The anterior insular cortex unilaterally controls feeding in response to aversive visceral stimuli in mice

The regulation of food intake is crucial for maintaining energy balance and overall health. Under normal conditions, this is primarily controlled by hypothalamic circuits. However, various pathological states, such as infections or exposure to toxins, can significantly reduce food intake, suggesting the existence of additional, emergency response circuits that override homeostatic mechanisms. The anterior insular cortex (aIC) plays a key role in integrating interoceptive states and emotional awareness, guiding behavioral responses accordingly. While the aIC's involvement in processing aversive stimuli is established, its specific role in modulating feeding behavior under these non-homeostatic conditions remains poorly understood. Previous studies have shown links between the insula and anorexigenic signals, and insular stimulation induces visceral sensations like nausea, while damage blunts malaise and blocks conditioned taste aversion. However, a direct role of the aIC in controlling food intake hasn't been definitively established. This study aimed to investigate the role of the aIC, specifically focusing on its influence on feeding responses to aversive visceral signals.

Extensive research points to the insular cortex, particularly the anterior insula (aIC), as a central region for processing salient stimuli and coordinating behavioral responses. The aIC receives a substantial amount of sensory input from the thalamus and serves as a primary cortical area for integrating visceral and gustatory information. Neuroimaging studies in humans have linked the aIC to responses to both food cues and noxious stimuli that trigger negative emotions like pain, anxiety, and disgust. Furthermore, research on eating disorders has revealed altered insula activation in conditions like bulimia and anorexia nervosa. Animal studies have confirmed the activation of the insular cortex in response to anorexigenic signals. Despite evidence suggesting insular cortex involvement in aversive signaling, the precise mechanisms governing its effects on feeding behavior have remained elusive. The present study addresses this gap by specifically investigating the role of the aIC in the context of aversive visceral stimuli-induced anorexia.

The study employed a multi-faceted approach combining various techniques to investigate the role of the aIC in feeding regulation. Firstly, immunostaining for Fos was used to assess neuronal activation patterns in response to intraperitoneal injection of anorexigenic agents (LiCl, LPS, Cisplatin) as well as orexigenic agents (ghrelin, CCK). Double immunohistochemical staining was employed to determine the neurochemical phenotype of the activated neurons. Fiber photometry was used for in vivo monitoring of neuronal activity (using GCaMP6f) in the right and left aIC in response to aversive stimuli. Optogenetic techniques (using ChR2 and eArch3.0) were utilized to either activate or inhibit CamKII+ neurons in the right and left aIC, with their effects on feeding behavior assessed in 24-h fasted and fed mice. Chemogenetic techniques (using hM3Dq and hM4Di) were used to achieve long-term activation or inhibition of aICCamKII neurons, examining their effects on feeding and body weight in response to anorexigenic agents (LiCl, LPS) and chemotherapy agent (Cisplatin). Finally, viral tracing (using AAV and rabies virus) was used to map the anatomical projections of aICCamKII neurons and to identify their downstream targets. Electrophysiological recordings in acute brain slices were performed to confirm the effect of optogenetic and chemogenetic manipulations on neuronal activity and to study the synaptic connections between aIC neurons and their targets in the hypothalamus. Behavioral assays included measurement of food intake, number of food approaches, time spent feeding, taste sensitivity, water consumption, elevated plus maze test, open field test, and real-time place preference assays. Statistical analysis involved various tests, including t-tests and ANOVAs, to assess the significance of observed effects.

The study revealed a significant lateralization of function within the aIC. The following key findings were reported: 1. **Aversive visceral stimuli specifically activate CamKII+ neurons in the caudal segment of the right aIC**, as indicated by increased Fos expression and elevated GCaMP6f fluorescence. The left aIC showed no such activation. 2. **Optogenetic activation of right aICCamKII neurons significantly suppressed food intake**, reducing both the appetitive and consummatory phases of feeding. This effect was specific to the caudal segment of the right aIC; activation of neighboring regions (rostral aIC, posterior IC) had no effect on feeding. 3. **Optogenetic inhibition of right aICCamKII neurons significantly increased food intake in fed mice** and induced feeding in satiated mice. This suggests that the right aICCamKII neurons normally exert an inhibitory influence on feeding. 4. **Chemogenetic activation of right aICCamKII neurons (using hM3Dq) caused a rapid and significant reduction in food intake and body weight in 24-h fasted mice**. This effect was long-lasting. 5. **Chemogenetic inhibition of right aICCamKII neurons (using hM4Di) prevented the anorexigenic effects of LiCl and LPS** and reversed Cisplatin-induced weight loss. These effects were not seen with left aIC manipulation. 6. **Viral tracing experiments revealed that right aICCamKII neurons directly project to vGluT2+ neurons in the lateral hypothalamus (LH)**, a brain region critical for regulating feeding. Light stimulation of the aIC terminals in the LH inhibited feeding. 7. **Activation of the right aICCamKII-LH pathway caused place avoidance**, indicating an aversive effect in addition to feeding suppression. 8. No significant effects were observed on other behaviours such as drinking, mating or anxiety-like behaviour when manipulating right or left aIC CamKII+ neurons, suggesting a specific role in feeding regulation. The manipulation of left aIC CamKII+ neurons resulted in no behavioral changes.

The findings of this study demonstrate a previously uncharacterized neural circuit mediating feeding responses to aversive visceral stimuli. The right aIC, specifically the caudal segment, acts as a critical node in this circuit, detecting aversive signals and suppressing food intake via direct projections to the LH. This is significant as it provides a neural mechanism for the anorexia observed in various pathological conditions. The lateralization of this effect to the right aIC is notable and consistent with findings in humans regarding the right hemisphere's dominance in negative emotional processing. This suggests a conserved mechanism across species. Future research should focus on elucidating the specific types of visceral inputs processed by the right aIC, and whether other cortical areas contribute to the regulation of feeding under non-homeostatic conditions. Investigating the downstream effects of LH neurons receiving inputs from the aIC would provide further understanding of the mechanisms underlying this feeding suppression.

This study identified a novel neural pathway from the right anterior insular cortex to the lateral hypothalamus that mediates the suppression of feeding in response to aversive visceral stimuli. This right-lateralized circuit plays a crucial role in regulating food intake during illness or other adverse conditions. The findings highlight the importance of considering laterality when investigating brain circuits and provides a valuable model for studying insular function and dysfunction.

The study primarily used male mice, limiting the generalizability of findings to female mice. The specific types of visceral afferents that trigger the aIC response and the exact nature of the synaptic interactions within the aIC-LH circuit need further investigation. The use of optogenetics and chemogenetics might have introduced some artificiality to neuronal activation patterns and long-term effects. A more comprehensive investigation of the downstream effects of LH activation is needed.