Interruption of continuous opioid exposure exacerbates drug-evoked adaptations in the mesolimbic dopamine system

Drug-evoked adaptations in the mesolimbic dopamine system are postulated to drive opioid abuse and addiction. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological and behavioral impact. We exposed male and female mice to morphine for one week, with administration patterns that were either intermittent (daily injections) or continuous (osmotic minipump infusion). We then interrupted continuous morphine exposure with either naloxone-precipitated or spontaneous withdrawal. Continuous morphine exposure caused tolerance to the psychomotor-activating effects of morphine, whereas both intermittent and interrupted morphine exposure caused long-lasting psychomotor sensitization. Given links between locomotor sensitization and mesolimbic dopamine signaling, we used fiber photometry and a genetically encoded dopamine sensor to conduct longitudinal measurements of dopamine dynamics in the nucleus accumbens. Locomotor sensitization caused by interrupted morphine exposure was accompanied by enhanced dopamine signaling in the nucleus accumbens. To further assess downstream consequences on striatal gene expression, we used next-generation RNA sequencing to perform genome-wide transcriptional profiling in the nucleus accumbens and dorsal striatum. The interruption of continuous morphine exposure exacerbated drug-evoked transcriptional changes in both nucleus accumbens and dorsal striatum, dramatically increasing differential gene expression and engaging unique signaling pathways. Our study indicates that opioid-evoked adaptations in brain function and behavior are critically dependent on the pattern of drug administration, and exacerbated by interruption of continuous exposure. Maintaining continuity of chronic opioid administration may, therefore, represent a strategy to minimize iatrogenic effects on brain reward circuits.

All pumps were implanted under anesthesia (5% isoflurane/95% oxygen) through a small incision on the rump, which was then closed with wound clips. Carprofen (5 mg/kg, s.c.) was given as an analgesic before surgery and for 3 days following pump implantation or removal. Behavioral testing began 24 hours after pump implantation (Day 1), to allow for recovery from surgical anesthesia.

Behavioral and Pharmacokinetic Assessments
We tested open-field locomotor activity in a clear plexiglass arena (ENV-510, Med Associates) housed within a sound-attenuating chamber. The location of the mouse within the arena was tracked in two dimensions by arrays of infrared beams, connected to a computer running Activity Monitor software (Med Associates). Mice were habituated to the chamber for one hour the day before initiating drug treatment, and then tested on the first and last days of chronic morphine treatment (30 minutes after morphine injections). For morphine challenges, the session length varied as a function of morphine dose: 60 mins (Saline and 2 mg/kg), 90 mins (6.32 mg/kg), or 120 mins (20 mg/kg) Thermal antinociception was tested on a 55°C hot plate (IITC Life Scientific). The day before initiating drug treatment, mice were habituated to the instrument for 60 seconds at room temperature. Immediately before the first drug exposure, we established baseline latency to either jump or lift and lick a hind paw at 55°C. Mice were then tested on the first and last days of chronic morphine exposure (30 minutes after morphine injections), with a maximal cutoff of 30 seconds to prevent tissue damage. The percent maximum possible effect was calculated as (test latency -baseline latency) / (30 sec -baseline latency) x 100.
In a subset of mice, we also collected facial vein blood samples on the first and last days of morphine exposure (5 minutes after injections), to determine serum morphine concentration. These samples were collected in heparinized tubes and centrifuged at 7500 RPM for 3 minutes at 14°C. Serum was transferred to a 5 ml vial and stored at -20°C until analysis.
A global withdrawal score [1] was assessed on days 1 and 6 of interrupted morphine exposure, during a 10-minute period after the first naloxone injection on that day. Mice were placed in a tall glass cylinder and filmed for offline analysis. An experimenter blind to treatment counted the total number of jumps, rearing and wet dog shakes. Ptosis, rhinorrhea, lacrimation, salivation, diarrhea and eye twitch were evaluated over 2-minute intervals, with a value of 1 being assigned if present during each interval (maximum score 5). Piloerection and locomotor activity were evaluated together over 2-minute intervals, and assigned a value of 0, 1, or 2 for each interval based on minimal, moderate and maximum response, respectively (maximum score 10). All values were added together for the entire 10 minute period to calculate the global withdrawal score.

Stereotaxic Surgery
Mice were anesthetized with a ketamine:xylazine cocktail (100:10 mg/kg), and a small hole was drilled above target coordinates for the nucleus accumbens core (AP +1.35, ML +1.10; DV -4.40) and ventral tegmental area (AP -3.0, ML +0.5; DV -4.5). Using a 33-gauge Hamilton syringe, AAV1-Syn-dLight1.3b was delivered to the nucleus accumbens and AAVdj-Syn-FLEX-ChrimsonR-tdTomato was delivered to the ventral tegmental area. Viral solutions were injected at a concentration of ~10 13 particles/mL, a volume of 0.75 μL, and a rate of 0.1 μL/min. The syringe tip was left in place at the injection site for 5 minutes, and then slowly retracted over the course of 5 minutes. This was followed by unilateral implantation of a 400 µm fiberoptic cannula (Doric Lenses: MFC_400/430-0.48_6mm_MF1.25_FLT) just above the site of virus injection in the nucleus accumbens (+0.02 mm), affixed to the skull using a dual-cure resin (Patterson Dental, Inc.). After surgery, mice were given 500 μL saline and 5 mg/kg carprofen (s.c.) daily for 3 days, and recovered a minimum of 2 weeks before fiber photometry recordings to allow for sufficient virus expression.

Fiber Photometry Recording and Analysis
Real-time optical signals were acquired using a RZ5P fiber photometry workstation (Tucker Davis Technologies) as previously described [2]. LEDs at 470 and 405 nm (ThorLabs) were modulated at distinct carrier frequencies (531 Hz and 211 Hz, respectively), and passed through a fluorescence mini cube (Doric Lenses) coupled into a patch cord (400 µm, 0.48 NA). For experiments involving red-shifted optogenetic stimulation with ChrimsonR, a 595 nm LED (ThorLabs) controlled using a pulse generator (Master-8, A.M.P.I.) was connected to the fluorescence mini cube and coupled to the same patch cord. The distal end of the patch cord was connected to the implanted fiberoptic cannula with a ceramic sleeve. Fluorescence was back-projected through the mini cube and focused onto a photoreceiver (Newport Model 2151). Signals were sampled at 6.1 kHz, demodulated in real-time, and saved for offline analysis.
Raw data from each recording session were processed as previously described [3]. Each channel was low-pass filtered (<2 Hz) and a linear least-squared model fit the control signal (405 nm) to the dopaminedependent signal (470 nm). Fluorescent signal (dF/F) was calculated as ([470 nm signal − fitted 405 nm signal]/[fitted 405 nm signal]). To measure slow fluctuations in fluorescent signal caused by injection of morphine or fentanyl, we first filtered the signal by calculating median fluorescence across a rolling 20 sec window, to remove transient events. This filtered signal was then averaged for the 30 minutes immediately before injection (baseline), and 30 mins immediately after morphine challenge or 5 mins immediately after fentanyl injection (i.e., area-under-the-curve averaged over time).
To measure spontaneous fluorescent transient events, we first corrected for photobleaching and other tonic signal fluctuations over time by subtracting the dF/F signal by the eighth percentile value over a rolling 20 sec window [4]. We identified spontaneous fluorescent transient events from this normalized signal as exceeding the median absolute mediation (MAD) by >2.91 deviations across a rolling 20 sec window [5]. For analysis of optogenetically-evoked signals, the onset and offset of the 595 nm LED were transmitted to the fiber photometry workstation as a TTL signal. The dLight signal on each stimulation trial was then normalized to a 2 sec baseline period immediately preceding stimulation, and the peak dLight signal during the 2 sec immediately following LED onset was averaged across the ten trials per frequency (0/5/10/20/40 Hz).

Quantitative RT-PCR
For quantitative RT-PCR, tissue was snap frozen on dry ice and stored at -80°C. RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. All RNA samples had A260/A280 purity ratio ≥ 2. Reverse transcription was performed using Superscript III (Invitrogen). For each sample, duplicate cDNA preparations were set up. Mouse β-actin mRNA was used as the endogenous control.
Quantitative RT-PCR using SYBR green (BioRad, Hercules, CA) was carried out with a Lightcycler 480 II (Roche) system with the following cycle parameters: 1 x (30 sec @ 95°C), 35 x (5 sec @ 95°C followed by 30 sec @ 60°C). Data were analyzed by comparing the C(t) values of the treatments tested using the ΔΔC(t) method.
Expression values of target genes were first normalized to the expression value of β-actin. The median of each cDNA replicate reactions was used to quantify the relative target gene expression. Primers were designed in Primer3 and validated in BLAST and are listed in Table S1.

RNA Sequencing
For RNA sequencing, tissue was stored in RNAlater (Qiagen) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) as previously described [6]. Total eukaryotic RNA isolates were quantified using a fluorimetric RiboGreen assay. Total RNA integrity was assessed using capillary electrophoresis (Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN) that was >8 for all samples. Total RNA samples were converted to Illumina sequencing libraries using the Truseq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Extracted mRNA was then oligo-dT purified using oligo-dT coated magnetic beads, fragmented and then reverse transcribed into cDNA. The cDNA was adenylated and then ligated to dualindexed (barcoded) adapters. Truseq libraries were then sequenced 50-bp paired-end run on the Illumina HiSeq 2500, generating roughly 20 million paired-end reads per run.
Raw Illumina reads were cleaned of low quality bases, adapter contamination, and low complexity sequence with Trimmomatic [7]. Cleaned reads were aligned to the Mus musculus reference genome, version GRCm38 with HISAT2 [8], with a list of putative splice sites derived from the GTF annotation file. Resulting BAM files were sorted by read name, and counts were generated with HTSeq [9] with the unstranded option. A modification to the GTF file from Ensembl was performed to consider only protein coding genes for the counts.
The expression counts matrix was filtered to remove genes with less than 150 total counts across all samples prior to differential expression testing. Nucleus accumbens and dorsal striatum samples were handled separately for all analyses.

Normalization and Differential Expression Tests
For differential expression analysis, filtered expression counts were normalized and variance-stabilized with DESeq2 [10]. The variance-stabilized counts for all genes passing the expression filter were decomposed with principal components analysis (PCA) to identify potential broad patterns in gene expression differences among samples. Stabilized and filtered counts were also centered about 0 for each gene and used as input for hierarchical clustering. One sample from the nucleus accumbens was identified as an outlier based on both PCA and hierarchical clustering, and was removed from further analyses. Normalized and stabilized counts were exported for other analyses. Differential expression testing was performed with normalized counts in DESeq2. No differences were observed between the saline-saline and saline-naloxone treatments (see Table S3), so these treatments were combined in a single "control" group for differential expression testing. Differentially expressed genes were identified among three comparisons: control vs. continuous, control vs. interrupted, and continuous vs.
interrupted. A false discovery rate (q) of 0.05 was used for Benjamini-Hochberg multiple testing corrections [11], with a fold change threshold of 15%. For the purpose of comparison with prior literature [12], we also repeated this analysis using a less stringent cutoff of p<0.05 (see Figure S6).

Pathway and Network Analyses
Differentially expressed genes from DESeq were used to identify overrepresented pathways with Ingenuity Pathway Analysis (IPA) core expression analysis (QIAGEN Inc). Filtered, normalized, and stabilized counts from DESeq2 were scaled to mean 0 and unit variance was used as input for a weighted gene coexpression network analysis (WGCNA) [13,14]. Scale-free topology assumptions and soft power thresholds for network edges were checked with routines within the WGCNA package. Coexpression networks were built separately for each brain region, and then region-specific networks were merged to form a consensus network, merging modules with a Pearson correlation of at least 0.3 between their eigengenes. Eigengene values for each sample were exported. Tests for associations between module eigengene expression value and experimental treatment were performed with a one-way ANOVA. An eigengene-treatment association was considered as significant if its Bonferroni-adjusted P-value was less than 0.05, with the number of modules tested taken as the number of independent hypothesis tests.

Statistical Analyses
Similar numbers of male and female animals were used in all experiments, except for RNA sequencing, which was only conducted in males to minimize variability. Individual data points from males (filled circles) and females (open circles) are distinguished in figures. Sex was included as a variable in factorial ANOVA models analyzed using IBM SPSS Statistics v24, with repeated measures on within-subject factors, but sex effects were not significant unless noted otherwise. For main effects or interactions involving repeated measures, the Huynh-Feldt correction was applied to control for potential violations of the sphericity assumption. This correction reduces the degrees of freedom, resulting in non-integer values. Significant interactions were decomposed by analyzing simple effects (i.e., the effect of one variable at each level of the other variable). Significant main effects were analyzed using LSD post-hoc tests. Effect sizes are expressed as partial eta-squared (η p 2 ) values.
The Type I error rate was set to α=0.05 (two-tailed) for all comparisons.

SUPPLEMENTARY FIGURES & FIGURE LEGENDS
Figure S1. Antinociception and pharmacokinetics after intermittent injection or continuous infusion of morphine. (A-B) Thermal antinociception on the hot-plate increased in a dose-dependent fashion after morphine injections (A) (n=14/group) and infusion (B) (n=16/group). Tolerance was observed at the highest dose, for both injection and infusion. As noted in Supplemental Table S2, there was a significant Day x Dose x Sex interaction for morphine injections on the hot plate (A). This interaction reflected a heightened antinociceptive response in female mice after 20mg/kg morphine injection (but not other doses), on Day 1 but not Day 7. (C-D) Serum morphine concentration increased in a dose-dependent fashion after injection (C) (n=5-6/group) or infusion (D) (n=8/group), with lower absolute levels after infusion (magnified in inset). All groups contained similar numbers of female and male mice; see Supplemental Table S2 for detailed statistical analyses. # p<0.05 for simple effect within group.  Table S2 for detailed statistical analyses.  Table S2 for detailed statistical analyses.  Table S2 for detailed statistical analyses.  Table S2 for detailed statistical analyses. *p<0.05 between groups, LSD post-hoc test.  Table S2 for detailed statistical analyses. *p<0.05 between groups, LSD post-hoc test.      Remaining supplementary tables are provided in Excel format.