Activation of differential brain regions after sensing and eating a high-fat diet in mice

Technical Summary: Activation of Differential Brain Regions After Sensing and Eating a High-Fat Diet in Mice

Problem Statement
While the neural mechanisms underlying homeostatic feeding (driven by low energy) are well-characterized, the specific circuits mediating "hedonic feeding" (consumption driven by pleasure/reward) remain less understood. Previous studies have identified brain regions such as the nucleus accumbens (NAc) and ventral tegmental area (VTA) as active during palatable food intake; however, these studies often fail to distinguish whether neural activation is driven by the "desire" (wanting) to obtain the food or the "pleasure" (liking) derived from consuming it. This study aims to dissect these distinct phases by isolating the neural activation caused by sensing a high-fat diet (HFD) without eating it versus the activation caused by the actual consumption of HFD.

Methodology
The researchers utilized male wild-type mice (8–12 weeks old) habituated to a high-fat diet (60% fat) for 10 days to stabilize intake levels. The experimental design involved three groups:

1. Control: No HFD exposure.
2. exHFD (Exposure without Eating): Mice were exposed to HFD placed inside a perforated acrylic enclosure within their home cage. This allowed the mice to perceive the HFD via sight and smell but prevented physical access and consumption. This phase was designed to measure "desire."
3. HFD (Eating): Mice were allowed to consume HFD freely for one hour.

To assess "desire" behavior, the time spent by mice in an interaction zone near the HFD enclosure was recorded using infrared sensors. Following these behavioral tests, brain tissues were processed for:

• c-Fos Immunohistochemistry (IHC): To identify activated neurons (c-Fos-immunoreactive cells) in specific regions, including the mesolimbic pathway (NAc, VTA), amygdala (Basolateral Amygdala [BLA], Central Amygdala [CeA]), and hypothalamus (Paraventricular Nucleus [PVN], Lateral Hypothalamus [LH], Arcuate Nucleus [ARH]).
• In Situ Hybridization Chain Reaction (HCR) combined with c-Fos IHC: To characterize the neurochemical identity of activated cells. Specific probes were used to detect:
  • vGLUT1 mRNA in the BLA (to identify glutamatergic neurons).
  • PENK (proenkephalin) and PDYN (prodynorphin) mRNAs in the NAc (to identify specific subpopulations of striatal neurons).

Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer tests for group comparisons, and paired t-tests for behavioral data.

Key Results

• Behavioral Validation: Mice exposed to HFD without the ability to eat it spent significantly more time in the interaction zone and made more entries into that zone compared to mice exposed to standard food, confirming the induction of a "desire" state.
• Differential Brain Activation:
  • Sensing HFD (Desire Phase): Significant increases in c-Fos-immunoreactive cells were observed in the Basolateral Amygdala (BLA), Paraventricular Nucleus (PVN), and Lateral Hypothalamus (LH) in the exHFD group compared to controls.
  • Eating HFD (Pleasure Phase): Significant increases in c-Fos-immunoreactive cells were observed in the LH, NAc, VTA, and CeA in the HFD group. Notably, the NAc, VTA, and CeA showed activation only after eating, not merely upon exposure.
  • Overlap: The LH was the only region activated by both sensing and eating HFD.
• Cellular Characterization:
  • BLA: Approximately 73% of the c-Fos-positive neurons activated by HFD exposure (without eating) were positive for vGLUT1, indicating that glutamatergic neurons in the BLA are primarily responsible for the "desire" response.
  • NAc: In mice that ate HFD, activated neurons included those expressing PENK (23% of activated cells) and PDYN (14–25% depending on the core/shell region). These markers are associated with specific dopamine receptor pathways (D2R and D1R, respectively).

Key Contributions
The study provides a spatial and temporal resolution of neural circuits involved in hedonic feeding by separating the "desire" and "pleasure" components:

1. Identification of "Desire" Circuits: The study identifies the PVN and glutamatergic neurons in the BLA as key neural substrates activated by the sensory perception of palatable food, independent of consumption.
2. Identification of "Pleasure" Circuits: The study confirms that the NAc, VTA, and CeA are specifically activated by the act of eating palatable food, aligning with the "pleasure" phase of the hedonic reaction.
3. Dual Role of the LH: The Lateral Hypothalamus is shown to be activated during both the anticipation (sensing) and consumption (eating) of HFD, suggesting a continuous role in the hedonic feeding process.
4. Neurochemical Specificity: The use of in situ HCR allowed for the specific identification of glutamatergic neurons in the BLA and enkephalin/dynorphin-expressing neurons in the NAc as the primary activated populations during these distinct phases.

Significance
The authors conclude that the neural mechanisms mediating the desire to obtain palatable food are distinct from those mediating the pleasure of consuming it. Specifically, the activation of PVN neurons and BLA glutamatergic neurons appears to drive the "wanting" or desire to consume HFD, whereas the activation of NAc, VTA, and CeA neurons is associated with the "pleasure" derived from eating. The LH appears to bridge these two processes. These findings offer a refined model of hedonic feeding, suggesting that "desire" and "pleasure" are regulated by discriminable neural mechanisms and brain regions, rather than a single, undifferentiated reward system.