Peripherally restricted oxytocin is sufficient to reduce food intake and motivation, while CNS entry is required for locomotor and taste avoidance effects.

OBJECTIVES: Oxytocin (OT) has a well-established role in reproductive behaviours; however, it recently emerged as an important regulator of energy homeostasis. In addition to central nervous system (CNS), OT is found in the plasma and OT receptors (OT-R) are found in peripheral tissues relevant to energy balance regulation. Here, we aim to determine whether peripheral OT-R activation is sufficient to alter energy intake and expenditure. METHODS AND RESULTS: We first show that systemic OT potently reduced food intake and food-motivated behaviour for a high-fat reward in male and female rats. As it is plausible that peripherally, intraperitoneally (IP) injected OT crosses the blood-brain barrier (BBB) to produce some of the metabolic effects within the CNS, we screened, with a novel fluorescently labelled-OT (fAF546-OT, Roxy), for the presence of IP-injected Roxy in CNS tissue relevant to feeding control and compared such with BBB-impermeable fluorescent OT-B12 (fCy5-OT-B12; BRoxy). While Roxy did penetrate the CNS, BRoxy did not. To evaluate the behavioural and thermoregulatory impact of exclusive activation of peripheral OT-R, we generated a novel BBB-impermeable OT (OT-B12 ), with equipotent binding at OT-R in vitro. In vivo, IP-injected OT and OT-B12 were equipotent at food intake suppression in rats of both sexes, suggesting that peripheral OT acts on peripheral OT-R to reduce feeding behaviour. Importantly, OT induced a potent conditioned taste avoidance, indistinguishable from that induced by LiCl, when applied peripherally. Remarkably, and in contrast to OT, OT-B12 did not induce any conditioned taste avoidance. Limiting the CNS entry of OT also resulted in a dose-dependent reduction of emesis in male shrews. While both OT and OT-B12 proved to have similar effects on body temperature, only OT resulted in home-cage locomotor depression. CONCLUSIONS: Together our data indicate that limiting systemic OT CNS penetrance preserves the anorexic effects of the peptide and reduces the clinically undesired side effects of OT: emesis, taste avoidance and locomotor depression. Thus, therapeutic targeting of peripheral OT-R may be a viable strategy to achieve appetite suppression with better patient outcomes.

Counting the Number of Glutamate Molecules in Single Synaptic Vesicles.

Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.

Glucagon-Like Peptide-1-, but not Growth and Differentiation Factor 15-, Receptor Activation Increases the Number of Interleukin-6-Expressing Cells in the External Lateral Parabrachial Nucleus.

Interleukin (IL)-6 in the hypothalamus and hindbrain is an important downstream mediator of suppression of body weight and food intake by glucagon-like peptide-1 (GLP-1) receptor stimulation. CNS GLP-1 is produced almost exclusively in prepro-glucagon neurons in the nucleus of the solitary tract. These neurons innervate energy balance-regulating areas, such as the external lateral parabrachial nucleus (PBNel); essential for induction of anorexia. Using a validated novel IL-6-reporter mouse strain, we investigated the interactions in PBNel between GLP-1, IL-6, and calcitonin gene-related peptide (CGRP, a well-known mediator of anorexia). We show that PBNel GLP-1R-containing cells highly (to about 80%) overlap with IL-6-containing cells on both protein and mRNA level. Intraperitoneal administration of a GLP-1 analogue exendin-4 to mice increased the proportion of IL-6-containing cells in PBNel 3-fold, while there was no effect in the rest of the lateral parabrachial nucleus. In contrast, injections of an anorexigenic peptide growth and differentiation factor 15 (GDF15) markedly increased the proportion of CGRP-containing cells, while IL-6-containing cells were not affected. In summary, GLP-1R are found on IL-6-producing cells in PBNel, and GLP-1R stimulation leads to an increase in the proportion of cells with IL-6-reporter fluorescence, supporting IL-6 mediation of GLP-1 effects on energy balance.

CNS ß3-adrenergic receptor activation regulates feeding behavior, white fat browning, and body weight.

Pharmacological ß3-adrenergic receptor (ß3AR) activation leads to increased mitochondrial biogenesis and activity in white adipose tissue (WAT), a process commonly referred to as "browning", and transiently increased insulin release. These effects are associated with improved metabolic function and weight loss. It is assumed that this impact of ß3AR agonists is mediated solely through activation of ß3ARs in adipose tissue. However, ß3ARs are also found in the brain, in areas such as the brain stem and the hypothalamus, which provide multisynaptic innervation to brown and white adipose depots. Thus, contrary to the current adipocentric view, the central nervous system (CNS) may also have the ability to regulate energy balance and metabolism through actions on central ß3ARs. Therefore, this study aimed to elucidate whether CNS ß3ARs can regulate browning of WAT and other aspects of metabolic regulation, such as food intake control and insulin release. We found that acute central injection of ß3AR agonist potently reduced food intake, body weight, and increased hypothalamic neuronal activity in rats. Acute central ß3AR stimulation was also accompanied by a transient increase in circulating insulin levels. Moreover, subchronic central ß3AR agonist treatment led to a browning response in both inguinal (IWAT) and gonadal WAT (GWAT), along with reduced GWAT and increased BAT mass. In high-fat, high-sugar-fed rats, subchronic central ß3AR stimulation reduced body weight, chow, lard, and sucrose water intake, in addition to increasing browning of IWAT and GWAT. Collectively, our results identify the brain as a new site of action for the anorexic and browning impact of ß3AR activation.

GLP-1 and weight loss: unraveling the diverse neural circuitry.

Glucagon-like peptide-1 (GLP-1) is currently one of the most promising biological systems for the development of effective obesity pharmacotherapies. Long-acting GLP-1 analogs potently reduce food intake and body weight, and recent discoveries reveal that peripheral administration of these drugs reduces food intake largely through humoral pathways involving direct action on brain GLP-1 receptors (GLP-1R). Thus, it is of critical importance to understand the neural systems through which GLP-1 and long-acting GLP-1 analogs reduce food intake and body weight. In this review, we discuss several neural, physiological, cellular and molecular, as well as behavioral mechanisms through which peripheral and central GLP-1R signaling reduces feeding. Particular attention is devoted to discussion regarding the numerous neural substrates through which GLP-1 and GLP-1 analogs act to reduce food intake and body weight, including various hypothalamic nuclei (arcuate nucleus of the hypothalamus, periventricular hypothalamus, lateral hypothalamic area), hindbrain nuclei (parabrachial nucleus, medial nucleus tractus solitarius), hippocampus (ventral subregion; vHP), and nuclei embedded within the mesolimbic reward circuitry [ventral tegmental area (VTA) and nucleus accumbens (NAc)]. In some of these nuclei [VTA, NAc, and vHP], GLP-1R activation reduces food intake and body weight without concomitant nausea responses, suggesting that targeting these specific pathways may be of particular interest for future obesity pharmacotherapy. The widely distributed neural systems through which GLP-1 and GLP-1 analogs act to reduce body weight highlight the complexity of the neural systems regulating energy balance, as well as the challenges for developing effective obesity pharmacotherapies that reduce feeding without producing parallel negative side effects.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6.

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

A vagal influence on schizophrenia? A nationwide retrospective cohort of vagotomized individuals.

Background and Objectives: Emerging preclinical evidence suggests that vagal signals contribute to the development of schizophrenia-related abnormalities in brain and behavior. Whether vagal communication in general, and its impairment in particular, is a risk factor for schizophrenia in humans remains, however, unclear. Vagotomy, the surgical lesion of the vagus nerve, was routinely performed as a treatment for peptic ulcer before modern treatment options were available. Hence, the primary aim of this study was to investigate whether vagotomy modulates the subsequent risk of developing schizophrenia. Moreover, given the existence of diverse vagotomy techniques (i.e., "truncal" or "selective"), our secondary goal was to test whether the extent of denervation modulates the risk of schizophrenia. Methods: Using a nationwide retrospective matched cohort design, we identified 8,315 vagotomized individuals from the Swedish National Patient Register during the period 1970-2020 and 40,855 non-vagotomized individuals matching for age, sex and type of peptic ulcer. The risk of being diagnosed with schizophrenia and associated psychoses (ICD10 codes F20-29) was analyzed using Cox proportional hazards regression models, including death as competing risk. Results: When considering all types of vagotomy together, vagotomy was not significantly associated with schizophrenia (HR: 0.91 [0.72; 1.16]). However, truncal vagotomy (which denervates all subdiaphragmatic organs) significantly increased the risk of developing schizophrenia by 69% (HR: 1.69 [1.08; 2.64]), whereas selective vagotomy (which only denervates the stomach) showed no significant association (HR: 0.80 [0.61; 1.04]). Discussion: Our results provide epidemiological support for the hypothesis that impairments in vagal functions could increase the risk of schizophrenia. Notably, the finding that truncal but not selective vagotomy is associated with an increased risk of schizophrenia raises the possibility that the activity of subdiaphragmatic non-gastric vagal branches may be of particular relevance for the development of schizophrenia.

Lateral parabrachial nucleus astrocytes control food intake.

Food intake behavior is under the tight control of the central nervous system. Most studies to date focus on the contribution of neurons to this behavior. However, although previously overlooked, astrocytes have recently been implicated to play a key role in feeding control. Most of the recent literature has focused on astrocytic contribution in the hypothalamus or the dorsal vagal complex. The contribution of astrocytes located in the lateral parabrachial nucleus (lPBN) to feeding behavior control remains poorly understood. Thus, here, we first investigated whether activation of lPBN astrocytes affects feeding behavior in male and female rats using chemogenetic activation. Astrocytic activation in the lPBN led to profound anorexia in both sexes, under both ad-libitum feeding schedule and after a fasting challenge. Astrocytes have a key contribution to glutamate homeostasis and can themselves release glutamate. Moreover, lPBN glutamate signaling is a key contributor to potent anorexia, which can be induced by lPBN activation. Thus, here, we determined whether glutamate signaling is necessary for lPBN astrocyte activation-induced anorexia, and found that pharmacological N-methyl D-aspartate (NMDA) receptor blockade attenuated the food intake reduction resulting from lPBN astrocyte activation. Since astrocytes have been shown to contribute to feeding control by modulating the feeding effect of peripheral feeding signals, we further investigated whether lPBN astrocyte activation is capable of modulating the anorexic effect of the gut/brain hormone, glucagon like peptide -1, as well as the orexigenic effect of the stomach hormone - ghrelin, and found that the feeding effect of both signals is modulated by lPBN astrocytic activation. Lastly, we found that lPBN astrocyte activation-induced anorexia is affected by a diet-induced obesity challenge, in a sex-divergent manner. Collectively, current findings uncover a novel role for lPBN astrocytes in feeding behavior control.

Elevated circulating adiponectin levels do not prevent anxiety-like behavior in a PCOS-like mouse model.

Polycystic ovary syndrome (PCOS) is associated with symptoms of moderate to severe anxiety and depression. Hyperandrogenism is a key feature together with lower levels of the adipocyte hormone adiponectin. Androgen exposure leads to anxiety-like behavior in female offspring while adiponectin is reported to be anxiolytic. Here we test the hypothesis that elevated adiponectin levels protect against the development of androgen-induced anxiety-like behavior. Pregnant mice overexpressing adiponectin (APNtg) and wildtypes were injected with vehicle or dihydrotestosterone to induce prenatal androgenization (PNA) in the offspring. Metabolic profiling and behavioral tests were performed in 4-month-old female offspring. PNA offspring spent more time in the closed arms of the elevated plus maze, indicating anxiety-like behavior. Intriguingly, neither maternal nor offspring adiponectin overexpression prevented an anxiety-like behavior in PNA-exposed offspring. However, adiponectin overexpression in dams had metabolic imprinting effects, shown as lower fat mass and glucose levels in their offspring. While serum adiponectin levels were elevated in APNtg mice, cerebrospinal fluid levels were similar between genotypes. Adiponectin overexpression improved metabolic functions but did not elicit anxiolytic effects in PNA-exposed offspring. These observations might be attributed to increased circulating but unchanged cerebrospinal fluid adiponectin levels in APNtg mice. Thus, increased adiponectin levels in the brain are likely needed to stimulate anxiolytic effects.

The glucagon-like peptide 1 (GLP-1) analogue, exendin-4, decreases the rewarding value of food: a new role for mesolimbic GLP-1 receptors.

The glucagon-like peptide 1 (GLP-1) system is a recently established target for type 2 diabetes treatment. In addition to regulating glucose homeostasis, GLP-1 also reduces food intake. Previous studies demonstrate that the anorexigenic effects of GLP-1 can be mediated through hypothalamic and brainstem circuits which regulate homeostatic feeding. Here, we demonstrate an entirely novel neurobiological mechanism for GLP-1-induced anorexia in rats, involving direct effects of a GLP-1 agonist, Exendin-4 (EX4) on food reward that are exerted at the level of the mesolimbic reward system. We assessed the impact of peripheral, central, and intramesolimbic EX4 on two models of food reward: conditioned place preference (CPP) and progressive ratio operant-conditioning. Food-reward behavior was reduced in the CPP test by EX4, as rats no longer preferred an environment previously paired to chocolate pellets. EX4 also decreased motivated behavior for sucrose in a progressive ratio operant-conditioning paradigm when administered peripherally. We show that this effect is mediated centrally, via GLP-1 receptors (GLP-1Rs). GLP-1Rs are expressed in several key nodes of the mesolimbic reward system; however, their function remains unexplored. Thus we sought to determine the neurobiological substrates underlying the food-reward effect. We found that the EX4-mediated inhibition of food reward could be driven from two key mesolimbic structures-ventral tegmental area and nucleus accumbens-without inducing concurrent malaise or locomotor impairment. The current findings, that activation of central GLP-1Rs strikingly suppresses food reward/motivation by interacting with the mesolimbic system, indicate an entirely novel mechanism by which the GLP-1R stimulation affects feeding-oriented behavior.

An appetite for aggressive behavior? Female rats, too, derive reward from winning aggressive interactions.

While aggression is an adaptive behavior mostly triggered by competition for resources, it can also in and of itself be rewarding. Based on the common notion that female rats are not aggressive, much of aggression research has been centered around males, leading to a gap in the understanding of the female aggression neurobiology. Therefore, we asked whether intact virgin female rats experience reward from an aggressive interaction and assessed aggression seeking behavior in rats of both sexes. To validate the involvement of reward signaling, we measured mesolimbic dopamine turnover and determined the necessity of dopamine signaling for expression of aggression-seeking. Together our data indicate that female rats exhibit aggressive behavior outside of maternal context, experience winning aggressive behaviors as rewarding, and do so to a similar extent as male rats and in a dopamine-dependent manner.

From the stomach to locus coeruleus: new neural substrate for ghrelin's effects on ingestive, motivated and anxiety-like behaviors.

Ghrelin, a stomach-derived orexigenic hormone, has a well-established role in energy homeostasis, food reward, and emotionality. Noradrenergic neurons of the locus coeruleus (LC) are known to play an important role in arousal, emotion, cognition, but recently have also been implicated in control of feeding behavior. Ghrelin receptors (the growth hormone secretagogue receptor, GHSR) may be found in the LC, but the behavioral effects of ghrelin signaling in this area are still unexplored. Here, we first determined whether GHSR are present in the rat LC, and demonstrate that GHSR are expressed on noradrenergic neurons in both sexes. We next investigated whether ghrelin controls ingestive and motivated behaviors as well as anxiety-like behavior by acting in the LC. To pursue this idea, we examined the effects of LC GHSR stimulation and blockade on food intake, operant responding for a palatable food reward and, anxiety-like behavior in the open field (OF) and acoustic startle response (ASR) tests in male and female rats. Our results demonstrate that intra-LC ghrelin administration increases chow intake and motivated behavior for sucrose in both sexes. Additionally, females, but not males, exhibited a potent anxiolytic response in the ASR. In order to determine whether activation of GHSR in the LC was necessary for feeding and anxiety behavior control, we utilized liver-expressed antimicrobial peptide 2 (LEAP2), a newly identified endogenous GHSR antagonist. LEAP2 delivered specifically into the LC was sufficient to reduce fasting-induced chow hyperphagia in both sexes, but food reward only in females. Moreover, blockade of GHSR in the LC increased anxiety-like behavior measured in the ASR test in both sexes. Taken together, these results indicate that ghrelin acts in the LC to alter ingestive, motivated and anxiety-like behaviors, with a degree of sex divergence.

Imaging obesity: fMRI, food reward, and feeding.

Animal studies have revealed brain regions that control homeostatic feeding, but the rampant overeating contributing to the obesity epidemic suggests the participation of "nonhomeostatic" control centers. Recent papers by Batterham et al. (2007) and Farooqi et al. (2007) link peptide YY(3-36) and leptin to the activation of nonhomeostatic brain regions.

Role of ghrelin in food reward: impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetylcholine receptor gene expression.

The decision to eat is strongly influenced by non-homeostatic factors such as food palatability. Indeed, the rewarding and motivational value of food can override homeostatic signals, leading to increased consumption and hence, obesity. Ghrelin, a gut-derived orexigenic hormone, has a prominent role in homeostatic feeding. Recently, however, it has emerged as a potent modulator of the mesolimbic dopaminergic reward pathway, suggesting a role for ghrelin in food reward. Here, we sought to determine whether ghrelin and its receptors are important for reinforcing motivation for natural sugar reward by examining the role of ghrelin receptor (GHS-R1A) stimulation and blockade for sucrose progressive ratio operant conditioning, a procedure used to measure motivational drive to obtain a reward. Peripherally and centrally administered ghrelin significantly increased operant responding and therefore, incentive motivation for sucrose. Utilizing the GHS-R1A antagonist JMV2959, we demonstrated that blockade of GHS-R1A signaling significantly decreased operant responding for sucrose. We further investigated ghrelin's effects on key mesolimbic reward nodes, the ventral tegmental area (VTA) and nucleus accumbens (NAcc), by evaluating the effects of chronic central ghrelin treatment on the expression of genes encoding major reward neurotransmitter receptors, namely dopamine and acetylcholine. Ghrelin treatment was associated with an increased dopamine receptor D5 and acetylcholine receptor nAChRß2 gene expression in the VTA and decreased expression of D1, D3, D5 and nAChRα3 in the NAcc. Our data indicate that ghrelin plays an important role in motivation and reinforcement for sucrose and impacts on the expression of dopamine and acetylcholine encoding genes in the mesolimbic reward circuitry. These findings suggest that ghrelin antagonists have therapeutic potential for the treatment of obesity and to suppress the overconsumption of sweet food.

Peripheral signals modifying food reward.

The pleasure derived from eating may feel like a simple emotion, but the decision to eat, and perhaps more importantly what to eat, involves central pathways linking energy homeostasis and reward and their regulation by metabolic and endocrine factors. Evidence is mounting that modulation of the hedonic aspects of energy balance is under the control of peripheral neuropeptides conventionally associated with homeostatic appetite control. Here, we describe the significance of reward in feeding, the neural substrates underlying the reward pathway and their modification by peptides released into the circulation from peripheral tissues.

Commonly-used rodent tests of anxiety-like behavior lack predictive validity for human sex differences.

Women are more likely to develop an anxiety disorder than men. Yet, preclinical models of anxiety were largely developed in male rodents, with poorly understood predictive validity for sex differences. Here, we investigate whether commonly-used anxiety-like behavior tests, elevated plus maze (EPM) and open field (OF), represent the human sex difference in adult Sprague-Dawley rats. When interpreted by EPM or OF, female rats displayed less anxiety-like behavior compared to males, as they spent twice as much time in the open arms of the EPM or the center of the OF compared to males. However, they also displayed vastly different levels of locomotor activity, possibly confounding interpretation of these locomotion-dependent tests. To exclude locomotion from the assessment, the acoustic startle response (ASR) test was used. When interpreted by the ASR test, females displayed more anxiety-like behavior compared to males, as indicated by a nearly two-fold higher startle amplitude. The observed sex differences were not driven by gonadal steroids. Overall, all but one of the tests fail to mirror the sex difference in anxiety reported in humans. Our findings suggest that the ASR might be a better fit in modelling female anxiety-like behavior.

From an Empty Stomach to Anxiolysis: Molecular and Behavioral Assessment of Sex Differences in the Ghrelin Axis of Rats.

Ghrelin, a stomach-produced hormone, is well-recognized for its role in promoting feeding, controlling energy homeostasis, and glucoregulation. Ghrelin's function to ensure survival extends beyond that: its release parallels that of corticosterone, and ghrelin administration and fasting have an anxiolytic and antidepressant effect. This clearly suggests a role in stress and anxiety. However, most studies of ghrelin's effects on anxiety have been conducted exclusively on male rodents. Here, we hypothesize that female rats are wired for higher ghrelin sensitivity compared to males. To test this, we systematically compared components of the ghrelin axis between male and female Sprague Dawley rats. Next, we evaluated whether anxiety-like behavior and feeding response to endogenous or exogenous ghrelin are sex divergent. In line with our hypothesis, we show that female rats have higher serum levels of ghrelin and lower levels of the endogenous antagonist LEAP-2, compared to males. Furthermore, circulating ghrelin levels were partly dependent on estradiol; ovariectomy drastically reduced circulating ghrelin levels, which were partly restored by estradiol replacement. In contrast, orchiectomy did not affect circulating plasma ghrelin. Additionally, females expressed higher levels of the endogenous ghrelin receptor GHSR1A in brain areas involved in feeding and anxiety: the lateral hypothalamus, hippocampus, and amygdala. Moreover, overnight fasting increased GHSR1A expression in the amygdala of females, but not males. To evaluate the behavioral consequences of these molecular differences, male and female rats were tested in the elevated plus maze (EPM), open field (OF), and acoustic startle response (ASR) after three complementary ghrelin manipulations: increased endogenous ghrelin levels through overnight fasting, systemic administration of ghrelin, or blockade of fasting-induced ghrelin signaling with a GHSR1A antagonist. Here, females exhibited a stronger anxiolytic response to fasting and ghrelin in the ASR, in line with our findings of sex differences in the ghrelin axis. Most importantly, after GHSR1A antagonist treatment, females but not males displayed an anxiogenic response in the ASR, and a more pronounced anxiogenesis in the EPM and OF compared to males. Collectively, female rats are wired for higher sensitivity to fasting-induced anxiolytic ghrelin signaling. Further, the sex differences in the ghrelin axis are modulated, at least partly, by gonadal steroids, specifically estradiol. Overall, ghrelin plays a more prominent role in the regulation of anxiety-like behavior of female rats.

Sex and Species Differences in the Development of Diet-Induced Obesity and Metabolic Disturbances in Rodents.

Prevalence and health consequences of obesity differ between men and women. Yet, most preclinical studies investigating the etiology of obesity have, to date, been conducted in male rodents. Notably, diet is a major determinant of obesity, but sex differences in rodent models of diet-induced obesity, and the mechanisms that underlie such differences, are still understudied. Here, we aim to determine whether time course and characteristics of diet-induced obesity differ between sexes in rats and mice, and to investigate the potential causes of the observed divergence. To achieve this, we offered the most commonly tested rodents of both sexes, SD rats and C57BL/6 mice, a free choice of 60 % high-fat diet (HFD) and regular chow; body weight, food intake, fat mass, brown adipose responses, locomotor activity and glucose tolerance were assessed in a similar manner in both species. Our results indicate that overall diet-induced hyperphagia is greater in males but that females display a higher preference for the HFD, irrespective of species. Female rats, compared to males, showed a delay in diet-induced weight gain and less metabolic complications. Although male rats increased brown adipose tissue thermogenesis in response to the HFD challenge, this was not sufficient to counteract increased adiposity. In contrast to rats, female and male mice presented with a dramatic adiposity and impaired glucose tolerance, and a decreased energy expenditure. Female mice showed a 5-fold increase in visceral fat, compared to 2-fold increase seen in male mice. Overall, we found that male and female rodents responded very differently to HFD challenge, and engaged different compensatory energy expenditure mechanisms. In addition, these sex differences are divergent in rats and mice. We conclude that SD rats have a better face validity for the lower prevalence of overweight in women, while C57BL/6 mice may better model the increased prevalence of morbid obesity in women.

A Cre-driver rat model for anatomical and functional analysis of glucagon (Gcg)-expressing cells in the brain and periphery.

OBJECTIVE: The glucagon gene (Gcg) encodes preproglucagon, which is cleaved to form glucagon-like peptide 1 (GLP1) and other mature signaling molecules implicated in metabolic functions. To date there are no transgenic rat models available for precise manipulation of GLP1-expressing cells in the brain and periphery. METHODS: To visualize and manipulate Gcg-expressing cells in rats, CRISPR/Cas9 was used to express iCre under control of the Gcg promoter. Gcg-Cre rats were bred with tdTomato reporter rats to tag Gcg-expressing cells. Cre-dependent AAVs and RNAscope in situ hybridization were used to evaluate the specificity of iCre expression by GLP1 neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt), and by intestinal and pancreatic secretory cells. Food intake was assessed in heterozygous (Het) Gcg-Cre rats after chemogenetic stimulation of cNTS GLP1 neurons expressing an excitatory DREADD. RESULTS: While genotype has minimal effect on body weight or composition in chow-fed Gcg-Cre rats, homozygous (Homo) rats have lower plasma glucose levels. In neonatal and adult Gcg-Cre/tdTom rats, reporter-labeled cells are present in the cNTS and IRt, and in additional brain regions (e.g., basolateral amygdala, piriform cortex) that lack detectable Gcg mRNA in adults but display transient developmental or persistently low Gcg expression. Compared to wildtype (WT) rats, hindbrain Gcg mRNA and GLP1 protein in brain and plasma are markedly reduced in Homo Gcg-Cre rats. Chemogenetic stimulation of cNTS GLP1 neurons reduced overnight chow intake in males but not females, the effect in males was blocked by antagonism of central GLP1 receptors, and hypophagia was enhanced when combined with a subthreshold dose of cholecystokinin-8 to stimulate gastrointestinal vagal afferents. CONCLUSIONS: Gcg-Cre rats are a novel and valuable experimental tool for analyzing the development, anatomy, and function of Gcg-expressing cells in the brain and periphery. In addition, Homo Gcg-Cre rats are a unique model for assessing the role of Gcg-encoded proteins in glucose homeostasis and energy metabolism.

Hindbrain insulin controls feeding behavior.

OBJECTIVE: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice. METHODS: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used. RESULTS: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis. CONCLUSIONS: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.