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ObjectiveThe sensory detection of food and food cues suppresses Agouti related peptide (AgRP) neuronal activity prior to consumption with greatest suppression in response to high caloric food or energy need. Although external sensory cues regulate AgRP neuronal activity, the interoceptive mechanisms priming an appropriate AgRP neural response to sensory information of caloric 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.MethodsWe 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.ResultsThe 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.ConclusionsOur 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.

An environment can have a powerful influence over appetite and feeding behaviour. For example, an environmental context, which reliably predicts food, will increase the appetitive food drive to the same environment context. Interestingly, mice are required to be hungry to develop such a context-induced feeding (CIF) response, suggesting the neural circuits sensitive to hunger play an important role to associate an internal energy state with a particular environment context. Hunger-sensing Agouti related peptide (AgRP) neurons are activated by circulating signals of energy deficit and reset to a silenced state by gut feedback mechanisms following food consumption. We hypothesised that AgRP neurons are both necessary and sufficient to drive CIF in the absence of hunger. While fasting increased CIF, chemogenetic inhibition of AgRP neurons during context acquisition prevented this effect. Intriguingly, chemogenetic activation of AgRP neurons during context acquisition did not increase CIF, suggesting precise temporal firing properties may be required. Indeed, photostimulation of AgRP neurons, only during context exposure (ON-OFF in context), increased CIF. Moreover, AgRP photostimulation prior to context exposure, coupled with the termination of photostimulation in the context in the absence of food consumption, was sufficient to drive a subsequent CIF. Our results suggest that AgRP neurons regulate the acquisition of CIF when the temporal firing properties are matched to context exposure. These results further highlight that acute AgRP inhibition is a salient neural event underscoring the effect of hunger on associative learning.

Growing evidence highlights a complex interaction between olfaction and metabolism with impaired olfactory function observed in obesity and increased olfactory sensitivity during hunger. The mechanisms linking metabolic state and olfaction remain unknown, but increased accessibility of hormones, such as ghrelin, and the diverse expression of hormone receptors such as those for ghrelin (GHSRs) in the olfactory system suggests an underappreciated neuroendocrine role. Here, we examined the hypothesis that GHSRs in the olfactory bulb (OB) link hunger with olfactory sensitivity to influence foraging behaviours and metabolism. Selective deletion of OBGHSRsin adult male mice was achieved with adeno-associated viral expression of cre-recombinase in the OB of floxed-Ghsrmice. OBGHSRdeletion significantly affected olfactory discrimination and habituation to both food and pheromone odours, with greatest effect under fasted conditions. Anxiety-like and depression-like behaviour was significantly greater after OBGHSRdeletion using 3 independent anxiety behavioural tasks and testing for anhedonia, whereas exploratory behaviour was reduced. No effect on spatial navigation and memory was observed. Although OBGHSRdeletion did not affect cumulative food intake, it significantly impacted feeding behaviour as evidenced by altered bout number and duration. Moreover, food-finding after fasting or ip ghrelin was attenuated. Intriguingly, OBGHSRdeletion caused an increase in body weight and fat mass, spared fat utilisation on a chow diet and impaired glucose metabolism indicating metabolic dysfunction. We conclude that OBGHSRsmaintain olfactory sensitivity, particularly during hunger, and facilitate behavioural adaptations that optimise food-seeking in anxiogenic environments, priming metabolic pathways in preparation for food consumption.

Anorexia nervosa (AN) has among the highest mortality rates of any psychiatric disorder and is characterised by cognitive inflexibility that persists after weight recovery and contributes to the low rates of recovery. What remains unknown is whether cognitive inflexibility predisposes individuals to AN, a question that is difficult to determine from human studies. Our previous work using the most well-established animal model of AN, 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 optimise the first fully-automated and experimenter-free touchscreen cognitive testing system for rats (n=20) 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 (n=60). Firstly, 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 does not predispose rats to pathological weight loss in ABA but instead that rats subsequently susceptible to weight loss performed better on the reversal learning task. Intriguingly, we show reciprocal links between ABA exposure and cognitive flexibility, with ABA exposed (but weight recovered) rats performing much worse that ABA naïve rats on the reversal learning task. 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 behavioural 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 a robust target for future studies using the ABA model to investigate potential novel pharmacotherapies for AN.

The ventromedial hypothalamic (VMH) nucleus is a well-established hub for energy and glucose homeostasis. In particular, VMH neurons are thought to be important for initiating the counterregulatory response to hypoglycemia, and ex vivo electrophysiology and immunohistochemistry data indicate a clear role for VMH neurons in sensing glucose concentration. However, the temporal response of VMH neurons to physiologically relevant changes in glucose availability in vivo has been hampered by a lack of available tools for measuring neuronal activity over time. Since the majority of neurons within the VMH are glutamatergic and can be targeted using the vesicular glutamate transporter Vglut2, we expressed cre-dependent GCaMP7s in Vglut2 cre mice and examined the response profile of VMH to intraperitoneal injections of glucose, insulin, and 2-deoxyglucose (2DG). We show that reduced available glucose via insulin-induced hypoglycemia and 2DG-induced glucoprivation, but not hyperglycemia induced by glucose injection, inhibits VMH Vglut2 neuronal population activity in vivo. Surprisingly, this inhibition was maintained for at least 45 minutes despite prolonged hypoglycemia and initiation of a counterregulatory response. Thus, although VMH stimulation, via pharmacological, electrical, or optogenetic approaches, is sufficient to drive a counterregulatory response, our data suggest VMH Vglut2 neurons are not the main drivers required to do so, since VMH Vglut2 neuronal population activity remains suppressed during hypoglycemia and glucoprivation.

A greater understanding of how the brain controls appetite is fundamental to developing new approaches for treating diseases characterized by dysfunctional feeding behavior, such as obesity and anorexia nervosa.By modeling neural network dynamics related to homeostatic state and body mass index, we identified a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans (n = 53). We then assessed the physiological role and dissected the function of this mPFC-LH circuit in mice.In vivo recordings of population calcium activity revealed that this glutamatergic mPFC-LH pathway is activated in response to acute stressors and inhibited during food consumption, suggesting a role in stress-related control over food intake. Consistent with this role, inhibition of this circuit increased feeding and sucrose seeking during mild stressors, but not under nonstressful conditions. Finally, chemogenetic or optogenetic activation of the mPFC-LH pathway is sufficient to suppress food intake and sucrose seeking in mice.These studies identify a glutamatergic mPFC-LH circuit as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake.

Hunger increases the motivation of an organism to seek out and consume highly palatable energy dense foods. While hunger-sensing Agouti-related peptide (AgRP) neurons influence this process, whether metabolic detection of homeostatic state via metabolic sensing in AgRP neurons potentiates motivation through the midbrain dopamine system is unexplored. Here, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat), a metabolic enzyme regulating glucose and fatty acid oxidation, as a model of impaired metabolic-sensing in AgRP neurons. We then tested the hypothesis that appropriate metabolic-sensing in AgRP neurons is required to increase food reward motivation by modulating accumbal or striatal dopamine release. Electrophysiological studies confirm that Crat deletion in AgRP neurons (KO) impairs normal ex vivo glucose-sensing, and in vivo photometry experiments show that AgRP neurons in KO mice do not exhibit normal responses to repeated palatable food presentation and consumption, highlighting that this model is appropriate to test the hypothesis. Fiber photometry experiments, using the dopamine sensor GRAB-DA, revealed that impaired metabolic-sensing reduces acute dopamine release (seconds) in the nucleus accumbens, but not the dorsal striatum, to palatable food consumption and during operant responding. Positron electron tomography (PET) methods indicated that impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ∼30 minutes in the dorsal striatum but not the ventral striatum, suggesting a role for AgRP neurons to restrict a long term post-ingestive dopamine response in the dorsal striatum. Finally, impaired metabolic-sensing in AgRP neurons suppresses motivated operant responding for sucrose rewards. Notably, these behavioural effects are potentiated in the hungry state and therefore highlight that metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.