Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome

Abstract

Substance use disorders are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based quantitative proteomics, along with a data-independent acquisition analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveal cocaine and morphine differentially alter diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between the phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. The proteomics data in this study are available via ProteomeXchange with identifier PXD042043.

Keywords: circadian rhythms; cocaine; morphine; nucleus accumbens; proteomics.

Figure 1.

Cocaine or morphine induces differential changes in the nucleus accumbens proteome across time of day. (A) Schematic overview of the treatment paradigm and tissue collection prior to liquid chromatography mass spectrometry (LC-MS) quantification. Mice were injected intraperitoneally (i.p.) for 7 days with either saline (10 ml/kg), cocaine (15 mg/kg at 10 ml/kg), or morphine (10 mg/kg at 10 ml/kg). Following exposure, NAc tissue was collected across 4 times of day (ZT 5, 10, 17, & 22; ZT0 = 7am) and processed for LC-MS. (B) Volcano plots depicting differentially expressed (DE) proteins during the day (left; ZT 5 & 10) and night (right; ZT 17 & 22) as a result of cocaine (top) or morphine (bottom). Horizontal dashed line represents the p value significance cutoff (-Log₁₀P of 1.3, or p=0.05), while the vertical dashed lines represent the fold change (FC) cutoff (Log₂FC < −0.1375 or > 0.1375, or FC ± 1.1). Red dots indicate DE proteins that meet both cutoffs. (C) Venn diagrams depicting overlap of cocaine and morphine DE proteins during the day and night that met both FC and p value cutoffs. Venn diagram circles are sized by the number of proteins, proportional within comparisons. Schematic created with BioRender.com.

Figure 2.

Pathway and biological process enrichment analysis for differentially expressed proteins in the NAc following cocaine or morphine. (A) Top pathways enriched among the DE proteins in the NAc by substance across time of day, as revealed by Ingenuity Pathway Analysis (IPA). A significance cutoff of −Log10(p value) of 1.3, or p<0.05, was used to determine pathway enrichment, depicted by the red line. (B) Gene Ontology (GO) Biological process enrichment via Metascape for the top DE proteins in the NAc by substance across time of day. Meta-analysis was used to compare process enrichment across substances by time of day, depicted via a Cytoscape enrichment network plot. Terms with p < 0.01, a minimum count of 3, and enrichment factor >1.5 were grouped into clusters based on their membership similarities. The cluster was named after the most statistically significant term within the cluster.). The nodes are represented as pie charts, where the size of the pie is proportional to the total number of proteins for that specific term, and color indicates the identity of the gene list, where the size of the slice represents the percentage of proteins enriched for each corresponding term. Similar terms are connected by purple lines (kappa score >0.3), and line thickness indicates degree of connectivity. “Cocaine Day” is not included in the plot due to a lack of enrichment.

Figure 3.

Cocaine or morphine alters rhythmicity in the NAc proteome. (A) Rank-Rank Hypergeometric Overlap (RRHO) analysis of the overlap between the cocaine and morphine specific rhythmic proteins in the NAc reveals limited overlap. Cosiner rhythmicity analysis was utilized to detect rhythmicity. (B) Table overview of the number of rhythmic proteins in the NAc that either gained or lost rhythmicity in the NAc, as a result of cocaine or morphine relative to saline treated mice. (C) Representative proteins with a gain (right) or loss of rhythmicity (left) in the NAc following cocaine (top) or morphine (bottom), relative to saline. Proteins with the greatest amplitude change were chosen. Dots indicate individual samples, with the y axis indicating expression and x axis indicating time of day (ZT 5, 10, 17, 22). Red line depicts the fitted curve as revealed in the cosiner rhythmicity analysis. (D) Venn diagrams depicting overlap of proteins that either gained (left) or lost (right) rhythmicity following cocaine or morphine. Overlapped proteins with gained or lost rhythmicity are listed on the right. Venn diagram circles are sized by the number of proteins, proportional within comparisons.

Figure 4.

Pathway and biological process enrichment analysis for proteins with a gain or loss of rhythmicity in the NAc following cocaine or morphine. (A) Top pathways enriched among the proteins with a gain or loss of rhythmicity in the NAc by drug treatment, as revealed by Ingenuity Pathway Analysis (IPA). A significance cutoff of −Log10(p value) of 1.3, or p<0.05, was used to determine pathway enrichment, depicted by the red line. (B) Gene Ontology (GO) Biological process enrichment via Metascape for the top proteins with gained or lost rhythmicity in the NAc by drug treatment. Meta-analysis was used to compare process enrichment across treatment groups by gain or loss of rhythmicity, depicted via a Cytoscape enrichment network plot. Plots were generated using the same parameters as in Figure 2.

Figure 5.

Overlap between rhythmic and DE proteins in the NAc as a result of cocaine or morphine. (A) Rank-Rank Hypergeometric Overlap (RRHO) analysis of the overlap between the cocaine-specific rhythmic proteins in the NAc and the DE proteins in the NAc by substance and across time of day. RRHO revealed limited overlap except in the DE Cocaine Day versus Cocaine Rhythmic comparison. (B) RRHO analysis of the overlap between the morphine-specific rhythmic proteins in the NAc and the DE proteins in the NAc by substance and across time of day. RRHO revealed limited overlap except in the DE Morphine Night versus Morphine Rhythmic comparison. (C) Representative overlapped morphine-specific rhythmic proteins with the largest downregulation fold change at night, plotted relative to saline. (D) Representative overlapped morphine-specific rhythmic proteins with the largest upregulation fold change at night, plotted relative to saline. Dots indicate individual samples, with the y axis indicating expression and x axis indicating time of day (ZT 5, 10, 17, 22). Red line depicts the fitted curve as revealed in the cosiner rhythmicity analysis. Cocaine-specific overlapped rhythmic and DE proteins were not shown due to no proteins meeting significance thresholds.