Exploration of Variation in the Mu Opioid Receptor Gene and Correlated Changes in Social Stress and Reward Behavior

Transcription

1 Exploration of Variation in the Mu Opioid Receptor Gene and Correlated Changes in Social Stress and Reward Behavior Background The common use of phrases such as broken heart or sinking feeling in my stomach readily demonstrate the universal nature of physical sensations associated with emotional or social pain. Similarly, feelings of social reward are often described as lightness or floating. This connection between the experience of emotional and physical pain is mirrored by similarities in the underlying neurobiology associated with these feelings. In fact, some areas of the brain are activated in exactly the same way in response to both physical pain and emotional pain. 1,2 A key neurobiological regulator of both pain and reward is the opioid system. Endogenous opioid release is associated with increased feelings of euphoria and reduced perception of pain. 3 Opioid release may mediate the relief and reward associated with social interactions, especially after social isolation, and may thereby serve to biologically motivate social contact-seeking. 3,4 This hypothesis has been referred to as the brain opioid theory of social attachment. 3,5,6 Opioids and social behavior. Given the close link between social and physical feelings of pain and reward, it is unsurprising that the opioid system, and its receptor subtypes (mu, kappa, and delta), play a role in the regulation of social interactions. Opioid receptors are found in brain regions associated with social behavior, emotional regulation, and reward. 7 Mother-infant, 8 10 peer, 11 and romantic attachments 12,13 are all mediated by the opioid system, highlighting the importance of this system for the maintenance of a myriad of relationship types. Mu opioid receptor gene variants and behavior. The mu opioid receptor gene (OPRM1) codes for one of the receptors for endogenous opioids. Polymorphisms in the OPRM1 gene have been documented in both humans 14 and nonhuman primates. 15 This polymorphism alters an amino acid in the N terminal arm of the mu receptor. 16 In humans, a guanine (G) base is substituted for an adenine (A) in the 118 th position of the gene (rs ), 14 while in rhesus macaques the homologous polymorphism is a G substituted for a cytosine (C) at the 77 th position. 15 HEK-293 cells expressing the G polymorphism demonstrate a 3.5 times greater affinity for endogenous opioids in vitro, indicating a gain-of-function outcome of this polymorphism. 16 Behaviorally, rhesus macaque monkeys with the G allele display higher levels of infant attachment to, and more persistent distress upon separation from, their mothers compared to non-g carriers. 15 Additionally, rhesus that are carriers of the G allele display lower basal and experimentally stimulated hormonal stress responses (measured by cortisol levels), as well as higher levels of aggression to a threat. 16 In rhesus macaques, the distribution of the G allele has been established to be at least 15%, 15 with one study demonstrating heterozygous carriers at 50%. 16 In humans, carriers of the G allele show more activation of brain regions involved in emotional and pain processing during social rejection, 14 however, are more likely to engage in affectionate relationships and report lower levels of social anhedonia, indicating more pleasure or reward from social interactions. 17 Overall, G carriers may be more sensitive to social cues and show larger responses, both hormonally and behaviorally, to changes in their social environment. Description of Project and Hypotheses In this project, I will survey marmoset monkeys for a polymorphism in the OPRM1 gene homologous to the A118G polymorphism in humans and C77G polymorphism in rhesus macaques. Marmoset monkeys fill a gap in the current understanding of this polymorphism s effects on social behavior because they exhibit a social system (social monogamy) more similar to humans than that of rhesus macaques (polygyny). Thus, the relationship between genetic variants and social behavior between mates in marmosets may be more reflective of the relationship between gene variants and romantic attachments in humans. All marmosets housed at the Callitrichid Research Center (CRC) at the University of Nebraska

2 will be genotyped to assess genetic variation within the OPRM1 gene. Once marmosets have been genotyped, they will undergo a behavioral test in which they will be separated from their mate for several hours to assess behavioral response to social separation, and then returned to their home environment, and mate, to measure behavior upon reunion (e.g., a proxy measure of reactivity to social reward). Behavioral responses to these two components of social behavior (isolation and reward) will be analyzed with a between-subjects design, by genotype. Urine samples will be collected prior to, during, and after the separation to assess hormonal stress response and recovery from the stressor. Analyses will indicate if genetic differences between marmosets are predictive of behavioral, or hormonal, differences during social stress and/or social reunion. If there is no variation observed at the OPRM1 gene, behavioral tests will still be conducted and analyzed for differences in social responses based on individual characteristics. Research Trajectory 1. If the OPRM1 polymorphism is present, behavioral responses of marmosets to social separation and reunion will be analyzed and compared across genotype to quantify behavioral differences correlated with gene differences. Hypothesis: Marmosets that are carriers of the equivalent of the G allele will demonstrate stronger behavioral responses to both social separation and reunion and greater hormonal reactivity. 2. If the OPRM1 polymorphism is not present, behavioral responses of marmosets to social separation and reunion will be analyzed according to metrics of the pair rather than across genotype groups (e.g., length of pairing, age of subject). Hypothesis: Marmosets that have been paired for longer periods of time are predicted to show larger behavioral and hormonal responses to separation and reunion. Methods DNA Extraction and Genotyping Using a DNA extraction kit (Qiagen DNeasy Blood & Tissue Kit), I will extract DNA following the manual s recommended guidelines and following protocols already established in the lab from biosamples (i.e., blood) collected from all marmosets (N = 50) housed at the CRC. I will then amplify the portion of genomic DNA containing the OPMR1 gene with polymerase chain reaction (PCR) using several sets of primer pairs designed to target the sense and anti-sense strand of DNA at exons of the OPMR1 gene. I will design primer sequences using the Primer Blast NIH tool and following published guidelines outlined for primer design. 18 PCR product can then be sequenced with the use of the University of Nebraska Medical Center s CORE gene sequencing facility. I will identify nucleotide polymorphisms or genotypes across individual marmosets via sequence chromatograms using Chromas and nucleotide position alignment using MEGA. Behavioral Observations Marmosets (N = 50) will undergo a 3-hour social separation from their pair mates to assess behavioral responses to social isolation. This paradigm has been modified from a longer social separation previously utilized in marmosets. 19 During social separation, the subject will be provided with food and water and will be monitored for behavioral indicators of anxiety or stress. Additionally, hormonal stress response (cortisol) to the separation will be assessed using urine samples collected prior to, throughout, and after the social separation. At the completion of the 3-hour separation subjects will be reunited with their mate. Social interactions between mates during the reunion will be recorded. This paradigm will therefore provide indicators of both behavioral responses to social stress (isolation) and reward (reunion with the mate). Behavioral responses to these situations can then be statistically analyzed in a between groups analysis of variance (ANOVA) comparing across genetic variants in the OPRM1 gene. Contribution to Graduate Studies It is becoming increasingly important to receive training across multiple disciplines as a graduate student. By conducting the research proposed in this GRACA, I will gain experience in the rapidly expanding

3 field of behavioral genetics. Through coursework (namely the Next Generation Sequencing course at UNO), and direct instruction from senior lab members (i.e. senior graduate students and lab postdoctoral researchers), I have the theoretical background for the genetic component of this proposal. Thus, the hands-on experience provided by this proposal allows me to expand my toolkit of skills, and broadens the potential for my success in future positions as a postdoctoral researcher. Contribution to Field The findings from the proposed research will contribute to the field in two ways. First, the OPRM1 polymorphism proposed for examination has homologous variants in both humans and rhesus macaques. Thus, if this polymorphism exists in marmoset monkeys as well, it highlights the potential evolutionary significance of this mutation, as gene variants with important functional consequences are more likely to be conserved across evolution. Second, this project aims to assess the relationship between genetic variants and social behavior, which may help to discern potential pathways of interest in disorders of social functioning, such as autism spectrum disorder, schizophrenia, anxiety, and depression. April May - June July August - September October - December January - February March IACUC approval Collect Samples Project Timeline Design Sequence Primers/ Samples PCR Behavioral Experiments Analyze Data RCAF Presentation Graduate Student and Advisor Roles I will be responsible for all the behavioral and genetic data collection and analysis. My faculty advisor, Dr. Jeffrey French, has served as a sounding board to discuss research ideas and will continue to be available for questions as the project progresses. Additionally, two post-doctoral researchers currently in the French lab have experience in the field of behavioral genetics and will be available for questions troubleshooting the genetic components of the proposal, including data analysis. Dr. French will work with me to produce a manuscript of the findings collected through the proposed research. Previous Funding and Outcomes Two previous GRACAs have been funded at UNO. The first GRACA, awarded in 2015, titled Differential Roles of Dopamine Receptors in Pair Bond Formation and Maintenance in Socially Monogamous Marmosets, focused on the role of the dopamine system, another component of the neural reward system, in marmoset pair bonding. Data from this GRACA was used as preliminary findings for a predoctoral fellowship application submitted to the National Institutes of Health in the Mental Health institute. This fellowship was awarded in Fall of The second GRACA, which I received in 2016, titled The Role of Social Context and Dopamine in Partner Fidelity in Marmoset Monkeys, focused on the way in which social context interacts with the dopamine-mediated reward system in the production of social behavior. I am currently working with my advisor on writing the results of this project into a manuscript to be submitted to Frontiers in Ecology and Evolutionary, with an expected submission date of January The current proposal builds off, but is distinct from, the two previously funded GRACA proposals through broadening the assessment of the reward system to opioids and incorporating genetic methods in addition to behavioral measures.

4 Budget Justification The following budget and supplies list covers the cost of running 50 samples for genetic testing, as well as a buffer of the cost of approximately 5 samples should any need to be rerun. Should any supplies cost more than anticipated, there will be additional funds available (through Dr. French) to cover unforeseen expenses. DNA extraction and genotyping supplies: Lab supplies: Taq DNA polymerase kit with PCR reagents available through Qiagen $ PCR purification kit available through Qiagen $ DNA isolation and purification kit available through Qiagen $ DNA primers approximately $5/pair $20 Genotype sequencing ~$5-8/ sample 50 samples + 5 repeats $500 Total cost for genetic supplies = $928 Lab consumables (e.g., gloves, tubes, additional in-house reagents, etc) $200 TOTAL SUPPLY COSTS = $1,128 Stipend: The remainder of the requested funds will be put towards a stipend. Data collection is anticipated to take hours. Summer stipend = $3,872 TOTAL REQUESTED BUDGET = $5,000

5 References 1. Eisenberger, N. I. & Lieberman, M. D. Why rejection hurts: a common neural alarm system for physical and social pain. Trends Cogn. Sci. 8, (2004). 2. Eisenberger, N. I. The pain of social disconnection: examining the shared neural underpinnings of physical and social pain. Nat. Rev. Neurosci. (2012). doi: /nrn Machin, A. J. & Dunbar, R. I.. The brain opioid theory of social attachment: a review of the evidence. Behaviour 148, (2011). 4. Panksepp, J., Nelson, E. & Bekkedal, M. Brain Systems for the Mediation of Social Separation- Distress and Social-Reward Evolutionary Antecedents and Neuropeptide Intermediaries. Ann. N. Y. Acad. Sci. 807, (1997). 5. Nelson, E. E. & Panksepp, J. Brain Substrates of Infant Mother Attachment: Contributions of Opioids, Oxytocin, and Norepinephrine. Neurosci. Biobehav. Rev. 22, (1998). 6. Insel, T. R. Is social attachment an addictive disorder? Physiol. Behav. 79, (2003). 7. Ragen, B. J., Freeman, S. M., Laredo, S. A., Mendoza, S. P. & Bales, K. L. µ and κ opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning. Neuroscience 290, (2015). 8. Kalin, N. H., Shelton, S. E. & Lynn, D. E. Opiate systems in mother and infant primates coordinate intimate contact during reunion. Psychoneuroendocrinology 20, (1995). 9. Kalin, N. H., Shelton, S. E. & Barksdale, C. M. Opiate modulation of separation-induced distress in non-human primates. Brain Res. 440, (1988). 10. Moles, A., Kieffer, B. L. & D Amato, F. R. Deficit in Attachment Behavior in Mice Lacking the µ- Opioid Receptor Gene. 304, (2004). 11. Keverne, E. B., Martensz, N. D. & Tuite, B. Beta-endorphin concentrations in cerebrospinal fluid of monkeys are influenced by grooming relationships. Psychoneuroendocrinology 14, (1989).

6 12. Ragen, B. J., Maninger, N., Mendoza, S. P., Jarcho, M. R. & Bales, K. L. Presence of a pair-mate regulates the behavioral and physiological effects of opioid manipulation in the monogamous titi monkey (Callicebus cupreus). Psychoneuroendocrinology 38, (2013). 13. Burkett, J. P., Spiegel, L. L., Inoue, K., Murphy, A. Z. & Young, L. J. Activation of µ-opioid receptors in the dorsal striatum is necessary for adult social attachment in monogamous prairie voles. Neuropsychopharmacology 36, (2011). 14. Way, B. M., Taylor, S. E. & Eisenberger, N. I. Variation in the µ-opioid receptor gene (OPRM1) is associated with dispositional and neural sensitivity to social rejection. Proc. Natl. Acad. Sci. 106, (2009). 15. Barr, C. S. et al. Variation at the mu-opioid receptor gene (OPRM1) influences attachment behavior in infant primates. Proc. Natl. Acad. Sci. 105, (2008). 16. Miller, G. M. et al. A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression. Mol. Psychiatry 9, (2004). 17. Troisi, A. et al. Social hedonic capacity is associated with the A118G polymorphism of the muopioid receptor gene ( OPRM1 ) in adult healthy volunteers and psychiatric patients. Soc. Neurosci. 6, (2011). 18. Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134 (2012). 19. Cavanaugh, J., Carp, S. B., Rock, C. M. & French, J. A. Oxytocin modulates behavioral and physiological responses to a stressor in marmoset monkeys. Psychoneuroendocrinology 66, (2016).

7 Department of PsychologyAH 419 University of Nebraska at Omaha 6001 Dodge Street Omaha, Nebraska PHN (402) FA (402) /8/2017 TO: FR: RE: GRACA Award Committee Jeffrey A. French, Varner Professor of Psychology and Biology Letter of reference, Sarah Carp I endorse the GRACA proposal written by my graduate student, Sarah Carp, entitled Exploration of Variation in the Mu Opioid Receptor Gene and Correlated Changes in Social Stress and Reward Behavior. Sarah developed the research question proposed in this GRACA independently. She did the required background research to appropriately determine the theoretical foundation for her question, as well as the appropriate data collection methods required to address that question. She prepared her proposal autonomously. Sarah has displayed an understanding of the balance of doing independent work while still coming to me for guidance on theoretical questions. In the time that she has been in my lab she has assisted on several research projects and shown dedication to her role as a graduate student. With the funding of her previous GRACA proposals she has developed an independent foci of research distinct from the other graduate students. I have gone through her proposed project with her, have read her proposal, and feel that she has developed a manageable project that she has all the necessary skills to complete in the timeline that she has proposed. She has thoughtfully planned necessary supplies and appropriately budgeted the requested resources. I, as well as postdoctoral fellows in my lab, will be available to Sarah throughout her project for questions regarding techniques, theory, and data analysis. We hold weekly lab meetings at which Sarah can discuss any questions or concerns that arise. Sarah has been highly productive as a scholar in her 3 years at UNO. She completed her masters degree in the Spring of 2017, and has entered into the PhD program with a clear idea of her research goals. She has published one first-authored article based on her undergraduate research, served as co-author on an empirical paper for experiments she assisted with at UNO, and a co-author with me on two invited review papers on nonhuman primate behavior. She is currently preparing another first-author empirical paper of her findings from a study funded by a previous GRACA proposal. Finally, Sarah is one of the few UNO students who has taken the initiative to seek national funding opportunities. She was successfully awarded the Ruth L. Kirschstein National Research Service Award from the National Institute of Mental Health, a highly competitive predoctoral fellowship, in the Fall of University of Nebraska at Omaha University of Nebraska Medical Center University of Nebraska Lincoln University of Nebraska at Kearney