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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.

La reproduction est intimement liée à la balance énergétique, particulièrement chez les espèces sauvages exposées à des variations larges de leur environnement. L’objectif de cette thèse était d’étudier les mécanismes centraux qui régissent la régulation saisonnière de ces deux fonctions chez la gerboise. Nos résultats montrent une augmentation coordonnée au printemps de l’expression des gènes codant pour les neuropeptides impliqués dans la régulation de la reproduction et de la prise alimentaire, et rapportent des effets opposés de deux peptides classiquement considérés comme régulant la reproduction, Kisspeptine et RFRP-3, sur la prise alimentaire de la gerboise femelle ; un effet inhibiteur de Kisspeptine ayant lieu uniquement au printemps, et un effet activateur de RFRP-3 s’observant pendant les deux saisons. De plus, nous proposons que Kisspeptine et RFRP-3 exercent leurs effets sur la prise alimentaire via des actions sur des structures cérébrales dédiées au contrôle métabolique, notamment POMC et NPY. Dans l’ensemble, ces résultats renforcent notre hypothèse d’une coordination centrale de l’activité de la reproduction et de la prise alimentaire chez la gerboise et suggèrent une modulation de cette coordination en fonction du sexe et de l’environnement saisonnier. / Reproduction is intimately related to energy balance, especially in wild species exposed to marked seasonal changes in their environment. The aim of this thesis was to study the central mechanisms governing the seasonal regulation of these two functions in the jerboa. Our results reveal a spring coordinated increase in the expression of genes encoding neuropeptides involved in the regulation of reproduction and food intake, and report opposite effects of two central regulators of reproduction, Kisspeptin and RFRP-3, on food intake in the female jerboa; an inhibitory effect of Kisspeptin that occurs only in spring, and activatory effect of RFRP-3 observed in both seasons. Moreover, we propose that Kisspeptin and RFRP-3 display their effects on food intake via actions on brain structures dedicated to metabolic regulation, mainly POMC and NPY. Overall, these results strengthen our hypothesis of a central coordination of the jerboa’s reproductive activity and food intake and suggest a modulation of this coordination that depends on sex and seasonal environment.

Das Ziel der vorliegenden Arbeit war es, zu untersuchen, ob der Neurotransmitter Neuropeptid Y (NPY) Einfluss auf das Schwellungsverhalten retinaler Gliazellen der Ratte in hypoosmolarem Medium hat. Des Weiteren war von besonderem Interesse, welche Rezeptortypen und welche intrazellulären Signalwege in die Wirkung von NPY involviert sein könnten. Verwendet wurden 50 adulte Long-Evants-Ratten. Zuerst wurde bei einem Teil der Ratten eine transiente retinale Ischämie in einem Auge der Ratten induziert. Das andere Auge blieb unbehandelt und diente zur Kontrolle. Drei Tage post op wurden die Ratten euthanasiert. Nach Enukleation der Bulbi wurde die Netzhaut auf einen Membranfilter aufgebracht und Schnitte (1 mm) angefertigt. Um die Müllerzellen der vitalen Retina mit Hilfe des Laser Scanning-Mikroskops darstellen zu können, wurde der Farbstoff Mitotracker Orange verwendet (UCKERMANN et al. 2004). Erst kürzlich konnte gezeigt werden, dass die Müllerzellen der postischämischen Retina der Ratte in hypotonem Medium schwellen (PANNICKE et al. 2004). Dazu wurden die akut isolierten retinalen Schnitte einer hypotonen Lösung ausgesetzt (60 % der Kontrollosmolarität). Im Rahmen dieser Arbeit konnte gezeigt werden, dass NPY die Müllerzellschwellung in hypotonem Medium in der postischämischen Netzhaut verhindert. Die pharmakologische Untersuchung des durch NPY aktivierten Signalweges erfolgte an gesunden Netzhäuten. Hier führt ein hypotones Medium bei gleichzeitiger Blockade der Kaliumkanäle (K+-Kanäle) durch Ba2+ zu einer Gliazellschwellung, die mit derjenigen in der postischämischen Retina vergleichbar ist (PANNICKE et al. 2004). NPY hemmt konzentrationsabhängig das Schwellen der Gliazellen der gesunden Netzhaut in hypoosmolarem Medium in Anwesenheit von Ba2+. Die gleiche Wirkung konnte mit dem selektiven Y1-Rezeptoragonisten hervorgerufen werden, während die Y2- und Y5-Rezeptoragonisten keine Wirkung zeigten. Außerdem hatte NPY in der Anwesenheit des selektiven Y1-Rezeptoragonisten BIBP3226 keine Wirkung. Inkubation mit dem membranpermeablen Ca2+-Chelator BAPTA-AM kehrte die Wirkung des NPY um, ebenso wie die Inkubation mit den Proteinkinase C (PKC)-Inhibitoren Staurosporin und Gö6976. Die Neurotransmitter Glutamat und Adenosin zeigten eine dem NPY vergleichbare hemmende Wirkung auf das Schwellen der Müllerzellsomata. Außerdem konnte eine Stimulierung von metabotropen Glutamatrezeptoren (mGlu) und Adenosin A1-Rezeptoren (A1R) nachgewiesen werden. Jedoch hob der selektiven Na+-Kanalblocker Tetrodotoxin die hemmende Wirkung von NPY auf. Die erzielten Ergebnisse deuten darauf hin, dass NPY einen neuronalen Y1-Rezeptor aktiviert, was zu einer Mobilisierung von Ca2+ aus intrazellulären Speichern und zur Aktivierung der Proteinkinase C (PKC) führt. Weiterhin erfolgt eine von neuronaler Aktivität und Ca2+ abhängige Freisetzung von Glutamat und die Aktivierung von (glialen) mGlu. Letztendlich kommt es vermutlich zur Aktivierung des A1R. Resümierend könnten diese Ergebnisse wichtig sein für die Entwicklung neuer therapeutischer Strategien zur Vermeidung von postischämischen und posttraumatischen Gliazellschwellungen. / The aim of the present study was to determine wether neuropeptide Y (NPY) has an effect on hypotonic glia cell swelling from the retina of the rat. Furthermore, the special interest was to determine which receptor subtypes and which intracellular pathways are involved in the effect of NPY. 50 adult Long-Evants-rats were taken. Transient retinal ischemia was induced in one eye of the rats, while the other eye remained untreated and served as control. Three days after reperfusion, the animals were killed. After enucleation of the bulbi, the isolated retina was fixed on a membrane filter and 1 mm thick slices were produced. The acutely isolated slices were loaded with the vital dye Mitotracker Orange in order to selectively stain Müller glial cells (UCKERMANN et al. 2004). The slices were examined using a confocal laser scanning microscope. As shown recently (PANNICKE et al. 2004), the somata in postischemic retinas corresponded with swelling after changing the extracellular perfusate into a hypotonic solution which contained 60 % of the control ionic strength. NPY significantly decreased the hypotonic glia cell swelling in postischemic retinas. The following experiments for the pharmacological examination of the NPY pathway where made with untreated rats. Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution; this swelling is comparable with the swelling of glia cells in postischemic retina (PANNICKE et al. 2004). Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution (PANNICKE et al. 2004). NPY significantly decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. NPY displayed a dose-dependent swelling effect. The Y1-receptor agonist inhibited dose-dependently the hypotonic glial cell swelling, while agonists for Y2- and Y5-receptors were largely ineffective. Incubation with the membrane permeable Ca2+-chelator BAPTA-AM reversed the swelling inhibiting effect of NPY, just as incubation with PKC-inhibitors staurosporine and Gö6976 did. A dependence of the NPY effect on release of Ca2+ from intracellular stores is also suggested by the effect of thimerosal. Glutamate and adenosine also decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. In addition, glutamate stimulates metabotropic glutamte receptors (mGluR) and adenosin activates purinergic receptors. However, the selective Na+-canal blocker tetrodotoxin (TTX) reversed the inhibiting effect of NPY on swelling, but not of glutamate and adenosine. The data suggest that NPY inhibits hypotonic glia cell swelling by activation of neuronal Y1-receptors via Ca2+-dependent release of glutamate. This effect is mediated by subsequent stimulation of glial glutamergic and purinergic receptors in Müller cells. The results may have importance for the development of new therapeutic strategies for inhibition of postischemic and posttraumatic glial cell swelling.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_vinicius_campos.pdf: 483644 bytes, checksum: 9caf292597102dea88bfa2da68dc9158 (MD5) Previous issue date: 2009-03-03 / Food intake in vertebrates is a complex process involving several endocrine and neural pathways. Among the orexigenic factors, the most notably are the neuropetides as the neuropeptide Y (NPY) that is a 36 amino acid peptide which plays a key role in food intake. Studies evaluating fish NPY expression showed that this peptide is involved in appetite stimulation. However, despite the recent advances, our present knowledge of the regulation of feeding behavior in fish is limited and based in a few fish species, and there is increasing evidence of speciesspecific differences. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression during 24 h by real-time RT-PCR. A 597 bp NPY cDNA was cloned from Brazilian flounder brain by 3´RACE method. NPY expression was detected in all peripheral tissues analyzed, but with predominant expression in the brain. No significant differences were observed in brain NPY gene expression over a 24 h evaluation period. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during 24 hours. These results indicated that NPY may play roles in flounder peripheral tissues and may be not involved short-term regulation of food intake at low-temperatures in Brazilian flounder. / O apetite e a ingestão de alimentos em vertebrados é um processo complexo que envolve várias rotas neurais e endócrinas. Dentre os fatores orexigênicos, destacamse os neuropeptídeos, como o neuropeptídeo Y (NPY), o qual é constituído de 36 aminoácidos e desempenha papel chave na regulação do apetite. Estudos avaliando a expressão do NPY em peixes demonstraram que este peptídeo está envolvido no estímulo da ingestão. Entretanto, apesar dos avanços recentes, o atual conhecimento sobre a regulação fisiológica do apetite é limitado e baseado em poucas espécies de peixes com evidencias de diferenças interespecíficas. O linguado Paralichthys orbignyanus tem sido considerado para a aqüicultura e com isso, torna-se importante compreender os mecanismos de regulação do apetite para incrementar o seu desempenho em cativeiro. Os objetivos deste trabalho compreenderam clonar o gene do NPY, avaliar os níveis de RNA mensageiro nos diferentes tecidos do linguado e também avaliar a expressão do NPY no cérebro durante 24 h por RT-PCR em tempo real. Um fragmento de 597 pb do cDNA do NPY foi clonado a partir do RNAm do cérebro do linguado através do método RACE 3 . A expressão do NPY foi detectada em todos os tecidos analisados (cérebro, baço, coração, intestino, estômago, brânquia, fígado, rim, músculo e testículo), mas com predominante expressão no cérebro. A expressão do NPY não cérebro demonstrou variações durante 24 horas de avaliação. Nenhuma correlação foi observada entre glicose, proteínas totais, colesterol e triglicerídeos plasmáticos. Estes resultados indicam que o NPY pode desenvolver funções em outros tecidos além do cérebro e também pode não estar associado na regulação da alimentação em curto prazo em baixas temperaturas.

Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

Dans le hilus du gyrus denté de l'hippocampe, les cellules moussues excitatrices et les interneurones GABAergiques constituent des acteurs clés du réseau. Cependant, en raison en partie de leur grande diversité, la fonction des interneurones GABAergiques du hilus dans la physiologie du gyrus denté reste peu détaillée. Nous avons utilisé des souris transgéniques GAD67-GFP, exprimant la GFP sous le contrôle du promoteur de la GAD67, et évalué les densités de neurones GABAergiques marqués pour la Calretinine (CR), Parvalbumine (PV), Somatostatine (SOM), Neuropeptide Y (NPY) et l'oxyde nitrique synthase (NOS-1) dans le hilus et la couche granulaire. Pour mieux caractériser les différentes populations d'interneurones, nous avons caractérisé les propriétés de 123 neurones en utilisant la technique de RT-PCR sur cellule unique sur des tranches de cerveaux de souris C57Bl6 âgées de 2 à 3 mois. Une analyse non supervisée en clusters basée sur 18 paramètres électrophysiologiques et 7 paramètres moléculaires a clairement mis en évidence un cluster de cellules moussues excitatrices (n=67) et 3 clusters de cellules GABAergiques (n=56). Les deux premiers clusters d'interneurones GABAergiques comprennent des neurones (n=18 et n=16) qui co-expriment le NPY et la SOM mais se différencient clairement par leurs propriétés élcetrophysiologiques. Le troisième cluster d'interneurones comprend des neurones à décharge rapide exprimant soit la PV (n=9) soit la NOS-1 (n=13). Cette caractérisation multiparamétrique supporte l'existence de classes fonctionnelles distinctes parmi les interneurones GABAergiques du gyrus denté

During the past few decades, obesity has risen significantly in the United States with recent estimates showing that 65% of Americans are overweight and 30% are obese. This increase is a major cause for concern because obesity is linked to many secondary health consequences that include type II diabetes, heart disease, and cancer. Current approaches to the obesity problem primarily have focused on controls of food intake and have been largely unsuccessful. Food, however, almost always has to be acquired (foraging) and frequently is stored for later consumption (hoarding). Therefore, a more comprehensive approach that includes studying the underlying mechanisms in human foraging and food hoarding behaviors could provide an additional target for pharmaceutical or behavioral manipulations in the treatment and possibly prevention of obesity. Neuropeptide Y (NPY) is a particular peptide that provides a potent orexigenic drive to alter foraging, food hoarding (appetitive ingestive behaviors) and food intake (consummatory ingestive behaviors) in variety of species. NPY is predominantly produced in the arcuate nucleus of the hypothalamus (ARC) and has extensive efferent projections throughout the brain. Two target nuclei of ARC-NPY, the paraventricular nucleus of the hypothalamus (PVH) and perifornical area (PFA), have been shown to mediate the effect of NPY on food intake in laboratory rats and mice, but nothing is known about the effect of ARC-NPY on foraging and food hoarding. In addition, the action of specific NPY receptor subtypes within these two nuclei for these behaviors is unknown. Even though ARC-NPY is one of the main sources of input into the PVH and PFA, it is not known if this NPY fiber projection mediates alterations in appetitive and consummatory ingestive behaviors. Therefore, the purpose of this dissertation is to test 1) if NPY within the PVH or PFA controls appetitive, as well as, consummatory ingestive behaviors, 2) if NPY Y1 receptors within the PVH or PFA differentially control appetitive or consummatory ingestive behaviors, and 3) if NPY from the ARC is necessary for the control of appetitive and consummatory ingestive behaviors.

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs. The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes. This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity. A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246. In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

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