Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson’s Dementia in Humans

Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl transferase-null (GOAT/) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients, and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression remained unchanged; however, GOAT+ cell number was reduced in PDD. We identify UAG as a regulator of hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic biomarker of dementia

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AbstractDiet-induced obesity (DIO) causes ghrelin resistance in hypothalamic Agouti-related peptide (AgRP) neurons. However, ghrelin promotes feeding through actions at both the hypothalamus and mesolimbic dopamine reward pathways. Therefore, we hypothesized that DIO would also establish ghrelin resistance in the ventral tegmental area (VTA), a major site of dopaminergic cell bodies important in reward processing. We observed reduced sucrose and saccharin consumption in Ghrelin KO vs Ghrelin WT mice. Moreover, DIO reduced saccharin consumption relative to chow-fed controls. These data suggest that the deletion of ghrelin and high fat diet both cause anhedonia. To assess if these are causally related, we tested whether DIO caused ghrelin resistance in a classic model of drug reward, conditioned place preference (CPP). Chow or high fat diet (HFD) mice were conditioned with ghrelin (1mg/kg in 10ml/kg ip) in the presence or absence of food in the conditioning chamber. We observed a CPP to ghrelin in chow-fed mice but not in HFD-fed mice. HFD-fed mice still showed a CPP for cocaine (20mg/kg), indicating that they maintained the ability to develop conditioned behaviour. The absence of food availability during ghrelin conditioning sessions induced a conditioned place aversion, an effect that was still present in both chow and HFD mice. Bilateral intra-VTA ghrelin injection (0.33μg/μl in 0.5μl) robustly increased feeding in both chow-fed and high fat diet (HFD)-fed mice; however, this was correlated with body weight only in the chow-fed mice. Our results suggest that DIO causes ghrelin resistance albeit not directly in the VTA. We suggest there is impaired ghrelin sensitivity in upstream pathways regulating reward pathways, highlighting a functional role for ghrelin linking appropriate metabolic sensing with reward processing

Food seeking in a risky environment:A method for evaluating risk and reward value in food seeking and consumption in mice

Most studies that measure food intake in mice do so in the home cage environment. This necessarily means that mice do not engage in food seeking before consumption, a behavior that is ubiquitous in free-living animals. We modified and validated several commonly used anxiety tests to include a palatable food reward within the anxiogenic zone. This allowed us to assess risk-taking behavior in food seeking in mice in response to different metabolic stimuli. We modified the open field test and the light/dark box by placing palatable peanut butter chips within a designated food zone inside the anxiogenic zone of each apparatus. We then assessed parameters of the interaction with the food reward. Fasted mice or mice treated with ghrelin showed increased consumption and increased time spent in the food zone immediately around the food reward compared to ad libitum fed mice or mice treated with saline. However, fasted mice treated with IP glucose before exposure to the behavioral arena showed reduced time in the food zone compared to fasted controls, indicating that acute metabolic signals can modify the assessment of safety in food seeking in a risky environment. The tests described in this study will be useful in assessing risk processing and incentive salience of food reward, which are intrinsic components of food acquisition outside of the laboratory environment, in a range of genetic and pharmacological models

Ghrelin signalling in AgRP neurons links metabolic state to the sensory regulation of AgRP neural activity

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

Acute inhibition of hunger-sensing AgRP neurons promotes context-specific learning in mice

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

Activation of thermogenesis in brown adipose tissue and dysregulated lipid metabolism associated with cancer cachexia in mice

Cancer cachexia/anorexia is a complex syndrome that involves profound metabolic imbalances and is directly implicated as a cause of death in at least 20% to 30% of all cancers. Brown adipose tissue (BAT) plays a key role in thermogenesis and energy balance and potentially contributes to the physiologic perturbations associated with cachexia. In this study, we investigated the impact of cachexia-inducing colorectal tumor on BAT in mice. We found that brown adipocytes were smaller and exhibited profound delipidation in cachectic tumor–bearing mice. Diurnal expression profiling of key regulators of lipid accumulation and fatty acid ?-oxidation and their corresponding target genes revealed dramatic molecular changes indicative of active BAT. Increased Ucp1, Pbe, and Cpt1? expression at specific points coincided with higher BAT temperatures during the dark cycle, suggestive of a temporal stimulation of thermogenesis in cachexia. These changes persisted when cachectic mice were acclimatized to 28°C confirming inappropriate stimulation of BAT despite thermoneutrality. Evidence of inflammatory signaling also was observed in the BAT as an energetically wasteful and maladaptive response to anorexia during the development of cachexia

Des-acyl ghrelin and ghrelin O-acyltransferase regulate hypothalamic-pituitary-adrenal axis activation and anxiety in response to acute stress

Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation status. It is therefore critical to assess and state the acylation status of plasma ghrelin in all studies. In this study we tested the effect of des-acyl ghrelin administration onthe hypothalamic-pituitaryadrenal axis and on anxiety-like behavior of mice lacking endogenous ghrelin and in ghrelin-O-acyltransferase (GOAT) knockout (KO) mice that have no endogenous acyl ghrelin and high endogenous des-acyl ghrelin. Our results show des-acyl ghrelin produces an anxiogenic effect under nonstressed conditions, but this switches to an anxiolytic effect under stress. Des-acyl ghrelin influences plasma corticosterone under both nonstressed and stressed conditions, although c-fos activation in the paraventricular nucleus of the hypothalamus is not different. By contrast, GOAT KO are anxious under both nonstressed and stressed conditions, although this is not due to corticosterone release from the adrenals but rather from impaired feedback actions in the paraventricular nucleus of the hypothalamus, as assessed by c-fos activation. These results reveal des-acyl ghrelin treatment and GOAT deletion have differential effects on the hypothalamic-pituitaryadrenal axis and anxiety-like behavior, suggesting that anxiety-like behavior in GOAT KO mice is not due to high plasma des-acyl ghrelin.Fil: Stark, Romana. Monash University; AustraliaFil: Santos, Vanessa V.. Monash University; AustraliaFil: Geenen, Bram. Radboud University Medical Center; Países BajosFil: Cabral, Agustina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Dinan, Tara. RMIT University; AustraliaFil: Bayliss, Jacqueline A.. Monash University; AustraliaFil: Lockie, Sarah H.. Monash University; AustraliaFil: Reichenbach, Alex. Monash University; AustraliaFil: Lemus, Moyra B.. Monash University; AustraliaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Spencer, Sarah J.. RMIT University; AustraliaFil: Kozicz, Tamas. Radboud University Medical Center; Países Bajos. University of Tulane; Estados UnidosFil: Andrews, Zane B.. Monash University; Australi

Human and mouse studies identify an anorexigenic circuit from prefrontal cortex to lateral hypothalamus

Code and data for human results generated for manuscript titled "Human and mouse studies identify an anorexigenic circuit from prefrontal cortex to lateral hypothalamus" (Clarke et al., submitted 2021