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.

CCK increases the transport of insulin into the brain.

Food intake occurs in bouts or meals, and numerous meal-generated signals have been identified that act to limit the size of ongoing meals. Hormones such as cholecystokinin (CCK) are secreted from the intestine as ingested food is being processed, and in addition to aiding the digestive process, they provide a signal to the brain that contributes to satiation, limiting the size of the meal. The potency of CCK to elicit satiation is enhanced by elevated levels of adiposity signals such as insulin. In the present experiments we asked whether CCK and insulin interact at the level of the blood-brain barrier (BBB). We first isolated rat brain capillary endothelial cells that comprise the BBB and found that they express the mRNA for both the CCK1R and the insulin receptor, providing a basis for a possible interaction. We then administered insulin intraperitoneally to another group of rats and 15min later administered CCK-8 intraperitoneally to half of those rats. After another 15min, CSF and blood samples were obtained and assayed for immunoreactive insulin. Plasma insulin was comparably elevated above baseline in both the CCK-8 and control groups, indicating that the CCK had no effect on circulating insulin levels given these parameters. In contrast, rats administered CCK had CSF-insulin levels that were more than twice as high as those of control rats. We conclude that circulating CCK greatly facilitates the transport of insulin into the brain, likely by acting directly at the BBB. These findings imply that in circumstances in which the plasma levels of both CCK and insulin are elevated, such as during and soon after meals, satiation is likely to be due, in part, to this newly-discovered synergy between CCK and insulin.

Regulation of the Motivation to Eat.

Although food intake is necessary to provide energy for all bodily activities, considering food intake as a motivated behavior is complex. Rather than being a simple unconditioned reflex to energy need, eating is mediated by diverse factors. These include homeostatic signals such as those related to body fat stores, to food available and being eaten, and to circulating energy-rich compounds like glucose and fatty acids. Eating is also greatly influenced by non-homeostatic signals that convey information related to learning and experience, hedonics, stress, the social situation, opportunity, and many other factors. Recent developments identifying the intricate nature of the relationships between homeostatic and non-homeostatic influences significantly add to the complexity underlying the neural basis of the motivation to eat. The future of research in the field of food intake would seem to lie in the identification of the neural circuitry and interactions between homeostatic and non-homeostatic influences.

Moderate voluntary exercise attenuates the metabolic syndrome in melanocortin-4 receptor-deficient rats showing central dopaminergic dysregulation.

OBJECTIVE: Melanocortin-4 receptors (MC4Rs) are highly expressed by dopamine-secreting neurons of the mesolimbic tract, but their functional role has not been fully resolved. Voluntary wheel running (VWR) induces adaptations in the mesolimbic dopamine system and has a myriad of long-term beneficial effects on health. In the present experiments we asked whether MC4R function regulates the effects of VWR, and whether VWR ameliorates MC4R-associated symptoms of the metabolic syndrome. METHODS: Electrically evoked dopamine release was measured in slice preparations from sedentary wild-type and MC4R-deficient Mc4r (K314X) (HOM) rats. VWR was assessed in wild-type and HOM rats, and in MC4R-deficient loxTB (Mc4r) mice, wild-type mice body weight-matched to loxTB (Mc4r) mice, and wild-type mice with intracerebroventricular administration of the MC4R antagonist SHU9119. Mesolimbic dopamine system function (gene/protein expression) and metabolic parameters were examined in wheel-running and sedentary wild-type and HOM rats. RESULTS: Sedentary obese HOM rats had increased electrically evoked dopamine release in several ventral tegmental area (VTA) projection sites compared to wild-type controls. MC4R loss-of-function decreased VWR, and this was partially independent of body weight. HOM wheel-runners had attenuated markers of intracellular D1-type dopamine receptor signaling despite increased dopamine flux in the VTA. VWR increased and decreased ΔFosB levels in the nucleus accumbens (NAc) of wild-type and HOM runners, respectively. VWR improved metabolic parameters in wild-type wheel-runners. Finally, moderate voluntary exercise corrected many aspects of the metabolic syndrome in HOM runners. CONCLUSIONS: Central dopamine dysregulation during VWR reinforces the link between MC4R function and molecular and behavioral responding to rewards. The data also suggest that exercise can be a successful lifestyle intervention in MC4R-haploinsufficient individuals despite reduced positive reinforcement during exercise training.