The transcriptional response to acute cocaine is inverted in male mice with a history of cocaine self-administration and withdrawal throughout the mesocorticolimbic system

Abstract

A large body of work has demonstrated that cocaine-induced changes in transcriptional regulation play a central role in the onset and maintenance of cocaine use disorder. An underappreciated aspect of this area of research, however, is that the pharmacodynamic properties of cocaine can change depending on an organism's previous drug-exposure history. In this study, we utilized RNA sequencing to characterize how the transcriptome-wide effects of acute cocaine exposure were altered by a history of cocaine self-administration and long-term withdrawal (30 days) in the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC) in male mice. First, we found that the gene expression patterns induced by a single cocaine injection (10 mg/kg) were discordant between cocaine-naïve mice and mice in withdrawal from cocaine self-administration. Specifically, the same genes that were upregulated by acute cocaine in cocaine-naïve mice were downregulated by the same dose of cocaine in mice undergoing long-term withdrawal; the same pattern of opposite regulation was observed for the genes downregulated by initial acute cocaine exposure. When we analyzed this dataset further, we found that the gene expression patterns that were induced by long-term withdrawal from cocaine self-administration showed a high degree of overlap with the gene expression patterns of acute cocaine exposure - even though animals had not consumed cocaine in 30 days. Interestingly, cocaine re-exposure at this withdrawal time point reversed this expression pattern. Finally, we found that this pattern was similar across the VTA, PFC, NAc, and within each brain region the same genes were induced by acute cocaine, re-induced during long-term withdrawal, and reversed by cocaine re-exposure. Together, we identified a longitudinal pattern of gene regulation that is conserved across the VTA, PFC, and NAc, and characterized the genes constituting this pattern in each brain region.

Keywords: Craving; Dorsal striatum; Gene expression; Incubation; RNA-sequencing; Ventral striatum.

Figure 1:. Cocaine self-administration alters cocaine-induced transcription across the mesocorticolimbic system.

(a) Mice underwent either saline or cocaine (0.5mg/kg/inj) self-administration (SA) for ten days followed by a 30-day withdrawal period. On day 30 mice were sacrificed 1 hour following either saline or cocaine injection (10 mg/kg, IP). To determine differential gene expression induced by acute cocaine (green), mice that administered saline and received a saline injection (cocaine-naïve mice) were compared to mice that self-administered saline and received a cocaine injection (first-time acute cocaine exposure). To determine differential gene expression induced by cocaine re-exposure (purple), mice that self-administered cocaine and received a cocaine injection (acute cocaine re-exposure) were compared to mice that self-administered cocaine and received a saline injection (30-day cocaine withdrawal) (b) Stratified Rank-rank hypergeometric overlap (RRHO) was used to compare the relationship between differential gene expression induced by acute cocaine and cocaine re-exposure in a threshold-free fashion. Results are displayed as a heatmap with more significantly overlapping gene expression denoted by warmer colors and less significantly overlapping gene expression denoted by cooler colors. Hotspots in the bottom left and top right quadrants of the heatmap indicate a concordant gene expression relationship between the two lists (i.e. the same genes are upregulated and downregulated in both lists) whereas hotspots in the bottom right and top left quadrants of the heatmap indicate a discordant gene expression relationship between the two lists (i.e. downregulated and upregulated genes in one condition are upregulated and downregulated, respectively, in the other list). Stratified RRHO comparisons of differential gene expression induced by acute cocaine and cocaine re-exposure were performed in the (c) ventral tegmental area (VTA; genes compared in x-axis and y-axis = 13,317, maximum −log10(hypergeometric p-value) = 63.5; number most significantly overlapping genes in top left quadrant = 3,486, number most significantly overlapping genes in bottom right quadrant = 2338), (d) nucleus accumbens (NAc; genes compared in x-axis and y-axis = 13,232, maximum −log10(hypergeometric p-value) = 129.3, number most significantly overlapping genes in top left quadrant = 3,044, number most significantly overlapping genes in bottom right quadrant = 2,580), (e) prefrontal cortex (PFC; genes compared in x-axis and y-axis = 13,092, maximum −log10(hypergeometric p-value) = 75.1, number most significantly overlapping genes in top left quadrant = 3,637, number most significantly overlapping genes in bottom right quadrant = 2,647), and (f) caudate putamen (CPU; maximum −log10(hypergeometric p-value) = 11.0). This analysis revealed that acute cocaine and cocaine re-exposure exhibited opposite patterns of transcriptional regulation of the same genes in the VTA, NAc, and PFC (c-e), but that this discordant gene expression relationship was not present in the CPU (f).

Figure 2:. Longitudinal comparisons of cocaine-induced transcription.

(a) Schematic of longitudinal comparisons for Stratified RRHO in the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC). Comparisons were first made between differential gene expression induced by acute cocaine and differential gene expression induced by two withdrawal timepoints: short-term (1 day; panel b) withdrawal from cocaine self-administration or long-term (30-day; panel c) withdrawal from cocaine self-administration. Differential gene expression induced by long-term cocaine withdrawal was then compared to differential gene expression induced by cocaine re-exposure (panel d). (b) Comparison of differential gene expression induced by acute cocaine to differential gene expression induced by short-term withdrawal revealed weak overall overlap across the VTA (i; maximum −log10(hypergeometric p-value)= 53.3), NAc (ii; maximum −log10(hypergeometric p-value) = 14.4) and PFC (iii; maximum −log10(hypergeometric p-value) = 10.5). (c) Comparison of differential gene expression induced by acute cocaine to differential gene expression induced by long-term withdrawal revealed a strong concordant gene expression relationship across the VTA (i; genes compared in x-axis and y-axis = 13,317, maximum −log10(hypergeometric p-value) = 293.2, number most significantly overlapping genes in bottom left quadrant = 4,329, number most significantly overlapping genes in top right quadrant = 4,022), NAc (ii; genes compared in x-axis and y-axis = 13,232, maximum −log10(hypergeometric p-value) = 103.7, number most significantly overlapping genes in bottom left quadrant = 2,054, number most significantly overlapping genes in top right quadrant = 3,920), and PFC (iii; genes compared in x-axis and y-axis = 13,092, maximum −log10(hypergeometric p-value) = 491.7, number most significantly overlapping genes in bottom left quadrant = 5,493, number most significantly overlapping genes in top right quadrant = 2,691). (d) Comparison of differential gene expression induced by long-term cocaine withdrawal to differential gene expression induced by cocaine re-exposure revealed strong discordant gene expression relationship across the VTA (i; genes compared in x-axis and y-axis = 13,317, maximum −log10(hypergeometric p-value) = 593.7, number most significantly overlapping genes in top left quadrant = 3,973, number most significantly overlapping genes in bottom right quadrant = 4,333), NAc (ii; genes compared in x-axis and y-axis = 13,232, maximum −log10(hypergeometric p-value) = 495.6, number most significantly overlapping genes in top left quadrant = 4,117, number most significantly overlapping genes in bottom right quadrant = 3,301), and PFC (iii; genes compared in x-axis and y-axis = 13,092, maximum −log10(hypergeometric p-value) = 615.5, number most significantly overlapping genes in top left quadrant = 3,510, number most significantly overlapping genes in bottom right quadrant = 4,641). Together, these results indicated that differential gene expression induced by acute cocaine is re-induced after long-term cocaine withdrawal (in the absence of drug) and reversed by cocaine re-exposure.

Figure 3:. The same genes are induced by acute cocaine, re-induced during withdrawal from self-administration, and reversed by cocaine re-exposure.

(a-b) Schematic of Stratified Rank-Rank Hypergeometric Overlap (RRHO) hotspot quadrants that contain intersecting genes. The results of our Stratified RRHO comparisons indicated that differential gene expression induced by acute cocaine is re-induced after long-term (30 day) cocaine withdrawal (in the absence of drug) and reversed by cocaine re-exposure across the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC). For this to be the case, however, the same genes would need to be present throughout this pattern within each of these brain regions. To determine if the same genes were induced at all three time points, we first extracted the most significantly overlapping genes within each hotspot quadrant of the Stratified RRHO comparisons of acute cocaine and cocaine re-exposure (dark gray), acute cocaine and long-term cocaine withdrawal (medium gray), and cocaine re-exposure and long-term cocaine withdrawal (light gray). We extracted the most significantly overlapping genes by selecting the combination of genes from the x-axis and the y-axis yielding the largest −log10(hypergeometric p-value) within each hotspot quadrant. We next examined the intersection of the most significantly overlapping genes from these three comparisons within the VTA, NAc, and PFC. Within each of these three brain regions, we identified two sets of genes that intersected above chance: (a) genes upregulated by acute cocaine (dark gray), upregulated by withdrawal (medium gray), and reversed/downregulated by cocaine re-exposure (light gray; intersection illustrated in red in venn diagrams below), or (b) genes downregulated by acute cocaine (dark gray), downregulated by withdrawal (medium gray), and reversed/upregulated by cocaine re-exposure (light gray; intersection illustrated in green in venn diagrams below). (c) Venn diagrams (left) denoting the intersection (red) of genes from hotspot quadrants illustrated in panel a, and line graphs (right) denoting the expression of these genes. (d) Venn diagrams (left) denoting the intersection (green) of genes from hotspot quadrants illustrated in panel b and line graphs (right) denoting the expression of these genes. The same pattern of above chance intersection was observed in the NAc (e, f) and PFC (g, h). Thus, the same sets of genes are induced by acute cocaine, re-induced by long-term withdrawal, and reversed by re-exposure across the VTA, NAc, and PFC. Line graphs reported as median log2(fold-change) ± interquartile range. Wilcoxin matched-pairs signed rank test was used to determine significance.

Figure 4:. Enriched biological pathways and predicted upstream regulators of longitudinally regulated gene sets.

(a) Schematic of identified gene set I (red) and gene set II (green). (b-d) Biological pathways from the Reactome Pathway Database (some Reactome titles were shortened for figure presentation, full names are available for all groups in supplementary data) enriched in longitudinal gene set I and longitudinal gene set II in the ventral tegmental area (b; VTA), nucleus accumbens (c; NAc), and prefrontal cortex (d; PFC) were characterized. (e-g) Predicted upstream transcriptional regulators of longitudinal gene set I ands longitudinal gene set II in the VTA, NAc, and PFC were identified using Causal Network Analysis and filtered to include non-pharmacological upstream regulators only. Predicted upstream transcriptional regulators of longitudinal gene set I and longitudinal gene set II were cross-referenced against the differential gene expression lists for acute cocaine, short-term (1 day) cocaine withdrawal, and long-term (30 day) cocaine withdrawal to identify significantly regulated upstream regulators at these time points. Expression of these upstream transcriptional regulators is displayed via volcano plots; the color of the points on these plots denotes whether transcriptional regulators are predicted to act upstream of longitudinal gene set I only, longitudinal gene set II only, or both longitudinal gene set I and II. (e) Expression of predicted upstream regulators induced by acute cocaine in the VTA (i), NAc (ii), and PFC (iii). (f) Expression of predicted upstream regulators induced following short-term withdrawal from cocaine self-administration in the VTA (i), NAc (ii), and PFC (iii). (g) Expression of predicted upstream regulators induced by long-term withdrawal from cocaine self-administration in the VTA (i), NAc (ii), and PFC (iii).