The hypocretin peptides are two hypothalamic peptides known to be involved in both sleeping and feeding behavior, however their specific roles in these domains are not well understood. The present study sought to determine the effect of chronic (72-hour) sleep deprivation and (48-hour) food deprivation on preprohypocretin (PPH), which is the precursor for the hypocretin peptides. PPH levels were visualized and quantified via in situ hybridization. A three-factor ANOVA ( group x dorsal/ventral x medial/lateral) revealed a significant effect of subregion, specifically dorsal/middle and ventral/medial exhibited elevated PPH levels, however there was no effect of group. A between group one-way ANOVA revealed no effect of group on PPH levels. It is theorized that four possible domains may be responsible for these results: presence of hypothalamic neuronal subpopulations, role of circadian rhythm, role of hypocretins in locomotive behavior and inextricably confounded variables. These are discussed at length.

Post-traumatic stress disorder (PTSD) is a psychiatric condition that can develop when people experience a stressful and life-threatening event. Clinical research indicates that the presence of a state of hyperarousal after a traumatic experience is the best predictor of a subsequent diagnosis of PTSD. The role of arousal peptides called orexins (hypocretins) in a PTSD-like condition produced by exposing rats to a single episode of footshocks (5× 2 s episodes of 1.5 mA) was investigated in this thesis. The first part of my thesis involves the characterization of the footshock model of PTSD and the second part examines the involvement of orexins in this footshock model. The following findings are reported. First, shock rats that exhibited a high level of anxiety to a novel tone (high responders, HR) the day after the footshock exposure subsequently displayed more avoidance when compared to shock rats that exhibited a low level of anxiety (low responders, LR). These results highlight the importance of individual differences in the reaction to a strong fear-inducing experience. Second, the orexin precursor peptide prepro-orexin (ppOX) mRNA was found to be elevated in rats at 6 and 14 days after exposure to footshocks. In addition, ppOX mRNA levels were found to be positively correlated with anxiety at 14 days post-shock. Third, pre-shock injections of the corticotropin releasing factor receptor antagonist antalarmin were found to attenuate the anxiety expressed to the shock chamber and eliminate the correlation between ppOX mRNA levels and anxiety. Fourth, systemic injections of the nonselective orexin receptor antagonist TCS-1102 was found to attenuate the anxiety expressed in rats at 14 days post-shock. Fifth, TCS-1102 was found to have anxiolytic effects that were specific for the HR. The results of these experiments provide evidence linking the orexin system to the anxiety produced by exposure of rats to footshocks. They also provide preclinical evidence in support of the use of orexin antagonists for the treatment of anxiety in PTSD.

Post-traumatic stress disorder (PTSD) is a psychiatric condition that can develop when people experience a stressful and life-threatening event. Clinical research indicates that the presence of a state of hyperarousal after a traumatic experience is the best predictor of a subsequent diagnosis of PTSD. The role of arousal peptides called orexins (hypocretins) in a PTSD-like condition produced by exposing rats to a single episode of footshocks (5× 2 s episodes of 1.5 mA) was investigated in this thesis. The first part of my thesis involves the characterization of the footshock model of PTSD and the second part examines the involvement of orexins in this footshock model. The following findings are reported. First, shock rats that exhibited a high level of anxiety to a novel tone (high responders, HR) the day after the footshock exposure subsequently displayed more avoidance when compared to shock rats that exhibited a low level of anxiety (low responders, LR). These results highlight the importance of individual differences in the reaction to a strong fear-inducing experience. Second, the orexin precursor peptide prepro-orexin (ppOX) mRNA was found to be elevated in rats at 6 and 14 days after exposure to footshocks. In addition, ppOX mRNA levels were found to be positively correlated with anxiety at 14 days post-shock. Third, pre-shock injections of the corticotropin releasing factor receptor antagonist antalarmin were found to attenuate the anxiety expressed to the shock chamber and eliminate the correlation between ppOX mRNA levels and anxiety. Fourth, systemic injections of the nonselective orexin receptor antagonist TCS-1102 was found to attenuate the anxiety expressed in rats at 14 days post-shock. Fifth, TCS-1102 was found to have anxiolytic effects that were specific for the HR. The results of these experiments provide evidence linking the orexin system to the anxiety produced by exposure of rats to footshocks. They also provide preclinical evidence in support of the use of orexin antagonists for the treatment of anxiety in PTSD.

Orexin A and B, two recently isolated hypothalamic peptides, have been reported to increase food consumption upon intracerebroventricular injections in rats. In addition to the hypothalamus, orexin A-immunoreactive fibers have been observed in several areas of the medulla that are associated with cardiovascular functions. The present study was undertaken to evaluate the hypothesis that orexins may influence cardiovascular response by interacting with neurons in the medulla. Intracisternal injections of orexins A (0.0056- 7.0 nmol) or B (0.028-0.28 nmol) dose dependently increased mean arterial pressure (MAP) by 4-27 mmHg and heart rate (HR) by 26-80 beats/min in urethan-anesthetized rats, with orexin A being more effective in this regard. MAP and HR were not changed by intravenous injection of orexins at higher concentrations. Microinjection of orexin A (14 pmol/50.6 nl) to the rostral ventrolateral medulla, which was confirmed by histological examination, increased MAP and HR. Our results indicate that, in addition to a role in positive feeding behavior, orexins may enhance cardiovascular response via an action on medullary neurons.

Abstract Orexins, or hypocretins, are peptides originally found in the hypothalamus, and have been shown to be involved in the stabilization and maintenance of sleep and wakefulness. In addition, these peptides are known for their actions on energy homeostasis by increased heat production or physical activity. Previous results suggest them to be also involved in peripheral actions on the regulation of intestinal secretion, depending on the subject’s nutritional status (fasted-fed). Orexin-A and Orexin-B peptides, are derived from the prepro-orexin precursor protein. These ligands bind to two G-protein-coupled receptors, orexin-1 and -2 -receptors. Despite intensive research, the role of orexins has not yet been clarified. The aim of the present study was to investigate the role of orexins and their receptors on sleep and wake patterns, energy homeostasis and intestinal secretion. The effects of orexins on sleep and wakefulness, and energy homeostasis were studied in a novel transgenic mouse line, overexpressing the human prepro-orexin gene. The overexpression of prepro-orexin and orexin-A was confirmed in the hypothalami of transgenic mice. The transgenic mice showed a significant reduction in their REM sleep during day and night time, and differences in their vigilance states in the light/dark transition periods. In addition, the mice demonstrated a significantly elevated day time food intake at room temperature, and an increased metabolic heat production independent of uncoupling protein 1 mediated thermogenesis in brown adipose tissue. Instead, transgenic mice showed increased levels of uncoupling protein 2 in white adipose tissue. Furthermore, transgenic mice significantly decreased their total locomotor activity during the first two nights in response to cold exposure (+4°C). The effect of orexins and their receptors on duodenal HCO3– secretion were studied after an overnight (16 h) food deprivation in an in situ perfused organ. Fasting decreased the expression of orexin receptors in rat duodenal mucosa and in acutely isolated duodenal enterocytes. Furthermore, food deprivation abolished OXA induced duodenal mucosal HCO3– secretion in rats, and intracellular calcium signalling in isolated rat and human duodenal enterocytes. In conclusion, the present thesis demonstrates that orexins inhibit REM sleep. In addition, peptides affect increasingly on metabolic heat production, independent of uncoupling protein 1 mediated thermogenesis. Furthermore, the orexin system has a significant role in duodenal bicarbonate secretion, which is regulated by the presence of food in the intestine.