Developmental changes in synaptic distribution in arcuate nucleus neurons.

Neurons coexpressing neuropeptide Y, agouti-related peptide, and GABA (NAG) play an important role in ingestive behavior and are located in the arcuate nucleus of the hypothalamus. NAG neurons receive both GABAergic and glutamatergic synaptic inputs, however, the developmental time course of synaptic input organization of NAG neurons in mice is unknown. In this study, we show that these neurons have low numbers of GABAergic synapses and that GABA is inhibitory to NAG neurons during early postnatal period. In contrast, glutamatergic inputs onto NAG neurons are relatively abundant by P13 and are comparatively similar to the levels observed in the adult. As mice reach adulthood (9-10 weeks), GABAergic tone onto NAG neurons increases. At this age, NAG neurons received similar numbers of inhibitory and EPSCs. To further differentiate age-associated changes in synaptic distribution, 17- to 18-week-old lean and diet-induced obesity (DIO) mice were studied. Surprisingly, NAG neurons from lean adult mice exhibit a reduction in the GABAergic synapses compared with younger adults. Conversely, DIO mice display reductions in the number of GABAergic and glutamatergic inputs onto NAG neurons. Based on these experiments, we propose that synaptic distribution in NAG neurons is continuously restructuring throughout development to accommodate the animals' energy requirements.

Developmental switch of leptin signaling in arcuate nucleus neurons.

Leptin is well known for its role in the regulation of energy homeostasis in adults, a mechanism that at least partially results from the inhibition of the activity of NPY/AgRP/GABA neurons (NAG) in the arcuate nucleus of the hypothalamus (ARH). During early postnatal development in the rodent, leptin promotes axonal outgrowth from ARH neurons, and preautonomic NAG neurons are particularly responsive to leptin's trophic effects. To begin to understand how leptin could simultaneously promote axonal outgrowth from and inhibit the activity of NAG neurons, we characterized the electrochemical effects of leptin on NAG neurons in mice during early development. Here, we show that NAG neurons do indeed express a functional leptin receptor throughout the early postnatal period in the mouse; however, at postnatal days 13-15, leptin causes membrane depolarization in NAG neurons, rather than the expected hyperpolarization. Leptin action on NAG neurons transitions from stimulatory to inhibitory in the periweaning period, in parallel with the acquisition of functional ATP-sensitive potassium channels. These findings are consistent with the idea that leptin provides an orexigenic drive through the NAG system to help rapidly growing pups meet their energy requirements.

Leptin stimulates neuropeptide Y and cocaine amphetamine-regulated transcript coexpressing neuronal activity in the dorsomedial hypothalamus in diet-induced obese mice.

Neuropeptide Y (NPY) neurons in both the arcuate nucleus of the hypothalamus (ARH) and the dorsomedial hypothalamus (DMH) have been implicated in food intake and obesity. However, while ARH NPY is highly expressed in the lean animal, DMH NPY mRNA expression is observed only after diet-induced obesity (DIO). Furthermore, while ARH NPY neurons are inhibited by leptin, the effect of this adipokine on DMH NPY neurons is unknown. In this study we show that in contrast to the consistent expression in the ARH, DMH NPY mRNA expression was undetectable until after 10 weeks in mice fed a high-fat diet, and peaked at 20 weeks. Surprisingly, electrophysiological experiments demonstrated that leptin directly depolarized and increased the firing rate of DMH NPY neurons in DIO mice. To further differentiate the regulation of DMH and ARH NPY populations, fasting decreased expression of DMH NPY expression, while it increased ARH NPY expression. However, treatment with a leptin receptor antagonist failed to alter DMH NPY expression, indicating that leptin may not be the critical factor regulating mRNA expression. Importantly, we also demonstrated that DMH NPY neurons coexpress cocaine amphetamine-regulated transcript (CART); however, CART mRNA expression in the DMH peaked earlier in the progression of DIO. This study demonstrates novel and important findings. First, NPY and CART are coexpressed in the same neurons within the DMH, and second, leptin stimulates DMH NPY neurons. These studies suggest that during the progression of DIO, there is an unknown signal that drives the expression of the orexigenic NPY signal within the DMH, and that the chronic hyperleptinemia increases the activity of these NPY/CART neurons.

Endogenous kisspeptin tone is a critical excitatory component of spontaneous GnRH activity and the GnRH response to NPY and CART.

BACKGROUND/AIMS: Kisspeptin is the major excitatory regulator of gonadotropin-releasing hormone (GnRH) neurons and is responsible for basal GnRH/LH release and the GnRH/LH surge. Although it is widely assumed, based on mutations in kisspeptin and Kiss1R, that kisspeptin acts to sustain basal GnRH neuronal activity, there have been no studies to investigate whether endogenous basal kisspeptin tone plays a direct role in basal spontaneous GnRH neuronal excitability. It is also of interest to examine possible interactions between endogenous kisspeptin tone and other neuropeptides that have direct effects on GnRH neurons, such as neuropeptide Y (NPY) or cocaine- and amphetamine-regulated transcript (CART), since the activity of all these neuropeptides changes during states of negative energy balance. METHODS: Loose cell-attached and whole-cell current patch-clamp recordings were made from GnRH-GFP neurons in hypothalamic slices from female and male rats. RESULTS: Kisspeptin activated GnRH neurons in a concentration-dependent manner with an EC50 of 3.32 ± 0.02 nM. Surprisingly, a kisspeptin antagonist, Peptide 347, suppressed spontaneous activity in GnRH neurons, demonstrating the essential nature of the endogenous kisspeptin tone. Furthermore, inhibition of endogenous kisspeptin tone blocked the direct activation of GnRH cells that occurs in response to antagonism of NPY Y5 receptor or by CART. CONCLUSIONS: Our electrophysiology studies suggest that basal endogenous kisspeptin tone is not only essential for spontaneous GnRH neuronal firing, but it is also required for the net excitatory effects of other neuropeptides, such as CART or NPY antagonism, on GnRH neurons. Therefore, endogenous kisspeptin tone could serve as the linchpin in GnRH activation or inhibition.

Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates.

Maternal obesity is thought to increase the offspring's risk of juvenile obesity and metabolic diseases; however, the mechanism(s) whereby excess maternal nutrition affects fetal development remain poorly understood. Here, we investigated in nonhuman primates the effect of chronic high-fat diet (HFD) on the development of fetal metabolic systems. We found that fetal offspring from both lean and obese mothers chronically consuming a HFD had a 3-fold increase in liver triglycerides (TGs). In addition, fetal offspring from HFD-fed mothers (O-HFD) showed increased evidence of hepatic oxidative stress early in the third trimester, consistent with the development of nonalcoholic fatty liver disease (NAFLD). O-HFD animals also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors. Furthermore, fetal glycerol levels were 2-fold higher in O-HFD animals than in control fetal offspring and correlated with maternal levels. The increased fetal hepatic TG levels persisted at P180, concurrent with a 2-fold increase in percent body fat. Importantly, reversing the maternal HFD to a low-fat diet during a subsequent pregnancy improved fetal hepatic TG levels and partially normalized gluconeogenic enzyme expression, without changing maternal body weight. These results suggest that a developing fetus is highly vulnerable to excess lipids, independent of maternal diabetes and/or obesity, and that exposure to this may increase the risk of pediatric NAFLD.

Chronic consumption of a high-fat diet during pregnancy causes perturbations in the serotonergic system and increased anxiety-like behavior in nonhuman primate offspring.

Childhood obesity is associated with increased risk of behavioral/psychological disorders including depression, anxiety, poor learning, and attention deficient disorder. As the majority of women of child-bearing age are overweight or obese and consume a diet high in dietary fat, it is critical to examine the consequences of maternal overnutrition on the development of brain circuitry that regulates offspring behavior. Using a nonhuman primate model of diet-induced obesity, we found that maternal high-fat diet (HFD) consumption caused perturbations in the central serotonergic system of fetal offspring. In addition, female infants from HFD-fed mothers exhibited increased anxiety in response to threatening novel objects. These findings have important clinical implications as they demonstrate that exposure to maternal HFD consumption during gestation, independent of obesity, increases the risk of developing behavioral disorders such as anxiety.

Perinatal exposure to high-fat diet programs energy balance, metabolism and behavior in adulthood.

The perinatal environment plays an important role in programming many aspects of physiology and behavior including metabolism, body weight set point, energy balance regulation and predisposition to mental health-related disorders such as anxiety, depression and attention deficit hyperactivity disorder. Maternal health and nutritional status heavily influence the early environment and have a long-term impact on critical central pathways, including the melanocortinergic, serotonergic system and dopaminergic systems. Evidence from a variety of animal models including rodents and nonhuman primates indicates that exposure to maternal high-fat diet (HFD) consumption programs offspring for increased risk of adult obesity. Hyperphagia and increased preference for fatty and sugary foods are implicated as mechanisms for the increased obesity risk. The effects of maternal HFD consumption on energy expenditure are unclear, and future studies need to address the impact of perinatal HFD exposure on this important component of energy balance regulation. Recent evidence from animal models also indicates that maternal HFD consumption increases the risk of offspring developing mental health-related disorders such as anxiety. Potential mechanisms for perinatal HFD programming of neural pathways include circulating factors, such as hormones (leptin, insulin), nutrients (fatty acids, triglycerides and glucose) and inflammatory cytokines. As maternal HFD consumption and obesity are common and rapidly increasing, we speculate that future generations will be at increased risk for both metabolic and mental health disorders. Thus, it is critical that future studies identify therapeutic strategies that are effective at preventing maternal HFD-induced malprogramming.

Suppression of basal spontaneous gonadotropin-releasing hormone neuronal activity during lactation: role of inhibitory effects of neuropeptide Y.

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 +/- 2.86 vs. 7.04 +/- 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 +/- 0.02 vs. 0.37 +/- 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from -56.7 +/- 1.94 to -62.1 +/- 1.83 mV; NPY's effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity.

Characterization of brainstem peptide YY (PYY) neurons.

Peptide YY (PYY), a member of the NPY superfamily of peptides, is predominantly synthesized by the colon and is thought to act on both the gut and brain to modulate energy homeostasis. Although neurons expressing PYY mRNA have also been reported in the brainstem, little is known about their physiological role and study of their projections has been problematic due to crossreactivity of PYY antibodies with NPY. In the present study we examined the localization of central PYY cell bodies in the mouse, rat, and monkey. In addition, efferent projections and afferent inputs of central PYY neurons were examined in rodents. Central PYY projections were examined by immunohistochemistry in the NPY knockout mouse, or with an NPY-preabsorbed PYY antibody in the rat to avoid any crossreactivity with NPY. In all species investigated PYY-immunoreactive (ir) cell bodies were localized exclusively to the gigantocellular reticular nucleus (Gi) of the rostral medulla. The highest density of PYY fibers was present within the solitary tract nucleus, specifically within the dorsal and lateral aspects. PYY fibers were also concentrated within the dorsal motor nucleus of the vagus and the hypoglossal nucleus. In addition, both orexin and melanin-concentrating hormone fibers made numerous close appositions with PYY cell bodies in the Gi. Collectively, the projection pattern and association with orexigenic neuropeptides suggest that brainstem PYY neurons may play a role in energy homeostasis through a coordinated effect on visceral, motor, and sympathetic output targets.

Melanocortinergic activation by melanotan II inhibits feeding and increases uncoupling protein 1 messenger ribonucleic acid in the developing rat.

The hypothalamic neurocircuitry that regulates energy homeostasis in adult rats is not fully developed until the third postnatal week. In particular, fibers from the hypothalamic arcuate nucleus, including both neuropeptide Y (NPY) and alpha-MSH fibers, do not begin to innervate downstream hypothalamic targets until the second postnatal week. However, alpha-MSH fibers from the brainstem and melanocortin receptors are present in the hypothalamus at birth. The present study investigated the melanocortin system in the early postnatal period by examining effects of the melanocortin receptor agonist melanotan II (MTII) on body weight, energy expenditure, and hypothalamic NPY expression. Rat pups were injected ip with MTII (3 mg/kg body weight) or saline on postnatal day (P) 5 to P6, P10-P11, or P15-P16 at 1700 and 0900 h and then killed at 1300 h. Stomach weight and brown adipose tissue uncoupling protein 1 mRNA were determined. In addition, we assessed central c-Fos activation 90 min after MTII administration and hypothalamic NPY mRNA after twice daily MTII administration from P5-P10 or P10-P15. MTII induced hypothalamic c-Fos activation as well as attenuating body weight gain in rat pups. Stomach weight was significantly decreased and uncoupling protein 1 mRNA was increased at all ages, indicating decreased food intake and increased energy expenditure, respectively. However, MTII had no effect on NPY mRNA levels in any hypothalamic region. These findings demonstrate that MTII can inhibit food intake and stimulate energy expenditure before the full development of hypothalamic feeding neurocircuitry. These effects do not appear to be mediated by changes in NPY expression.

Excess weight gain during the early postnatal period is associated with permanent reprogramming of brown adipose tissue adaptive thermogenesis.

Excess weight gain during the early postnatal period increases the risk of persistent obesity into adulthood and impacts on the subsequent risk for metabolic and cardiovascular diseases. The current study investigated the long-term effect of early excess weight gain, through reduced nursing litter size, on body weight regulation and its relation to brown adipose tissue (BAT) thermogenesis. Animals raised in a small litter (SL, three pups per litter) were compared with those raised in a normal litter size (NL, eight pups per litter). BAT from young adult NL and SL rats, maintained under either ambient or cold conditions, were used for gene expression, morphological, and functional analysis. Compared with NL, SL rats showed excess weight gain, and adult SL animals had a reduced thermogenic capacity as displayed by lower levels of uncoupling protein 1 (UCP1). When exposed to cold, BAT from SL rats was less active and demonstrated reduced responsiveness to cold. Furthermore, reduction in transcript abundance of several lipid lipases and transcriptional regulators was observed in SL rats either at ambient temperature or under cold conditions. Finally, the expression of sympathetic beta 3-adrenergic receptor and the response to the sympathetic receptor agonist isoproterenol were decreased in SL rats. Overall, these observations provide the first evidence that postnatal excess weight gain results in abnormalities in BAT thermogenesis and sympathetic outflow, which likely increases susceptibility to obesity in adulthood.

GLP-1R Signaling Directly Activates Arcuate Nucleus Kisspeptin Action in Brain Slices but Does not Rescue Luteinizing Hormone Inhibition in Ovariectomized Mice During Negative Energy Balance.

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status, and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide 1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. Because GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased after a 48-h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin(9-39), in ad libitum-fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Melanocortin 4 receptor-mediated hyperphagia and activation of neuropeptide Y expression in the dorsomedial hypothalamus during lactation.

In several hyperphagic models, including lactation, in which hypothalamic melanocortin signaling is reduced, a novel expression of NPY mRNA in the dorsomedial hypothalamus (DMH) has been observed, suggesting that melanocortin signaling and the induced NPY in the DMH may constitute unique neurocircuitry in mediating energy balance. Using lactating rats as a model, the present study first showed that in the DMH abundant alpha-MSH and agouti-related protein fibers are in close apposition to NPY-positive cells. However, no NPY and MC4R (a melanocortin receptor) double-labeled neurons were observed. These data suggested that melanocortin input may synapse on presynaptic terminals that then synapse on DMH NPY cells. To study the function of DMH MC4Rs in energy balance, an MC3/4R-selective agonist, melanotan II (MTII), was injected bilaterally into the DMH. MTII injection significantly suppressed feeding induced by 24 hr fasting or suckling-induced hyperphagia. Furthermore, MTII treatment greatly attenuated suckling-induced NPY expression in the DMH. MTII treatment also stimulated uncoupling protein 1 activity in the brown adipose tissue of suckling female rats, indicative of increased sympathetic outflow. In summary, the present study demonstrated that the melanocortin system in the DMH not only plays an important role in inducing NPY expression in the DMH of lactating rats but also in regulating energy homeostasis, at least in part, by modulating appetite and energy expenditure.

Orexin neurons express a functional pancreatic polypeptide Y4 receptor.

The receptor subtypes that mediate the effects of neuropeptide Y (NPY) on food intake have not been clearly defined. The NPY Y4 receptor has been identified recently as a potential mediator of the regulation of food intake. The purpose of the present study was to characterize the central site of action of the Y4 receptor using a combination of neuroanatomical and physiological approaches. Using immunocytochemistry, Y4-like immunoreactivity was found to be colocalized with orexin cell bodies in the lateral hypothalamic area (LHA) and orexin fibers throughout the brain. In situ hybridization confirmed the expression of Y4 mRNA in orexin neurons. To determine the functional interaction between Y4 receptors and orexin neurons, we examined the effects of rat pancreatic polypeptide (rPP), a Y4-selective ligand, or NPY, a nonselective ligand, administered directly into the LHA on the stimulation of food and water intake and c-Fos expression. Both rPP and NPY significantly increased food and water intake when they were administered into the LHA, although NPY was a more potent stimulator of food intake. Furthermore, both NPY and rPP significantly stimulated c-Fos expression in the LHA. However, whereas rPP stimulated c-Fos expression in orexin neurons, NPY did not. Neither rPP nor NPY stimulated c-Fos in melanin-concentrating hormone neurons, but both activated neurons of an unknown phenotype in the LHA. These results suggest that a functional Y4 receptor is expressed on orexin neurons and that these neurons are activated in response to a ligand with high affinity for the Y4 receptor (rPP). Although these data suggest a role for central Y4 receptors, the endogenous ligand for this receptor has yet to be clearly established.

Deoxyribonucleic acid microarray analysis of gene expression pattern in the arcuate nucleus/ventromedial nucleus of hypothalamus during lactation.

Lactation is characterized by extreme hyperphagia and negative energy balance resulting from a large energy drain due to milk production and by a suppression of cyclic ovarian function. Increases in neuropeptide Y and agouti-related protein and a decrease in proopiomelanocortin expression in the arcuate nucleus of hypothalamus (ARH) may contribute to the hyperphagia to maintain energy balance and to the suppression of LH secretion associated with lactation. However, little is known about the full extent of neuroendocrine changes in the ARH that may contribute to the various adaptations occurring during lactation. To address this issue, we used Affymetrix microarray to acquire a reliable profile of the lactation-induced transcriptional changes in micropunches containing the ARH and a portion of the ventromedial nucleus of the hypothalamus. Using high stringency criteria, 12 genes were identified as being differentially regulated during lactation, and an additional 10 genes and three transcribed sequences were identified using moderate stringency criteria. Changes in neuropeptide Y, enkephalin, tyrosine hydroxylase, and dynorphin, genes previously shown to be differentially regulated during lactation, provide validation for the microarray analysis. New genes identified as being differentially expressed include those related to neurotransmission, growth factors, signal transduction, and structure remodeling. These data identify new genes in ARH/ventromedial nucleus of the hypothalamus that may play an important role in the adaptations of lactation related to hyperphagia, milk production, and the suppression of cyclic reproductive function and may contribute to elucidating a framework for integrating changes in energy intake with the regulation of reproductive function during lactation.

Melanin concentrating hormone (MCH): a novel neural pathway for regulation of GnRH neurons.

The link between the state of energy balance and reproductive function is well known. Thus, signals denoting negative energy balance and the accompanying hyperphagic drive are likely to be factors in the suppression of gonadotropin releasing hormone (GnRH) activity. We have previously found that appetite-regulating systems, such as neuropeptide Y (NPY) in the arcuate nucleus (ARH) and orexin in the lateral hypothalamic area (LHA), send fiber projections that come in close apposition with GnRH neurons. Furthermore, the appropriate receptors, NPY Y5 and OR-1, respectively, are coexpressed on GnRH neurons, providing neuroanatomical evidence for a direct link between the NPY and orexin systems and GnRH neurons. Therefore, these orexigenic neuropeptide systems are potential candidates that convey information about energy balance to GnRH neurons. The current studies focused on melanin concentrating hormone (MCH), another orexigenic neuropeptide system located in the LHA that is sensitive to energy balance. The results showed that MCH fiber projections came in close apposition with approximately 85-90% of GnRH cell bodies throughout the preoptic area and anterior hypothalamic area in the rat. In addition, the MCH receptor (MCHR1) was coexpressed on about 50-55% of GnRH neurons. These findings present evidence for a possible direct neuroanatomical pathway by which MCH may play a role in the regulation of GnRH neuronal function. Thus, MCH is another potential signal that may serve to integrate energy balance and reproductive function.

Development of metabolic systems.

In the normal adult rodent and primate, arcuate nucleus (ARH) neurons function as conduits for transmitting metabolic hormonal signals into the hypothalamic circuitry that modulates feeding and energy expenditure. We and others have shown that ARH projections do not fully develop until the 3rd postnatal week in the rodent. This is in stark contrast to the nonhuman primate (NHP) in which ARH projections develop during the 3rd trimester of pregnancy. This species difference suggests that maternal diet and health are likely key factors for the development of ARH projections in the primate, whereas the postnatal environment (i.e., diet) would be more important in the rodent. Furthermore, pertubations in these circuits during critical periods of development may have long-term consequences on feeding behavior and body weight management. Our group has used a rat model of overfeeding and underfeeding specifically during the postnatal period to begin to investigate the metabolic adaptions that may cause developmental abnormalities in the hypothalamic circuitry. While the overfed animals become obese as adults and the underfed maintain a lean phenotype, both models display low basal insulin and IGFII levels as adults. Furthermore, both models have abnormal expression of several key genes in peripheral metabolic tissue that are suggestive of changes in sympathetic outflow. Human studies show that gestational diabetes can also contribute to the development of obesity and diabetes in children; however, the mechanism is unknown. Since the critical periods for the development of hypothalamic circuits are different between rodents and primates our group has begun studies using NHP model to determine if maternal obesity/diabetes causes abnormalities in the development of metabolic systems, including the brain, in the offspring. To do this we have placed female NHPs on either a control diet or a high fat/calorie diet to induce obesity and diabetes. We have characterized the onset of insulin resistance and hyperleptinemia in these animals over the last 2(1/2) years and have collected offspring. Ongoing studies will investigate the metabolic abnormalities in these offspring.

Maternal high-fat diet and obesity impact palatable food intake and dopamine signaling in nonhuman primate offspring.

OBJECTIVE: To utilize a nonhuman primate model to examine the impact of maternal high-fat diet (HFD) consumption and pre-pregnancy obesity on offspring intake of palatable food and to examine whether maternal HFD consumption impaired development of the dopamine system, critical for the regulation of hedonic feeding. METHODS: The impact of exposure to maternal HFD and obesity on offspring consumption of diets of varying composition was assessed after weaning. The influence of maternal HFD consumption on the development of the prefrontal cortex-dopaminergic system at 13 months of age was also examined. RESULTS: During a preference test, offspring exposed to maternal HFD consumption and obesity displayed increased intake of food high in fat and sugar content relative to offspring from lean control mothers. Maternal HFD consumption suppressed offspring dopamine signaling (as assessed by immunohistochemistry) relative to control offspring. Specifically, there was decreased abundance of dopamine fibers and of dopamine receptor 1 and 2 proteins. CONCLUSIONS: This study reveals that offspring exposed to both maternal HFD consumption and maternal obesity during early development are at increased risk for obesity due to overconsumption of palatable energy-dense food, a behavior that may be related to reduced central dopamine signaling.

Regulation of hypothalamic neuropeptide Y messenger ribonucleic acid expression during lactation: role of prolactin.

In the present study, we investigated the role of prolactin (PRL) in the suckling-induced increase in hypothalamic neuropeptide Y (NPY) gene expression in the dorsomedial nucleus of the hypothalamus (DMH) and the caudal portion of the arcuate nucleus of the hypothalamus (ARH-C). Lactating rats were deprived of their eight-pup litters on d 9 postpartum. After 48 h, the animals were randomly divided into two groups: nonsuckled controls and eight pups suckling for 24 h. In addition, some of the suckled animals received two injections of bromocriptine (0.5 mg/rat per injection) to inhibit suckling-induced PRL secretion. Some bromocriptine-treated rats also received ovine PRL (1 mg/rat per injection). In situ hybridization was performed to measure NPY mRNA levels. Suckling for 24 h induced a significant increase in NPY mRNA levels in the DMH and ARH-C. Bromocriptine treatment greatly attenuated the increase of NPY mRNA in the DMH but not in the ARH. Injections of ovine PRL in bromocriptine-treated rats greatly restored DMH NPY mRNA levels but had no additional effects on the ARH NPY expression. Double-label in situ hybridization for NPY and PRL receptor (PRL-R) in the lactating rat brains showed that NPY-positive neurons in the DMH also express PRL-R mRNA. On the contrary, few ARH NPY neurons expressed PRL-R. These data suggest that PRL could act directly on DMH NPY neurons to modulate NPY gene expression during lactation. Thus, the results from the present study demonstrate that NPY neurons in the DMH and ARH are differentially regulated by PRL during lactation.

Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers.

The orexins are produced in neurons of the lateral hypothalamic area and implicated in the regulation of both feeding and reproductive function. Orexins stimulate LH secretion in steroid-primed ovariectomized female rats and suppress LH secretion in nonprimed ovariectomized rats. The aim of the present study was to characterize the neuroanatomical pathway by which orexin might modulate LH secretion in the rat. Using double- and triple-label immunofluorescence coupled with confocal microscopy, we found that 75-85% of GnRH neurons were contacted by orexin fibers, and triple labeling with synaptophysin provided additional confirmation of close contacts. Furthermore, about 85% of GnRH neurons were colocalized with the orexin receptor 1 (OX-R1), and the OX-R1-expressing GnRH neurons were contacted by orexin terminals, providing the basis for a functional neuroanatomical pathway. GnRH nerve terminals in the median eminence, however, do not express OX-R1. An additional study investigated the coexpression of neuropeptide Y Y4-like receptors and orexin fibers in relation to GnRH neurons. There is evidence that Y4 receptor stimulation results in LH release, and studies from our laboratory show Y4-like immunoreactivity in the majority of orexin cell bodies in the lateral hypothalamic area and some orexin fibers scattered throughout the hypothalamus. The present study found that, although Y4-positive orexin fibers are in present in the area of GnRH neurons, they never come in close contact with GnRH neurons. Together, these data suggest that Y4 receptor modulation of LH release is likely to be indirect through orexin cell bodies and that orexin modulates GnRH neurons directly via OX-R1.