Characterization of excitatory and inhibitory neuron activation in the mouse medial prefrontal cortex following palatable food ingestion and food driven exploratory behavior

Abstract

The medial prefrontal cortex (mPFC) is implicated in aspects of executive function, that include the modulation of attentional and memory processes involved in goal selection. Food-seeking behavior has been shown to involve activation of the mPFC, both during the execution of strategies designed to obtain food and during the consumption of food itself. As these behaviors likely require differential engagement of the prefrontal cortex, we hypothesized that the pattern of neuronal activation would also be behavior dependent. In this study we describe, for the first time, the expression of Fos in different layers and cell types of the infralimbic/dorsal peduncular and prelimbic/anterior cingulate subdivisions of mouse mPFC following both the consumption of palatable food and following exploratory activity of the animal directed at obtaining food reward. While both manipulations led to increases of Fos expression in principal excitatory neurons relative to control, food-directed exploratory activity produced a significantly greater increase in Fos expression than observed in the food intake condition. Consequently, we hypothesized that mPFC interneuron activation would also be differentially engaged by these manipulations. Interestingly, Fos expression patterns differed substantially between treatments and interneuron subtype, illustrating how the differential engagement of subsets of mPFC interneurons depends on the behavioral state. In our experiments, both vasoactive intestinal peptide- and parvalbumin-expressing neurons showed enhanced Fos expression only during the food-dependent exploratory task and not during food intake. Conversely, elevations in arcuate and paraventricular hypothalamic fos expression were only observed following food intake and not following food driven exploration. Our data suggest that select activation of these cell types may be required to support high cognitive demand states such as observed during exploration while being dispensable during the ingestion of freely available food.

Keywords: infralimbic; palatable food seeking and ingestion; parvalbumin; prefrontal cortex; prelimbic; somatostatin; vasoactive intestinal peptide.

FIGURE 1

(A) Consumption of the palatable HFD (elevated fat and sugar content), but not of regular rodent chow is indicative of a hedonic rather than a metabolic drive. Food was offered shortly after light onset for 30 min starting at ZT = 0.5–1.5 h. **p < 0.001 (n = 6/group). (B) Introduction of HFD at ZT = 0.5–1.5 h led to a significant increase in amount of time spent on feeding when offered in an open dish bottom (middle black bar) or on food-driven exploration when introduced inside a closed perforated dish (right bar), whereas mice in the control group spent much less time investigating the empty dish (left bar). *p < 0.05 compared to the control group offered an empty dish bottom (n = 5 per group).

FIGURE 2

Compared to controls (A), hedonic feeding on HFD (B) and, to a greater degree, food-driven exploration (C) increased Fos immunreactivity throughout the medial prefrontal cortex from dorsal to ventral to include the anterior cingulate (AC), prelimbic (PL), infralimbic (IL), and dorsal peduncular (DP) subdivisions. The numbers 5/6, 2/3, and 1 approximate the cortical layers within the photomicrographs taken from the left hemisphere mPFC. The increase in Fos immunreactivity was evident in both deep and superficial layers of these regions (B,C). The coronal diagram in the lower left depicts the corresponding area and includes the dividing line for purpose of analysis of the dorsal and ventral halves. Scale bar in A = 100 μm, and applies to all panels.

FIGURE 3

Many Fos-labeled cell nuclei co-express the nuclear protein special AT-rich sequence-binding protein 2 (SATB2), a marker expressed in principal excitatory neurons but not inhibitory neurons. Photomicrographs show fluorescent labeling for Fos-ir (in A, with Cy3) and for SATB2-ir (in B, with Cy2, as well as merged channels in C) in layers 2–3 of the PL. Double-labeled cells are indicated with yellow arrows, whereas those only labeled for Fos (and thus negative for SATB2) are indicated with red arrows (putative inhibitory neurons). Some of the SATB2-labeled cells lacking c-Fos labeling are indicated with green arrowheads. (D) The number of double-labeled cells (SATB2-positive neurons that show Fos labeling) increased throughout the mPFC in the HFD feeding group, and even more so in the exploratory group in pursuit of the HFD (n = 5/group). *p < 0.05, **p <0.005, differences with respect to the control group. ^#p < 0.05, ^##p < 0.005, differences between feeding and exploring groups. Scale bar in A = 25 μm, and also applies to B,C.

FIGURE 4

Expression of Fos protein in inhibitory interneurons immunostained for parvalbumin (PV; in A,B), somatostatin (SOM; in C,D), and vasoactive intestinal polypeptide (VIP; in E,F) within the infralimbic (IL; in A,C,E) and prelimbic cortex (PL; in B,D,F). Double-labeled cells that show brown cytoplasmic stain and a black nucleus are indicated with black arrows. Single-labeled PV+ cells that lack a black nuclear staining are indicated with white arrows (A,B). Scale bar in A = 25 μm, and applies to all panels.

FIGURE 5

Fos expression in inhibitory interneurons and differences among the experimental groups vary among the three major non-overlapping subpopulations in the medial PFC (PV in A,B, SOM in C,D, VIP in E,F). A,C,E depict the number of double-labeled cells counted, whereas B,D,F show the percentage of the interneuron subpopulations that showed Fos-ir. A,B: HFD exposure led to an increase in Fos-ir in PV neurons most of which were in the deep layers of the DP/IL and PL/AC, and this increase was robust only in the food-driven exploration group. C,D: SOM-labeled neurons in layers 1–3 of the PL/AC showed comparable increases in Fos-ir among the HFD feeding and exploring groups, but no differences between groups were apparent in the other parts of the mPFC, where about half of all SOM-labeled cells were Fos-positive. E,F: Among the VIP neurons, a robust increase in Fos-ir occurred in the food-driven exploration group, whereas HFD feeding led to a small but insignificant rise. *p < 0.05, **p < 0.005, differences with respect to the control group;^#p < 0.05, ^##p < 0.005, differences between feeding and exploring groups.

FIGURE 6

HFD consumption, but not food-driven exploration, substantially increases Fos in the paraventricular nucleus (PVN) and the arcuate nucleus (Arc) of the hypothalamus when compared to the control group (exposed to empty petri dish). Representative photomicrographs are shown of the PVN (A–C) and the Arc (E–G) from control (A,E), HFD fed (B,F) and animals following food driven exploratory behavior (C,G). HFD feeding led to a significant increase in Fos in the PVN (D, **p < 0.05) and the Arc (H, *p < 0.05). No change in fos expression was observed in the PVN of the food driven exploratory behavior group (D, dish [HFD]) while a small, yet statistically insignificant increase was noticeable in the Arc (H, +). Scale bars in A,E = 100 μm and also applies to panels B,C and F,G.