Neural Action of Androgens in the Suprachiasmatic Nucleus Brain Clock By Lindsay Coome A thesis submitted in conformity with the requirements for the degree of Master of Arts Graduate Department of Psychology University of Toronto © Copyright by Lindsay Coome, 2013 Neural Action of Androgens in the Suprachiasmatic Nucleus Brain Clock Lindsay Coome Master of Arts Graduate Department of Psychology University of Toronto 2013 Abstract The suprachiasmatic nucleus (SCN) of the hypothalamus is the locus of a master circadian clock that is critical in the temporal organization of circadian activity. The SCN coordinates the rhythmic secretion of gonadal hormones, and in turn, reproductive hormones may act on their receptors within the SCN to alter circadian function. Using transgenic mice that over-express androgen receptor (AR) only in neurons, the current study investigated the influence of neural AR on the function of the SCN. In particular, it addressed the effects of androgens on circadian behaviours as well as physiological responses to light within the SCN by measuring Fos response after a phase-shifting light pulse. It was found that transgenic mice demonstrate a smaller increase in Fos expression in response to a light pulse than do wildtypes. Interpretations of our findings, including the possible functional significance of AR within the SCN, are discussed. ii Acknowledgements I would first like to thank my supervisor, Dr. Ashley Monks, for his wisdom, guidance, and tremendous patience throughout the year. His support and encouragement have allowed me to grow as a scientist and as a person. I would also like to thank my subsidiary advisor Dr. Joel Levine, for allowing me to benefit from his time and expertise, and Dr. Robert Gerlai, for joining my thesis committee and showing genuine interest in my work. I would like to thank all the members of the Monks and Holmes labs, particularly Ashlyn Swift-Gallant, for her friendship and unwavering belief in my abilities. Finally, I’d like to thank my parents for a lifetime of love and encouragement, without which none of this would be possible. iii Table of Contents Abstract ...............................................................................................................................ii Acknowledgements ............................................................................................................iii List of Tables ......................................................................................................................vi List of Figures ...................................................................................................................vii Introduction.......................................................................................................................... 1 1.1 Overview of Circadian Rhythms ...............................................................................1 1.2 The Circadian System ................................................................................................1 1.2.1 The SCN as the Master Clock......................................................................... 1 1.2.2 The Molecular Basis of the Clock ..................................................................3 1.2.3 Organization of the SCN ................................................................................3 1.2.4 Diffusible and Neural Signals......................................................................... 4 1.3 Circadian Rhythms and Hormones............................................................................ 6 1.3.1 Circadian Regulation of Neuroendocrine Secretions .....................................6 1.3.2 Endocrine Influences on the Circadian System ..............................................6 1.3.3 The Influence of Ovarian Hormones on Circadian Behaviour .......................6 1.3.4 The Influence of Androgens on Circadian Behaviour ....................................7 1.4 The Role of AR in the SCN .....................................................................................10 1.5 The Present Study.................................................................................................... 12 Methods .............................................................................................................................13 2.1 Animals and Housing ...............................................................................................13 2.2 Experimental Design ...............................................................................................14 2.3 Gonadectomy (GDX) and Hormone Replacement ..................................................14 2.3.1 Steroid Implants............................................................................................ 14 2.3.2 Gonadectomy................................................................................................ 15 2.4 Behavioural Testing .................................................................................................15 2.5 Neural Activation Following Exposure to Light .....................................................15 2.6 c-Fos Immunohistochemistry ..................................................................................16 iv 2.7 Androgen Receptor Immunohistochemistry ............................................................16 2.8 Data Analysis ...........................................................................................................17 2.8.1 Brain Analyses ..............................................................................................17 2.8.2 Behavioural Analyses ...................................................................................17 2.8.3 Statistical Analyses .......................................................................................18 Results ...............................................................................................................................18 3.1 Somatic Measures.................................................................................................... 18 3.2 Behaviour................................................................................................................. 19 3.3 Neural Activation..................................................................................................... 19 Discussion.......................................................................................................................... 20 References .........................................................................................................................26 Tables .................................................................................................................................33 Figure Captions.................................................................................................................. 37 Figures ...............................................................................................................................38 v List of Tables Table 1. Number of animals per condition for behavioural studies. Table 2. Number of animals per condition for analysis of neural activation. Table 3. Group Means and Standard Error of the Means (SEM) for number of Fos-ir cells. Table 4. Group Means and Standard Error of the Means (SEM) for number of beam breaks. vi List of Figures Figure 1. Total number of beam breaks recorded over 5 days. Figure 2. Number of Fos-ir cells in bilateral SCN. Figure 3. Androgen receptor immunoreactive cells of Nestin-AR mutants and wildtype littermates. vii 1 Introduction 1.1 Overview of Circadian Rhythms To ensure optimal function, animals have evolved to restrict many behaviours, including locomotor activity, feeding, and reproductive behaviours, to specific temporal niches. Much of this temporal variation in behaviour is controlled by biological clocks. Although many of the observed rhythms in physiology and behaviour are mediated by internal timing mechanisms, other aspects of rhythmicity are sensitive to environmental time cues, such as the daily light-dark cycle. Daily rhythms that are driven by external signals in the environment are called diurnal rhythms, and disappear under constant environmental conditions. Rhythms that are endogenously driven are referred to as circadian rhythms, and persist in the absence of external cues. Under constant conditions devoid of any external time cues, it has been found that circadian rhythms continue to be expressed with a period that approximates, but is rarely identical to, 24 hours (Pittendrigh, 1960). Some animals have free-running internal clocks with a period longer than 24 hours, while others, such as mice, free-run with a period shorter than 24 hours. The fact that the circadian period under free-running conditions is not exactly 24 hours suggests that the circadian pacemaker can be synchronized to external time cues (Arble, Copinschi, Vitaterna, Van Cauter, and Turek, 2011). Light is the primary environmental time cue, or “zeitgeber”, that synchronizes biological rhythms, however other zeitgebers include temperature, food availability, noise, and social cues (Bruce, 1960). Internal clocks must be adjusted on a daily basis to stay synchronized to local time, and this synchronization of the internal clock to a periodic cue in the environment is known as entrainment. In the absence of zeitgeber cues, circadian rhythms are said to be free- running. 1.2 The Circadian System 1.2.1 The SCN as the Master Clock The circadian system is necessary