Chemogenetic activation ofarcuate nucleus NPY and NPY/AgRP neurons increases feeding behaviour in mice.

Neuropeptide Y (NPY) plays a crucial role in controlling energy homeostasis and feeding behaviour. The role of NPY neurons located in the arcuate nucleus of the hypothalamus (Arc) in responding to homeostatic signals has been the focus of much investigation, but most studies have used AgRP promoter-driven models, which do not fully encompass Arc NPY neurons. To directly investigate NPY-expressing versus AgRP-expressing Arc neurons function, we utilised chemogenetic techniques in NPY-Cre and AgRP-Cre animals to activate Arc NPY or AgRP neurons in the presence of food and food-related stimuli. Our findings suggest that chemogenetic activation of the broader population of Arc NPY neurons, including AgRP-positive and AgRP-negative NPY neurons, has equivalent effects on feeding behaviour as activation of Arc AgRP neurons. Our results demonstrate that these Arc NPY neurons respond specifically to caloric signals and do not respond to non-caloric signals, in line with what has been observed in AgRP neurons. Activating Arc NPY neurons significantly increases food consumption and influences macronutrient selection to prefer fat intake.

Investigating GABA Neuron-Specific Androgen Receptor Knockout in two Hyperandrogenic Models of PCOS.

Androgen excess is a hallmark feature of polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Clinical and preclinical evidence links developmental or chronic exposure to hyperandrogenism with programming and evoking the reproductive and metabolic traits of PCOS. While critical androgen targets remain to be determined, central GABAergic neurons are postulated to be involved. Here, we tested the hypothesis that androgen signaling in GABAergic neurons is critical in PCOS pathogenesis in 2 well-characterized hyperandrogenic mouse models of PCOS. Using cre-lox transgenics, GABA-specific androgen receptor knockout (GABARKO) mice were generated and exposed to either acute prenatal androgen excess (PNA) or chronic peripubertal androgen excess (PPA). Females were phenotyped for reproductive and metabolic features associated with each model and brains of PNA mice were assessed for elevated GABAergic input to gonadotropin-releasing hormone (GnRH) neurons. Reproductive and metabolic dysfunction induced by PPA, including acyclicity, absence of corpora lutea, obesity, adipocyte hypertrophy, and impaired glucose homeostasis, was not different between GABARKO and wild-type (WT) mice. In PNA mice, acyclicity remained in GABARKO mice while ovarian morphology and luteinizing hormone secretion was not significantly impacted by PNA or genotype. However, PNA predictably increased the density of putative GABAergic synapses to GnRH neurons in adult WT mice, and this PNA-induced plasticity was absent in GABARKO mice. Together, these findings suggest that while direct androgen signaling in GABA neurons is largely not required for the development of PCOS-like traits in androgenized models of PCOS, developmental programming of GnRH neuron innervation is dependent upon androgen signaling in GABA neurons.

Excision of the endothelial blood-brain barrier insulin receptor does not alter spatial cognition in mice fed either a chow or high-fat diet.

Insulin is transported across the blood-brain barrier (BBB) endothelium to regulate aspects of metabolism and cognition. Brain insulin resistance often results from high-fat diet (HFD) consumption and is thought to contribute to spatial cognition deficits. To target BBB insulin function, we used Cre-LoxP genetic excision of the insulin receptor (InsR) from endothelial cells in adult male mice. We hypothesized that this excision would impair spatial cognition, and that high-fat diet consumption would exacerbate these effects. Excision of the endothelial InsR did not impair performance in two spatial cognition tasks, the Y-Maze and Morris Water Maze, in tests held both before and after 14 weeks of access to high-fat (or chow control) diet. The HFD increased body weight gain and induced glucose intolerance but did not impair spatial cognition. Endothelial InsR excision tended to increase body weight and reduce sensitivity to peripheral insulin, but these metabolic effects were not associated with impairments to spatial cognition and did not interact with HFD exposure. Instead, all mice showed intact spatial cognitive performance regardless of whether they had been fed chow or a HFD, and whether the InsR had been excised or not. Overall, the results indicate that loss of the endothelial InsR does not impact spatial cognition, which is in line with pharmacological evidence that other mechanisms at the BBB facilitate insulin transport and allow it to exert its pro-cognitive effects.

Deficiency in the neural cell adhesion molecule 2 (NCAM2) reduces axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), affects axonal organization in the hippocampus, and leads to behavioral deficits.

The neural cell adhesion molecule 2 (NCAM2) regulates axonal organization in the central nervous system via mechanisms that have remained poorly understood. We now show that NCAM2 increases axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protease that regulates axonal guidance. In brains of NCAM2-deficient mice, BACE1 levels are reduced in hippocampal mossy fiber projections, and the infrapyramidal bundle of these projections is shortened. This abnormal axonal organization correlates with impaired short-term spatial memory and cognitive flexibility in NCAM2-deficient male and female mice. Self-grooming, rearing, digging and olfactory acuity are increased in NCAM2-deficient male mice, when compared with littermate wild-type mice of the same sex. NCAM2-deficient female mice also show increased self-grooming, but are reduced in rearing, and do not differ from female wild-type mice in olfactory acuity and digging behavior. Our results indicate that errors in axonal guidance and organization caused by impaired BACE1 function can underlie the manifestation of neurodevelopmental disorders, including autism as found in humans with deletions of the NCAM2 gene.

The Effect of Dietary Fat and Sucrose on Cognitive Functioning in Mice Lacking Insulin Signaling in Neuropeptide Y Neurons.

Obesogenic diets can produce hippocampal insulin resistance and impairments to hippocampal-dependent cognition. This study investigated the effect of disrupted insulin signaling in Neuropeptide Y (NPY) neurons on diet-induced deficits in hippocampal-dependent memory. Wild-type mice and mice that had a targeted knockout of insulin receptors on NPY cells (IRlox/lox;NPYCre/+) were given ad libitum access to a high-fat diet (high fat; HF), 10% sucrose solution (high sugar; HS), both high-fat diet and sucrose solution (high fat, high sugar; HFHS), or a normal fat control chow for 12 weeks. Mice were tested in the Morris Water Maze (MWM), a hippocampal-dependent spatial memory task. Glucose homeostasis was assessed via a glucose tolerance test. Independent of genotype, consumption of HF, but not HS, diet increased energy intake, body weight, and plasma leptin, and impaired glucose tolerance. Disrupted insulin signaling in NPY cells and dietary interventions did not significantly affect the ability of mice to learn the location of the platform in the MWM. However, for IRlox/lox control mice, consumption of HF, but not HS, diet resulted in reduced time spent in the target quadrant during the probe trial, suggesting a hippocampal-dependent memory deficit. IRlox/lox;NPYCre/+ mice had poor performance in the probe trial regardless of diet, suggesting a floor effect. This study did not find adverse effects of chronic sucrose intake on metabolic outcomes or hippocampal-dependent memory. These data also suggest that the effects of HF diet on hippocampal-dependent memory may be dependent on insulin signaling in hippocampal NPY cells.

The effect of insulin receptor deletion in neuropeptide Y neurons on hippocampal dependent cognitive function in aging mice.

Insulin is known to act in the central nervous system to regulate several physiological and behavioural outcomes, including energy balance, glucose homeostasis and cognitive functioning. However, the neuronal populations through which insulin enhances cognitive performance remain unidentified. Insulin receptors are found in neuropeptide-Y (NPY) expressing neurons, which are abundant in the hypothalamus and hippocampus; regions involved in feeding behaviour and spatial memory, respectively. Here we show that mice with a tissue specific knockout of insulin receptors in NPY expressing neurons (IRl⁢o⁢x/l⁢o⁢x; NPYC⁢r⁢e⁣/+) display an impaired performance in the probe trial of the Morris Water Maze compared with control mice at both the 6 and the 12, but not at the 24 months time point, consistent with a crucial role of insulin and NPY in cognitive functioning. By 24 months of age all groups demonstrated similar reductions in spatial memory performance. Together, these data suggest that the mechanisms through which insulin influences cognitive functioning are, at least in part, via insulin receptor signaling in NPY expressing neurons. These results also highlight that cognitive impairments observed in aging may be due to impaired insulin signaling.

The continued need for animals to advance brain research.

Policymakers aim to move toward animal-free alternatives for scientific research and have introduced very strict regulations for animal research. We argue that, for neuroscience research, until viable and translational alternatives become available and the value of these alternatives has been proven, the use of animals should not be compromised.

Inhibition of the Renin-Angiotensin System Reduces Gene Expression of Inflammatory Mediators in Adipose Tissue Independent of Energy Balance.

Obesity is a growing health problem worldwide. The renin-angiotensin system (RAS) is present in adipose tissue, and evidence suggests that it is involved in both diet-induced obesity and the inflammation associated with obesity. The present experiments determined the effect of (1) different angiotensin-converting enzyme (ACE) inhibitors (captopril, perindopril, enalapril) and angiotensin receptor blockers (ARBs: telmisartan, losartan) on adiposity of mice fed a high-fat diet for 28 days (2); acute treatment with the ACE-inhibitor captopril on gene expression of inflammatory markers in mice fed a high-fat diet (HFD); and (3) short-term (2 days) and chronic (28 days) treatment of ACE-inhibition on energy expenditure (EE) and energy balance in mice fed HFD ad libitum (AL), as well as receiving HFD limited to the amount of calories eaten by controls (pair-fed (PF) group). Body weight, food intake, adiposity and plasma leptin were lower in ACE inhibitor or ARB-treated groups over 28 days compared with HFD untreated mice. Short-term treatment with captopril led to increased EE relative to the level in the PF group. After 28 days, EE was lower in both captopril-treated and PF mice compared with AL, but the effect was greater in the captopril-treated group. Adiponectin was elevated in captopril-treated mice, but not in PF mice, after both 2 and 28 days. Additionally, acute RAS blockade in HFD-fed mice reduced mRNA expression for MCP-1, IL-6, TLR4, and leptin in adipose tissue relative to values in untreated groups. These data demonstrate that ACE inhibition and angiotensin receptor blockade reduce food intake to produce weight loss and suggest that the anti-inflammatory effects of ACE inhibition may be independent of weight loss.

The regulation of food intake by insulin in the central nervous system.

Food intake and energy expenditure are regulated by peripheral signals providing feedback on nutrient status and adiposity to the central nervous system. One of these signals is the pancreatic hormone, insulin. Unlike peripheral administration of insulin, which often causes weight gain, central administration of insulin leads to a reduction in food intake and body weight when administered long-term. This is a result of feedback processes in regions of the brain that regulate food intake. Within the hypothalamus, the arcuate nucleus (ARC) contains subpopulations of neurones that produce orexinergic neuropeptides agouti-related peptide (AgRP)/neuropeptide Y (NPY) and anorexigenic neuropeptides, pro-opiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular infusion of insulin down-regulates the expression of AgRP/NPY at the same time as up-regulating expression of POMC/CART. Recent evidence suggests that insulin activity within the amygdala may play an important role in regulating energy balance. Insulin infusion into the central nucleus of the amygdala (CeA) can decrease food intake, possibly by modulating activity of NPY and other neurone subpopulations. Insulin signalling within the CeA can also influence stress-induced obesity. Overall, it is evident that the CeA is a critical target for insulin signalling and the regulation of energy balance.

Neurokinin 3 Receptor Antagonism Ameliorates Key Metabolic Features in a Hyperandrogenic PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition characterized by a range of endocrine, reproductive, and metabolic abnormalities. At present, management of women with PCOS is suboptimal as treatment is only symptomatic. Clinical and experimental advances in our understanding of PCOS etiology support a pivotal role for androgen neuroendocrine actions in PCOS pathogenesis. Hyperandrogenism is a key PCOS trait and androgen actions play a role in regulating the kisspeptin-/neurokinin B-/dynorphin (KNDy) system. This study aimed to investigate if targeted antagonism of neurokinin B signaling through the neurokinin 3 receptor (NK3R) would reverse PCOS traits in a dihydrotestosterone (DHT)-induced mouse model of PCOS. After 3 months, DHT exposure induced key reproductive PCOS traits of cycle irregularity and ovulatory dysfunction, and PCOS-like metabolic traits including increased body weight; white and brown fat pad weights; fasting serum triglyceride and glucose levels, and blood glucose incremental area under the curve. Treatment with a NK3R antagonist (MLE4901) did not impact the observed reproductive defects. In contrast, following NK3R antagonist treatment, PCOS-like females displayed decreased total body weight, adiposity, and adipocyte hypertrophy, but increased respiratory exchange ratio, suggesting NK3R antagonism altered the metabolic status of the PCOS-like females. NK3R antagonism did not improve circulating serum triglyceride or fasted glucose levels. Collectively, these findings demonstrate that NK3R antagonism may be beneficial in the treatment of adverse metabolic features associated with PCOS and support neuroendocrine targeting in the development of novel therapeutic strategies for PCOS.

Physiological and subjective validation of a novel stress procedure: The Simple Singing Stress Procedure.

Laboratory stress-induction procedures have been critical in illuminating the effects of stress on human health, cognition, and functioning. Here, we present a novel stress induction procedure, the Simple Singing Stress Procedure (SSSP), that overcomes some of the practical challenges and conceptual limitations of existing procedures in measuring the causal influence of stress on psychological variables. In the stress condition of the SSSP, participants were instructed to sing a song in front of the experimenter while being video- and audio-recorded. Participants were also informed that they would have to sing again at the end of the experiment, and that this second performance would later be assessed by a panel of experimenters. Participants in a no-stress condition instead read lyrics in each phase. Our findings revealed that participants in the stress condition showed significantly higher blood pressure immediately following the initial singing session, as well as heightened salivary cortisol at a latency consistent with the initial singing session, than those in the no-stress condition. Our stress procedure also generated elevations in self-reported stress ratings immediately after the first singing session and subsequently in anticipation of the second singing session, relative to the no-stress condition. Collectively, these findings suggest that the SSSP is a simple and effective stress induction procedure that may be a promising alternative to existing protocols.

Impaired Fluid Intake, but Not Sodium Appetite, in Aged Rats Is Mediated by the Cyclooxygenase-Prostaglandin E2 Pathway.

Aging results in decreased fluid intake following dehydration and other dipsogenic stimuli; similar reductions in sodium intake have also been observed with aging. Given that cyclooxygenase (COX)-derived prostanoids are elevated in aged rats in the midbrain and proinflammatory prostanoids are known to decrease fluid intake in dehydrated rats, the aim of this study was to determine if the reductions of fluid intake and sodium intake in aging are mediated by proinflammatory eicosanoid signaling. Therefore, we examined the effect of acute COX inhibition in adult (4 months-old) and aged (30 months-old) rats prior to ingestive behavior challenges. COX inhibition, using acetylsalicylic acid (ASA), increased fluid intake in aged, but not adult, rats in response to 24-h dehydration. ASA had no effect on salt intake following sodium depletion and ASA did not change basal fluid or sodium consumption in either age group. Hypothalamic COX-1 and -2, prostaglandin E synthase (PGES) and inducible nitric oxide synthase (iNOS) mRNA expression were all elevated in aged animals, leading to elevated PGE2 levels. COX expression in the hypothalamus was reduced by ASA treatment in rats of both ages resulting in reduced PGE2 levels in aged ASA treated animals. These data indicate that the reduced fluid intake that occurs in aging is due to increased COX-PGE2-mediated inflammation. However, the reduced sodium intake in these animals appears to occur via an alternate mechanism.

Amygdala NPY Circuits Promote the Development of Accelerated Obesity under Chronic Stress Conditions.

Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.

Female primary and secondary psychopathic variants show distinct endocrine and psychophysiological profiles.

Research with predominantly male samples supports primary and secondary developmental pathways to psychopathy that are phenotypically indistinguishable on aggressive and antisocial behavior. The aim of this study was to examine whether female variants of psychopathy show divergent endocrine (i.e., cortisol, dehydroepiandrosterone [DHEA], testosterone, and their ratios) and psychophysiological (i.e., heart rate variability [HRV]) reactivity to social provocation. We also tested whether variants differed on reactive aggression when performing a competitive reaction time task against the fictitious participant who previously insulted them. Latent profile analyses on 101 undergraduate women oversampled for high psychopathic traits identified a high-anxious, maltreated secondary variant (n=64) and a low-anxious primary variant (n=37). Although variants did not differ on aggression, secondary variants showed higher cortisol, testosterone, cortisol-to-DHEA ratios, and HRV following social provocation relative to primary variants. Findings suggest that the neurobiological mechanisms underpinning aggression in psychopathy may differ between women on primary versus secondary developmental pathways.

How and why do gastrointestinal peptides influence food intake?

Despite the ability of some gastrointestinal hormones to reliably reduce meal size when administered prior to a meal, it is not understood why the repeated administration or genetic knockout of these hormones appear largely ineffective in reducing food intake and body weight. Here, we review evidence that the ability of GI peptides such as cholecystokinin (CCK) to elicit satiation is a consequence of prior learning. Evidence includes first, that the ability of some of these signals to modify food intake depends upon past experience and is malleable with new experience. Additionally, the ability of CCK and other gut signals to reduce food intake may not be hard-wired; i.e., any so-called "satiation" signal that reduces food intake in a single-meal situation may not continue to do so over repeated trials. The individual will respond to the signal only so long as it provides reliable information about caloric content. If a particular signal becomes unreliable, the individual will rely on other signals to end meals. Thus, gut peptides/hormones have important metabolic effects such as mediating absorption, digestion, and many aspects of the distribution of ingested nutrients throughout the body; and, if they have been reliably associated with natural stimuli that mediate satiation, they also inform behavior.

The Effect of Intrahippocampal Insulin Infusion on Spatial Cognitive Function and Markers of Neuroinflammation in Diet-induced Obesity.

Obesity and high fat diet consumption contribute to the development of metabolic disorders, insulin resistance, neuroinflammation, and cognitive impairments. CNS administration of insulin into the brain can attenuate these cognitive impairments. The present study investigated whether hippocampal-dependent spatial memory impairments in a dietary induced mouse model of obesity could be improved by the direct administration of insulin into the hippocampus and whether this was associated with markers of hippocampal inflammation. C57Bl/6J mice consumed a low fat or high fat diet for 16 weeks and continuous intrahippocampal saline or insulin infusion for the final 4 weeks, during a period of behavioral testing, before gene expression analysis was performed. The high fat diet group demonstrated poorer spatial memory performance in the Morris water maze and Y-maze, supporting the hypothesis that high fat diet leads to hippocampal dependent cognitive impairment. Insulin infusion into the hippocampus reversed the deficit of high fat diet consumption on both of the tasks. Increased expression of inflammatory markers was detected in the hippocampus in the high fat diet group and expression of these markers was ameliorated in insulin infused mice. This demonstrates that CNS insulin can improve hippocampal-dependent memory and that hippocampal inflammation may be a factor in the development of cognitive deficits associated with diet-induced obesity. Furthermore, these data suggest that insulin may act to attenuate high fat diet induced cognitive deficits by reducing neuroinflammation.

Insulin controls food intake and energy balance via NPY neurons.

OBJECTIVES: Insulin signaling in the brain has been implicated in the control of satiety, glucose homeostasis and energy balance. However, insulin signaling is dispensable in energy homeostasis controlling AgRP or POMC neurons and it is unclear which other neurons regulate these effects. Here we describe an ancient insulin/NPY neuronal network that governs energy homeostasis across phyla. METHODS: To address the role of insulin action specifically in NPY neurons, we generated a variety of models by selectively removing insulin signaling in NPY neurons in flies and mice and testing the consequences on energy homeostasis. RESULTS: By specifically targeting the insulin receptor in both fly and mouse NPY expressing neurons, we found NPY-specific insulin signaling controls food intake and energy expenditure, and lack of insulin signaling in NPY neurons leads to increased energy stores and an obese phenotype. Additionally, the lack of insulin signaling in NPY neurons leads to a dysregulation of GH/IGF-1 axis and to altered insulin sensitivity. CONCLUSIONS: Taken together, these results suggest that insulin actions in NPY neurons is critical for maintaining energy balance and an impairment of this pathway may be causally linked to the development of metabolic diseases.

Disturbances of thirst and fluid balance associated with aging.

During heat waves, significant mortality and morbidity occurs in elderly populations due to heat-stress and dehydration. The dehydration is primarily attributable to inadequate water intake caused by dysfunction of the central nervous system mechanisms controlling thirst. The phenomenon of a reduced thirst in response to dehydration in aging was first observed decades ago and has been examined extensively since. The reduced thirst and ingestive behavior have been reported consistently in response to hyperosmotic stimuli, hypovolemic stimuli and dehydration in both elderly humans and animal models of aging. There are also data to suggest that sodium appetite is reduced in aged rats, potentially indicating a common etiology. Accompanying the behavioral changes in water and sodium intake that occur with aging there are also alterations to a number of hormonal systems involved in body fluid and electrolyte homeostasis. These changes include reductions in activity of the renin-angiotensin system and increases in circulating atrial natriuretic peptide and arginine vasopressin. While there is substantial evidence to suggest that the behavioral and physiological mechanisms responsible for body fluid and sodium homeostasis are impaired in aging, the precise etiology of reduced thirst remain to be determined.

Using the cerebrospinal fluid to understand ingestive behavior.

The cerebrospinal fluid (CSF) offers a window into the workings of the brain and blood-brain barrier (BBB). Molecules that enter into the central nervous system (CNS) by passive diffusion or receptor-mediated transport through the choroid plexus often appear in the CSF prior to acting within the brain. Other molecules enter the CNS by passing through the BBB into the brain's interstitial fluid prior to appearing in the CSF. This pattern is also often observed for molecules synthesized by neurons or glia within the CNS. The CSF is therefore an important conduit for the entry and clearance of molecules into/from the CNS and thereby constitutes an important window onto brain activity and barrier function. Assessing the CSF basally, under experimental conditions, or in the context of challenges or metabolic diseases can provide powerful insights about brain function. Here, we review important findings made by our labs, as influenced by the late Randall Sakai, by interrogating the CSF.