DESCRIPTION:
Male and female C57BL/6J mice, mu opioid receptor (Oprm1) knockout mice, and dopamine transporter (DAT)-IRES-Cre knock-in mice were obtained from The Jackson Laboratory or bred in-house. Mice were 6-12 weeks old at the beginning of each experiment. Osmotic minipumps (Alzet Model 2001) were implanted in mice weighing up to 25g, which is the upper limit for administration of 63.2 mg/kg/day morphine using these minipumps. After adjusting morphine concentration for body weight, minipumps were filled with 300 μL of solution and primed overnight at 40°C. Miniaturized programmable infusion pumps (iPrecio SMP-300) were implanted in mice weighing at least 20g. The pump reservoir was filled with saline (SAL) or morphine (MOR) (~50 mg/mL) according to manufacturer’s instructions, and then wirelessly programmed to infuse with one of two patterns: (1) a continuous pattern with sustained infusion for 7 consecutive days, or (2) a “discontinuous” pattern with alternating 24-hour periods of drug infusion and pump inactivity for 13 days. To interrupt continuous morphine infusion while controlling pharmacokinetic variables, we administered two daily naloxone (NLX) injections separated by an interval of 2h (Fig. 2a), as previously described. We selected a high dose of naloxone (10mg/kg) to fully interrupt activation of opioid receptors by morphine, as pilot studies showed this naloxone dose had more robust effects than lower doses. We assessed the downstream consequences of continuous and interrupted morphine exposure on gene expression in the nucleus accumbens. To minimize variability related to sex differences, we used only male mice in this experiment, since interrupted morphine caused more robust locomotor sensitization in males (n=5–6 male mice/group). At the end of chronic treatment, we dissected the nucleus accumbens as well as the dorsal striatum (Fig. 4a), and used next-generation RNA sequencing to perform genome-wide transcriptional profiling. Truseq libraries were sequenced 50-bp paired-end run on the Illumina HiSeq 2500, generating roughly 20 million paired-end reads per run. Cleaned reads were aligned to the Mus musculus reference genome, version GRCm38 with HISAT2. Nucleus accumbens and dorsal striatum samples were handled separately for all analyses. For differential expression analysis, filtered expression counts were normalized and variance-stabilized with DESeq2. One sample from the nucleus accumbens was identified as an outlier based on both PCA and hierarchical clustering, and was removed from further analyses. We defined differential gene expression with a fold change threshold of 15%, while controlling false discovery rate at q<0.05. From supplementary table S3.
LABEL:
DEG mouse DS control_pvalue
SCORE TYPE:
P-Value
THRESHOLD:
<= 0.5
GENES IN THRESHOLD:
267
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ABSTRACT:
Genes in threshold: 267
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