Mechanisms regulating the circannual rhythm of hibernation Item Type Thesis Authors Frare, Carla Download date 10/10/2021 05:57:14 Link to Item http://hdl.handle.net/11122/10618 MECHANISMS REGULATING THE CIRCANNUAL RHYTHM OF HIBERNATION By Carla Frare, B.S., M.Sc. A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry and Neuroscience University of Alaska Fairbanks August 2019 APPROVED: Kelly L. Drew, Committee Chair Abel Bult-Ito, Committee Member Thomas K. Green, Committee Member Thomas B. Kuhn, Committee Member Thomas K. Green, Chair Department of Chemistry and Biochemistry Leah W. Berman, Dean College of Natural Science and Mathematics Micheal A. Castellini, Dean of the Graduate School Abstract Hibernation is a unique adaptation to conserve energy entering a hypometabolic (low metabolic rate) and hypothermic (low body temperature) state called torpor. Torpor is characterized by a drop in metabolism to 1-2% of basal metabolic rate and a decrease in body temperature to one to two degrees above ambient temperature. Metabolic rate is restored to basal metabolic rate and body temperature increases from 2-3°C to 36°C during the regularly timed arousal. The adenosine A1 receptor agonists promote the onset of hibernation and torpor in different species, through a yet undefined neuronal circuit. In the Arctic ground squirrel, CHA, an adenosine A1 receptor agonist, induces hibernation during the winter- hibernation season but not in summer even when the environmental conditions are kept constant (ambient temperature of 20C and a light cycle of 4L:20D). Thus, the phenomenon of CHA- induced hibernation is entrained to an endogenous circannual rhythm. In this work, I aim to identify the changes in neuronal activation that reflect the circannual rhythm regulating the seasonal difference in response to CHA. Arctic ground squirrels, housed at constant ambient temperature (2°C) and light cycle (4L:20D), were implanted with body temperature transmitters. I collected tissue during Summer, Fall, Winter and Torpor conditions for seasonal analysis. For treatment analysis, I collected tissue form animals treated with CHA or vehicle in Summer and Winter. Primarily, I used immunohistochemistry to identify cell groups affected by season and treatment. I used cFos to identify neuronal activity and other immunohistochemical markers to identify neuronal phenotypes, based on specific cytoplasmic proteins. An overall seasonal decrease in thermogenesis, measured as reduced neuronal activity in the thermoregulatory pathways, and increase in vasoconstriction reflected the higher order processing necessary for CHA-induced hibernation. CHA inhibited the histaminergic neurons in the hypothalamus suppressing wakefulness and dis-inhibited the nucleus tractus solitarius, further suppressing thermogenesis. Preliminary data also suggested a seasonal change in the adenosine metabolic pathway, which may have increased adenosine receptor sensitivity during the hibernation season. Our results suggest that histaminergic neurons in the hypothalamus and the nucleus tractus solitarius are likely targets to manipulate metabolic demand in the clinical setting inducing therapeutic hypothermia or increasing metabolic rate. iii Al mio papa', to my dad La nebbia a gl'irti colli piovigginando sale, e sotto il maestrale urla e biancheggia il mar; ma per le vie del borgo dal ribollir de' tini va l'aspro odor de i vini l'anime a rallegrar. Gira su' ceppi accesi lo spiedo scoppiettando: sta il cacciator fischiando sull'uscio a rimirar tra le rossastre nubi stormi d'uccelli neri, com'esuli pensieri, nel vespero migrar. San Martino da Rime Nuove, Giosue' Carducci, 1883. iv Table of Content Page Abstract........................................................................................................................................................ iii Table of Content ........................................................................................................................................... v List of Figures ............................................................................................................................................... ix List of Tables ................................................................................................................................................ xi List of Appendices ....................................................................................................................................... xiii Acknowledgment ........................................................................................................................................xiv Chapter 1: General introduction................................................................................................................... 1 1.1 The phenomenon of hibernation........................................................................................................ 1 1.2 Medical application............................................................................................................................. 3 1.2.1 Obesity ......................................................................................................................................... 3 1.2.2 Therapeutic hypothermia ............................................................................................................ 4 1.3 Adenosine, a potential mediator of hibernation ................................................................................ 5 1.3.1 Adenosine biosynthesis in the brain ............................................................................................ 5 1.3.2 Adenosine and energy metabolism ............................................................................................. 6 1.3.3 Adenosine and sleep .................................................................................................................... 6 1.3.4 Adenosine, hibernation and torpor ............................................................................................. 7 1.4 Research objectives ............................................................................................................................ 8 1.5 References ........................................................................................................................................ 12 Chapter 2: The raphe pallidus and the hypothalamic-pituitary-thyroid axis gate seasonal changes in thermoregulation in the hibernating Arctic Ground Squirrel (Urocitellus Parryii). ................................... 17 2.1 Abstract ............................................................................................................................................. 17 2.2 Introduction ...................................................................................................................................... 18 2.3 Materials and Methods..................................................................................................................... 19 2.3.1 Animals....................................................................................................................................... 19 2.3.2 Seasonal changes in the HPT-axis .............................................................................................. 19 2.3.3 Changes in the HPT-axis after CHA-induced cooling.................................................................. 20 2.3.4 Drugs ..........................................................................................................................................20 2.3.5 Brain tissue processing............................................................................................................... 21 2.3.6 Immunohistochemistry .............................................................................................................. 21 v 2.3.7 Image analysis ............................................................................................................................ 22 2.3.8 Thyroid tissue processing and image analysis .......................................................................... 22 2.3.9 TH analysis.................................................................................................................................. 23 2.3.10 Statistical analysis .................................................................................................................... 24 2.4 Results ............................................................................................................................................... 24 2.4.1 Seasonal changes in the HPT-axis ............................................................................................. 24 2.4.2 CHA-induced cooling .................................................................................................................. 25 2.4.3 Changes in the HPT-axis after CHA-induced cooling.................................................................. 26 2.4.4 Seasonal changes in the SNS and in CHA-induced cooling ....................................................... 26 2.5 Discussion.......................................................................................................................................... 27 2.6 Author contributions......................................................................................................................... 30 2.7 Funding ............................................................................................................................................