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[Preprint]. 2024 Feb 8:2023.11.02.565378.
doi: 10.1101/2023.11.02.565378.

Differential genetic expression within reward-specific ensembles in mice

Carl G Litif  1 , Levi T Flom  1 , Kathryn L Sandum  1 , Skylar L Hodgins  1 , Lucio Vaccaro  1 , Jerry A Stitzel  2   3 , Nicolas A Blouin  4 , Maria Constanza Mannino  4 , Jason P Gigley  4 , Todd A Schoborg  4 , Ana-Clara Bobadilla  1   5
Affiliations

Differential genetic expression within reward-specific ensembles in mice

Carl G Litif et al. bioRxiv. .

Abstract

Maladaptive reward seeking is a hallmark of cocaine use disorder. To develop therapeutic targets, it is critical to understand the neurobiological changes specific to cocaine-seeking without altering the seeking of natural rewards, e.g., sucrose. The prefrontal cortex (PFC) and the nucleus accumbens core (NAcore) are known regions associated with cocaine- and sucrose-seeking ensembles, i.e., a sparse population of co-activated neurons. Within ensembles, transcriptomic alterations in the PFC and NAcore underlie the learning and persistence of cocaine- and sucrose-seeking behavior. However, transcriptomes exclusively driving cocaine seeking independent from sucrose seeking have not yet been defined using a within-subject approach. Using Ai14:cFos-TRAP2 transgenic mice in a dual cocaine and sucrose self-administration model, we fluorescently sorted (FACS) and characterized (RNAseq) the transcriptomes defining cocaine- and sucrose-seeking ensembles. We found reward- and region-specific transcriptomic changes that will help develop clinically relevant genetic approaches to decrease cocaine-seeking behavior without altering non-drug reward-based positive reinforcement.

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Figures

Figure 1 |
Figure 1 |. Polyreward self-administration of cocaine and sucrose and cell sorting of reward-specific ensembles.
a. Behavioral training timeline for inducing cocaine and sucrose seeking for downstream fluorescently activated cell sorting and transcriptomic characterization of reward-specific ensembles from the nucleus accumbens core (NAcore) and prefrontal cortex (PFC). b. During SA, mice interacted significantly more with the cocaine-seeking and sucrose-reinforced active NP (repeated measures two-way ANOVA for SA Active NP vs. SA Inactive NP over 20 sessions; NP F(1, 84) = 70.85, P<0.0001; session F(5.147, 432.3) = 3.343, P=0.0052; interaction F(19, 1596) = 2.004, P=0.0063). c. During EXT, mice diminished differentiation between the active NP paired with cocaine or sucrose SA (repeated measures two-way ANOVA for EXT Cocaine NP vs. EXT Sucrose NP over 20 sessions; NP F(1, 84) = 1.005, P=0.3190; session F(1.758, 147.7) = 28.98, P<0.0001; interaction F(9, 756) = 4.886, P<0.0001). d. Induction of cocaine-seeking and sucrose-seeking behavior displayed by significantly increased interaction with the active NP in comparison to the inactive NP during both C-RST (paired t-test for C-RST Cocaine NP vs. C-RST Sucrose NP; P<0.0001) and S-RST (paired t-test for S-RST Sucrose NP vs. S-RST Cocaine NP; P<0.0001) in addition to significantly increased interaction with the active NP in comparison to the level of active NP interaction in the last EXT session before C-RST (paired t-test for C-RST Cocaine NP vs. C-EXT Cocaine NP; P<0.0001) or S-RST (paired t-test for S-RST Sucrose NP vs. S-EXT Sucrose NP; P<0.0001). There was no difference when comparing levels of interaction with the active NP between C-RST and S-RST sessions (paired t-test for C-RST Cocaine NP vs. S-RST Sucrose NP; P=0.2399). e. Flow cytometric quadrant plot for negative control absent of antibody for cocaine-seeking and sucrose-seeking ensembles. f,g. Flow cytometric quadrant plots for male (f) and female (g) with percentages of cocaine-seeking neurons (C; bottom-right; tdTomato+/cFos−), sucrose-seeking neurons (S; top left; cFos+/tdTomato−) and overlapping neurons between cocaine-seeking and sucrose-seeking (O; top right: tdTomato+/cFos+) and non-ensemble (bottom-left; tdTomato−/cFos−) “nontag” neurons. Quadrant plots are a representative for male (Figure S2d) and female replicates (Figure S2e).
Figure 2 |
Figure 2 |. Cocaine-seeking and sucrose-seeking ensembles have overlapping and discrete genetic factors within the nucleus accumbens core (NAcore).
a. Schematic of NAcore region for RNA extraction. b. Cell deconvolution for NAcore samples (cocaine-seeking ensemble = C; sucrose-seeking ensemble= S; overlapping ensemble= O; non-ensemble = N) to determine percent of neurons (Rbfox3/NeuN), microglia (Aif1/Iba1), oligodendrocytes (Olig1/Olig1), Schwann (Ngfr/p75ntr), and astrocyte (s100b/S100b) cell types. c. Neuron cell-type deconvolution for NAcore samples to determine percent of dopaminergic (Drd1, Drd2, Foxa2), GABAergic (Gad1, Slc6a1, Gabbr1), glutaminergic (Glul, Grin1, Slc17a7), serotoninergic (Ptprc, Slc6a4, Fev), cholinergic (Ache, Chat) cell types. d. Volcano plots representing reward-specific DEGs scaled by −log10(FDR) (y-axis; factor representing significance) and log2(fold-change) (log2FC; x-axis; factor representing gene expression) within the NAcore. e. Four-way Venn diagrams comparing differentially expressed genes (DEG; in reference to naïve mice) in cocaine-seeking, sucrose-seeking, and overlapping ensembles in addition to the non-ensemble population within the NAcore. Upside down triangles denote the NAcore DEGs defined as cocaine-seeking (red triangle), sucrose-seeking (aqua triangle), or overlapping (purple triangle) for downstream characterization. f. Heatmap of DEGs specific to cocaine-seeking, sucrose-seeking, and overlapping ensembles in the NAcore. Colored triangles denote the NAcore DEGs (from comparison in Figure 2e) used for comparison with log2FC of the same gene in other reward-seeking ensembles. g,h,i. Heatmap for the ten highest and ten lowest expressed DEGs specific to cocaine-seeking (g), sucrose-seeking (h), and overlapping ensembles (i) in the NAcore. Colored triangles denote the NAcore DEGs (from comparison in Figure 2e) used for comparison with log2FC of the same gene in other reward-seeking ensembles.
Figure 3 |
Figure 3 |. Cocaine-seeking and sucrose-seeking ensembles have overlapping and discrete genetic factors within the prefrontal cortex (PFC).
a. Schematic of PFC region for RNA extraction. b. Cell deconvolution for PFC samples (cocaine-seeking ensemble = C; sucrose-seeking ensemble= S; overlapping ensemble= O; non-ensemble = N). b. Cell deconvolution for PFC samples (cocaine-seeking ensemble = C; sucrose-seeking ensemble= S; overlapping ensemble= O; non-ensemble = N) to determine percent of neurons (Rbfox3/NeuN), microglia (Aif1/Iba1), oligodendrocytes (Olig1/Olig1), Schwann (Ngfr/p75ntr), and astrocyte (s100b/S100b) cell types. c. Neuron cell-type deconvolution for PFC samples to determine percent of dopaminergic (Drd1, Drd2, Foxa2), GABAergic (Gad1, Slc6a1, Gabbr1), glutaminergic (Glul, Grin1, Slc17a7), serotoninergic (Ptprc, Slc6a4, Fev), cholinergic (Ache, Chat) cell types. e. Four-way Venn diagrams comparing differentially expressed genes (DEG; in reference to naïve mice) in cocaine-seeking, sucrose-seeking, and overlapping ensembles in addition to the non-ensemble population within the PFC. Upside down diamonds denote the PFC DEGs defined as cocaine-seeking (red diamond), sucrose-seeking (aqua diamond), or overlapping (purple diamond) for downstream characterization. f. Heatmap of DEGs specific to cocaine-seeking, sucrose-seeking, and overlapping ensembles in the PFC. Colored diamonds denote the PFC DEGs (from comparison in Figure 3e) used for comparison with log2FC of the same gene in other reward-seeking ensembles. g,h,i. Heatmap for the ten highest and ten lowest expressed DEGs specific to cocaine-seeking (g), sucrose-seeking (h), and overlapping ensembles (i) in the PFC. Colored diamonds denote the PFC DEGs (from comparison in Figure 3e) used for comparison with log2FC of the same gene in other reward-seeking ensembles.
Figure 4 |
Figure 4 |. Cocaine-seeking and sucrose-seeking ensembles have similar and discrete genetic ontology within the nucleus accumbens core (NAcore).
a. Network representation for KEGG gene set enrichment analysis (g:Profiler) of reward-specific DEGs in the NAcore (from comparison in 2e). Colored circular nodes represent enriched KEGG pathways colored by reward (cocaine-seeking ensembles = red; sucrose-seeking ensembles = aqua; overlapping ensembles = purple; shared by multiple types of reward seeking ensembles = multicolored fractions). Lines connecting circular nodes represent similarity of genes within two KEGG pathways. b. Heatmap of all DEGs annotated in the KEGG “dopaminergic synapse” pathway enriched in cocaine-seeking in the NAcore from Figure 4a with log2FC comparisons of genes in all reward-seeking ensembles (cocaine-seeking ensemble = C; sucrose-seeking ensemble= S; overlapping ensemble= O).
Figure 5 |
Figure 5 |. Cocaine-seeking and sucrose-seeking ensembles have similar and discrete genetic ontology within the prefrontal cortex (PFC).
a. Network representation for KEGG gene set enrichment analysis (g:Profiler) of reward-specific DEGs in the PFC (from comparison in 3e). Colored circular nodes represent enriched KEGG pathways colored by reward (cocaine-seeking ensembles = red; sucrose-seeking ensembles = aqua; overlapping ensembles = purple; shared by multiple types of reward seeking ensembles = multicolored fractions). Lines connecting circular nodes represent similarity of genes within two KEGG pathways. b. Heatmap of all DEGs annotated in the KEGG “cocaine addiction” pathway enriched in cocaine-seeking in the PFC from Figure 5a with log2FC comparisons of genes in all reward-seeking ensembles (cocaine-seeking ensemble = C; sucrose-seeking ensemble= S; overlapping ensemble= O).
Figure 6 |
Figure 6 |. Cocaine-seeking and sucrose-seeking ensembles have a similar and discrete genetic expression in the nucleus accumbens core (NAcore) and prefrontal cortex (PFC).
a. Schematic of NAcore and PFC regions. b,c,d. Venn diagrams to compare reward-specific DEGs between the NAcore and PFC for cocaine-seeking (b), sucrose-seeking (c), and overlapping (d) ensembles. Colored triangles represent reward-specific DEGs from comparison 2e for the NAcore. Colored circles denote the shared NAcore-PFC DEGs in cocaine-seeking (red circle), sucrose-seeking (aqua circle), or overlapping (purple circle) ensembles for downstream characterization. e,f,g,h. Heatmap for DEGs specific to cocaine-seeking (e), sucrose-seeking (f), ten highest and ten lowest expressed DEGs in sucrose-seeking (g), and overlapping ensembles (h) in the PFC. Colored circles denote the reward-specific shared NAcore-PFC DEGs (from comparison in Figure 6b–d) used for comparison with log2FC of the same gene in other reward-seeking ensembles in the NAcore and PFC. i. Network representation for KEGG gene set enrichment analysis (g:Profiler) of reward-specific DEGs shared between the NAcore and PFC (from comparison in Figure 6b–d). Colored circular nodes represent enriched KEGG pathways colored by reward (cocaine-seeking ensembles = red; sucrose-seeking ensembles = aqua; overlapping ensembles = purple; shared by multiple types of reward seeking ensembles = multicolored fractions). Lines connecting circular nodes represent similarity of genes within two KEGG pathways.
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