Relaxin and the Paraventricular Nucleus of the Hypothalamus by Megan Susan McGlashan A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Biomedical sciences Guelph, Ontario, Canada © Megan S. McGlashan, August, 2013 ABSTRACT RELAXIN AND THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS Megan Susan McGlashan Advisor: University of Guelph, 2013 Professor A. J. S. Summerlee The hormone relaxin regulates the release of the magnocellular hormones, oxytocin and vasopressin, from the central nervous system. Studies have yet to determine whether relaxin regulates magnocellular hormone release through the circumventricular organs alone, or whether relaxin can act on the brain regions containing the magnocellular neurons as well. The paraventricular nucleus of the hypothalamus was isolated from other brain regions and maintained in vitro, in order evaluate the effects of the relaxin and relaxin-3 on the somatodendritic release of oxytocin and vasopressin. At 50 nM concentrations, relaxin induced oxytocin release, while relaxin-3 inhibited oxytocin release. Neither relaxin nor relaxin-3 had an effect on the vasopressin release, however the RXFP3 specific agonist, R3/I5, induced vasopressin release. The effect of the relaxin peptides on the electrical activity of neurons in the paraventricular nucleus was also evaluated. Relaxin depolarized magnocellular neurons while relaxin-3 hyperpolarized the neurons. Relaxin and relaxin-3 appear to have differential effects on the magnocellular neurons of the paraventricular nucleus. iii ACKNOWLEDGEMENTS When we first enter graduate school, we are very much children in the ways of research. As the saying goes, it takes a village to raise a child, so it was for my master’s degree. I would like to thank my village. This thesis was made possible because of you. I would like to thank my advisor, Alastair Summerlee. Thank you for your support, for your guidance, and for teaching me that research is as much about people as it is about bubbling buffer in a lab. Above all I would like to thank you for your continued confidence in my abilities. Thank to my lab family. Jordan- thank you for seeing my potential. Roman- your motivational talks were pivotal in my success. Lindsay- thank you for your support and for inspiring me through your perseverance and success. I would also like to thank the members of my advisory committee- John LaMarre, Brian Wilson and Bettina Kalisch. Your advice was invaluable. Also, thank you to Craig Bailey for being an enthusiastic and capable teacher; learning electrophysiology was far easier than it was reputed to be. Finally, I would like to thank my incredible family. You are responsible for teaching me that education is not just steps between childhood and adulthood, rather it is a lifetime affair. This thesis is as much yours as it is mine. I could not have succeeded without you. Thank you. iv DECLARATION OF WORK PERFORMED I declare that all the work described in this thesis was carried out by the author alone in the laboratories of Dr. Summerlee and Dr. Bailey in the Department of Biomedical Sciences at the University of Guelph, Guelph, Ontario, Canada. v TABLE OF CONTENTS ACKNOWLEDGEMENTS. iii DECLARATION OF WORK PERFORMED. iv TABLE OF CONTENTS. v LIST OF FIGURES. vii LIST OF TABLES. vii LIST OF ABBREVIATIONS . viii LITERATURE REVIEW . 1 Relaxin family of peptides . 1 Historical perspective . 1 Structure of relaxin peptides. 3 Receptors. 5 Signalling pathways . 7 Relaxin-3 . 9 Paraventricular Nucleus of the Hypothalamus . 12 Paraventricular Nucleus of the Hypothalamus . 12 The HNS as model of hormone release in the brain . 14 Heterogeneity within the parvocellular divisions . 15 Electrical properties of neuron types . 17 Relaxin and the hypothalamic-neurohypophyseal system . 18 INTRODUCTION AND PURPOSE OF EXPERIMENTS . 21 METHODS . 23 Animals . 23 Experiment 1. 23 Development of the in vitro release protocol. 23 Perfusion aCSF system . 24 Static aCSF system . 25 Static HEPES buffer system . 27 Experiment 2. 28 vi Patch-clamp recording. 28 RESULTS. 31 Experiment 1: The effect of relaxin-3 on the release of oxytocin and vasopressin from isolated PVN. 31 Perfusion aCSF system. 32 Static aCSF system. 32 Oxytocin. 33 Vasopressin. 34 Static HEPES buffer system. 37 Oxytocin. .37 Vasopressin . .38 Experiment 2: The effect of relaxin peptides on the intracellular electrical activity of isolated neurons from the PVN in vitro. 42 The effect of relaxin-3 and relaxin-2 on the membrane potential of magnocellular neurons . 42 The effect of relaxin-3 and relaxin-2 on the membrane potential of parvocellular neurons. 45 DISCUSSION. 47 The regulation of somatodendritic release in the PVN. 54 The function of relaxin-3 . 58 Technical considerations. .59 CONCLUSION. .63 REFERENCES . 64 vii LIST OF FIGURES Figure 1 Oxytocin release from PVN slices perfused with human relaxin-2. 33 Figure 2 No effect of human relaxin-3 or R3/I5 on oxytocin release from the pvn. 35 Figure 3 The effect of human relaxin-3 and R3/I5 on vasopressin release from the pvn. 36 Figure 4 The effects of relaxin peptides on oxytocin release from PVN of young rats. 39 Figure 5 The effects of relaxin peptides on vasopressin release from PVN of young rats. 40 Figure 6 Identification of neuron types within the PVN by expression of low threshold spikes or outward rectification . 43 Figure 7 The effects of human relaxin-3 and human relaxin-2 on the membrane potential of magnocellular neurons in the paraventricular nucleus. 44 Figure 8 The effects of human relaxin-3 and human relaxin-2 on the membrane potential of neuroendocrine parvocellular neurons in the paraventricular nucleus. 46 LIST OF TABLES Table 1 Change in oxytocin and vasopressin release from isolated PVN when incubated in HEPES buffer with relaxin peptides. 41 viii LIST OF ABBREVIATIONS Abbreviation Term α-MSH Alpha-melanocyte stimulating hormone ACTH Adrenocorticotropic hormone cAMP Adenosine 3’, 5’-cyclic monophosphate cGMP Guanosine 3’, 5’-cyclic monophosphate DAG Diacyl glycerol CRH Corticotropin-releasing hormone GABA Gamma-aminobutyric acid GRH Gonadotropin-releasing hormone GPCR G-protein coupled receptor HNS Hypothalamic-neurohypophysial system IA current A-type potassium current INSL Insulin-like IP3 Inositol trisphosphate LCDVs Large dense cored vesicles LGR Leucine-rich repeat G-protein coupled receptor LTS Lower threshold spikes MAPK Mitogen-activated protein kinase MMP Matrix metalloproteinase mRNA Messenger ribonucleic acid ix NE Neuroendocrine NF- kB Nuclear factor kappa B NO Nitric oxide NOS Nitric oxide synthase PA Pre-autonomic PI3K Phosphoinositide-3 kinase PKA Protein Kinase A PKC Protein Kinase C PVN Paraventricular nucleus of the hypothalamus RXFP Relaxin family peptide receptor SON Supraoptic nucleus TGF-β Transforming growth factor-β TRH Thyroid-releasing hormone 1 LITERATURE REVIEW 1. Relaxin family of peptides 1.1 Historical perspective Relaxin was first identified in 1926 when Fredrick Hisaw observed a loosening of the pelvic symphysis of female guinea pigs following an injection of ovarian extracts derived from pregnant guinea pigs (Hisaw 1926). The protein responsible for this relaxation was later isolated and named relaxin (Fevold et al. 1930). Relaxin was assumed to be a hormone of pregnancy but there was little progress made towards understanding its actions until a technique to extract and purify relaxin from ovarian extracts was developed in 1974 (Sherwood and O’Byrne 1974) and a sensitive radioimmunoassay was developed in 1979 (Sherwood and Crnekovic 1979). Once relaxin could be purified, researchers were able to determine its structure, sequence and binding sites (James et al. 1977, Schwabe et al. 1976; 1977). The relaxin-1 gene was cloned in 1983 (Hudson et al. 1983) followed by the identification of a second human relaxin gene, relaxin-2 (Hudson et al. 1984). Human relaxin-1 is found in primates only, while human relaxin-2 is equivalent to relaxin-1 (relaxin) in non-primates (Sherwood 2004). In fact, we now recognize seven members of the relaxin family: relaxin-1 (Hudson et al. 1983); relaxin-2 (Hudson et la. 1984); relaxin-3 (Bathgate et al. 2002a); and multiple insulin-like peptides (INSL), including INSL3 (Adham et al. 1993); INSL4 (Chassin et al. 1995); INSL5 (Conklin et al. 1999); and, INSL6 (Lok et al. 2000). It is commonly 2 accepted to refer to relaxin-2 simply as relaxin because it was the first relaxin to be identified. Originally, relaxin was considered to be a reproductive hormone, but it has subsequently been shown to be produced in a variety of other tissues, and it is now known to have functions in a number of different systems including the brain, heart, cardiovascular system, skin, kidney, prostate etc. (See review: Sherwood 1994; Bathgate et al. 2013). Summerlee and colleagues (1984) were the first to report an action of relaxin in the brain. They reported an action for intravenously administered relaxin in suppressing reflex milk-ejection in anaesthetized.