Corticostriatal ensemble dynamics across heroin self-administration to reinstatement

Abstract

Corticostriatal projection neurons from prelimbic medial prefrontal cortex to the nucleus accumbens core critically regulate drug-seeking behaviors, yet the underlying encoding dynamics whereby these neurons contribute to drug seeking remain elusive. Here we use two-photon calcium imaging to visualize the activity of corticostriatal neurons in mice from the onset of heroin use to relapse. We find that the activity of these neurons is highly heterogeneous during heroin self-administration and seeking, with at least 8 distinct neuronal ensembles that display both excitatory and inhibitory encoding dynamics. These neuronal ensembles are particularly apparent during relapse, where excitatory responses are amplified compared to heroin self-administration. Moreover, we find that optogenetic inhibition of corticostriatal projection neurons attenuates heroin seeking regardless of the relapse trigger. Our results reveal the precise corticostriatal activity dynamics underlying drug-seeking behaviors and support a key role for this circuit in mediating relapse to drug seeking.

Figure 1.. PrL→NAcc circuit is necessary for reinstatement of heroin seeking.

(A) Schematic showing head-fixed behavioral apparatus. (B) Behavioral paradigm for intravenous head-fixed heroin self-administration. (C) Grouped data for acquisition of heroin self-administration. Mice learn to discriminate between the active and inactive levers, with greater active lever presses across acquisition (n=19 mice; lever: F_1,36 = 119.7, p<0.001). (D) Grouped data for extinction training. Mice decreased activer lever pressing following a minimum of 10 days of extinction training (n =19 mice; averaged first 3-days of extinction vs last 3 days of extinction: t₁₈=3.39, p=0.003). (E) Viral approach used to optogenetically inhibit PrL neurons that project to the NAcc. (F) Representative images showing eNpHR3.0-eYFP expression and optical fiber placement in the PrL (top), and eNpHR3.0-eYFP expressing fibers in the NAcc (bottom). (G) Optogenetic inhibition of PrL→NAcc neurons prevents cue-, drug- and stress-induced reinstatement of heroin seeking, measured as active lever presses (n= 6–10/group; cue reinstatement: interaction: F_1,17=21.36, p<0.001, group comparisons: ext: p=0.9935, rein: p<0.001; drug reinstatement: interaction: F_1,17=28.70, p<0.001, group comparisons: ext: p=0.9477, rein: p<0.001; stress reinstatement: interaction: F_1,12=21.24, p<0.001 group comparisons: ext: p=0.9965, rein: p<0.001). AC, anterior commissure; Ext, extinction; PrL, IL, infralimbic cortex; prelimbic cortex; NAcc, nucleus accumbens core; ns, not significant; Rein, reinstatement; SA, self-administration. Data are mean ± SEM. **p<0.01, ***p<0.001.

Figure 2.. PrL→NAcc calcium activity is heterogenous across heroin self-administration.

(A) Viral approach used for two-photon calcium imaging of PrL→NAcc neurons. (B) Example field of view (FOV) in cyan and second FOV in magenta separated by 50μm. (C) Example extracted signals of calcium activity of cells in FOV1 during habituation to head-fixed chamber. (D) Head-fixed apparatus used for two-photon imaging and heroin self-administration (SA). (E) Heroin SA data for imaging animals. Imaging days shown in yellow. Mice learned to press the active and not inactive lever (n=14 mice; lever: F_1,26 = 59.45, p<0.001). (F-H) Averaged traces (top) and single-cell heatmaps (bottom) reveal PrL→NAcc activity during early (F; n=987 neurons; 14 mice), middle (G; n = 901 neurons; 14 mice), and late (H; n=851 neurons, 14 mice) SA sessions. Average fluorescence (top) increased in the 3 seconds following lever press compared to a 3 second baseline period (early: t₂₂=24.94, p<0.001; mid: t₂₂=31.47, p<0.001; late: t₂₂=24.77, p<0.001). (I) Pie charts (left) and averaged traces (right) for each phase of heroin SA show excited (orange) and inhibited (blue) neurons with significant area under the receiver operator characteristic (auROC) scores (p<0.05). The proportion of excited neurons varied across day of heroin SA (Χ²₂= 7.721, p=0.021) while the proportion of inhibited neurons were similar between day of heroin SA (Χ²₂= 2.416, p=0.299). FOV, field of view; PrL, prelimbic cortex; NAcc, nucleus accumbens core; ns, not significant; SA, self-administration. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 3.. PrL→NAcc calcium activity is heterogeneous during reinstatement of heroin seeking.

(A-C) Active lever presses during cue- (A), drug- (B), and stress-induced (C) reinstatement tests where active lever presses increased above previous extinction session (n=7–12 mice; cue: t₁₁=5.867, p=0.0001; drug: t₁₀=4.1414, p=0.0013; stress: t₆=4.197, p=0.0057). (D-E) Averaged traces (top) and single-cell heatmaps (bottom) reveal PrL→NAcc neuronal activity during cue- (D; n=350 neurons, 12 mice), drug-(E; n=313 neurons, 11 mice) and stress-induced reinstatement (F; n=160 neurons, 7 mice). (G-I) Pie-charts (top) and averaged traces (bottom) for cue- (G), drug- (H), and stress-induced (I) reinstatement tests show excited (orange) and inhibited (blue) neurons with significant area under the receiver operator characteristic (auROC) scores (p<0.05). The proportion of excited neurons varied with reinstatement test (Χ²₂=12.06, p=0.002), as did the proportion of inhibited neurons (Χ²₂=19.11, p<0.001). PrL, prelimbic cortex; NAcc, nucleus accumbens core; ns, not significant; Rein, reinstatement. Data are mean ± SEM. *p<0.01, ***p<0.001.

Figure 4.. PrL→NAcc neuronal ensembles are stable across trials and differentially encode distinct aspects of heroin seeking.

(A) Spectral clustering reveals4 excited ensembles (total n=658, orange, top half of heat map), and 4 inhibited ensembles (total n=442, blue, bottom half of heatmap) across all imaging sessions (n=8) and all animals (n=14 mice), with distinct activity patterns around the lever press (averaged line graphs, right). (B) Averaged traces for neurons that do not have significant responses (black) and for each ensemble for early trials (first 33% of trials, left) and late trials (last 33% of trials, right) (C) Significantly excited or inhibited neurons exhibit stable auROC scores within session (Pearson-R value=0.361, p<0.001), while (D) neurons that do not have significant responses exhibit negatively correlated auROC scores between early and late trials (Pearson-R value=−0.046, p=0.007). auROC, area under the receiver operator characteristic; PrL, prelimbic cortex, NAcc, nucleus accumbens core ns, not significant. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001