Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

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.

Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 μM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.

Orexine sind im Hypothalamus gebildete Neuropeptide, die Vigilanz fördern und eine entscheidende Rolle in der Energie-Homöostase und der Aufrechterhaltung von Schlaf-Wach-Rhythmen spielen. Das Orexin-System dient dazu, Signale wie Stress oder Hunger aus den afferenten Gehirnregionen in Verhaltensweisen wie erhöhte Wachsamkeit, Nahrungsaufnahme oder einen erhöhten sympathischen Tonus umzusetzen. Da die Panikstörung durch eine erhöhte Erregungsbereitschaft charakterisiert ist, ist ein Einfluss des Orexin-Systems auf die Entwicklung der Panikstörung denkbar. Zudem legen (insbesondere präklinische) Studien eine Rolle des Orexin-Systems, insbesondere des Orexin-Rezeptor-1 in der Pathophysiologie von Panik-ähnlicher Angst nahe. Vor diesem Hintergrund wurde die Leithypothese untersucht, ob die Panikstörung mit dem Ile408Val-Polymorphismus (rs2271933) in dem für den Orexin- Rezeptor-1 kodierenden HCRTR1-Gen assoziiert sein könnte. In der vorliegenden Studie wurden zwei unabhängige Stichproben mit 131 Panikpatienten und 131 Alters- und Geschlechts-gematchten Kontrollen („Discovery Sample“) sowie 292 Panikpatienten und 292 gematchten Kontrollen („Replication Sample“) für den HCRTR1 Ile408Val-Polymorphismus mittels eines Restriktionsfragment-Längen-Polymorphismus (RFLP)-Assays genotypisiert. Die experimentelle Untersuchung umfasste im Detail die Amplifikation des den Polymorphismus umgebenden Genabschnittes durch Polymerase-Ketten-Reaktion (PCR) in der aus EDTA-Blut extrahierten DNA, einen Restriktionsverdau mit dem Enzym BsmBI und anschließend die optische Darstellung und Auswertung der entstandenen Fragmente mittels Gelelektrophorese. Die Genotyp- und Allelverteilung wurde zwischen Patienten und Kontrollen sowie in der Subgruppe der weiblichen Patienten/Kontrollen mittels des Chi-Quadrat Tests vergleichend analysiert. Im „Discovery Sample“ trat das T-Allel bei den Patienten signifikant häufiger als bei den Kontrollen auf (p = 0,027). Im „Replication Sample“ konnte die Assoziation des T- Alles bei Patienten mit Panikstörung bestätigt werden (p = 0,005). Diese Assoziation war spezifisch für die weibliche Subgruppe (p = 0,002). Die vorliegenden Ergebnisse legen eine Assoziation der Panikstörung mit dem Ile408Val-Polymorphismus im HCRTR1-Gen nahe, wobei das T-Allel in zwei unabhängigen Stichproben als möglicherweise frauenspezifisches Risikoallel identifiziert wurde. Diese Ergebnisse könnten - eine weitere Replikation in großen Stichproben und die funktionelle Charakterisierung dieses Polymorphismus vorausgesetzt - dazu führen, innovative Therapien in Form von Orexin-1-Rezeptor Antagonisten zu entwickeln und diese auf Basis genetischer HCRTR1- Risikoallelkonstellationen in Zukunft ggf. auch in einem personalisierten Ansatz anzuwenden. / Preclinical studies point to a pivotal role of the orexin 1 (OX1/HCRTR1) receptor in arousal and fear learning and therefore suggest the HCRTR1 gene as a prime candidate in panic disorder (PD) with/without agoraphobia (AG) and PD/AG-related intermediate phenotypes. Here, a multilevel approach was applied to test the HCRTR1 C/T Ile408Val gene variant (rs2271933) for association with PD/AG in two independent case- control samples (total n = 423 patients with panic disorder and n = 423 healthy subjects). The HCRTR1 rs2271933 T allele was associated with PD/AG in both samples independently, particularly in the female subsample. In sum, the present results provide converging evidence for an involvement of HCRTR1 gene variation in the etiology of PD/ AG, supporting future therapeutic approaches targeting the orexin-related arousal system.

Alzheimer’s disease (AD) is a neurodegenerative disease which affects over 500,000 people in the UK. Worldwide 44 million people are affected by AD and other dementias. Most cases occur over the age of 65 and is characterised by gradual and increasing loss of cognitive function and behavioural abnormalities. The main causes are a build-up of the toxic protein amyloid-β (Aβ) and hyperphosphorylation of the microtubule stabilising protein: tau, leading to neurofibrillary tangles (NFT). These two hallmarks of disease result in neuronal damage and cell death causing associated symptoms and eventually death. Orexins (OX) are neuropeptides which function to regulate the sleep-wake cycle and feeding behaviour. They are produced from a prepro-orexin (PPO) molecule and cleaved into two isoforms: orexin-A (OXA) and orexin-B (OXB). OXA and OXB are the ligands for two G-protein coupled receptors (GPCR): orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). 50-80,000 OX producing neurons project to many areas of the brain including the lateral hypothalamus (LHA), locus coeruleus (LC), tuberomammillary nucleus (TMN), paraventricular nucleus (PVN) and raphe nuclei and from these areas regulate feeding and appetite and the sleep wake cycle through their receptors. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity including the control of feeding behaviour. It is the ligand for the GPCR GPR103, both of which are widely expressed in the brain and also in the retina, testes, thyroid, pituitary and prostate. GPR103 also shares 48 and 47% protein sequence homology with OX1R and OX2R respectively. It is in these tissues where it can exert other physiological functions including regulation of feeding, control of the gonadotropic axis and bone formation. The exact expression and signalling characteristics and physiological actions of QRFP/GPR103 are still poorly understood. It is through the physiological functions of the orexigenic system and the clinical symptoms observed in AD which suggests a possible link between the two. For example, in AD one of the main reasons for institutionalisation is the severely dysregulated sleep pattern that is experienced by sufferers. They experience increased nocturnal activity and early awakenings as well as hypersomnia and excessive daytime sleepiness; all of which is beyond what someone of the same age experiences. As well as this AD patients suffer from significant weight loss and a significant negative correlation has been identified between progression of disease and appetite. All of this points towards an involvement of the orexigenic system in AD. AD patients have been found to have a 40% loss of immunoreactive OX neurons and have severe reductions in circulating OXA. This led us to believe that the OX system is of vital importance in AD and could be targeted to ameliorate symptoms. Studies have implicated OX and OXR in memory processes, appetite regulation, and severe disturbances of the sleep-wake cycle all of which are phenotypes of AD. Given that they play a key role in energy homeostasis and physiological behaviour, we hypothesise that OXs and their receptors are implicated in the pathophysiology of AD. Therefore, in this study we will investigate the detailed expression and signalling characteristics of OXR and GPR103 in vitro and in clinical samples In this study we neuronally differentiated two human neuroblastoma cell lines: IMR32 and SH-SY5Y. Neuronally acquired phenotype was confirmed through increased neurite length, increased expression of key neuronal proteins and increases in microtubule-associated protein tau (MAPT), neurogenin1 (NG1) and neuron-specific enolase (NSE) as well as a reduction in the neuronal marker of immaturity; nestin (NES). OXR and GPR103 were confirmed in both cell lines after differentiation at mRNA and protein level and were shown to be fully functional through phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). We also identified possible cross talk of GPR103 with the OXR though addition of selective OXR antagonists, which blocked QRFP induced ERK1/2 phosphorylation. We show for the first time that addition of Aβ42 and zinc sulphate to mimic AD in vitro, results in a significant reduction of OX1R and GPR103 in the cell lines SH-SY5Y and we have performed the first comprehensive study in clinical AD patients which demonstrate a loss of OX1R, OX2R and GPR103 at mRNA and protein level compared to age matched controls in the hippocampus. We performed microarray analysis which identified many genes and pathways regulated by the OXA, OXB and QRFP; including corticotropin-releasing hormone receptor (CRHR1), regulated in development and DNA damage responses 1 (REDD1), erythropoietin (EPO), Bcl-2-like protein 1 (BCL2L11), myb proto-oncogene protein (c-myb), vasoactive intestinal peptide (VIP), endothelin 1 (EDN1) as well as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and hypoxia-inducible factor-1α (HIF-1α) pathways. These genes are all implicated in neuroprotection, particularly in AD. This represents the first comprehensive gene expression data in a neuroblastoma cell line for these orexigenic proteins. Collectively these data suggest a potential role of the orexigenic system in neuroprotection and a functional loss of the receptors in AD patients which could confer a loss of neuroprotection through the orexigenic system. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.

In most birds, changing photoperiods from winter to spring and from summer to fall have two consequences: increased feeding followed by migratory activity. To date, the neural system controlling the activation of migratory activity remains unknown, though behavioral observations point to a possible mechanism. During the migration season, diurnal songbirds show extensive disruption of their sleep/wake cycle, sleeping during the day and flying at night. In mammals, similarly altered cycles of activity result from blocking orexin expression in the hypothalamus. It is possible that decreased orexin expression is associated with migratory activity in songbirds. In addition, changes in ingestive behaviors and fuel availability may also be associated with disruptions in the sleep/wake cycles of migratory birds. The studies in my dissertation will address these issues through three main specific aims. First, I will determine that orexin systems are conserved in vertebrate brains. Second, I will test the association between orexin and migratory activity in songbirds. Third, I will confirm the association between fuel availability, orexin expression and migratory activity in songbirds. / text

No description available.

Although the perifornical lateral hypothalamic area (PeFLH), which contains orexin/hypocretin (OX) neurons, plays an important role in arousal-related behaviors, its neuromodulatory inputs are incompletely understood. The present study examined the role of glutamatergic inputs to the PeFLH in various arousal-related behaviors. Adult male rats received a microinjection of the ionotropic glutamate receptor agonists AMPA (1 and 2 mM) or NMDA (1 and 10 mM), or vehicle into the PeFLH, and were placed in an open field; 90 min later, rats were perfused for immunohistochemistry for OX and c-Fos as a marker of neuronal activation. AMPA injections dose-dependently increased locomotion, rearing, and drinking. NMDA injections (at 10 mM) increased locomotion and feeding. All these behaviors (except feeding) were positively correlated with the number of c-Fos/OX-immunoreactive neurons. These results support the role of ionotropic glutamate receptors on OX (and other) neurons in the PeFLH in the regulation of locomotor and ingestive behaviors.

<p>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.</p><p>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 IP₃increase 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.</p>

Thesis advisor: Gorica Petrovich / Thesis advisor: Christina Reppucci / In order to understand the neurobiological basis for the phenomenon in which environmental cues override physiological cues to influence the behavioral control of feeding, we utilized an animal model for fear-cue induced inhibition of eating. Female rats that had learned to associate a tone with foot-shocks showed inhibition of eating across three extinction tests, whereas male rats that had received tone-shock pairings extinguished their inhibition of eating after the first test day. We assessed activation of orexin (ORX), a neuropeptide involved in eating and arousal, in the lateral hypothalamic area (LHA) of the brains of male and female control and experimental rats during the final test day. Female rats exhibited greater recruitment of ORX neurons in the LHA than male rats; there was no difference in ORX activation between control and experimental groups of either sex, indicating that ORX is involved in sex differences in fear-cue induced inhibition of eating. / Thesis (BS) — Boston College, 2013. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Psychology.