A total of 21 male Sprague Dawley rats 13.91+1.34 weeks old and weighing 250–300 g, were obtained from the Experimental Animal Center of Zhejiang Province. The rats were subsequently housed in a temperature (22–24°C)- and humidity (40–50%)-controlled room with a reversed 12-h light-dark cycle (lights on 7:00 p.m.-7:00 a.m.) with ad libitum access to food and water. Trainings were performed between 9:00 a.m. and 5:00 p.m. Rats were randomly divided into three equal groups (n=7): Group A, self-administered delivery of heroin; group B, yoked delivery of heroin; and group C, yoked delivery of saline. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Rats were implanted with chronically indwelling intravenous catheters under sodium pentobarbital (50 mg/kg, i.m; Sigma-Aldrich, St. Louis, MO, USA) anesthesia. A silicon catheter (length, 4 cm; inner diameter, 0.51 mm; outer diameter, 0.94 mm) was subsequently inserted into the right external jugular vein and secured with thread. The other end of the catheter (length, 10 cm; PE20) exited the rats from an incision on the back of the body. Catheters were flushed daily with 0.3 ml benzylpenicillin (200,000 units) and 0.3 ml heparin (50 U/ml) to prevent bacterial infection and maintain catheter patency, and were capped daily. Rats were allowed to recover for at least 7 days.

Heroin was obtained from the National Institute of Forensic Science of China (Shanghai, China). A total of 21 custom-made Plexiglas operant boxes (working area, 30×30×30 cm) were used. Each operant box was equipped with two nose-pokes, with an LED light and an infrared probe inside each. Heroin self-administration sessions were conducted daily for 21 consecutive days under a fixed ratio 1 (FR1) schedule. In brief, rats were moved to the operant chambers, and their connectors were attached to the infusion lines. Each trial began with illumination of the house-light. Rats received a single heroin infusion at a dose of 0.05 mg/kg/infusion paired with a 2-sec active nose-poke light, following completion of the ratio requirement in the active nose-poke. The house-light was turned off during heroin infusions. A 20-sec inter-trial interval followed, after which another trial began. Responding in the inactive nose-poke period had no consequences. The session ended after 4 h or 50 heroin infusions, whichever occurred first. Rats were returned to their individual cages shortly after the session.

The remaining two groups served as the yoked heroin and yoked saline controls. Yoked heroin rats received amounts of heroin equal to those of the rats in the self-administration group over the same time course; however, their exposure to heroin was not explicitly associated with nose-poke responses. Yoked saline control rats received the same volume of saline at the same frequency over a 4-h period. Rats were deeply anaesthetized with sodium pentobarbital (80 mg/kg intraperitoneally) and decapitated after the final behavioral training session. The VTA was isolated using a rat brain matrix. The entire VTA was removed from the decapitated rats, immediately flash frozen in liquid nitrogen, and subsequently stored at −80°C until further use.

ChIP assays were performed using standard procedures (31,32). Frozen VTA tissue was pulverized into a powder using a hammer and liquid nitrogen. A total of 60 mg VTA tissue was prepared for each group of 7 rats. The tissue was cross-linked in formaldehyde at a final concentration of 0.7%. All experimental reagents were included in the Magna ChIP-Seq™ kit (17–1010, EMD Millipore, Billerica, MA, USA). Sonication was conducted using a Bioruptor UCD-200 (Diagenode, Liège, Belgium) under the optimal conditions to shear cross-linked DNA to fragments of 100–600 base pairs in length. In brief, chromatin samples were sheared for 5, 3, 3, 3 and 2 cycles of 30 sec on/30 sec off for a total of 16 cycles and paused for 1 min with a short centrifugation during the intervals. Equal amounts of chromatin lysate (50 µl) were diluted with ChIP buffer H (Auto Histone ChIP-seq kit; C01010022; Diagenode) to a final volume of 200 µl, ensuring that the final concentration of SDS was <1%. An aliquot (1%; 2 µl) of the pre-immunoprecipitated lysate was saved as ‘input’ for subsequent normalization. A SX-8 G IP-Star^® automated system (Diagenode) was used for the immunoprecipitation reaction, with H4K5 (9672S; Cell Signaling Technology, Inc.) as the target antibody and normal rabbit IgG as a negative control, due to the rabbit source of the antibody. The DNA, associated with acetylated histones, was extracted using a Diagenode Auto IPure kit (C03010010; Diagenode), and subsequently resuspended in 50 µl buffer C from the kit.

The level of specific histone modification at the gene of interest was determined by measuring the amount of acetylated histone-associated DNA by quantitative polymerase chain reaction (qPCR) using a LightCycler^® 480 (Roche Diagnostics, Mannheim, Germany). To avoid errors that may occur from differences in the loading quantity of the samples, GAPDH was taken as the internal reference for qPCR. The qPCR reaction was performed in a total volume of 10 µl and contained 5 µl of 2X SYBR Green Master Mix, 0.2 µl primers (from a stock at 10 µM each), 1.8 µl of ddH₂O and 3 µl of ChIP or input sample. Triplicate qPCR reactions per ChIP sample were performed. The primers used were as follows: BRG1, forward 5′-ACAGAGCCTTGCAGAGCA-3′ and reverse 5′-GAGGAAAGTGAAGCCGAGA-3′; GAPDH, forward 5′-CGTAGCTCAGGCCTCTGCGCCCTT-3′ and reverse 5′-CTGGCACTGCACAAGAAGATGCGGCTG-3′. qPCR conditions included a pre-incubation stage at 95°C for 3 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and primer extension at 72°C for 30 sec, then a melting curve (90°C for 10 sec, 60°C for 30 sec and 90°C 10 sec) with final cooling at 40°C for 30 sec. The results of qPCR for enrichment were performed using a relative standard curve method to compare DNA from a mock IP using IgG versus DNA immunoprecipitated with the ChIP antibody.

The recovery rate was calculated by referring to the Auto Histone ChIP-seq kit (Diagenode, Belgium) and the manufacturer's instructions as recovery rate % (ChIP/total input)=2^[(Ct (x% input)-log (x%)/log2)-Ct (ChIP)] × 100%. Fold enrichment over IgG was calculated by the recovery of BRG1/GAPDH. Ct (ChIP) and Ct (x% input) are threshold values obtained from the exponential phase of qPCR for the IP'd DNA and input sample, respectively. The compensatory factor [log (x%)/log2] is used to take into account the dilution, 1:x, of the input. The recovery is the % (ChIP/total input). Comparisons among the three groups or between two groups were calculated by one-way analysis of variance and Student's t-test using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The association between histone acetylation and heroin addiction was evaluated in three groups of rats. Group A contained rats that received heroin by self-administration, whereas the rats of group B and group C were administered heroin and saline, respectively, in a yoked passive manner.

VTA tissue was cross-linked with 0.7% formaldehyde, and the DNA-histone complex was sheared to fragments of 100–600 bp in length via sonication (Fig. 1A). ChIP assays were then performed using an antibody against H4K5, and the amount of DNA associated with the modified histone was quantified using qPCR. The results demonstrated that the IgG was not amplified by qPCR, which indicated that the experimental system was reliable. Example curves for qPCR are presented in Fig. 1B. The results of quality control showed that the experimental results meet the requirements.

Recovery was calculated, which indicated the percentage of the total input BRG1 that was recovered by ChIP. The results demonstrated that the quantity of BRG1 combined with H4K5 modification differed among the three groups; however, recovery was consistently low (0.52%, 0.78%, and 0.74% for groups A, B and C respectively; Fig. 2A) and the P-value was 0.063. Notably, a significant difference was detected between the rats that received heroin by self-administration and those that received heroin in a passive manner (A and B, P=0.031; Fig. 2A). BRG1 was less associated with H4K5 histone modification in the heroin self-administration group, as compared with the other two groups (A and B, P=0.031; A and C, P=0.067). No other significant differences were detected between the passive heroin groups and the saline-administered group (P=0.760) (1). The results of the recovery rate suggested that the group of heroin self-administration had less H4K5 associated with BRG1.

To avoid the errors that occurred by differences in the quantity of the samples loaded, GAPDH was taken as the internal reference for qPCR. Fold enrichment indicated the ratio of BRG1:GAPDH was associated with H4K5 histone modification. The results demonstrated that the heroin-treated rats exhibited lower fold changes than those receiving saline (2.44, 2.28, 4.16 for groups A, B and C respectively; Fig. 2B). Furthermore, there was a significant difference among the three groups (P=0.019), specifically between group C and the other two groups (A vs. C, P=0.013; B vs. C, P=0.009; and A vs. B, P=0.731; Fig. 2B). The fold enrichments demonstrated that H4K5 histone acetylation of BRG1 in the VTA may be associated with heroin administration but not associated with the intake methods.