Comparison of prefrontal cortex sucrose seeking ensembles engaged in multiple seeking sessions: Context is key

Abstract

Encoding of memories, including those associated with prior drug or reward, is thought to take place within distinct populations of neurons, termed ensembles. Neuronal ensembles for drug- and reward-seeking have been identified in regions of the medial prefrontal cortex, but much of our understanding of these ensembles is based on experiments that take place in a single reward-associated environment and measure ensemble encoding over short durations of time. In contrast, reward seeking behavior is evident across different reward-associated environments and persists over time. Using TetTag mice and Fos immunohistochemistry, we examined the relationship between persistent sucrose-seeking and ensemble encoding in mice that undergo seeking sessions in the same or different sucrose self-administration contexts 2 weeks apart. We found that prelimbic (PrL) and anterior cingulate cortex ensembles tagged in the first seeking session were highly sensitive to the context in which a second seeking session took place: reactivation of these ensembles was reduced in the same context but elevated in a distinct sucrose self-administration context. Correlational analyses revealed that ensemble reactivation in the PrL was proportional to the persistence of sucrose seeking behavior across sessions in differing ways in female mice. In the same context, reactivation was proportional to the persistence of non-reinforced operant responses, whereas in a distinct context, reactivation was proportional to the persistence of non-reinforced head entries into the sucrose receptacle. This study underlines the importance of the medial prefrontal cortex importance in maintaining a reward-seeking ensemble over time and identifies context-dependent changes in behavioral correlates of ensemble reactivation.

Keywords: infralimbic cortex; natural reward; prelimbic cortex; self-administration; sex differences.

Figure 1:. Immunohistochemistry and image analysis allows for unbiased quantification of ensembles across sucrose seeking sessions.

(A) EGFP and (B) c-Fos immunoreactivity were detected via confocal microscopy. (C) Image shows EGFP and c-Fos channels merged. Scale bar is 100 um. (D-F) Unbiased identification of EGFP⁺ (D) and c-Fos⁺ (E) nuclei using spot rendering (Imaris). EGFP⁺ nuclei are shown in green, and Fos⁺ nuclei are shown in magenta. (F) Isolation of double-labeled spots for quantification, shown in blue. Green and magenta arrows indicate EGFP⁺ and c-Fos⁺ nuclei, respectively. Blue arrows indicate double-labeled nuclei (G) Visual representation for two outcome measures used in this study, % EGFP reactivation and % c-Fos co-allocation.

Figure 2:. Timeline of experiment and behavioral data for sucrose self-administration training and seeking sessions (Experiment 1: single context).

(A) Experimental timeline (see methods for details). (B) Control and experimental mice had similar response patterns in the initial seven days of sucrose self-administration (SA). (C) In the final three days of self-administration, there was consistent preference for the active operandum, but no difference between control and experimental mice. (D) Active responses were similar between experimental groups on days 7 and 20/21 of abstinence. n=6/control, 16/experimental, analyzed by 3-factor (experimental group, session, operandum) mixed model ANOVA.

Figure 3:. Ensemble reactivation across seeking sessions is lower in experimental than control in PrL, ACC and OFC.

(A) Diagram depicting regions of interest in the mouse mPFC. Coronal section at approximately bregma 1.9mm. (B-D, left) EGFP⁺ and c-Fos⁺ cell counts showed no difference between experimental and control groups in ACC, PrL or OFC. (B-D) Left: %EGFP reactivation shows lower ensemble reactivation in experimental than control animals in PrL(*p=0.011), ACC (*p=0.013), and OFC (p=0.060). Right: Average % c-Fos co-allocation shows no difference between groups. Image in panel A modified from (Paxinos & Franklin, 2001). n=6/control, 16/experimental, analyzed by two-tailed t-tests. Bars represent mean ± SEM. *p≤0.05.

Figure 4:. Persistence of active lever presses or nose pokes across seeking sessions correlates with increased ensemble reactivation and decreased c-Fos co-allocation in some mPFC regions.

(A) In PrL, linear regression of individual active persistence ratios and the percentage of ensemble reactivation across contexts shows a near-significant significant positive relationship in females. (B) In OFC, linear regression shows a significant negative relationship between the active response persistence ratio and the percent of c-Fos⁺ cells that are co-allocated to both seeking days. An opposite though non-significant trend is seen in males. n=8/sex, analyzed by Pearson correlation. M: male, F: female.

Figure 5.. Timeline of experiment and behavioral data for sucrose self-administration training and seeking sessions (Experiment 2: two contexts).

(A) Experimental timeline. (B-C) Left: Control and experimental mice had similar response patterns in the first seven days of sucrose SA training in context 1 or 2 respectively. Right: In the final three days of SA, consistent preference for the active operandum was observed similarly in both control and experimental groups. (D) Active responses during sucrose seeking sessions on day 7 and day 20/21 of abstinence were similar between control and experimental groups. n=10/control, 26/experimental, analyzed by 3-factor (experimental group, session, operandum) mixed model ANOVA. Bars represent mean ± SEM. *p≤0.05

Figure 6:. Ensemble size and reactivity across contexts differs in experimental vs control groups throughout mPFC and dHC.

(A) Diagram showing regions of interest in the mPFC and dHC. (B-E) Left: Population-wide cell counts of EGFP⁺ and c-Fos⁺ cells in mPFC regions show no difference in cell counts between experimental and control groups in any mPFC region. Right: Average percent of EGFP+ cells double-labeled as c-Fos⁺ showed higher ensemble reactivation in ACC and PrL. Average percent of c-Fos⁺ cells double-labeled with EGFP shows higher c-Fos co-allocation in PrL and OFC.(G-I) Left: Population averages of EGFP+ cell counts in dHC regions show no difference between number of active cells on day 7 seeking between experimental and control groups. Average c-Fos⁺ cell count is higher in each region in the experimental group. Right: Average percent EGFP reactivation is higher in CA3 in experimental group vs control. Average percent c-Fos co-allocation is lower in CA1 and CA3 in experimental vs control group. Image in panel A modified from (Paxinos & Franklin, 2001). n=10/control, 26/experimental, analyzed by two-tailed t-tests. Bars represent mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001

Figure 7:. Head-entries into food port correlate with number of active cells during dual-context sucrose seeking.

(A) Linear regression shows head-entries during day 7 seeking session correlate positively with the number of EGFP⁺ cells in the ACC in combined-sexes group. (B-C) Linear regression shows positive correlations between day 21 head entries and number of c-Fos⁺ cells in IL in males and combined-sexes group (B) and in CA3 in females (C). n=18/male, 16 female (ACC, control and experimental groups combined); n=9-12/male, 9-10/female (IL, CA3 numbers differ due to tissue loss/damage), analyzed by Pearson correlation. M: male, F: female. †p≤0.08, *p≤0.05, **p≤0.01.

Figure 8:. Head-entry persistence across sucrose seeking in dual contexts correlates positively with ensemble reactivation in mPFC and dHC regions.

(A) Linear regression shows head-entry persistence correlates positively with ensemble reactivation in the PrL, (B) IL, and (C) in CA3 in combined-sexes group, with similar trends in females alone. Head-entry persistence was calculated by dividing an individual’s head-entries into the food port during day 21 seeking by those on day 7 seeking. n=12/male, 10/female, analyzed by Pearson correlation. M: male, F: female. †p≤0.08, *p≤0.05.

Figure 9:. Summary of reactivation and co-allocation correlations with persistence ratios calculated from active operant responses (cue reactivity) or head entries into the sucrose receptacle (goal-directed seeking).

Analyzed by Pearson or Spearman correlation. M: male, F: female, ρ: Spearman rho, ☨p≤0.08, *p≤0.05.