When most omnivores and herbivores become sodium depleted they engage in the motivated behavior of sodium appetite (AKA salt appetite), or the seeking out and ingestion of salty substances. Sodium appetite is associated with psychological processes that serve to enhance the incentive and rewarding value of salty substances in order to attract animals to salty substances and reinforce the ingestion of them. The experience of sodium depletion also produces long-lasting changes in behavior; one of the most apparent changes being a seemingly life-long increase in hypertonic salt intake which indicates sodium appetite is sensitized. Two neural circuits have been implicated in the sensitization of sodium appetite: 1) a forebrain neural circuit that regulates body fluid homeostasis, and 2) the mesolimbic dopamine system which mediates motivated behaviors. This dissertation has three aims that serve the overall purpose of providing a better understanding of the neurobiological mechanisms that mediate the sensitization of sodium appetite. The first aim is to develop a model of sodium depletion that is amenable to pharmacological manipulation in order to determine whether the -blockade of N-methyl-d-aspartate receptors, which are critical for neural plasticity, will prevent the sensitization of sodium appetite. The second aim is to determine whether sensitization is associated with relatively long-term molecular changes in forebrain areas that regulate body fluid homeostasis. The third aim is to identify how forebrain areas involved in body fluid homeostasis may connect to and influence activity in the mesolimbic dopamine system.

<p>The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca²⁺, which has been shown to be due to influx of extracellular Ca²⁺ as a primary response.</p><p>Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX₁ than on OX₂ receptors.</p><p>When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca²⁺-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX₁ receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency G_S-coupling and a high-potency PKC coupling.</p><p>In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca²⁺-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.</p>

The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca2+, which has been shown to be due to influx of extracellular Ca2+ as a primary response. Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX1 than on OX2 receptors. When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca2+-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX1 receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency GS-coupling and a high-potency PKC coupling. In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca2+-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.

The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX1 and OX2 receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors. Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca2+ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca2+ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX1R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX1R signaling, but also SOCs amplified PLC activity. A technique to block OX1R-induced IP3 increase and subsequent Ca2+ release was devised, leaving ROC influx as the only source of Ca2+ elevation upon OX1R activation. This block had no effect on OX1R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca2+ elevating process in OX1R signaling. OX1Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP3. At even lower orexin-A concentrations, OX1Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.

Elektroenzephalografische (EEG-) Untersuchungen belegen eine veränderte Wachheitsregulation bei uni- und bipolar affektiven Störungen. Orexin- A (syn. Hypocretin-1, HCRT-1) ist ein in der Modulation der Schlaf- Wach- Rhythmik zentraler Neurotransmitter, dessen Defizienz zu Narkolepsie führt. Ziel der vorliegenden Arbeit war die Untersuchung der Konzentration von Orexin- A im Liquor cerebrospinalis (CSF) bei unipolarer Depression (MDD), Manie bei bipolarer Störung (BD) und gesunden Probanden (GP) sowie die Untersuchung des Zusammenhangs zwischen CSF- Orexin- A und der am Tag der Punktion bestimmten EEG- basierten Vigilanzregulation. Die CSF- Orexin- A- Konzentrationen wurde mittels Fluoreszenzimmunoassay (FIA) gemessen und die EEG- basierte Vigilanz mit Hilfe des Vigilanz Algorithmus Leipzig (VIGALL) erhoben. Es zeigten sich keine signifikanten Mittelwertunterschiede von CSF- Orexin- A zwischen 17 Patienten mit MDD (74,32±17,81 pg/ml), 5 Patienten mit BD (77,3±20,7 pg/ml) und 10 GP (82,82±22,06 pg/ml). Auch fand sich kein signifikanter Zusammenhang zwischen den CSF- Orexin- A-Konzentrationen und den EEG- Vigilanzstadien bzw. Stadienwechsel in einer Subgruppe von 13 Patienten mit MDD und 7 GP. Die Ergebnisse sprechen gegen eine im CSF messbare Störung der Orexin- A- Regulation bei affektiven Störungen und lassen die Beantwortung der Frage nach dem Zusammenhang von Orexin- Spiegeln und EEG-basierter Vigilanzregulation weiterhin offen. Diesbezüglich sind weiterführende Analysen an größeren Stichproben unmedizierter Patienten zu fordern.

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Psychostimulant dependence remains a major health and economic problem, leading to premature death and costing $181 billion annually in health care, crime, and lost productivity costs. Currently, no pharmacotherapies are available to effectively treat psychostimulant dependence. Psychostimulants cause changes in neural circuits involved in reward and affect, but addiction neurocircuitry is incompletely understood and new targets for therapeutic intervention are needed. Lateral hypothalamic orexins (hypocretins) have been shown to have functional roles in arousal, reward processing, attention, motivation, and impulsivity. The opioid peptide dynorphin, co-localized with orexin, has critical roles in producing negative affective emotion states through interactions with, among others, stress circuitry. Orexin-dynorphin neurons project to neural substrates governing positive and negative motivated behavior, including the bed nucleus of the stria terminalis (BNST), amygdala, locus coeruleus and ventral tegmental area (VTA). Orexin and dynorphin modulate post-synaptic membrane activity through opposing signaling mechanisms; while orexins bind to predominantly excitatory orexin-1 and -2 G-protein coupled receptors, dynorphins bind to predominantly inhibitory G-protein coupled kappa opioid receptors (KORs). Multiple psychopathologies, including anxiety and substance abuse disorders, are characterized in part by alterations in orexin-dynorphin signaling. While these peptides have been shown to co-localize within single presynaptic vesicles and exert opposing effects on post-synaptic membrane potentials, the utility of producing oppositely-behaving peptides and the implications on psychopathologies remains unknown. The present studies were conducted to explore the role of orexin and dynorphin activity in cocaine’s rewarding effects as well as the negative effects of withdrawal. To accomplish this, we measured 1. Effects of orexin and cocaine administration on impulsive behaviors that increase the likelihood of psychostimulant addiction, using 5-choice serial reaction time task in concert with systemic and site directed pharmacology. 2. Effects of orexin and dynorphin on motivation for cocaine administration and intracranial self-stimulation. Using immunohistochemistry, ultrasonic vocalizations, and fast scan cyclic voltammetry we explored possible dopaminergic mechanisms of orexin and dynorphin contributions to reward. Lastly 3. Effects of orexin, dynorphin and chronic cocaine on withdrawal-induced anhedonia using intracranial self-stimulation, elevated plus maze, and correlations with immunohistochemistry and plasma corticosterone levels to explore further mechanisms. The results of this dissertation support our hypothesis that orexin receptor activity contributes to cocaine-induced impulsivity, motivation to self-administer cocaine and the reinforcing effects of psychostimulants. Dynorphin activity contributes to anhedonia and anxiety seen during drug abstinence after chronic exposure. Orexin and dynorphin exert these effects, in part, by modulating activity of dopaminergic neurons projecting from the ventral tegmental area to the nucleus accumbens. / Biomedical Sciences

L’objectif de notre étude est d’améliorer la connaissance des réseaux neuronaux responsables de la régulation du sommeil paradoxal (SP). Dans ce but, nous avons combiné des techniques de polysomnographie, de neuroanatomie fonctionnelle, de pharmacologie et de traçage de voies nerveuses. Nous avons ainsi mis en évidence une population de neurones GABAergiques à la jonction entre la substance grise périaqueductale ventrolatérale et la partie dorsale du noyau profond du mésencéphale (vlPAG/dDpMe), capable de bloquer l’entrée en SP. Nous avons également démontré l’existence de nombreuses populations de neurones GABAergiques pontiques activés au cours du SP. De plus, nos travaux ont révélé la présence de nombreux neurones GABAergiques, dont les neurones à MCH, actifs en SP dans plusieurs régions hypothalamiques. Enfin, nous avons établi une cartographie des neurones à MCH et à Hcrt envoyant des projections sur les neurones histaminergiques du noyau tubéromammillaire ventral (VTM), promoteurs de l’éveil. L’ensemble de notre travail de thèse a permis d’affiner le modèle des réseaux neuronaux impliqués dans la régulation du SP, particulièrement en confirmant le rôle du GABA dans ces mécanismes de régulation / The aim of our study is to improve the knowledge of the neural networks responsible for paradoxical sleep (PS) regulation. To this end, we combined polysomnographic recordings, functional neuroanatomy, pharmacology and tract-tracing of nervous pathways. We thus highlighted a population of GABAergic neurons at the junction between the ventroleral periaqueductal gray and the dorsal part of the deep mesencephalic nucleus (vlPAG/dDpMe), able to gate PS genesis. We also showed the existence in the pons of several GABAergic neurons populations activated during PS. Moreover, our work revealed the presence of a large number of GABAergic neurons including the MCH neurons, activated in PS in several hypothalamic areas. Finally, we mapped the MCH, Hcrt neurons that send projections to the wake-promoting histaminergic neurons of the ventral tuberomammillary nucleus (VTM). Our thesis work made it possible to refine the model of neural networks involved in PS regulation, particularly by confirming the role of GABA in these mechanisms of regulation.

The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases. Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D. In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase. We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.

Made available in DSpace on 2016-06-02T19:23:01Z (GMT). No. of bitstreams: 1 6358.pdf: 1461364 bytes, checksum: 88df144bc77432e6c6026f8cd985fa7a (MD5) Previous issue date: 2014-08-29 / Universidade Federal de Minas Gerais / The hypocretin or orexin A plays an important role in the modulation of respiratory control in mammals, but there are no data available for the role of Orexins in the peripheral and central chemoreception of non-mammalian vertebrates. Thus, the present study was designed to evaluate the location of orexinergic neurons in toads (Rhinella schneideri). In addition, we investigate if the orexinergic system of this species is important to hypoxic (5% O2 and N2 for balance) and hypercarbic (5% CO2, 21% O2 and N2 for balance) drive to breath. We assessed the role of the orexinergic system on respiratory responses by using intracerebroventricular injection of SB-334867 (orexin A receptor antagonist) during the light and the dark phase. Our results demonstrated that orexinergic neurons of Rhinella schneideri are located in the suprachiasmatic nucleus of diencephalon. Additionally, the injection of the orexin antagonist attenuated the ventilatory response to hypercarbia during the dark phase by acting on tidal volume and breathing frequency, while in the light phase, there was an attenuation in the ventilatory response to hypoxia by acting just in the tidal volume. We conclude that central orexin A contributes to hypercarbic and to hypoxic chemoreflex in toads Rhinella schneideri. / A orexina ou hipocretina exerce uma importante modulação no controle respiratório em mamíferos, mas nenhum estudo verificou a participação das orexinas na quimiorrecepção central e periférica em vertebrados não-mamíferos. Em vista disso, o objetivo do presente estudo foi avaliar a imunorreatividade para orexina para localização dos neurônios orexinérgicos no encéfalo de sapos (Rhinella schneideri) e investigar se o sistema orexinérgico desta espécie participa nas respostas respiratórias à hipóxia (5% O2 e N2 balanço) e à hipercarbia (5% CO2, 21%O2 e N2 balanço). A participação da neurotransmissão orexinérgica na regulação respiratória em sapos (Rhinella schneideri) foi avaliada por meio de microinjeções intracerebroventriculares (i.c.v.) de SB-334867 (antagonista de receptores OX1R) em duas doses (5 mM e 10mM) no ventrículo lateral durante as fases clara e escura desses animais. Nossos resultados mostram que os neurônios orixinérgicos de sapos Rhinella schneideri estão localizados no núcleo supraquiasmático no diencéfalo. Adicionalmente, a injeção do antagonista orexinérgico atenua a resposta ventilatória à hipercarbia na fase escura, devido tanto à diminuição do volume corrente quanto da frequência respiratória, enquanto, na fase clara, atenua a resposta ventilatória à hipóxia devido a uma redução no volume corrente. Concluímos, portanto, que a Orexina A participa na modulação dos quimiorreflexos central e periférico em sapos Rhinella schneideri.