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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 86-97. / Microfiche. / xii, 97 leaves ill. 29 cm

Typescript. / Bibliography: leaves 158-169. / Photocopy. / Microfilm. / xviii, 169 leaves, bound ill. 29 cm

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.

Thesis (Ph.D.)--University of Wyoming, 2008. / Title from PDF title page (viewed on June 24, 2009). Includes bibliographical references (p. 158-174).

Thesis (M.A.)--University of Delaware, 2008. / Principal faculty advisor: Mary Dozier, Dept. of Psychology. Includes bibliographical references.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity. / Medicine, Faculty of / Graduate

Background & Aims: Vasoactive agents with endoscopic therapy are used to treat acute variceal bleeding (AVB). There are two main groups of vasoactive agents: terlipressin and vasopressin (T-V), and octreotide and somatostatin (O-S). However, the benefit/harm balance is unclear. Our aim was to assess the efficacy and safety of T-V versus O-S for the management of AVB. Methods: We performed a systematic search for randomized controlled trials (RCTs) in PubMed, Scopus, and CENTRAL. Our main outcomes were mortality and adverse events. Secondary outcomes were bleeding control, rebleeding, blood transfusion, hospital stay. We evaluated the certainty of evidence using GRADE methodology. Results: We included 21 RCTs. The risk of mortality (RR: 1.01; 95%CI: 0.83-1.22), bleeding control (RR: 0.96; 95%CI: 0.91-1.02; I2=53%), early rebleeding (RR: 0.91; 95%CI: 0.66-1.24: I2=0%), late rebleeding (RR: 0.94; 95 CI: 0.56-1.60; I2=0%), blood transfusion (MD: 0.04; 95%CI:-0.31-0.39; I2=68%) and hospital stay (MD:-1.06; 95%CI:-2.80-0.69; I2=0%) were similar between T-V and O-S groups. Only 15 studies reported adverse events, which were significantly higher in the T-V compared to the O-S group (RR 2.39; 95%CI: 1.58-3.63; I2=57%). The certainty of evidence was moderate for the main outcomes, and low or very low for others. Conclusions: In cirrhotic patients with AVB, those treated with T-V had similar mortality risk compared to O-S. However, the use of T-V showed an increased risk of adverse events compared to O-S. / Revisión por pares

Anxiety disorders pose a great burden onto society and economy and can have devastating consequences for affected individuals. Treatment options are still limited to psychopharmacotherapy originally developed for the treatment of depression and behavioral therapy. A combination of genetic traits together with aversive events is most likely the cause of these diseases. Gene x environment studies are trying to find a link between genetic traits and specific negative circumstances. In a first study, we focused on social anxiety disorder (SAD), which is the second most-common anxiety disorder after specific phobias. We used a social fear conditioning (SFC) paradigm, which is able to mimic the disease in a mouse model. We wanted to investigate protein levels, as well as mRNA expression of immediate early genes (IEGs), to determine brain areas affected by the paradigm. We also included genes of the vasopressin (AVP)-, oxytocin (OXT)-, neuropeptide Y (NPY)-, and the serotonin system, to investigate the effects of SFC on neurotransmitter gene expression levels in brain regions related to social as well as fear-related behavior. AVP and OXT regulate a lot of different social and anxiety-related behaviors, both positive and negative. Finding a link between different neurotransmitter systems in the development of anxiety disorders could help to identify potential targets for new treatment approaches, which are desperately needed, because the rate of patients not responding to available treatment is very high. We were able to show altered gene expression of the IEGs cFos and Fosl2, as well as a change in number and density of cFOS-positive cells in the dorsal hippocampus, indicating an influence of SFC on neuronal activity. Our results reveal a possible involvement of anterior dentate gyrus (DG), as well as cornu ammonis area 1 (CA1) and CA3 in the dorsal hippocampus during the expression of social fear. Contrary to our hypothesis, we were not able to see changes in neuronal activity through expression changes of IEGs in the amygdala. Significant higher IEG immunoreactivity and gene expression in the dorsal hippocampus of animals without fear conditioning (SFC-), compared to animals with fear conditioning (SFC+), indicate an involvement of different hippocampal regions in two possible scenarios. Either as elevated gene expression in SFC- animals compared to SFC+ animals or as reduction in SFC+ animals compared to SFC- animals. However, this question cannot be answered without an additional control of basal IEG-activity without social interaction. The NPY system in general and the neuropeptide y receptor type 2 in particular seem to be involved in regulating the response to social fear, mostly through the septum region. In addition to that, a possible role for the induction of social fear response could be identified in the serotonergic system and especially the serotonin receptor 2a of the PVN. In a second study we focused on changes in the serotonergic system. A polymorphism in the human serotonin transporter (5-HTT) gene is associated with higher risks for the development of anxiety disorders. This makes the 5-HTT a widely used target to study possible causes and the development of anxiety disorders. In mice, a genetically induced knockout of the 5-Htt gene is associated with increased anxiety-like behavior. High amounts of stress during pregnancy, also known as prenatal stress, significantly increase the risk to develop psychiatric disorders for the unborn child. We utilized a prenatal stress paradigm in mice heterozygous for the 5-Htt gene. Some of the animals which had been subjected to prenatal stress showed noticeably “unsocial” interaction behavior towards conspecifics. Again, we were searching for links between the serotonergic system and AVP- and OXT systems. Through quantitative gene expression analysis, we were able to show that both AVP and OXT neuromodulator systems are affected through prenatal stress in female mice, but not in male mice. The 5-Htt genotype seems to be only slightly influential to AVP, OXT or any other neurotransmitter system investigated. Gene expression of AVP and OXT brain systems is highly influenced through the estrous cycle stages of female mice. Additionally, we analyzed the AVP and OXT neuropeptide levels of mice with different 5-Htt genotypes and in both sexes, in order to see whether the production of AVP and OXT is influenced by 5-Htt genotype. On neuropeptide level, we were able to identify a sex difference for vasopressin-immunoreactive (ir) cells in the PVN, with male mice harboring significantly more positive cells than female mice. / Angsterkrankungen sind eine große Belastung für Gesellschaft und Wirtschaft und können verheerende Folgen für Betroffene haben. Behandlungsmöglichkeiten sind nach wie vor auf Psychopharmakotherapie, welche ursprünglich für die Behandlung von Depressionen entwickelt wurde, und Verhaltenstherapie beschränkt. Eine Kombination aus bestimmten genetischen Eigenschaften zusammen mit aversiven Lebensereignissen sind die wahrscheinlichste Ursache für die Entstehung dieser Erkrankungen. Gen x Umweltstudien versuchen dabei, Verbindungen zwischen genetischen Merkmalen und spezifischen negativen Ereignissen zu finden. In einer ersten Studie haben wir uns auf die soziale Phobie konzentriert, welche die zweithäufigste Angsterkrankung nach spezifischen Phobien ist. Wir haben ein soziales Furchtkonditionierungs-Paradigma (social fear conditioning, SFC), verwendet, welches in der Lage ist, die soziale Phobie im Tiermodell nachzustellen. Wir haben nach einer Verbindung zwischen dem serotonergen System und den zwei Systemen der Neuromodulatoren Vasopressin (AVP) und Oxytocin (OXT) gesucht. Diese Neuropeptide beeinflussen im Gehirn als Neuromodulatoren das Verhalten, und regulieren sowohl positive als auch negative Aspekte des Sozial- und Angstverhaltens. Eine gegenseitige Beeinflussung dieser Neurotransmittersysteme bei der Entstehung von Angsterkrankungen zu identifizieren könnte dabei helfen, potentielle Ziele für neue Behandlungsansätze zu finden. Diese werden dringend benötigt, da der prozentuale Anteil der Patienten, für die es keine wirksame Behandlung gibt, hoch ist. Wir haben Proteinebene und mRNA Expression von unmittelbar frühen Genen (immediate early genes, IEGs) analysiert, um zu ermitteln, in welchen Hirnregionen die neuronale Aktivität durch das Paradigma beeinflusst wird. Außerdem wurde in dieser Studie eine Untersuchung der Gene von AVP-, OXT-, Neuropeptid Y (NPY)-Systemen, sowie von Genen des serotonergen Transmissionssystems eingeschlossen. Damit sollten die Auswirkungen von SFC auf die Genexpression in Hirnregionen, die mit Sozial- sowie Angstverhalten in Verbindung stehen, ermittelt werden. Wir konnten sowohl eine veränderte Genexpression von verschiedenen IEGs wie cFos und Fosl2, als auch Veränderungen in Zahl und Dichte von cFOS-positiven Zellen feststellen, was einen Einfluss von SFC auf neuronale Aktivität andeutet. Unsere Ergebnisse offenbaren eine mögliche Beteiligung des Gyrus dentatus (DG), sowie der Cornu ammonis area 1 (CA1) und CA3 im dorsalen Hippocampus bei der Expression von sozialer Angst. Entgegen unseren Vermutungen waren in der Amygdala keine Veränderungen der neuronalen Aktivität durch Expressionsänderungen der IEGs nachzuweisen. Signifikant höhere IEG-Immunreaktivität und -Genexpression im dorsalen Hippocampus von Tieren ohne Furchtkonditionierung (SFC-) im Vergleich zu Tieren mit Furchtkonditionierung (SFC+) weisen auf zwei mögliche Szenarien hin. Entweder handelt es sich um eine verstärkte Expression in SFC--Tieren im Vergleich zu SFC+-Tieren, oder die Expression in SFC+-Tieren ist im Vergleich zu SFC--Tieren erniedrigt. Ohne eine zusätzliche Kontrolle der basalen mRNA Konzentration und des Proteinvorkommens der IEGs in einer Kontrollgruppe ohne soziale Interaktionsmöglichkeit kann diese Frage allerdings nicht beantwortet werden. Das NPY-System generell und der NPY-Rezeptor 2 im Speziellen scheinen in die Regulation der Reaktion auf soziale Angst involviert zu sein, und dies hauptsächlich im Septum. Zusätzlich konnte eine mögliche Rolle für das serotonerge System und insbesondere den Serotonin Rezeptor 2a im Nucleus paraventricularis (PVN) bei der Reaktion auf soziale Angst identifiziert werden. In einer zweiten Studie haben wir uns auf Veränderungen des serotonergen Systems konzentriert. Ein Polymorphismus im humanen Serotonintransporter Gen (5-HTT) konnte mit einem höheren Risiko für Angsterkrankungen assoziiert werden. Dies macht den 5-HTT zu einem weit verbreiteten Ziel zur Erforschung von möglichen Ursachen und der Entwicklung von Angsterkrankungen. In Mäusen ist ein gentechnisch induzierter knockout des 5-Htt Gens mit erhöhtem Angstverhalten assoziiert. Ein hohes Stresslevel während der Schwangerschaft, auch als pränataler Stress bekannt, erhöht das Risiko für spätere psychiatrische Erkrankungen des noch ungeborenen Kindes signifikant. In unserer Studie haben wir ein pränatales Stress-Paradigma in Mäusen mit einer Defizienz des 5-Htt Gens verwendet. In einer vorangegangenen Studie hatten sich bereits einige der Tiere, die pränatalem Stress ausgesetzt waren, in der Interaktion mit anderen Tieren auffällig „unsozial“ verhalten, bzw. geringes Sozialverhalten gezeigt. Wir haben erneut mithilfe von Genexpressionsstudien nach einer Verbindung zwischen dem serotonergen System und den AVP- und OXT-Systemen gesucht. Zusätzlich haben wir AVP und OXT in Mäusen mit verschiedenen 5-Htt Genotypen und in beiden Geschlechtern auf Neuropeptidebene analysiert, um zu sehen, ob die Produktion von AVP und OXT durch den 5-Htt Genotyp und das Geschlecht beeinflusst ist. Im Zuge der quantitativen Genexpressionsstudie konnten wir zeigen, dass die AVP- und OXT- Neuropeptidsysteme in weiblichen, aber nicht in männlichen Mäusen, durch Pränatalstress beeinflusst werden. Der 5-Htt Genotyp scheint AVP, OXT und andere untersuchte Neurotransmittersysteme nur geringfügig zu beeinflussen. In Weibchen ist die Genexpression von Oxt und Oxtr teilweise stark durch den Östruszyklus beeinflusst. Auf Neuropeptidebene konnten wir einen Geschlechterunterschied bzgl. der durchschnittlichen Anzahl AVP-positiver Zellen im PVN feststellen; männliche Tiere hatten signifikant mehr positive Zellen als weibliche Tiere.