Drugs of abuse hijack a mesolimbic pathway that processes homeostatic need

Abstract

Drugs of abuse are thought to promote addiction in part by "hijacking" brain reward systems, but the underlying mechanisms remain undefined. Using whole-brain FOS mapping and in vivo single-neuron calcium imaging, we found that drugs of abuse augment dopaminoceptive ensemble activity in the nucleus accumbens (NAc) and disorganize overlapping ensemble responses to natural rewards in a cell type-specific manner. Combining FOS-Seq, CRISPR-perturbation, and single-nucleus RNA sequencing, we identified Rheb as a molecular substrate that regulates cell type-specific signal transduction in NAc while enabling drugs to suppress natural reward consumption. Mapping NAc-projecting regions activated by drugs of abuse revealed input-specific effects on natural reward consumption. These findings characterize the dynamic, molecular and circuit basis of a common reward pathway, wherein drugs of abuse interfere with the fulfillment of innate needs.

Fig. 1.. Whole-brain FOS mapping identifies shared and distinct brain regions activated by repeated exposure to cocaine and morphine.

(a), Schematic of the experimental design for repeated exposure to drug rewards vs. saline. Comparisons of (b), Cumulative food intake (g), Cumulative water intake (g), Weight (%) over the 5-day treatment (n = 10, 10, 10 for saline, cocaine, morphine group, respectively, two-way ANOVA with Dunnett’s multiple comparisons). (c), Schematic of the experiment design for repeated exposure to drug rewards vs. saline followed by whole-brain clearing and mapping to Allen Brain Atlas. (d), Heatmap overview of brain areas showing significant FOS induction across three groups (One-way ANOVA for each brain area with cut-off p < 0.05 classified as statistically significant, followed by K-means clustering). (e), Scatter plot of FOS levels in cortical areas in response to cocaine vs. morphine (left). Scatter plot of FOS levels in subcortical areas in response to cocaine vs. morphine (right). Common response: areas showed significant changes (P < 0.05) of FOS+ counts in cocaine and morphine groups compared to the saline group; Cocaine or Morphine specific: areas only showed significant changes of FOS+ counts in either the cocaine or morphine group compared to the saline group. (f), Similarity of FOS responses across different phases of drug exposure (top). Heatmap representations of brain areas after acute or repeated exposure to cocaine or morphine or after spontaneous withdrawal (bottom). All error bars represent mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. An overlapping set of dopaminoceptive neurons exhibit preferential activation in response to drugs of abuse over natural rewards.

(a), Schematic of the experimental design for comparing neuronal responses to natural vs. drug rewards. (b), Venn diagram of D1 MSNs activated by food, water, or cocaine; n = 111 neurons pooled from 3 mice across all sessions recorded. (c), Distribution of average peak responses of D1 MSNs activated by food, water or cocaine. (d), Distribution of preferential activation strength of D1 MSNs between food/water vs. cocaine. (e), Comparison of peak responses of the neurons activated by food/water and cocaine (n = 44 neurons, two-tailed Wilcoxon test). (f), Venn diagram of activated D2 MSNs among food, water, cocaine; n = 46 neurons pooled from 3 mice across all sessions recorded. (g), Distribution of averaged peak responses of the D2 MSNs activated by food, water and cocaine. (h), Distribution of preferential activation strength of D2 MSNs between food/water vs. cocaine. (i), Comparison of the peak responses of the neurons activated by food/water and cocaine (n = 5 neurons, two-tailed Wilcoxon test). (j), Venn diagram of D1 MSNs activated by food, water, or morphine; n = 85 neurons pooled from 3 mice across all sessions recorded. (k), Distribution of average peak responses of the D1 MSNs activated by food, water and morphine. (l), Distribution of preferential activation strength of D1 MSNs between food/water vs. morphine. (m), Comparison of the peak responses of the above neurons activated by food/water and morphine (n = 33 neurons, two-tailed Wilcoxon test). (n), Venn diagram of D2 MSNs activated by food, water, or morphine. n = 170 neurons pooled from 3 mice across all sessions recorded. (o), Distribution of average peak responses of the D2 MSNs activated by food, water and morphine. (p), Distribution of preferential activation strength of D2 MSNs between food/water vs. morphine. (q), Comparison of the peak responses of the neurons activated by food/water and morphine (n = 38 neurons, two-tailed Wilcoxon test). All error bars represent mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. Repeated cocaine or morphine exposure augments the activity of subsets of dopaminoceptive neurons.

(a), Schematic of tensor component analysis (TCA). (b), Cocaine-induced D1 neural state 1 and linear regression of the representation of neural state 1 across all sessions. (c), Cocaine-induced D1 neural state 2 and linear regression of the representation of neural state 2 across all sessions. (d), Loading factors of neurons contributing to state 1 relative to state 2 (n = 119 neurons merged from 3 mice across all 15 sessions). (e), Comparison of D1 MSNs positively contributing to cocaine-induced neural state 1 between session 1 and 5 (n = 46 neurons, two-tailed Wilcoxon test). (f), Cocaine-induced D2 neural state 1 and linear regression of the representation of neural state 1 across all sessions. (g), Cocaine-induced D2 neural state 2 and linear regression of the representation of neural state 2 across sessions. (h), Loading factors of neurons contributing to state 1 relative to state 2 (n = 45 neurons merged from 3 mice across all 15 sessions). (i), Comparison of D2 MSNs positively contributing to cocaine-induced neural state 1 between session 1 and 5 (n = 26 neurons, Wilcoxon). (j), Morphine-induced D1 neural state 1 and linear regression of the representation of neural state 1 across all sessions. (k), Morphine-induced D1 neural state 2 and linear regression of the representation of neural state 2 across all sessions. (l), Loading factors of neurons contributing to state 1 relative to state 2 (n = 95 neurons merged from 3 mice across all 15 sessions). (m), Comparison of D1 MSNs positively contributing to morphine-induced neural state 1 between session 1 and 5 (n = 68 neurons, Wilcoxon). (n), Morphine-induced D2 neural state 1 and linear regression of the representation of neural state 1 across all sessions. (o), Morphine-induced D2 neural state 2 and linear regression of the representation of neural state 2 across all sessions. (p), Loading factors of neurons contributing to state 1 relative to state 2 (n = 155 neurons merged from 3 mice across all 15 sessions). (q), Comparison of D2 MSNs positively contributing to morphine-induced neural state 1 between session 1 and 5 (n = 97 neurons, two-tailed Wilcoxon test). All error bars represent mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Disorganized cell-type-specific responses to natural rewards following cocaine or morphine withdrawal.

(a, b) Representative heatmaps of D1 neuronal responses (top panels) to food prior to cocaine exposure or following cocaine withdrawal (n = 176 matched neurons), and non-negative matrix factorization (NMF) representation of neuronal states labeled by k-means clustering (bottom panels) (c), Percentage of D1 MSNs activated by food or water (n = 914 matched neurons, 13 matched sessions, 3 mice). (d), Variances explained by top 3 principal components (PCs) during food and water consumption (n = 13 matched sessions, 3 mice). (e), D1 responses to food and water (n = 914 matched neurons). (f, g) Representative heatmaps of D2 responses to food prior to cocaine exposure or following cocaine withdrawal (n = 118 matched neurons), and NMF representation of neuronal states. (h), Percentage of D2 MSNs activated by food or water (n = 488 neurons, 12 sessions, 3 mice). (i), Variances explained by top 3 PCs during food and water consumption (n = 12 sessions, 3 mice). (j), D2 responses to food and water pre/post cocaine (n = 488 matched neurons). (k, l), Representative heatmaps of D1 responses to food prior to morphine exposure or following morphine withdrawal (n = matched 52 neurons), and NMF representation of neuronal states. (m), Percentage of D1 MSNs activated by food or water pre/post morphine (n = 587 matched neurons, 10 sessions, 3 mice). (n), Variances explained by top 3 PCs during food and water consumption (n = 10 sessions, 3 mice). (o), D1 responses (n = 587 matched neurons). (p, n), Representative heatmaps of D2 responses to food prior to morphine exposure or following morphine withdrawal (n = 284 matched neurons) (n), and NMF representation of neuronal states. Percentage of D2 MSNs activated by food or water (n = 1174 neurons, 11 sessions, 3 mice). (s), Variances explained by top 3 PCs during food and water consumption (n = 11 sessions from 3 mice). (t), D2 responses to food and water (n = 1174 neurons). The Fisher’s exact test was used to compare the percentages of neurons. The two-tailed paired t-test was used to compare variance ratios. The two-tailed Wilcoxon test was used to compare the neuronal responses. Error bars represent mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.. Rheb regulates cell-type-specific signal transduction and is necessary for the ability of repeated cocaine and morphine exposure to suppress natural reward consumption.

(a), Schematic of the in silico FOS-Seq approach to identify genes associated with brain-wide FOS patterns. Genes with Pearson Correlation Coefficient > 0.15 or < −0.15 and p < 0.05 are classified as positively correlated or negatively correlated genes. Adjusted P-values are FDR-corrected at 5% threshold. (b), Volcano plot of genes associated with repeated exposure to cocaine. (c), Volcano plot of genes associated with repeated exposure to morphine. (d), Scatter plot of Pearson coefficient from genes associated with repeated exposure to cocaine vs. Pearson coefficient from genes associated with repeated exposure to morphine. (e), Schematic of in vivo NAc region-specific knockout of Rheb gene by co-expressing Cre and Rheb-sgRNAs or their control scrambled-sgRNAs in NAc core in LSL-Cas9 transgenic mice. (f), Immunohistochemistry validation of pS6 levels in the NAc from the Rheb knockout (Rheb-KO) group vs. the Control group at baseline (scale bar: 100 μm). (g), Quantification of total pS6 fluorescent intensity in the NAc (n = 6 sections per group, with 2 sections per animal, 3 animals per group. The 2 sections were each chosen from anterior and posterior NAc, with at least 200 μm apart). (h), Schematic of snRNA-seq after CRISPR perturbations (n = 7001 cells mapped with either sgRNA). (i), Distribution of Drd1+ cells in the UMAP. (j), Differentially expressed genes in Rheb-KO cells vs. control cells in the D1-MSNs1 cluster (n = 598, 390 cells respectively). (k), Distribution of Drd2+ cells in the UMAP. (l), Differentially expressed genes in Rheb-KO cells vs. control cells in the D2-MSNs1 cluster (n = 449, 375 cells respectively). (m), Venn diagram of genes significantly regulated by Rheb-KO between D1-MSNs1 and D2-MSNs1 clusters. (n, o), Comparisons of 5-day averaged daily food intake (g), and daily water intake (g), as well as weight (%) after 5-day drug exposure in the Rheb-KO group treated with saline for 5 days followed by another 5-day cocaine or morphine treatment (n = 10, 8 for the Control and Rheb-KO groups, respectively. Two-way ANOVA with Šídák’s multiple comparisons). All error bars represent mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.