Incubation of palatable food craving is associated with brain-wide neuronal activation in mice

Abstract

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed "incubation of craving." Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

Keywords: Fos; addiction; incubation; mice; whole-brain analysis.

Fig. 1.

Incubation of palatable food seeking in male CD1 mice. (A) Experimental overview. (B) Timeline of food self-administration training, abstinence, and relapse tests. (C) Food self-administration training. Mice learned to self-administer palatable food pellets over seven sessions. Mean (± SEM) number of active and inactive lever presses (Left), food pellets earned (Middle Left), timeout presses (Middle Right), and food-port entries (Right) during each 1-h session. *Significant difference (P < 0.05) between active and inactive lever presses (n = 46). (D) Relapse (incubation) test. Responding on active but not inactive lever progressively increased during abstinence. Mean (± SEM) number of active and inactive lever presses (Left), CS presentations (Middle Left), timeout presses (Middle Right), and food-port entries (Right) during the 30-min relapse test session. *Significant difference (P < 0.05) from day 1. (E) Time course of relapse behavior. Binned 10-min time course of active and inactive lever presses (Left), CS presentations (Middle Left), timeout presses (Middle Right), and food-port entries (Right). *Significant difference (P < 0.05) from day 1. ^#Significant difference (P < 0.05) from day 15. See Dataset S1 for a detailed listing of all statistical outputs relating to this figure.

Fig. 2.

Unbiased brain-wide activity mapping of incubated relapse to palatable food seeking using ClearMap. (A) Mean (± SEM) Fos+ cell counts across the whole brain and for 10 major anatomical subdivisions. (B) Brain-wide changes in activation during abstinence. Raw Fos+ cell counts for each region are z-score-normalized to the homecage group distribution for statistical analysis. Mean z-scored cell counts for 10 major anatomical subdivisions showing time-dependent changes in activation pattern in multiple brain regions induced by the relapse tests (Left). *Significant differences (FDR-adjusted P < 0.1) one-way ANOVA. Heat map of individual z-scored Fos+ cell counts, one-way ANOVA FDR-adjusted P values, and Tukey’s HSD pairwise group comparison P values for 56 subregions across the analyzed brain volume (Right). Individual data are sorted by group and ranked in descending order of activation level within each group. Subregions are organized by 10 parent anatomical subdivisions and ranked in descending order of mean day-60 group activation level. See Dataset S2 for a detailed listing of all statistical results associated with this figure.

Fig. 3.

Targeted analysis of corticostriatal coronal subvolume (AP +1.55 to AP +1.75 relative to Bregma) using SMART2 (A) Spatial map of Fos+ cell density. Grayscale intensity of individual points (20- × 20- × 200-μm voxel) represents mean cell density in cells per mm³ within AP +1.55 to AP +1.75 coronal subvolume. (B, Left) Fos+ cell counts for five major anatomical regions within the subvolume. (B, Right) Z-score-normalized counts (normalized to homecage group distribution) for five major anatomical regions within the subvolume. *Significant differences (FDR-adjusted P < 0.1) one-way ANOVAs. (C) Changes in activation across the subvolume during abstinence. Fos+ cell counts for each region are z-score-normalized to homecage group distribution for statistical analysis. Heat map of individual z-scored Fos+ cell counts (Left), one-way ANOVA FDR-adjusted P values (Middle), and Tukey’s HSD pairwise group comparisons P values (Right) for 18 subregions within the analyzed coronal subvolume. Subregions are organized by five parent anatomical subdivisions and ranked in descending order of mean day-60 group activation level. (D) Layer- and subdivision-specific changes in activation. Heat map of one-way ANOVA FDR-adjusted P values and Tukey’s HSD pairwise group comparison P values for selected subregion layers or subdivisions within isocortex (Left) or olfactory areas (Right). See Dataset S3 for a detailed listing of all statistical results associated with this figure.

Fig. 4.

Targeted analysis of thalamocortical coronal subvolume (AP −1.08 to AP −1.28 relative to Bregma) using SMART2. (A) Spatial map of Fos+ cell density. Grayscale intensity of individual points (20- × 20- × 200-μm voxel) represents mean cell density in cells per mm³ within AP −1.08 to AP −1.28 coronal subvolume. (B, Left) Fos+ cell counts for eight major anatomical regions within the subvolume. (B, Right) Z-score-normalized counts (normalized to homecage group distribution) for five major anatomical regions within the subvolume. *Significant differences (FDR-adjusted P < 0.1) one-way ANOVAs. (C) Changes in activation across the subvolume. Fos+ cell counts for each region are z-score-normalized to homecage group distribution for statistical analysis. Heat map of individual z-scored Fos+ cell counts (Left), one-way ANOVA FDR-adjusted P values (Middle), and Tukey’s HSD pairwise group comparisons P values (Right) for 28 subregions within the analyzed coronal subvolume. Subregions are organized by eight parent anatomical subdivisions and ranked in descending order of mean day 60 group activation level. (D) Layer- and subdivision-specific changes in activation. Heat map of one-way ANOVA FDR-adjusted P values and Tukey’s HSD pairwise group comparison P values for selected subregion layers or subdivisions within cortical subplate (Left) or striatum (Right). See Dataset S4 for a detailed listing of all statistical results related to this figure.