Phenylethanolamine N-methyltransferase (Pnmt) is the enzyme in the catecholamine pathway responsible for converting norepinephrine to epinephrine. Pnmt is present in numerous areas; however, the scope of its expression in the mouse brain is not fully understood. A genetic mouse model was generated by the Ebert lab that exhibited the selective destruction of all Pnmt+ cells through the induction of apoptosis by Diphtheria Toxin A. Unexpected phenotypic defects arose that are characterized by metabolic weight deficits and motor ataxia. The distribution of Pnmt+ neurons was examined throughout the hypothalamus and cerebellum to generate an anatomical map of current and historical Pnmt expression using various histochemical methods. Historical Pnmt expression appears more extensive than current expression levels at the adult stage, indicating that certain cells in the mouse brain may have experienced transient Pnmt expression. The presence of Pnmt in these regions suggests that the destruction of these neurons may play a role in the phenotypic defects observed in the ablation mouse model. Gaining a more comprehensive understanding of the potential role of Pnmt in these areas may elucidate new drug targets or novel methods to treat obesity and motor control disorders such as ataxia.

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Chickens from lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations serve as unique models to study eating disorders. The LWS have different severities of anorexia while all HWS become obese. Over the past decade our groups has demonstrated that these lines have differential food intake threshold responses to a range of intracerebroventricular (ICV) injected neurotransmitters. The major brain region regulating homeostatic regulation of appetite is the hypothalamus, and hence this dissertation was focused on understanding how the hypothalamus is different between LWS and HWS lines. Experiments 1 and 2 were performed as follows: whole hypothalamus as well as individual hypothalamic nuclei, respectively, were collected from 5 day-old chicks that had been fasted for 180 min or had free access to food. The hypothalamic nuclei included those primarily associated with appetite including the lateral hypothalamus, paraventricular nucleus (PVN), ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus (ARC). Total RNA was isolated, reverse transcribed, and real time PCR performed. Hypothalamic expression of anorexigenic factors was greater in LWS than HWS, those factors including calcitonin, corticotropin-releasing factor receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3 (MC3R), and mesotocin. The gene expression data from individual hypothalamic nuclei revealed that mesotocin from the PVN may play an important role in the inhibition of appetite in the LWS. Experiment 3 was then designed to evaluate the effects of stress on food intake: besides the differences in hypothalamic gene expression between the lines, they also have different feeding responses when stressed: ICV injection of neuropeptide Y (0.2 nmol, NPY) did not increase food intake in LWS on day 5 after stress exposure. Experiment 4 was thus designed to study the molecular mechanisms underlying conditional feeding responses to exogenous NPY after stress in the LWS. The melanocortin system (AgRP and MC3R) changed in the hypothalamus after stress in the LWS, and hence may be responsible for the loss of responsiveness to exogenous NPY in stressed LWS. Experiment 5 was designed to evaluate whether hypothalamic differences exist at the protein level: label-free liquid chromatography coupled to tandem-mass spectrometry was used to measure the abundance of proteins in the hypothalamus. Hypothalamus was obtained from fed and 180 minute-fasted 5 day-old male LWS and HWS chicks. Proteins involved in energy metabolism were different between the lines. Differences were also found in proteins involved in GABA synthesis and uptake as well as protein ubiquitination. In conclusion, these results suggest that different feeding behaviors of LWS and HWS may be due to differences in gene and protein expression in the hypothalamus. / Ph. D.

Understanding the central nervous systems role in appetite regulation is crucial to cure the obesity epidemic, which is more prevalent than any disease in the United States. Central appetite regulators, known as neuropeptides, are pivotal in understanding appetite regulation. Neuropeptide Y (NPY), a 36 amino acid peptide, plays a major role in regulating the hunger signals from the brain. In all vertebrates studied, it is a strong orexigenic neurotransmitter located throughout multiple nuclei of the hypothalamus. Peripheral hormones associated with hunger are able to activate NPY neurons in the arcuate nucleus, which leads to a cascade of events that activate orexigenic neurons throughout the hypothalamus. Although extensive research has gone into understanding the role of NPY in appetite regulation, the effects of macronutrient composition of diets on NPY function have not been elucidated in non-mammalian species. This research investigates how food intake is affected by dietary macronutrient composition in broiler type chickens that are fed three varying macronutrient diets: high carbohydrate (22% CP, 3000kcal/kg) a broiler starter diet, high fat (60% ME from lard), high protein 30%CP). All diets were formulated to be isocaloric. When chicks are fed the high fat diet central NPY administration has a greater effect on feed intake compared to both the basal and high protein diet. Regardless of what diet the chick is fed from hatch, if they are switched to one of the other two diets post central administration of NPY the high fat diet stimulated feed intake for the longest duration. Although, NPY had the strongest orexigenic effect on chicks fed the high fat diet, in a choice diet situation broiler chicks chose the high protein diet, independent of central NPY administration. / Master of Science

Energy homeostasis is essential for survival across all vertebrate species and involves a multitude of physiological systems that are regulated by both central and peripheral neural signaling. The hypothalamus is responsible for integrating and processing these signals and thus is regarded as the regulatory center for balancing energy homeostasis. Eating disorders, such as compulsive eating behavior associated with obesity, and anorexia, are significant public health concerns worldwide. Thus, studying appetite regulation is necessary to provide novel information for the design of solutions for health concerns that stem from altered energy intake. Such information is also relevant for improving chicken health and productivity in an agricultural setting. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch and hypothalamic nuclei, including the lateral hypothalamus (LH), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and arcuate nucleus (ARC), were collected 5 days later. Real-time PCR was used to measure the mRNA abundance of appetite-associated neuropeptides and receptors in each nucleus. The results showed that the two lines displayed distinct gene expression profiles in response to stress. In particular, the PVN of the LWS was significantly affected by stress, and expression of several anorexigenic factors was up-regulated including corticotropin-releasing factor (CRF), CRF receptor sub-types 1 and 2 (CRFR1 and CRFR2, respectively), melanocortin receptor 4, and urocortin 3, suggesting that stress-induced anorexia in the LWS may result from overriding anorexigenic signaling in the PVN, primarily through CRF signaling. This CRF signaling-associated hypothesis was further supported by results showing that the original phenotypes were restored when the LWS chicks were treated with astressin (CRF receptor antagonist) before exposure to stress. In Experiments 2 and 3, we attempted to determine the mechanisms of CRF's anorexigenic effect in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species, however the hypothalamic mechanisms underlying this anorexigenic effect were different. ICV injection of CRF increased c-Fos expression in the PVN, VMH, dorsomedial nucleus (DMN), and ARC in chicks while it only affected the PVN and LH in quail. Hypothalamic gene expression results suggested that CRF decreased neuropeptide Y receptor sub-type 1 (NPYR1) in chicks while it increased proopiomelanocortin (POMC), MC4R, CRF, and CRFR2 in quail. These results suggested that the anorexigenic effect of CRF may involve a dampened neuropeptide Y (NPY) system in chicks whereas it is associated with activated CRF and melanocortin systems in quail. At the nucleus level in chicks, CRF injection decreased NPY system-associated gene expression (ARC and DMN) and increased CRF (ARC and PVN) and mesotocin (MT) (VMH)-associated mRNAs, suggesting that orexigenic signaling through NPY was overridden by the heightened anorexigenic tone through CRF and MT, which led to the inhibition of food intake. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the anorexigenic effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that NPK injection activated the ARC and PVN, which was associated with increased mRNAs for a group of anorexigenic factors including CRF, UCN3, cocaine and amphetamine-regulated transcript (CART), and POMC, and decreased expression of several orexigenic factors, such as NPY and agouti-related peptide (AgRP). In Experiment 5, ICV injection of AM activated the ARC, the nucleus in which POMC and CART mRNAs were increased. In conclusion, these experiments revealed novel hypothalamic mechanisms underlying stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives. / Ph. D. / Appetite regulation is important for survival across all vertebrate species and the hypothalamus is the regulatory center for control of feeding behavior. Thus, studying the functions of the hypothalamus on appetite regulation provide novel insight into the eating disorders, such as obesity and anorexia, a worldwide health issue. Also, such information is relevant for improving productivity in the modern chicken industry. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch. The results suggested the two lines displayed distinct appetite-associated gene expression profiles in response to stress in the hypothalamus. In particular, stress-induced anorexia in the LWS may result from potent feeding-inhibitory factor corticotropin-releasing factor (CRF). Thus, in Experiments 2 and 3, we attempted to determine the mechanisms of CRF's inhibitory effect on food intake in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species. However, the inhibitory effect of CRF was primarily associated with a dampened neuropeptide Y (NPY) system which is a potent stimulatory factor for feeding behavior in chickens, whereas it may involve activated CRF and melanocortin systems in quail. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the inhibitory effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that the feeding-inhibitory effect of NPK was associated with a group of increased feeding-inhibitory factors such as CRF and cocaine and amphetamine-regulated transcript (CART) and decreased feeding-stimulatory factors, such as NPY and agouti-related peptide (AgRP) in the hypothalamus. In Experiment 5, AM increased gene expression of CART and proopiomelanocortin (POMC). Overall, these experiments suggested the roles of the hypothalamus in stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives.

Yes / Wnt signalling and its downstream effectors are well known for their roles in embryogenesis and tumourigenesis, including the regulation of cell proliferation, survival and differentiation. In the nervous system, Wnt signalling has been described mainly during embryonic development, although accumulating evidence suggests that it also plays a major role in adult brain morphogenesis and function. Studies have predominantly concentrated on memory formation in the hippocampus, although recent data indicate that Wnt signalling is also critical for neuroendocrine control of the developed hypothalamus, a brain centre that is key in energy balance regulation and whose dysfunction is implicated in metabolic disorders such as type 2 diabetes and obesity. Based on scattered findings that report the presence of Wnt molecules in the tanycytes and ependymal cells lining the third ventricle and arcuate nucleus neurones of the hypothalamus, their potential importance in key regions of food intake and body weight regulation has been investigated in recent studies. The present review brings together current knowledge on Wnt signalling in the hypothalamus of adult animals and discusses the evidence suggesting a key role for members of the Wnt signalling family in glucose and energy balance regulation in the hypothalamus in diet-induced and genetically obese (leptin deficient) mice. Aspects of Wnt signalling in seasonal (photoperiod sensitive) rodents are also highlighted, given the recent evidence indicating that the Wnt pathway in the hypothalamus is not only regulated by diet and leptin, but also by photoperiod in seasonal animals, which is connected to natural adaptive changes in food intake and body weight. Thus, Wnt signalling appears to be critical as a modulator for normal functioning of the physiological state in the healthy adult brain, and is also crucial for normal glucose and energy homeostasis where its dysregulation can lead to a range of metabolic disorders.