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Objective: Although the metabolic state of an organism affects olfactory function, the precise mechanisms and their impact on behavior and metabolism remain unknown. Here, we assess whether ghrelin receptors (GHSRs) in the olfactory bulb (OB) increase olfactory function and influence foraging behaviors and metabolism. Methods: We performed a detailed behavioural and metabolic analysis in mice lacking GHSRs in the OB (OBGHSR deletion). We also analsyed OB scRNA-seq and spatial transcriptomic datasets to assess GHSR+ cells in the main and accessory olfactory bulbs, as well as the anterior olfactory nucleus. Results: OBGHSR deletion affected olfactory discrimination and habituation to both food and non-food odors. Anxiety-like and depression-like behaviors were significantly greater after OBGHSR deletion, whereas exploratory behavior was reduced, with the greatest effect under fasted conditions. OBGHSR deletion impacted feeding behavior as evidenced by altered bout number and duration, as well as buried food-seeking. OBGHSR deletion increased body weight and fat mass, spared fat utilisation on a chow diet and impaired glucose metabolism indicating metabolic dysfunction. Cross referenced analysis of OB scRNA-seq and spatial transcriptomic datasets revealed GHSR+ glutamate neurons in the main and accessory olfactory bulbs, as well as the anterior olfactory nucleus. Ablation of glutamate neurons in the OB reduced ghrelin-induced food finding and phenocopied results seen after OBGHSR deletion. Conclusions: OBGHSRs help to maintain olfactory function, particularly during hunger, and facilitate behavioral adaptations that optimise food-seeking in anxiogenic environments, priming metabolic pathways in preparation for food consumption.

Objective: The sensory detection of food and food cues suppresses Agouti related peptide (AgRP) neuronal activity prior to consumption with greatest suppression occurring in response to highly caloric food or interoceptive energy need. However, the interoceptive mechanisms priming an appropriate AgRP neural response to external sensory information of food availability remain unexplored. Since hunger increases plasma ghrelin, we hypothesized that ghrelin receptor (GHSR) signalling on AgRP neurons is a key interoceptive mechanism integrating energy need with external sensory cues predicting caloric availability. Methods: We used in vivo photometry to measure the effects of ghrelin administration or fasting on AgRP neural activity with GCaMP6s and dopamine release in the nucleus accumbens with GRAB-DA in mice lacking ghrelin receptors in AgRP neurons. Results: The deletion of GHSR on AgRP neurons prevented ghrelin-induced food intake, motivation and AgRP activity. The presentation of food (peanut butter pellet) or a wooden dowel suppressed AgRP activity in fasted WT but not mice lacking GHSRs in AgRP neurons. Similarly, peanut butter and a wooden dowel increased dopamine release in the nucleus accumbens after ip ghrelin injection in WT but not mice lacking GHSRs in AgRP neurons. No difference in dopamine release was observed in fasted mice. Finally, ip ghrelin administration did not directly increase dopamine neural activity in the ventral tegmental area. Conclusions: Our results suggest that AgRP GHSRs integrate an interoceptive state of energy need with external sensory information to produce an optimal change in AgRP neural activity. Thus, ghrelin signalling on AgRP neurons is more than just a feedback signal to increase AgRP activity during hunger.

Objective: An environmental context, which reliably predicts food availability, can increase the appetitive food drive within the same environment context. However, hunger is required for the development of such a context-induced feeding (CIF) response, suggesting the neural circuits sensitive to hunger link an internal energy state with a particular environment context. Since Agouti related peptide (AgRP) neurons are activated by energy deficit, we hypothesised that AgRP neurons are both necessary and sufficient to drive CIF. Methods: To examine the role of AgRP neurons in the CIF process, we used fibre photometry with GCaMP7f, chemogenetic activation of AgRP neurons, as well as optogenetic control of AgRP neurons to facilitate acute temporal control not permitted with chemogenetics. Results: A CIF response at test was only observed when mice were fasted during context training and AgRP population activity at test showed an attenuated inhibitory response to food, suggesting increased food-seeking and/or decreased satiety signalling drives the increased feeding response at test. Intriguingly, chemogenetic activation of AgRP neurons during context training did not increase CIF, suggesting precise temporal firing properties may be required. Indeed, termination of AgRP neuronal photostimulation during context training (ON–OFF in context), in the presence or absence of food, increased CIF. Moreover, photoinhibition of AgRP neurons during context training in fasted mice was sufficient to drive a subsequent CIF in the absence of food. Conclusions: Our results suggest that AgRP neurons regulate the acquisition of CIF when the acute inhibition of AgRP activity is temporally matched to context exposure. These results establish acute AgRP inhibition as a salient neural event underscoring the effect of hunger on associative learning.

Anorexia nervosa has among the highest mortality rates of any psychiatric disorder and is characterized by cognitive inflexibility that persists after weight recovery and contributes to the chronic nature of the condition. What remains unknown is whether cognitive inflexibility predisposes individuals to anorexia nervosa, a question that is difficult to address in human studies. Our previous work using the most well-established animal model of anorexia nervosa, known as activity-based anorexia (ABA) identified a neurobiological link between cognitive inflexibility and susceptibility to pathological weight loss in female rats. However, testing flexible learning prior to exposure to ABA in the same animals has been thus far impossible due to the length of training required and the necessity of daily handling, which can itself influence the development of ABA. Here, we describe experiments that validate and optimize the first fully-automated and experimenter-free touchscreen cognitive testing system for rats and use this novel system to examine the reciprocal links between reversal learning (an assay of cognitive flexibility) and weight loss in the ABA model. First, we show substantially reduced testing time and increased throughput compared to conventional touchscreen testing methods because animals engage in test sessions at their own direction and can complete multiple sessions per day without experimenter involvement. We also show that, contrary to expectations, cognitive inflexibility measured by this reversal learning task does not predispose rats to pathological weight loss in ABA. Instead, rats that were predisposed to weight loss in ABA were more quickly able to learn this reversal task prior to ABA exposure. Intriguingly, we show reciprocal links between ABA exposure and cognitive flexibility, with ABA-exposed (but weight-recovered) rats performing much worse than ABA naïve rats on the reversal learning task, an impairment that did not occur to the same extent in rats exposed to food restriction conditions alone. On the other hand, animals that had been trained on reversal learning were better able to resist weight loss upon subsequent exposure to the ABA model. We also uncovered some stable behavioral differences between ABA susceptible versus resistant rats during touchscreen test sessions using machine learning tools that highlight possible predictors of anorectic phenotypes. These findings shed new light on the relationship between cognitive inflexibility and pathological weight loss and provide targets for future studies using the ABA model to investigate potential novel pharmacotherapies for anorexia nervosa.

Agouti-related peptide (AgRP) neurons increase motivation for food, however, whether metabolic sensing of homeostatic state in AgRP neurons potentiates motivation by interacting with dopamine reward systems is unexplored. As a model of impaired metabolic-sensing, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat) in mice. We hypothesised that metabolic sensing in AgRP neurons is required to increase motivation for food reward by modulating accumbal or striatal dopamine release. Studies confirmed that Crat deletion in AgRP neurons (KO) impaired ex vivo glucose-sensing, as well as in vivo responses to peripheral glucose injection or repeated palatable food presentation and consumption. Impaired metabolic-sensing in AgRP neurons reduced acute dopamine release (seconds) to palatable food consumption and during operant responding, as assessed by GRAB-DA photometry in the nucleus accumbens, but not the dorsal striatum. Impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ~30 min in the dorsal striatum but not the nucleus accumbens. Impaired metabolic sensing in AgRP neurons suppressed motivated operant responding for sucrose rewards during fasting. Thus, metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.