Developmental changes within the hypothalamus are necessary for maturation of the reproductive neuroendocrine axis. Recent reports have implicated several neuronal networks in this process, but genes involved in their regulation have not been elucidated. Using a well-established model for nutritional induction of precocious puberty, objectives were to 1) use microarray technology to examine changes in gene expression within the arcuate nucleus (ARC) of the hypothalamus in pre-pubertal heifers fed high or low-concentrate diets, and 2) determine if high-concentrate diets are required for nutritional induction of precocious puberty. In Experiment 1, early-weaned, cross-bred heifers were fed either a high-forage/low-gain (HF/LG; 0.45 kg/d) or a highconcentrate/ high-gain (HC/HG; 0.91 kg/d) diet for 91 d. Analysis of microarray data indicated that 346 genes were differentially expressed (P < 0.05) between HC/HG and HF/LG heifers. Expression of three key metabolic genes [neuropeptide Y (NPY), agoutirelated protein (AGRP), and growth hormone receptor (GHR)] observed to be differentially expressed in the microarray analysis was investigated further by quantitative PCR. Real-time RT-PCR indicated that expression of NPY, AGRP and GHR was lower (P < 0.05) in HC/HG compared to HF/LG heifers. In contrast, concentrations of insulin (P < 0.05), IGF-1 (P < 0.002) and leptin (P = 0.1) were greater in HC/HG compared to HF/LG. For Experiment 2, 48 heifers were used in 2 replicates (24 heifers/replicate) in a 2 x 2 factorial design to examine the roles of diet type (HF vs HC) and rate of gain (LG, 0.45 kg/d vs HG, 0.91 kg/d) on age at puberty. Heifers were fed HC/HG, HC/LG, HF/HG or HF/LG (n = 12/group) for 14 wk, and then switched to a common growth diet (0.68 kg/d) until puberty. Heifers in both HG groups reached puberty at a younger age (54.5 ± 1.8 wk) than heifers in both LG groups (60.2 ± 1.9 wk; P < 0.04). A marked increase (P < 0.01) in serum concentrations of leptin occurred in HC/HG heifers between 24 and 30 wk of age. This increase in circulating leptin was not observed in other groups. Overall, results indicate that nutritional regulation of reproductive neuroendocrine development involves the control of NPY, AGRP and GHR expression. The abrupt increase noted for circulating leptin in heifers fed HC/HG diets, if timed and sustained appropriately, could represent an important temporal cue for activation of the neuroendocrine system and the onset of puberty.

Distribution of orexin-A-like immunoreactivity (ORX-LI) in rat brains was investigated with the use of a rabbit polyclonal antibody against the full length peptide orexin A. Virtually all the ORX-LI cell bodies were observed in the lateral hypothalamus at the level of median eminence. The large majority of ORX-LI neurons appeared spherical or fusiform, 20-30 μm in diameter and issued two to five cell processes with few secondary branchings. Numerous ORX-LI fibers were observed in subregions of the hypothalamus. ORX- LI cell processes were sparsely distributed in the cortex, hippocampus and thalamus. Many varicose ORX-LI cell processes were situated close to the 3rd and lateral ventricles, some of which appeared to be protruding into the lumen. As a corollary, orexin A may be released into the ventricles and interact with neurons in distant targets, in addition to influencing the activity of neurons with which ORX-LI axons make synaptic contacts.

Understanding how appetite is regulated, via exogenous or endogenous factors, is essential to animal agriculture in order to maximize production capabilities, as well as in human medicine to generate ways to treat conditions such as eating disorders or obesity. The aim of this thesis was to evaluate the effects of ferulic acid (FA), an exogenous factor found within plant cells, and oxyntomodulin (OXM), an endogenous hormone generated in the gastrointestinal tract, on food intake in avian models, as well as elucidate the hypothalamic mechanisms responsible. In broiler chicks (Gallus gallus), FA administered peripherally (IP) resulted in a transient yet potent reduction of food intake. A behavior analysis revealed that FA-treated chicks defecated fewer times than control birds. Within the arcuate nucleus (ARC) there was an increase in c-Fos immunoreactivity, indicating neuronal activation, in FA-treated chicks. Within the hypothalamus, there was a decrease in mRNA abundance of galanin, ghrelin, melanocortin receptor 3, and pro-opiomelanocortin (POMC), however within the ARC there was a decrease in POMC and an increase in c-Fos mRNA after FA treatment. OXM, a proglucagon-derived peptide produced in the gastrointestinal tract, administered intracerebroventricularly (ICV) or IP in Japanese quail (Coturnix japonica), resulted in a decrease in food intake for 3 hours post-injection. There was an increase in c-Fos immunoreactivity within the ARC as well as the dorsomedial nucleus (DMN) in quail ICV injected with OXM. In conclusion, these novel data provide insights on the similarities and differences between factors that can affect appetite regulation via anorexigenic effects. / Master of Science / Exogenous and endogenous factors affect appetite regulation. Exogenous factors originate in feed components, additives, and other environmental factors that can affect bodily functions but are derived from an external source. Endogenous factors are made within the body, such as hormones and neurotransmitters, usually in response to a stimulus, and serve to communicate signals both locally and distantly in the body. Ferulic acid (FA), a natural exogenous factor originating within plant cells, is found in commonly consumed plant-based foods. When administered peripherally into broiler chicks (meat-type birds), FA caused a direct and potent, yet quickly diminishing, decrease in food intake via activation of cells within the hypothalamus, the region of the brain that is responsible for appetite regulation. Oxyntomodulin (OXM), an endogenous peptide hormone generated within the gastrointestinal tract in response to the digestion of nutrients, is known to decrease food intake in humans, rodents, and the broiler chick. However, its effects in Japanese quail, a model closer to a "wild-type" bird, are unknown. Quail injected peripherally (outside the brain) or intracerebroventricularly (ICV; into lateral ventricle of brain) with OXM showed a reduction in food intake that was more persistent than FA's effects with the effects also mediated via activation within the hypothalamus, although through slightly different molecular mechanisms. Understanding different factors that can regulate appetite in animals is necessary for agricultural applications to maximize production and improve health and welfare, as well as in humans to elucidate methods to treat appetite-related conditions, such as eating disorders and obesity.

Domestication in chickens has made feed-restriction a necessity if broiler breeder hens should reach sexual maturity and be fertile. This is claimed to cause chronic hunger. To measure hunger the gene expression of the appetite regulators agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and adenosine monophosphate-activated protein kinase (AMPK) of the melanocortin system was quantified with qPCR. This was done in feed-restricted Red Junglefowl and compared with the gene expression of two strains of feed-restricted broilers, Ross 308 and Rowan Ranger, to detect possible effects on domestication on appetite regulation. POMC-expression was upregulated 2-fold in the feed-restricted Red Junglefowl. POMC-expression was downregulated by half in the feed-restricted Ross 308. AgRP/NPY-expression was upregulated 4-fold in feed-restricted Rowan Rangers. A comparison between the control groups (ad libitum fed) of the breeds showed that the NPY-expression was lower in Ross 308 and Rowan Ranger compared with the ancestor. Results show no difference in body weight of ad libitum fed and feed-restricted Red Junglefowl. Conclusions were that the feed-restricted Red Junglefowl was not properly restricted in food supply since no difference in body weight between the treatment groups was detected. The upregulation of POMC in the feed-restricted Red Junglefowl could be stress-linked influenced by the feeding type (scattering of food in litter). No conclusions of the impact of domestication on chicken’s appetite could be drawn. Domestication has probably had its impact by altering other signaling pathways of the melanocortin system than in the arcuate nucleus.

Leptin is an adipokine that acts centrally to regulate feeding behaviour, energy expenditure and autonomic function via activation of its receptor (ObRb) in nuclei in the central nervous system (CNS). This thesis investigates the involvement of two sensory circumventricular organs (CVOs), the subfornical organ (SFO) and area postrema (AP), in mediating the central effects of leptin using a variety of experimental approaches. We first show that acute electrical stimulation of the SFO elicits feeding in satiated rats, supporting a role for this specialized CNS structure in the control of food intake. We then demonstrate, using RT-PCR, the presence of ObRb mRNA in SFO and, using whole cell current clamp electrophysiology, reveal that leptin influences the excitability of individual SFO neurons, causing both excitatory and inhibitory responses. Furthermore, we find that leptin activates the same SFO neurons activated by amylin. Given the association between obesity and hypertension and the well-established role of the SFO in cardiovascular regulation, we show that leptin microinjection into the SFO decreases blood pressure in young rats, effects that are abolished in leptin-resistant, diet induced obese rats, suggesting that leptin-insensitivity in the SFO of obese, leptin-resistant, individuals may contribute to obesity-related hypertension. Our studies also show that the medullary AP expresses ObRb and that leptin influences the excitability of AP neurons. Furthermore, we show that leptin and amylin act on the same subpopulation of neurons in the AP. Finally, our preliminary AP/SFO lesion studies reveal that animals with these lesions exhibit a profound decrease in body weight and food intake and no longer exhibit decreases in body weight in response to peripheral leptin administration. In summary, the data presented in this thesis suggest the SFO and AP to be important in body weight homeostasis and in mediating the central effects of leptin. In addition, these areas appear to be important in the integration of multiple signals derived from peripheral sources. Furthermore, the fact that the SFO appears to be involved in leptin effects on both energy balance and cardiovascular regulation attest to the integrative nature of the SFO in the control of diverse physiological functions. / Thesis (Ph.D, Physiology) -- Queen's University, 2012-10-15 14:57:15.387

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.

Le kisspeptine est un peptide, encodé par le gène Kiss1, qui joue un rôle majeur dans le contrôle central de la fonction de reproduction en régulant la sécrétion du GnRH après la naissance. Les neurones exprimant Kiss1 apparaissent avant la naissance exclusivement dans le noyau arqué. Les objectifs de cette thèse étaient de décrire comment le système s‟organise avant la naissance en fonction du sexe à une période où il existe chez le mâle un pic prénatal de testostérone, puis de déterminer si cette organisation pouvait être influencée par les stéroïdes sexuels. Nos résultats ont permis 1) de caractériser précisément chez la souris la mise en place des cellules kisspeptine en fonction du sexe et d‟identifier un gradient de différenciation antéro-postérieur sexe-indépendant 2) de mettre en évidence une interaction réciproque avec les neurones à GnRH, chez les mâles et les femelles, suggérant ainsi une certaine maturité du système 3) de montrer la diversité des récepteurs aux stéroïdes sexuels déjà exprimés par ces cellules avant la naissance ainsi que l‟émergence d‟un dimorphisme sexuel 4) de démontrer la sensibilité et la réponse morphologique à l'oestradiol de ces cellules in vitro après la mise au point du premier modèle de culture primaire de cellules kisspeptine. / Kisspeptin neurons express the Kiss1 gene encoding kisspeptin, a potent neuropeptide secretagogue of gonadotropin releasing hormone (GnRH) that plays a fundamental role in regulation of reproductive life cycles after birth. Neurons expressing Kiss1 appear before birth only in the arcuate nucleus. The overall purpose of this study is to understand how kisspeptin neurons are set up during fetal development, if and how estrogen signaling in these cells could interfere with their development at a time of a prenatal testosterone peak in male. Our finding showed 1) precisely the placement of kisspeptin cells as a function of sex and to identify a sex-independent anteroposterior differentiation gradient 2) a reciprocal interaction with GnRH neurons, in males and females, thus suggesting a certain maturity of the system 3) the diversity of sexual steroids receptors already expressed by these cells before birth as well as the emergence of a sexual dimorphism 4) sensitivity and morphological response to estradiol of these cells in vitro after the development of the first primary culture model of kisspeptin cells.

Neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30) are 23- and 30-amino acid peptides recently isolated from the porcine hypothalamus. Immunohistochemical studies using a rabbit polyclonal antiserum against the rat NPW23 peptide revealed a limited distribution in the rat brain. NPW23-immunoreactive (irNPW) cells were detected in the paraventricular nucleus (PVH), mainly in the parvocellular division, supraoptic nucleus (SO), accessory neurosecretory nuclei, dorsal and lateral hypothalamic areas, perifornical nucleus, arcuate nucleus, and anterior and posterior pituitary; whereas, irNPW fibers were noted in the PVH and SO, retrochiasmatic nucleus, dorsal and lateral hypothalamic areas, median eminence, amygdala, and posterior pituitary. The pattern of distribution of irNPW in the hypothalamus corroborates a possible role of NPW on prolactin release and feeding behavior reported by others.

Insulin-like 6 immunoreactivity (irINSL6) was detected in Leydig cells of the mouse testis. In the brain, labeled somata were detected mainly in the caudal hypothalamus and midbrain. Double labeling the brainstem sections revealed that irINSL6 somata were 5-hydroxytryptamine (5-HT) positive. The presence of irINSL6 in discrete populations of hypothalamic and brainstem neurons and in Leydig cells of the testis suggests a diverse biological function of this novel peptide.

Yes / The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.