; Zijlstra et al. 2009). In addition, locomotor sensitization in mice occurs to both these drugs.

Opiate and psychostimulant addiction are often perceived as variants of the same disorder. However, Badiani et al. (2011) have recently argued that opiate addiction and psychostimulant addiction are behaviourally and neurobiologically distinct. Therefore, it is important to estimate to what extent molecular effects of opioids and psychostimulants are common or different. The approach that searches for similarities between various drugs of abuse has long been recognized (Leshner & Koob 1999; Piechota et al. 2010; Valjent et al. 2004). The genes whose expression changes similarly after the administration of HER and METH could be responsible for their common effects, such as sensitization or anhedonia. In contrast, genes whose expression differs after administration of these substances could be responsible for the drug-specific effects. This is of particular interest because transcriptional responses to acute administration of these drugs overlap only partially and differ in their time dynamics (Piechota et al. 2010).

Multiple researchers have used microarray analysis to study the molecular effects of chronic treatment with morphine (Anghel et al. 2010; Desjardins et al. 2008; Grice et al. 2007; Korostynski et al. 2007; McClung et al. 2005), HER (Kuntz-Melcavage et al. 2009), cocaine (Lynch et al. 2008; Renthal et al. 2009) or METH (Yang et al. 2008). However, these studies focused only on individual substances or single time-points. Therefore, it is difficult to determine when the transcriptional changes occur and whether the changes are transient or persistent. Exploring the dynamics of the changes in the gene expression profile during chronic treatment and withdrawal could provide an answer for these questions. The next unanswered question is whether the changes are common for various drugs of abuse. Our previous research focused on comparing the acute effects of various drugs of abuse (Piechota et al. 2010). While acute treatment with drugs from the same pharmacological class (e.g. the psychostimulants cocaine and METH) provoked highly similar changes in the striatal transcriptome, drugs from distinct pharmacological classes have relatively distinct transcriptional profiles (e.g. METH and HER).

In this study, we utilized whole-genome microarray profiling to evaluate common alterations and their time-course in the striatal transcriptome following chronic treatment with METH and HER.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

Animals

Adult male (8–10 weeks old) C57BL/6J inbred mice (Charles River, Sulzfeld, Germany) were housed five to seven per cage (31 × 16 × 14 cm), under a 12-h dark/light cycle, with free access to food and water. Animals weighing 20–30 g were used throughout the experiments. The animal protocols used in this study were approved by the local Bioethics Commission at the Institute of Pharmacology, Polish Academy of Sciences (Krakow, Poland). We used independent cohorts of animals for each experiment performed. Only weight and locomotor sensitization measurements were taken using the same animals.

Drug treatment

Animals were injected intraperitoneally with saline (SAL) (Polfa, Lublin, Poland), HER (Institute of Pharmacology PAS) or d-METH (Sigma-Aldrich, Poznan, Poland) twice a day for consecutive 12 days as described in Fig. 1. Body weight was measured to estimate the influence of chronic drug treatment and withdrawal on the overall physical condition (Figure S1). Mice were sacrificed by decapitation after 14 h after the last dose following 1, 3, 6 or 12 days of drug treatment and 1, 3, 6 or 12 days of withdrawal. The initial doses of drugs display stimulatory and rewarding properties and were based on our previous studies (Piechota et al. 2010).

image

Figure 1. Schedule of chronic HER or METH injections and withdrawal. The bar graph represents doses of HER (scale on the left) or METH (scale on the right) injected throughout the chronic drug treatment. The animals were injected twice a day. Doses were increased up to four times the initial dose on the last day of treatment. The initial doses of HER and METH are equally rewarding for C57BL/6J mice (Piechota et al. 2010). Arrows represent time-points at which tissues were collected for microarray studies (14 h after the last drug injection).

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Locomotor activity test

In the locomotor activity test, mice were individually placed in the centre of a test cage containing photocells (20 × 10 × 12 cm). After 1 h of acclimatization, mice were injected with SAL, HER (10 mg/kg) or METH (2 mg/kg) and during the next 3 h locomotor activity assessed by the number of beam interruptions was measured. Each animal was tested three times: after acute treatment (first drug injection during locomotor activity test), after chronic treatment (SAL, HER or METH) and after 12 days of withdrawal (SAL, HER or METH).

Naloxone-precipitated withdrawal

Physical dependence was evaluated by measuring the behavioural manifestations of naloxone-precipitated withdrawal in mice treated with chronic HER or METH. Each mouse was subcutaneously (s.c.) injected with naloxone (4 mg/kg) either 14 h or 12 days after the last drug treatment and then placed in a plexiglass tube. The number of jumps (defined as the removal of all four paws from the surface) was scored over the following 15 min.

Circadian activity

The animals were kept in the IntelliCage home cages (NewBehavior, Zurich, Switzerland) containing four single animal-sized corners. Corner entries were recorded by the system, which allowed the assessment of animal activity over a long period of time. To assess circadian activity, the corner entries were binned daily into 48 intervals of 30-min each. For each interval, entry counts were averaged over several consecutive days to generate the average animal activity at a given time-point. Data were recorded starting 7 days after the animals were introduced to the cage to allow for adaptation to the new environment and establish a consistent behavioural pattern.

Tissue collection and RNA preparation

The brain was transected along the sagittal fissure, the septum and the medial frontal cortex were pulled away to expose the interior of the lateral ventricle and the caudate putamen. A vertical cut was then made ventrally at the level of the anterior commissure and the tissue surrounding the commissure (nucleus accumbens) was pulled out together with the portion of caudate putamen situated rostrally to the vertical cut using fine forceps. Tissue samples (referred to hereafter as the striatum) were placed in RNAlater reagent (Qiagen Inc., Valencia, CA, USA) and preserved at −70°C. Samples were thawed at room temperature and homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was isolated following the manufacturer's protocol and further purified using the RNeasy Mini Kit (Qiagen Inc.). The total RNA concentration was measured using a NanoDrop ND-1000 Spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). RNA quality was determined by chip-based capillary electrophoresis using an RNA 6000 Nano LabChip Kit and Agilent Bioanalyser 2100 (Agilent, Palo Alto, CA, USA), according to the manufacturer's instructions. RNA from two mice was pooled to create a sample for each microarray.

Gene expression profiling

A starting amount of 200 ng high-quality total RNA (equally pooled from two animals) was used to generate cDNA and cRNA with the Illumina TotalPrep RNA Amplification Kit (Illumina Inc., San Diego, CA, USA). The procedure consisted of reverse transcription with an oligo(dT) primer bearing a T7 promoter using Array-Script. The obtained cDNA became a template for in vitro transcription with T7 RNA polymerase and biotin uridine-5′-triphosphate (UTP), which generated multiple copies of biotinylated cRNA. The purity and concentration of the cRNA were checked using an ND-1000 Spectrometer. Quality cRNA was then hybridized with Illumina's direct hybridization array kit (Illumina). Each cRNA sample (1.5 µg) was hybridized overnight to the MouseWG-6 BeadChip arrays (Illumina) in a multiple-step procedure according to the manufacturer's instructions; the chips were washed, dried and scanned on the BeadArray Reader (Illumina). Raw microarray data were generated using BeadStudio v3.0 (Illumina). The microarray experiment included eight time-points (four time-points for the drug treatment, four for withdrawal) for each of three treatments (SAL, HER and METH). Three biological replicates of the microarrays were prepared per each time-point for every experimental group. Six additional arrays (naÏve) were used at predrug point. A total of 78 Illumina MouseWG-6 v2 were used. The microarray experimental design involved pooling two animals per array.

Microarray data analysis

Microarray quality control was performed using BeadArray R package v1.10.0. The following parameters were checked on all 78 arrays: number of outliers, number of beads and percent of detected probes. After background subtraction, the data were normalized using quantile normalization and log2 transformed. Statistical analysis of the results was performed using a two-way ANOVA (analysis of variance) (for the factors: drug and time) followed by correction for multiple testing using false discovery rate (FDR) (Benjamini & Hochberg 1995). The naÏve samples were not used for ANOVA analysis. Tukey's honestly significance difference (HSD) test was used for analysis of pairwise comparisons. For clustering and heatmap visualization, the data were z-score standardized using R scale function. Hierarchical clustering was performed using the measure of Euclidean distance and average distance linkage methods. For functional classification, other microarray datasets were utilized, including data from the genome-wide expression profiling of diurnally regulated genes (Yang et al. 2008), the ‘Gene Atlas' data set, which contains expression patterns from a diverse set of tissues and cell types (Wu et al. 2009) and data from detailed time-courses of transcriptome alterations following the acute administration of drugs of abuse in mice (Piechota et al. 2010).

Validation of microarray data by qPCR

We performed qPCR measurements for a set of genes representative of the identified gene groups. Reverse transcription was performed with Omniscript Reverse Transcriptase enzyme (Qiagen) at 37°C for 60 min. The reaction was carried out in the presence of the RNase inhibitor rRNAsin (Promega, Madison, WI, USA), and an oligo(dT16) primer (Qiagen) was used to selectively amplify mRNA. qPCR reactions were performed using Assay-On-Demand TaqMan probes according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA) and were run on an iCycler (Bio-Rad, Foster City, CA, USA). For each reaction, approximately 50 ng of cDNA synthesized from a total RNA template (isolated from an individual animal) was used (n = 5–6). To minimize the contribution of contaminating genomic DNA, primers were designed to span exon junctions. In addition, control reactions without reverse transcription enzyme for each assay were performed. The amplification efficiency for each assay was determined by running a standard dilution curve. The expression of the Hprt1 transcript, which had a stable mRNA level, was quantified to control for variations in cDNA levels. The cycle threshold values were calculated automatically by iCycler IQ 3.0a software with default parameters. The abundance of RNA was calculated as 2−(thresholdcycle).

Statistical analysis

Body weight and locomotor sensitization were analysed using two-way ANOVA with repeated measures followed by Bonferroni corrected paired, two-sided, two sample Student's t tests with time as a repeated measures factor and drug treatment as a second factor. Drug–dose effects were analysed using Pearson correlation. Overlap of gene lists was analysed using Fishers's exact test. Data for qPCR validation were analysed using one-way ANOVA followed by Bonferroni corrected unpaired, one-sided Student's t tests with treatment as a factor. Circadian effect on sensitization was analysed using unpaired, two-sided, two sample Student's t test was used. Data for naloxone-precipitated withdrawal were analysed using three-way ANOVA followed by Bonferroni corrected Welch's t test with time (days), drug treatment and diurnal time (diurnal/nocturnal) as factors. All statistical analyses were performed in R software version 2.11.1.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

Chronic METH and HER treatment induced a sensitization to their effects in ambulatory movements

To determine whether our treatment regimen caused persistent neuroplastic adaptations, drug-induced locomotor movement was measured (Bradberry 2007). Measurements were taken at three time-points: following the first injection (primary dose), following the primary dose the day after chronic treatment and following the primary dose after 12 days of withdrawal (n = 4–6). Changes in drug-induced locomotor activation are shown in Fig. 2. Chronic METH treatment did not result in sensitization (t = 0.28, df = 5, P = 1). However, 12 days of withdrawal from METH resulted in locomotor sensitization (t = 3.51, df = 5, P = 0.039). Chronic HER treatment resulted in a mild increase in the response to HER (t = 3.08, df = 5, P = 0.054). Withdrawal from HER resulted in evident sensitization (t = 7.51, df = 5, P < 0.001). The results including SAL-treated animals are available in Figure S2.

image

Figure 2. Acquisition of locomotor sensitization to HER or METH after chronic treatment and withdrawal. The line graphs represent the locomotor response to METH (2 mg/kg) or HER (10 mg/kg) at three time-points. Responses to the first injection (acute), 14 h after the last injection (chronic), 12 days after the last injection (withdrawal). Dark line shows mean ± standard error of the mean. Light grey single lines show single animals. Significant differences based on a Bonferroni corrected paired ttests are indicated by asterisks (***P < 0.001 and *P < 0.05).

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Similar effects of chronic METH and HER on the striatal transcriptome

Statistical analysis with a two-way ANOVA (drug × time factors) followed by FDR correction for multiple testing (FDR <5% which corresponds to F > 12.1; df = 2,48; P < 5.5 × 10−5) identified 51 probes with significant drug factor, 318 probes with significant time factor and 3 probes with significant interaction (Figure S3). All probes with uncorrected significance (P < 0.05) are available in Table S1. To determine how many genes were regulated after each treatment, Tukey's HSD test was used. The analysis showed that 36 probes were altered in response to HER treatment (FDR corrected Tukey's HSD <5%). Twenty probes were altered in response to METH treatment. Sixteen probes were common for both HER and METH treatment. One probe was differentially regulated between HER and METH. Overlap between lists of HER and METH-induced genes was significant (Fisher's exact test; total number of probes = 45 280; P < 10−15).

For further analyses, 33 significantly altered probes representing 27 unique genes were selected using additional criteria (FDR for the drug factor <5%, log2 ratio vs saline for at least one time-point >0.35 or <−0.35, expression level of at least three samples >8.5). Direction of gene expression changes was consistent between the two drugs for all selected probes.

Post-hoc analysis using Tukey's HSD showed that the transcriptome alterations were the most evident after 12 days of drug treatment (Fig. 3). During the next 12 days of protracted abstinence period altered genes returned to normal level and none of the 33 identified microarray probes remained altered after 12 days of withdrawal (Tukey's HSD, P < 0.05).

image

Figure 3. Transient effects of chronic HER or METH on the striatal transcriptome. The barplots represent number of genes significantly regulated by HER or METH in each day of chronic treatment or withdrawal (Tukey's HSD P < 0.05).

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The complete list of significantly altered genes is presented in Table 1. The data are available at the Gene Expression Omnibus accession GSE30305.

Table 1.  Genes with expression altered during chronic treatment and withdrawal from METH or HER in the mouse striatum
Gene symbolProbe IDTwo-way ANOVA FDR value for drug factorHighest fold change (log2 ratio)Time-point at which highest fold change occurred
Gm129ILMN_3029489FDR < 0.00011.08HER treatment day 12
DbpILMN_2616226FDR < 0.00010.58HER treatment day 12
Ano2ILMN_2802590FDR < 0.00010.93METH treatment day 12
Gm129ILMN_3102736FDR < 0.00011.05HER treatment day 12
Spink8ILMN_1220865FDR < 0.00010.74METH withdrawal day 3
Plin4ILMN_2588249FDR < 0.00011.85HER treatment day 3
Coq10bILMN_31044620.000120.53METH withdrawal day 1
Gm129ILMN_12360790.000120.93HER treatment day 12
Trim9ILMN_12215460.00038−0.42METH treatment day 6
Fam40bILMN_31503130.000380.58HER treatment day 12
Per1ILMN_28134870.000380.66HER withdrawal day 3
Homer1ILMN_24860120.00064−1.09HER treatment day 12
Nr1d1ILMN_12290910.000960.58HER treatment day 12
Fkbp5ILMN_27182660.003560.38HER treatment day 12
Nr1d1ILMN_27496690.003560.59HER treatment day 12
PdynILMN_27838730.003560.58METH treatment day 12
Fgfr2ILMN_12250710.00432−0.38HER treatment day 6
Per2ILMN_29878620.005080.69METH withdrawal day 1
Sult1a1ILMN_27453700.006830.93HER treatment day 3
Per1ILMN_28134840.009120.60HER withdrawal day 3
InmtILMN_12314450.009120.56HER treatment day 6
PdynILMN_27678710.009270.44METH treatment day 12
Per2ILMN_29878630.010340.69METH withdrawal day 1
HnrnpmILMN_12390210.01433−0.38HER treatment day 12
Rgs2ILMN_26666220.024900.36METH treatment day 12
Adipor2ILMN_12602190.025800.43METH treatment day 6
Tmem125ILMN_27031380.02597−0.36HER withdrawal day 6
Hif3aILMN_26496710.025971.01HER treatment day 12
Irf9ILMN_12334610.029790.39METH withdrawal day 1
DtnaILMN_12218050.02996−0.51HER withdrawal day 1
Gbp3ILMN_29180020.039290.59METH withdrawal day 1
CartptILMN_27197550.039290.44HER withdrawal day 3
CryabILMN_28402130.04771−0.69HER treatment day 12

Effects of METH and HER treatment were observable 14 h after injection

The 14 h time-point was selected to avoid the effects of acute METH and HER injection. Previously, we reported that almost all effects of acute injection were normalized after 8 h (Piechota et al. 2010). However, 7 of the 27 genes regulated by chronic drug administration were also reported to be regulated by acute drug injection in our previous report. Genes regulated by acute injection are marked in the right panel of Fig. 4.

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Figure 4. Gene expression changes during chronic HER or METH treatment and withdrawal. Microarray results are shown as a heat map (left side) and include genes with genome-wide significance based on a two-way ANOVA of the drug factor. Coloured rectangles represent transcript abundance of the gene labelled on the right after 1, 3, 6 and 12 days of chronic treatment or withdrawal from the drug indicated above. The intensity of the colour is proportional to the standardized values (between −2.5 and 2.5) from each microarray, as indicated on the bar below the heat map image. Clustering was performed using Euclidean distance according to the scale on the left. Bioinformatic analysis for the identified genes using previously published datasets is presented as a heat map (right side). Regulation by a single HER or METH injection at the early time-points is indicated in red, as reported by Piechota et al. (2010). Regulation of the circadian rhythm is indicated in red, as reported by Yang et al. (2007). Enrichment in the nucleus accumbens is indicated in red, as reported by SymAtlas (GNF Mouse GeneAtlas V3, Wu et al. 2009).

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Our experimental scheme included an increasing dose paradigm to avoid the effects of tolerance. Thus gene expression was measured at 10, 20, 30 and 40 mg/kg doses of HER and at 2, 4, 6 and 8 mg/kg doses of METH followed by four measurements during withdrawal (0 mg/kg dose). We next determined how much of the variance in the expression level of these nine transcripts was explained by the dose (see Figure S4 for details). A dose response effect explained a significant portion of the variance in the three genes identified for METH treatment (Pearson correlation coefficient, r > 0.7, n = 8, P < 0.05; Fkbp5, Hif3a and Adipor2) and six genes identified for HER treatment (r > 0.7, n = 8, P < 0.05; Fkbp5, Plin4, Hif3a, Adipor2, Sult1a1 and Homer1). Three genes regulated by acute administration had no significant dose response association for any of the substances (Per1, Per2 and Spink8).

Alteration of circadian genes by chronic METH and HER treatment

We noticed that three of the genes induced by chronic drug administration are widely known as circadian rhythm genes (Per1, Per2 and Nr1d1) (Albrecht et al. 1997; Shearman et al. 1997; Yin et al. 2006). Therefore, we utilized data from the genome-wide expression profiling of diurnally regulated genes to identify circadian genes among the genes regulated by chronic HER or METH administration (Yang et al. 2007). A total of 17 genes were found to be circadian genes (log2 ratiomax vs. min> 0.5), 8 genes of which were found to be robustly regulated by circadian rhythms (log2 ratiomax vs. min> 1; Per1, Per2, Nr1d1, Gm129, Dbp, Ano2, Coq10b and Homer1). Genes regulated by circadian rhythms are marked in the right panel of Fig. 4.

Chronic METH and HER treatment activated genes enriched in the nucleus accumbens

Hierarchical clustering of the probes significantly regulated by chronic METH and HER administration showed five major groups of genes (Fig. 4). Three clusters consisted of circadian genes (e.g. Gm129 and Cryab). One cluster consisted of genes regulated by single drug administration (e.g. Fkbp5 and Sult1a1). Only one gene cluster consisted of genes that could not be classified as circadian rhythm dependent or regulated by acute treatment. Two genes from this cluster (Pdyn and Cartpt) are widely known to be highly enriched in the striatum or the nucleus accumbens (Höllt et al. 1980; Koylu et al. 1998). Therefore, we utilized data from the ‘SymAtlas’ data set (GNF Mouse GeneAtlas V3), which contain expression patterns from a diverse set of tissues and cell types to identify nucleus accumbens enriched genes (Wu et al. 2009). All genes from the cluster containing Cartpt and Pdyn were enriched in the nucleus accumbens compared with other brain regions (Fold > 7), and four of the five genes from this cluster (Pdyn, Cartp, Inmt and Fam40b) have higher levels in the nucleus accumbens (ventral striatum) than in the caudate putamen (dorsal striatum). Genes enriched in the nucleus accumbens are marked in the right panel of Fig. 4.

Transcriptional effects of METH and HER display significant differences between the diurnal and nocturnal phase

Alteration of circadian genes by chronic METH and HER treatment led us to ask whether these changes were the result of the injection schedule. For the microarray experiment animals were injected twice daily during the diurnal phase (Fig. 5b). Thus we tested whether the changes in gene expression found in the microarray experiment persist if METH and HER were administered during the nocturnal phase. We injected the drugs at two time-points immediately before the peaks of nocturnal activity, as presented in Fig. 5b. We selected four circadian genes (Per1, Per2, Gm129 and Dbp), two genes with significant dose response effects (Fkbp5 and Plin4) and two genes enriched in the nucleus accumbens by chronic drug treatment (Pdyn and Cartpt) for qPCR validation (n = 4–9). The analysis confirmed that levels of Gm129 (F = 6.41, df = 2,14; P = 0.011), Dbp (F = 6.74, df = 2,14; P = 0.008), Per2 (F = 3.77, df = 2,14; P = 0.049), Per1 (F = 4.49, df = 2,14; P = 0.031), Fkbp5 (F = 4.23, df = 2,14; P = 0.036), Plin4 (F = 31.88, df = 2,13; P < 0.001) and Pdyn (F = 4.26, df = 2,13; P = 0.037) transcripts were altered if the animals were chronically treated during the diurnal phase. The Cartpt transcript displayed an insignificant tendency (F = 1.75, df = 2,13; P = 0.212) in a direction consistent with the microarray results. The levels of the circadian genes Gm129 (F = 0.34, df = 2,24; P = 0.713), Dbp (F = 0.84, df = 2,24; P = 0.443), Per2 (F = 2.88, df = 2,23, P = 0.076) and Per1 (F = 0.56, df = 2,23; P = 0.575) remained unchanged if the animals were chronically treated during the nocturnal phase. However, the levels of Fkbp5 (F = 3.77, df = 2,24; P = 0.037) and Pdyn (F = 13.71, df = 2,24, P < 0.001) were altered by nocturnal HER or METH injections. All inspected genes remained unchanged after 12 days of withdrawal (P > 0.15). The results from the qPCR measurements are presented in Fig. 5a.

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Figure 5. Validation of drug-induced alterations in the gene expression profile. (a) Bar graphs summarizing the qPCR-based measurements of changes in the expression of selected genes following the indicated treatment. Data are presented as the fold change over the SAL control group ± standard deviation (n = 5–8). Significant differences in the main effects based on a multivariate ANOVA for drug treatment are indicated by hash symbols (###P < 0.001, ##P < 0.01 and #P < 0.05) and from the post-hoc analysis using a Bonferroni corrected t-test (vs. appropriate SAL control) by asterisks (***P < 0.001, **P < 0.01 and *P < 0.05). Digits inside bars indicate sample size. (b) Timing of the METH and HER injections. The line graph represents the average home cage circadian activity of the C57BL/6J mice for 30 min intervals counted by the IntelliCage system. Zeitgeber (light) time is marked on the x-axis. White arrows show the time-points of diurnal injections. Black arrows show the time-points of nocturnal injections. The nocturnal treatment injection time-points were selected to stimulate animals during their highest activity and were designed to avoid disturbing the circadian rhythm of the animals.

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Phase of the circadian rhythm in which METH and HER are injected affected sensitization

The results of qPCR prompted us to test whether the locomotor sensitization depend on the phase of the cycle in which the substances are administered. For this purpose, locomotor measurements were taken for the nocturnally treated animals in the same scheme used for the diurnally treated animals. The measurements were taken at three time-points: after the first injection, 14 h after chronic treatment and after 12 days of withdrawal (n = 4–6).

Changes in the drug-induced locomotor activation are shown in Figure S2 (bottom panel). Chronic METH treatment resulted in an increase in locomotor response to METH (t = 3.44, df = 5; P = 0.037). Withdrawal from METH resulted in even higher sensitization (t = 8.03, df = 5; P < 0.001). Chronic HER treatment resulted in an increase in the response to HER (t = 4.81, df = 3; P = 0.034). After 12 days of withdrawal from HER, the locomotor sensitization remained at the same level (t = 10.26, df = 3; P = 0.004).

Naloxone produced withdrawal symptoms 14 h after the last dose of chronic drug treatment

Previous work has described the involvement of dynorphin in opioid withdrawal symptoms (Takemori et al. 1992). Expression of the dynorphin coding gene (Pdyn) was upregulated 14 h after the last dose of both METH and HER in both nocturnal and diurnal-treated animals. Therefore, we investigated whether naloxone injection was able to elicit withdrawal symptoms following chronic METH treatment. The standard scheme for the naloxone-precipitated withdrawal procedure involves the injection of naloxone 3 h after the last drug injection. However, we decided to measure withdrawal symptoms at the same time-points at which we measure expression, 14 h after the last injection and after 12 days of withdrawal. The effects of naloxone-precipitated withdrawal are presented in Fig. 6. Analysis using a multifactorial ANOVA showed significant effects of the drug (F = 4.22, df = 2,41; P = 0.021) and the length of the withdrawal period (F = 5.59, df = 1,41; P = 0.023) and no effects of circadian rhythm (F = 0.37, df = 1,41; P = 0.543). Post-hoc analysis showed that mice chronically treated with HER jumped more following naloxone injection than SAL-treated animals (t = 2.64, df = 10; P = 0.012). A similar tendency was observed for animals chronically treated with METH (t = 1.37, df = 13; P = 0.096).

image

Figure 6. Naloxone-precipitated withdrawal in mice following chronic treatment with METH or HER. Bar graphs summarizing the mean and standard deviation of jumps in response to naloxone (s.c., 4 mg/kg) after the indicated treatment (n = 4–7). HER-treated mice exhibited significant withdrawal signs 14 h after the last injection (P < 0.05). METH-treated mice did not exhibit significant withdrawal signs 14 h after the last injection. Digits inside bars indicate sample size.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

Molecular effects of chronic METH and HER treatment display high similarity

Despite the fact that METH and HER have relatively distinct transcriptome profile changes after acute administration (Piechota et al. 2010), transcriptome changes following chronic treatment with METH and HER show a striking similarity. Diurnal chronic treatment with each of the substances leads to changes in the level of circadian genes (e.g. Gm129, Dbp, Per1 and Per2), which is consistent with previous studies (Perreau-Lenz & Spanagel 2008; Wang et al. 2006).

Both METH and HER caused a dose-dependent induction of genes (e.g. Fkbp5 and Plin4), which, as we have shown previously, are regulated by the hypothalamic pituitary adrenal axis (Piechota et al. 2010). The last dose of chronic treatment is four times higher than the first dose. The induction of glucocorticoid-dependent genes, which disappears within 8 h if the low dose is used, may remain elevated for an extended period of time (14 h) if the higher dose is used. In addition, these genes have a long mRNA half-life (Fkbp5, 11 h; Sult1a1, 18 h; and Hif3a, 21 h) (Sharova et al. 2009). In principle, we used the time-point 14 h after the last injection to avoid the direct effects of the drugs. However, in the case of METH and HER, it seems that these effects occurred at the higher doses. These effects are independent of the circadian rhythm phase at which the substance is administered.

Interestingly, both substances induced a group of genes (Pdyn, Cartpt, Inmt, Fam40b and Rgs2) that was not caused by the alteration in circadian activity or the dose response effect. The level of these genes gradually increases during chronic treatment and then descends to the basal level during the withdrawal. These genes exhibit highly specific expression throughout the whole striatum (Rgs2) or in the nucleus accumbens (Cartpt and Inmt) (Wu et al. 2009).

A nucleus accumbens-specific gene network is activated by chronic METH and HER treatment and may correspond to homeostatic changes

Genes from a cluster enriched in the nucleus accumbens were previously shown to be regulated by chronic treatment with drugs of abuse (i.e. Cartpt, Pdyn and Rgs2) (Dandekar et al. 2008; Hurd et al. 1992; Kuntz-Melcavage et al. 2009; Zió-lkowska et al. 2006). This group of genes appears to be the most interesting in terms of functionality. The Pdyn gene has been linked with the emergence of sensitization and physical dependence (Beadles-Bohling & Wiren 2005; Shin et al. 2009). We hypothesize that if Pdyn contributes significantly to the formation of physical dependence (Koob 1992; Newton et al. 2004; Zijlstra et al. 2009), it would be possible to obtain naloxone-induced withdrawal after chronic METH treatment. However, our study did not provide a definitive answer to this question. Another gene from this group, Cartpt, is linked most often with the regulation of food intake (Kristensen et al. 1998). However, in the striatum, its role is to attenuate excessive excitation (Kim et al. 2003). On the basis of the induction of Pdyn and Cartpt genes, we hypothesize that chronic METH and HER activate the transcriptional programme to inhibit the dopamine input to the striatum. Therefore, it is possible that it is a homeostatic response. This is also supported by evidence from the Rgs2 gene, which may also be involved in the action of chronically administered drugs of abuse (Burchett et al. 1999). Chronic exposure to psychostimulants generally increases the levels of Rgs2 transcripts (Lomazzi et al. 2008). Rgs2 has the potential to regulate dopamine D1 signalling via direct inhibition of adenylyl cyclase (Sinnarajah et al. 2001). The next gene from this group, indolethylamine N-methyltransferase (Inmt), catalyses the N-methylation of tryptamine and structurally related compounds (Thompson & Weinshilboum 1998). In particular, Inmt can catalyse the transmethylation of tryptamine into dimethyltryptamine (Mandel et al. 1971), which binds non-selectively to serotonin receptors (Keiser et al. 2009). Thus this induction may be responsible for some of the psychotic effects of the withdrawal from drugs of abuse (Zorick et al. 2010). The function of the last gene from this group, Fam40b, is still unknown. Distribution of this gene is limited to the striatum, the CA2 field of the hippocampus and the Purkinje cells (Ng et al. 2009), which may imply that this gene has important neuronal functions.

Changes in the striatal transcriptome after chronic METH or HER treatment are transient

We were interested in which changes in the striatal transcriptome might be responsible for the long-term changes that result from the chronic administration of drugs of abuse. For example, sensitization may last for the entire length of an animal's life (Pierce & Kalivas 1997). Our research, however, did not identify permanent transcriptome changes in the striatum. Changes in the striatal transcriptome developed during treatment and returned to basal levels after 12 days of withdrawal. Thus, we did not find genes whose expression levels are directly associated with locomotor sensitization. We identified changes in gene expression (Pdyn, Cartpt, Rgs2, Inmt and Fam40b) that are related to the repeated administration of the drugs and the early stages of spontaneous withdrawal. These changes might be directly connected to the short-term effects of chronic treatment with drugs of abuse, such as withdrawal syndrome, irritability and anhedonia. Moreover, it is possible that the changes in expression of these genes are involved in the remodelling of neuronal structures and connections in the ventral striatum and thus indirectly responsible for long-lasting changes in behaviour (Evrard et al. 2006).

Complex interaction between drugs of abuse and circadian rhythm

Drugs of abuse influence both molecular and behavioural rhythms (Falcón & McClung 2008). Our data have shown that chronic treatment with METH and HER during the diurnal phase leads to an induction of circadian genes (e.g. Per1, Per2, Gm129 and Dbp). It is in agreement with work of other researchers who also observed alterations in the levels of circadian genes after chronic treatment with drugs of abuse (Perreau-Lenz & Spanagel 2008). Both METH and HER cause locomotor activation in mice, which are nocturnal animals. Therefore, if drug is administered during the diurnal phase, mice are pharmacologically forced to stay awake during their natural sleeping time. Such forced behavioural changes may lead to a deregulation of the diurnal cycle causing alteration in levels of circadian genes mRNA. However, some researchers consider the direct rather than the indirect effect of drugs of abuse on circadian rhythm (Falcón & McClung 2008). Changes in circadian genes return to the baseline in 3 days, which is probably associated with the normalization of circadian rhythm (Stinus et al. 1998). Our results show that if animals were treated during the nocturnal phase, immediately before the peaks of their natural locomotor activity, the alteration in the levels of circadian genes was not observed.

Another aspect of interaction between drugs of abuse and circadian rhythm is that the response to drug exposure is different over the light/dark cycle (Arvanitogiannis et al. 2000; Falcón & McClung 2008). Our studies have shown that locomotor sensitization caused by nocturnal treatment did not require withdrawal incubation, whereas the sensitization caused by diurnal treatment required a long period of withdrawal. It is in agreement with a previous study showing that the most sensitized response occurs during the middle of the dark cycle (Gaytan et al. 1999). Moreover, rodents display circadian rhythm of self-administration with a greater intake during the dark phase (Roberts et al. 2002). However, the diurnal pattern of intake can be abolished when animals are given high doses of the drug or access to more trials (Roberts et al. 2002).

Moreover, two circadian genes, Per1 and Per2, that are altered by the chronic drug treatment, are also induced by acute drug treatment (McClung & Nestler 2003; Piechota et al. 2010; Yuferov et al. 2003). These two genes influence cocaine-induced sensitization and reward in an opposite manner (Abarca et al. 2002). All these findings suggest again that interplay between circadian rhythm and drugs of abuse is complex.

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

Our work is the first to present a detailed time-course of the striatal transcriptome changes during chronic treatment and withdrawal from METH and HER. First, we found a high similarity in the drug-induced changes in both behaviour and gene transcription during chronic treatment with two substances with distinct pharmacological profiles, METH (psychostimulant) and HER (opioid). Second, our study highlights a group of genes that are induced by chronic treatment with drugs of abuse and are enriched in the nucleus accumbens (Pdyn, Cartpt, Fam40b, Rgs2 and Inmt), which may be responsible for some of the effects of addictive substances, such as physical dependence and anhedonia. Third, we show that striatal transcriptome alterations are transient and persisted up to 6 days after withdrawal. Behavioural sensitization, however, was maintained throughout the 12-day withdrawal period for both HER and METH. Therefore, we suggest that these transient gene expression alterations during drug treatment and in the early period of withdrawal are involved in the establishment of persistent neuroplastic alterations. Fourth, our study emphasizes that there is a complex interaction between the circadian system and drug response.

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  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

This work was supported by Polish MSHE grants NN405 274137, IUVENTUS Plus, NCN 2011/01/N/NZ2/04827 and POIG De-Me-Ter 3.1.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. References
  8. Acknowledgments
  9. Supporting Information

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1:Animal weight loss during chronic HER or METH treatment and withdrawal. The line graphs represent the weight of animals at three time-points: before the first injection (naive), 14 h after the last injection (chronic), and 12 days after the last injection (withdrawal) of METH (left), HER (middle) or SAL (right) using the diurnal (up) or nocturnal (down) treatment scheme. Single lines show single animals. Chronic diurnal treatment with both METH (P < 0.01) and HER (P < 0.05) caused a reduction in body weight. A more severe loss of body weight was observed following nocturnal chronic treatment with both METH (P < 0.001) and HER (P < 0.01).

Figure S2:Acquisition of locomotor sensitization to HER or METH after chronic treatment and withdrawal. The line graphs show the locomotor response to METH (2 mg/kg, left), HER (10 mg/kg, middle) or SAL (right) at three time-points. Responses to the first injection (Naive), 14 h after the last injection (Chronic), 12 days after the last injection (Withdrawal) using the diurnal (up) or nocturnal (down) treatment scheme are reported. Single lines show single animals.

Figure S3:Numbers of genes regulated by drug treatment. The Venn diagram represents numbers of probes significant for the main factors (drug, time) and interaction (Two-way ANOVA, FDR < 5%).

Figure S4:Dose-dependent effects of treatment on Hif3a gene and lack of the effect on Gm129 gene. Barplots show fold change of gene expression for every day of treatment and withdrawal in which the expression was measured. Scatterplots show dose effect on gene expression alteration of Hif3a (P < 0.05) and Gm129 (P > 0.05) genes.

Figure S5:Naloxone-precipitated withdrawal in mice following chronic treatment with METH or HER. Bar graphs summarising the mean and standard deviation of jumps in response to naloxone (s.c., 4 mg/kg) after the indicated treatment (n = 4–7). HER-treated mice exhibited significant withdrawal signs 14 h after the last injection (P < 0.05). METH-treated mice did not exhibit significant withdrawal signs 14 h after the last injection.

Table S1: Probes with uncorrected significance (P < 0.05).

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