Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a γ- aminopropyltriethoxysilane solution and ending with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeatability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5-10 μm and, depending on the length of the pipette inserted into the CNS, diameters of the pipette shaft will approach 40-50 μm. Once removed, the microprobe is then incubated with the radiolabeled peptide. Binding of the radiolabeled peptide will occur to the antibody sites not occupied by the endogenously released peptide. The images of the microprobes on sensitive autoradiographic film are analyzed for differences in the optical density along a specified length of probe. Areas of lighter density signify sites along the microprobe where endogenous peptide was biologically released during the physiological intervention. Knowing the exact location of the probe tip in vivo in the CNS permits identification of neurophysiological sites corresponding along the length of the microprobe where the peptide was released.

To investigate the possible GABAergic nature of somatostatin-immunoreactive neurons of the larval tiger salamander retina, somatostatin immunocytochemistry was combined with either γ-aminobutyric acid (GABA) immunocytochemistry or autoradiography of GABA high-affinity uptake. A total of 1,062 somatostatin cells were visualized in these studies. Double-label immunocytochemistry revealed that 96.3% of somatostatin-immunoreactive cells expressed GABA immunoreactivity. Double-label studies combining somatostatin immunocytochemistry with autoradiography of GABA high-affinity uptake revealed a slightly lower percentage (93%) of colocalization. Double-labelled cells were identified as Type 1, Type 2 and displaced amacrine cells. The small percentage of somatostatin-immunoreactive cells that did not co-label for GABA were identified as Type 1 amacrine cells. An analysis of retinal sections processed for double-label immunocytochemistry revealed that approximately 5% of GABA-immunoreactive cells in the amacrine and ganglion cell layers co-label for somatostatin. Somatostatin immunocytochemistry was combined with autoradiography of glycine high-affinity uptake to examine whether tiger salamander somatostatin-amacrine cells express this glycine marker. A total of 100 somatostatin-immunoreactive amacrine cells were visualized in double-label preparations. None of these cells were observed to exhibit glycine high-affinity uptake.

Sex steroids are important hormones produced in the body to regulate an individual's physiology and behavior in preparation for reproduction. Aromatase (Cyp19a1) is the steroidogenic enzyme that converts aromatizable androgens into bioactive estrogens, and hence is in a pivotal position to mediate reproductive processes and sexual behavior. In mice, evidence from whole body aromatase knockout and estrogen receptor knockout lines has revealed the critical roles of estrogen signalling in ovarian development, gonadotropin signalling, ovulation, and sexual behavior. While the ovaries have high aromatase activity levels and are a major source of circulating estrogens in the female body, they can also be locally produced in tissues via tissue-specific aromatase expression. Of particular interest, the importance of brain-derived estrogens for reproductive processes and sexual behavior is still under study. Teleosts are an amenable model system for understanding the role of brain-derived estrogens in reproduction as they have two aromatase paralogs, cyp19a1a and cyp19a1b, that are highly expressed in the ovary and brain, respectively. In this thesis, I sought to identify the importance of brain-derived estrogens for sexual behaviors and reproductive health in zebrafish using cyp19a1⁻ᐟ⁻ mutant lines and a transgenic cyp19a1b-GFP line. In dyadic sexual behavior assays, female cyp19a1b⁻ᐟ⁻ mutants took 4.1 times longer to initiate spawning behavior with wildtype (WT) males compared to WT females. This suggested a potential impairment in mate identification and assessment in the female cyp19a1b⁻ᐟ⁻ mutants. The importance of the cyp19a1 paralogs for early larval development was revealed by 4 times higher progeny mortality from cyp19a1b⁻ᐟ⁻ mutant female and cyp19a1a⁻ᐟ⁻;cyp19a1b⁻ᐟ⁻ mutant male pairings compared to WT pairings. Gene expression analyses identified significantly lower levels of important neuroendocrine genes including the estrogen receptors and the nonapeptides, arginine vasopressin (avp) and oxytocin, in the telencephalon and hypothalamus of cyp19a1b⁻ᐟ⁻ mutant female compared to WT female brains. I performed acute intraperitoneal injections of Avp and Oxytocin, as well as mixtures of their respective receptor antagonists, in cyp19a1b⁻ᐟ⁻ mutant females to determine if the behavioral impairments could be rescued in adult fish. Arginine vasopressin was found to uniquely recover the delayed oviposition in female cyp19a1b⁻ᐟ⁻ mutants. Immunohistochemistry experiments using the transgenic cyp19a1b-GFP zebrafish line with a zebrafish-validated Avp antibody revealed a close neuroanatomical proximity and contact points between radial glial cell Cyp19a1b-expressing fibres and Avp-immunopositive neurons in preoptic areas. These findings suggest that brain-derived estrogens, via Cyp19a1b activity, might affect female sexual behavior by diffusing to nearby neurons to regulate Avp signalling levels in preoptic areas. Together, these findings establish the importance of cyp19a1b for female zebrafish sexual behavior and identify a positive link between Cyp19a1b and Avp. Future study can help to characterize the estrogen-dependent pathways involved in regulating Avp signalling in the female brain and the extent of evolutionary conservation of this regulation pathway for female sexual behavior across vertebrates.

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Peptides that regulate feeding behavior via the brain may also regulate energy storage and expenditure in the adipose tissue, a system collectively known as the "brain-fat axis". Neuropeptide Y (NPY) is orexigenic and promotes adipogenesis in both birds and mammals, although mechanisms in adipose tissue are unclear. The first objective was thus to evaluate effects of NPY on chick preadipocyte proliferation and differentiation. Preadipocytes were treated with NPY and gene expression and cellular proliferation were evaluated. Cells were also treated with NPY during differentiation and harvested during the later stages. With increased gene expression of proliferation markers in preadipocytes, and during differentiation increased expression of adipogenesis-associated factors, increased lipid accumulation, and increased activity of an adipogenic enzyme, glycerol-3-phosphate dehydrogenase, results suggest that NPY may enhance preadipocyte activity and adipogenesis and promotes lipid accumulation throughout chicken adipocyte differentiation. Another appetite-regulatory peptide, alpha-melanocyte stimulating hormone (α-MSH), is anorexigenic and mediates lipolysis in adipose tissue, but effects on fat in avians are unreported. The second objective was thus to determine the effects of exogenous α-MSH on adipose tissue physiology in broiler chicks. Chicks were intraperitoneally injected with α-MSH and adipose tissue and plasma collected. Cells isolated from abdominal fat of a different set of chicks were treated with α-MSH. Results suggest that α-MSH increases lipolysis and reduces adipogenesis in chick adipose tissue. Collectively, results of this research provide insights on how appetite-regulatory peptides like NPY and α-MSH affect adipose tissue physiology, thereby playing important roles in regulating whole-body energy balance. / Master of Science

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.

<p>Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors.</p><p>In rat a receptor called PP1, later renamed Y₄, was cloned and characterized. It displays the highest amino acid sequence identity to the Y₁ receptor. Rat Y₄ differs extensively from human Y₄, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y₄are the most diverged orthologues in the NPY receptor family.</p><p>In guinea pig, the y₆ receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig.</p><p>In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y₁, Y₄ and y₆, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. </p><p>The discoveries of the rat Y₄ and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.</p>