Pair bonding: what mediates its formation and maintenance? by Amanda M. Dios A Dissertation submitted to Graduate School-Newark Rutgers, The State University of New Jersey for the degree of Doctor of Philosophy in Psychology written under the direction of Mei-Fang Cheng and approved by ___________________________________ Mei-Fang Cheng ___________________________________ Michael Shifflet ___________________________________ Michael Stephen J. Hanson ___________________________________Shifflet Mauricio Delgado ___________________________________ Mark Hauber Newark, NJ May 2015 Copyright page: © [2015] Amanda Dios ALL RIGHTS RESERVED ABSTRACT OF THE DISSERTATION Pair bonding: what mediates its formation and maintenance? BY: AMANDA DIOS Dissertation Director: Mei Fang Cheng Pair bonding is an exclusive mating relationship associating the memory of a mate with the potential successful completion of a breeding cycle. In evolutionary biology, pair bonding has been studied as a mating strategy and this biological phenomenon has been associated with survival related reproductive behaviors, however, behaviors that function in the formation of bonding and those that are established as a result of bonding are seldom distinguished, making it difficult to assess how pair bonding is represented in the brain. This thesis assesses pair bonding in ring doves, an animal that has a predictable sequence leading to a successful breeding cycle. Through a series of lesion, immunohistochemistry, and behavior studies we sought to understand the processing and execution of this fitness-critical complex behavior, by assessing the neural basis of pair bonding, understanding which behavioral events initiate the formation of the bond and how bonding affects an animal's decisions involving a mate. We found a neuro- marker for pair bonding that is more accurate than current methods of measuring bonding. Lesions to this region, the nucleus taeniae, disrupted pair bonding in doves, suggesting that the nucleus taeniae is causally linked to pair bonding. Using the neural marker for pair bonding, we found that ring doves formed a pair bond following the performance of courtship (nest coo) ii behavior, rather than the completion of the entire breeding cycle as previously determined (Morris and Erickson 1971). The nest coo phase of the breeding cycle triggers the hypothalamic- pituitary response in female doves culminating in ovulation (Cheng et al 1998; Cheng and Balthazart 1982), suggesting that doves are using this stage to predict the successful completion of the breeding cycle with their mate, a concept consistent with pair bonding’s role in survival behavior. We found that quality of the female, whether she is a bonded mate versus a novel female, affects the amount of courtship behavior performed by the males. Male doves that have formed a pair bond perform less amount of courtship (nest coo) behavior towards their mate than male doves that are not bonded do towards a strange female, consistent with the observation that bonded doves can skip earlier parts of the courtship routine because they have already successfully completed a breeding cycle. Paradoxically, we show that this attenuation of nest coo behavior, in response to the value of the female stimulus (bonded mate versus strange female) , diminishes the production of new neurons following lesions to the VMN, the brain region moderating reproductive behavior, thereby slowing the process of synaptogenesis and, in turn, recovery of behavior after damage to this area. iii Preface: The idea for my thesis stemmed from casual conversations with my advisor regarding what it means to be pair bonded. Although pair bonding manifests itself differently across varying pair bonded species, the ultimate goal of forming a bond is the same, to ensure the reproductive success of the individual. If this was the case, pair bonding could be more than just a conglomeration of behaviors that occurs following sensory input from the bonded mate. Ultimately my thesis led me to ask: can pair bonding be represented in the brain as its own concept and, therefore, be measured in its own right, rather than measured by the behaviors which it produces? I set out to answer this question, the product of which can be found in the sections that follow. I could not have accomplished this undertaking without the guidance and support of my advisor, Mei Fang Cheng, who allowed me to follow my interests and offered advice and encouragement throughout my time in graduate school, in particular while I was writing my thesis. I would also like to thank my committee members, Michael Shifflet, Stephen Hanson, Mauricio Delgado, and Mark Hauber for their input during the production of my thesis and our laboratory’s undergraduate research assistant, Cody Curatolo, who helped administer a portion of the behavior tests in my thesis. iv Acknowledgements: The completion of my thesis would not have been possible without the support of my family. I would like to thank my mother, Maria Dios, for encouraging my curiosity, inspiring me to take chances, and instilling in me the value of hard work. Thank you to my sister, Cristina Dios, for encouraging me to pursue a career in a field that I love. Thank you to Guy Sterling for his support, my aunt, Obdulia Mendez, for always being a phone call away, and my grandmother, Maria D. Enriquez, for reminding me that the opportunities that I have today would not be possible without the determination of the people before me. Thank you to my friends and fellow graduate students Dana Mastrovito, David Lorenzi, and Kasia Garland, who spent hours entertaining my discussions about pair bonding. Thank you to my mentors, especially my advisor, Mei-Fang Cheng, for encouraging my interest in the sciences and allowing me the opportunity to follow it. v Table of Contents: Chapter Title Page Title page i Copyright ii Abstract iii Preface v Acknowledgements vi Introduction 1 Functional properties of amygdala and taeniae: emotional memories 7 Amygdala and taeniae connectivity 13 Reproductive behavior in doves 18 1 Chapter 1: Is there a neural marker for pair bonding 25 1.1 Introduction 25 1.2 Experiment 1: Methods 28 1.3 Experiment 2: Methods 32 1.4 Results 33 1.5 Discussion 36 1.6 Addendum 40 2 Chapter 2: Does the nucleus taeniae mediate pair bonding behavior? 47 2.1 Introduction 47 2.2 Experiment 1: Does a taeniae lesion destroy a dove’s preference 51 for its mate Methods 51 Results 54 2.3 Experiment 2: Do bonded male doves court (nest coo) less 56 towards their mates than non-bnded doves do towards a stimulus female? Methods 57 Results 58 2.4 Experiment 3: Do taeniae lesions affect intensity of nest coo 59 (courtship) behavior due to bond status? Methods 59 Results 61 2.5 Experiment 4: Is a brief exposure (no courtship behavior) 62 sufficient to produce difference in ZENK expression in doves that are bonded versus non-bonded Methods 62 Results 64 vi 2.6 Discussion 64 3 Chapter 3: Breeding cycle and its relationship to pair bonding 68 3.1 Introduction 68 3.2 Methods 70 3.3 Results 73 3.4 Discussion 74 4 Chapter 4: The nucleus accumbens and pair bonding in ring doves 77 4.1 Introduction 77 4.2 Experiment 1: Pair bond maintenance 79 Methods 79 Results 83 4.3 Experiment 2: Pair bond formation 86 Methods 86 Results 89 4.4 Discussion 90 5 Chapter 5: The impact of pair bond status on the rate of neurogenesis 94 induced by VMN damage 5.1 Introduction 94 5.2 Experiment 1: Effect of housing stimulus (female) of varying 97 bond status on VMN lesion induced new neuron production and behavior during recovery period following lesion Methods 97 Results 101 5.3 Experiment 2: Male’s courtship behavior during recovery period 105 following VMN lesion: effect of female’s behavior during testing period Methods 105 Results 106 5.4 Experiment 3: Effect of standardizing testing stimulus (exposing 109 all males to a novel female during behavior tests) on neurogenesis, recovery of courtship function, and mate preference Methods 110 Results 111 5.5 Experiment 4: Effect of destruction of memory of mate, via 115 taeniae lesion, on neurogenesis and courtship behavior during recovery period following VMN lesion Methods 115 Results 117 5.6 Discussion 121 General Discussion 127 vii Bibliography 138 viii List of tables: Chapter Title Page Introduction Amygdala and taeniae connectivity in birds and mammals: table 1 16 1 Chapter 1: Is there a neural marker for pair bonding Preference test table: table 1 34 5 Chapter 5: The impact of pair bond status on the rate of neurogenesis induced by VMN damage Summary of experiments 97 ix List of illustrations: Chapter Title Page 1 Chapter 1 Figure 1: Preference test 29 Figure 2: Classification for discriminant analysis 35 Addendum Figure 1: ZENK labeled cells in taeniae 43 Figure 1: ZENK labeled cells in nucleus accumbens 44 2 Chapter 2: Does the nucleus taeniae mediate pair bonding behavior? Experiment 1: Does a taeniae lesion destroy a dove’s preference for its mate Figure 1: Cumulative lesion site area 55 Figure 2: Image of lesion site 55 Figure 3: Percentage of time spent with mate 56 Figure 4: Amount of bow and nest coo behavior 56 Experiment 2: Do bonded male doves court (nest coo) less towards their mates than non-bnded doves do towards a stimulus female? Figure 5: Amount of nest coo behavior 58 Experiment 3: Do taeniae lesions affect intensity of nest coo (courtship) behavior due to bond status? Figure 6: Cumulative lesion site area 60 Figure 7: Image of lesion site 61 Figure 8: Amount of nest coo behavior 61 3 Chapter