1. The Physiology of Oxytocin and Its Role in Social Behavior and Health Philip McCabe Department of Psychology Health Psychology Program; Neuroscience Program University of Miami Coral Gables, FL

2. Oxytocin Oxtocin (OT) means “Quick Birth” OT found in placental mammals, some marsupial mammals, and the ratfish Related peptides found in most other species (e.g., isotocin)

3. Oxytocin: Chemical Properties 9 amino acid peptide Molecular weight is 1007 Differs from vasopressin (AVP) by amino acids at positions 3 and 8

4. Oxytocin: Biosynthesis Human OT gene mapped to chromosome 20p13 Gene transcribes inactive precursor, OT- Neurophysin I, which is hydrolyzed by enzymes into smaller fragments, including OT and Neurophysin Gimpl & Fahrenholz, 2001

5. Hypothalamic Source of OT OT synthesized in hypothalamic magnocellular neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) OT also found in some parvocellular neurons in PVN

6. Hypothalamic OT Cell Bodies 3V PVN SON Optic Chiasm Ludwig & Leng, Nature Reviews Neuroscience, 2006

7. Neurohypophyseal Mechanism

8. OT: Beyond Reproduction Classic OT functions related to parturition and milk ejection in females OT found in equivalent concentrations in the posterior pituitary and plasma of both sexes Suggests OT has functions beyond female reproductive functions

9. Plasma Titers of OT Human0.13pg/ml-414pg/ml Monkey5.0pg/ml-275pg/ml Cow1.0pg/ml-10.0pg/ml Rabbit8.0pg/ml-3000pg/ml Rat1.0pg/ml-700pg/ml Guinea Pig1.0pg/ml-25pg/ml Vole250pg/ml-500pg/ml Mouse1.0pg/ml-300pg/ml

10. OT Release and Metabolism Evidence that OT is released in a pulsatile fashion Turnover rate of OT is fast Half life in plasma reported between 2-12 minutes (most estimates between 2-7 minutes) Half life in brain is longer (approx. 30 minutes) OT is metabolized by an enzyme, oxytocinase At physiological concentrations, OT does not appear to cross the blood-brain-barrier

11. OT Tissue Expression OT is also expressed in a variety of tissues, including: Ovaries/Corpus Luteum, Uterus, Placenta, Prostate Gland, Testes/Leydig Cells, Thymus, Adrenal Medulla, Heart, Aorta, Cancer Tumors, Brain OT may have local autocrine/paracrine actions

12. Oxytocin Receptor (OTR) OTR is a 389 amino acid polypeptide 7 transmembrane domains G-protein coupled receptor OTR gene is present as single copy mapped to chromosome 3 in humans Forms a subfamily of structurally related receptors with 3 vasopressin receptor subtypes (V1a, V1b, and V2) Gimpl & Fahrenholz, 2001

13. Oxytocin Receptor Gimpl & Fahrenholz, 2001

14. OTR Selectivity OTR is relatively non-selective OTR has only a 10-fold higher affinity for OT than AVP AVP acts as partial agonist for OTR Need 100-fold greater concentration of AVP than OT to get a comparable response Gimpl & Fahrenholz, 2001

15. OTR Signaling Pathways OTRs are functionally coupled to G q/11α class GTP binding proteins that stimulate phospholipase C-β isoforms Leads to production of inositol triphosphate, which triggers Ca ++ release from intracellular stores, and diacylglycerol This stimulates protein kinase C, which phosphorylates target proteins and activates the ERK1/2 pathway Intracellular Ca ++ leads to production of nitric oxide and cGMP

16. OTR Signaling Pathways Devost, Wrzal & Zingg, 2008

17. OTR Regulation OTRs go through dramatic tissue-specific up and down regulation (as much as 10-100 fold) Regulation occurs at the transcriptional, translational and protein levels Down regulation of OTR can occur in seconds/minutes as receptor uncouples from the G-protein and undergoes endocytosis, internalization and sequestration Devost, Wrzal & Zingg, 2008

18. OTR Regulation OTRs require two elements for high affinity binding: divalent cations (Mg ++ or Mn ++ ) and cholesterol (membrane stabilization) Estrogen up regulates the expression of OTRs, whereas progesterone inhibits the action of OTRs

19. OTRs: Acute-Phase Response Elements? Promoter region of OTR gene contains IL-6 response elements and acute- phase response elements Suggests that the acute induction of OTR expression could be similar to induction of acute-phase response genes induced by infection or inflammation

20. OTR Tissue Expression OTRs are expressed in a variety of tissues, including: Specific brain regions, uterus, mammary glands, pituitary gland, prostate gland, heart, blood vessels, kidney, pancreas, adrenal gland, cancerous tumors (e.g., breast, uterus, brain, lung), lymphocytes, macrophages and adipocytes No OTRs observed in liver

21. Role of OT in Social Behavior and Brain Activity Beginning in 1970’s, OT implicated in coordination of behaviors in mothers necessary for survival of offspring (i.e., maternal behavior) Accumulating evidence that OT acts in the brain to modulate constellation of behaviors associated with sociality (social cognition & affiliative behavior in both sexes; see Ross & Young, 2009 for review)

22. Maternal Care in Rodents OT injected intracerebroventricularly (i.c.v.) in virgin rats elicited maternal behavior toward pups within 2 hours (facilitated by estrogen priming) i.c.v. administration of OT antagonists (OTAs) blocked maternal behavior in rats who just gave birth OT plays more important role in the initiation of maternal behavior than the maintenance of these behaviors OT knockout mice (OTKO) and OTR knockouts (OTRKO) support these pharmacological findings

23. Ovine Maternal Bonding Sheep form strong selective mother-lamb social bonds, and mothers will reject lambs who are not their offspring This effect seems to be dependent on olfactory memory i.c.v. OT induces maternal behavior in less than one minute in estrogen-primed nonpregnant ewes, and facilitates olfactory memory by modulating norepinephrine and synaptic plasticity in the olfactory bulb

24. Alloparental Behavior in Voles Unlike most rodents and mammals, prairie voles are socially monogamous, and form selective preference for one mate (i.e., pair bond) Prairie voles also display biparental care, and will “baby sit” vole pups not their own There is evidence that OTR density in the nucleus accumbens (NAcc) of the brain is related to this alloparental care

25. OTR Density in NAcc and Alloparental Behavior ( Olazabal & Young, 2006 )

26. Social Bonding in Adult Voles i.c.v. infusion of OT during 6 hr cohabitation with male induces partner preference in unmated female voles OTA administration in mated females blocks pair bonding i.c.v. AVP in males induces partner preference AVP antagonist blocks pair bonding in males Role of OT in male pair bonding not clear Ross & Young, 2009

27. OT and AVP in Pair Bonding Young, Wang & Insel, 1998

28. Pair Bonding and NAcc OTRs Monogamous prairie voles have greater density of OTRs in NAcc than non-monogamous species of voles OTA injected into NAcc in female prairie voles prevented the formation of a partner preference Over-expression of OTRs in NAcc enhanced pair bonds in prairie voles, but not other species of voles Therefore, increased OTR in NAcc alone is not sufficient to produce species differences in pair bonding Ross & Young, 2009

29. Pair Bonding and NAcc OTRs Insel & Young, 2001

30. Why Is NAcc OT Important? NAcc receives dopamine input from midbrain ventral tegmental area, and is part of mesolimbic dopamine reward/reinforcement pathway It has been shown in female prairie voles that dopamine and OT systems interact in NAcc to promote pair bonding (i.e., reinforcement of the mating experience) It has been suggested that ability of female to form attachment with male partner evolutionarily arose from a modification of the neural machinery involved in regulating maternal behavior Ross & Young, 2009

31. Social Recognition in Rodents It has been hypothesized that pair bond formation is due to an association between the rewarding mating experience and the olfactory signature of the partner (i.e., social recognition) i.c.v. OT increases the amount of time a male rat remembers a conspecific

32. Brain Mechanisms in Social Recognition Brain regions implicated in rodent social memory are: Ventral Hippocampus, Septal Nuclei, Medial Preoptic Area and Olfactory Bulb Although OT injected into these regions enhances memory, OTA administration does not block memory performance, and therefore the brain mechanisms for social recognition are not clear

33. OTKO Mice and Social Memory OTKO mice show deficits in social memory, but not general memory deficits (e.g., habituation to non-social odors) A single i.c.v. injection of OT before initial exposure completely rescues the deficit in social recognition Effect seems to involve the medial amygdala OT may act to enhance the saliency of social stimuli and to encode social memories, which facilitates social relationships Ross & Young, 2009

34. Neural Circuitry of OT System Cell bodies of OT neurons are almost exclusively confined to the hypothalamic PVN and SON OT fibers are located throughout the CNS: Dorsal Medial Hypothalamus, Several Thalamic Nuclei, NAcc, Hippocampus, Entorhinal Cortex, Septal Nuclei, Amygdala, Olfactory Bulbs, Periaqueductal Grey, Substantial Nigra, Locus Coeruleus, Raphe Nuclei, Nucleus Tractus Solitarius, Dorsal Vagal Nucleus, Spinal Cord

35. Central OT Projections B.B. McEwen, 2004

36. Central OT Projections Prevailing view is that separate populations of OT neurons project to posterior pituitary and to CNS structures SON and PVN magnocellular neurons project to the posterior pituitary PVN parvocellular neurons are the source of centrally projecting fibers Evidence that central and peripheral OT release can be dissociated

37. Dendritic Release of OT Peptidergic neurons can release peptide from entire cell surface, and peptide can diffuse long distances due to longer CNS half-life OT can be released by dendrites independent of neuronal firing Dendritically-released OT can have local autocrine/paracrine actions, or can diffuse to distant brain sites Ludwig & Leng, 2006

38. Differential Regulation of Dendritic and Axonal OT Secretion Ludwig & Leng, 2006

39. Role of OT in Stress and Emotion A variety of stressors and stress paradigms result in increased plasma OT: Noxious Stimuli (e.g., footshock) Shaker Stress Forced Swimming Immobilization Stress Fear Conditioning And increased secretion of CNS OT: Forced Swimming (increased PVN & SON OT) Social Defeat (increased SON OT) Shaker Stress (increased PVN OT)

40. Role of OT in Stress Given that OT is released in brain as a function of various types of stress, what is the significance of this local hypothalamic release? Since hypothalamic OT is released at the same time as activation of the HPA axis, does brain OT regulate HPA function? Neumann, 2008

41. Central OT Regulation of HPA Axis i.c.v. OT infusions in rats reduces plasma corticosterone release to wide variety of physical, emotional and pharmacological stressors in both male and female rodents Suggests OT can modulate HPA axis, probably via inhibition of CRH in PVN Elevated brain OT may serve to buffer the organism from stress Neumann, 2008

42. Central OT Regulation of Anxiety-Related Behavior i.c.v. administration of OT in both female and male rats exerted an anxiolytic effect (assessed by elevated plus maze) These effects were localized to the central nucleus of the amygdala and PVN Over expression of amygdaloid OTR in virgin female rats reduced anxiety-related behavior compared to control virgin females Neumann, 2008

43. Stress Responses and Anxiety Related Behavior in OTKO Mice Deletion of OT gene in females led to increased anxiety-related behavior and elevated plasma corticosterone during anticipation of shaker stress In OTKO males, elevated corticosterone was seen following overnight food and water deprivation Both OTKO and OTRKO mice exhibited heightened aggression compared to wild type mice Amico et al., 2008

44. Central OT, Stress and Emotion Increased central OT leads to reduced anxiety and calmness accompanied by blunted plasma glucocorticoid responses It appears that the OT system is activated in response to particular stressors, and serves to attenuate both the physiological stress response and the emotional component of the response

45. Role of OT in Inflammation OT shown to reduce inflammation and oxidative stress in several models: Carrageenan-Induced Inflammation (hindpaw) Wound Healing/Burns Sepsis Induction Colonic Inflammation Renal Injury Suggests that OT is an endogenous anti- inflammatory and anti-oxidant molecule Does OT play a role in the attenuation of disease via anti-oxidant and anti-inflammatory mechanisms?

46. Behavior & Atherosclerosis In our lab, we sought to examine the influence of social environment and emotional behavior on the progression of atherosclerosis Developed an animal model of disease that would allow us to study more easily the pathophysiological mechanisms in atherosclerosis

47. Watanabe Heritable Hyperlipidemic Rabbit (WHHL) Model for human familial hypercholesterolemia Spontaneous genetic mutation in LDL receptor synthesis Extremely high plasma lipids from birth Aortic atherosclerosis begins at 2 months, severe in all animals by 7 months, CHD develops by 8-10 months, death occurs after 1 year Since disease occurs spontaneously, can examine factors that slow the progression of disease as well as factors that advance disease progression

48. Social Environment and Progression of Atherosclerosis in the WHHL (McCabe et al., 2002, Circulation) 33 male WHHLs, 3 months of age Assigned to one of 3 social conditions: Unstable (paired with unfamiliar rabbit 4hrs/day, pairing rearranged each week) Stable (paired with littermate 4hrs/day, pairing maintained throughout study) Individually-caged (housed alone, no contact with other animals) Study ran from 3 to 7 months of age

49. Area of Atherosclerosis as a Function of Social Environment

50. Study Conclusions Stable social environment, characterized by increased affiliative behavior and decreased agonistic behavior, slows the progression of atherosclerosis by approximately 50% in animals genetically predisposed to disease Group differences in disease could not be explained by differences in lipids, glucocorticoids, or gonadal steroids Is disease attenuation in the Stable group related to oxytocin’s antioxidant and antiflammatory effects on vascular tissue?

51. Pathophysiology of Atherosclerosis From Ross, R., NEJM, 1999

52. LDL LDL Endothelium Vessel Lumen Monocyte Macrophage Adhesion Molecules Foam Cells Intima Oxidized LDL Cytokines Further Inflammation Cell Proliferation Matrix Degradation Cytokines Growth Factors Metalloproteinases MCP-1MCP-1 NAD(P)H Oxidase Pathophysiological Processes in Atherosclerosis INFLAMMATION OXIDATIVE STRESS Adapted from P. Barter, Lipids Online

53. Oxytocin and Vascular Cells: In Vitro studies (Szeto et al., Am. J. Physiol.: Endocr. Metab., 2008) We have cultured human aortic endothelial cells, monocytes, macrophages and vascular smooth muscle cells Incubated cells with physiological concentrations of oxytocin Assessed the influence of oxytocin on oxidative stress (via NAD(P)H oxidase activity) and inflammation (via cytokine secretion)

54. Oxytocin Receptor Protein Expression

55. Oxytocin Inhibits Vascular Oxidative Stress in Monocytes & Macrophages THP-1 MonocytesTHP-1 Macrophages * * * * * * * * * * * * * * * *

56. NADPH Oxidase in Aortic Endothelial & Aortic Smooth Muscle Cells * * * * * * * * * * * * * Aortic Endothelial CellsAortic Smooth Muscle Cells

57. OT Attenuates IL-6 Secretion from Vascular Cells in vitro

58. Chronic OT Infusion in WHHL Rabbits OT (n=14) or saline (n=14) infused chronically via osmotic minipumps for 4 months beginning at 3 months of age Rabbits housed individually for entire study Aortas and Adipose tissue removed at endpoint. Ex vivo secretion of IL-6 by adipocytes measured

59. Vehicle Oxytocin *** Chronic in vivo OT infusion in WHHL rabbits

60. Oxytocin and Fat Cells Adipose tissue is a major source of proinflammatory cytokines Increased adiposity is a risk factor for a number of inflammatory diseases, including cardiovascular disease Does oxytocin influence the secretion of proinflammatory cytokines from adipocytes?

61. Epididymal Fat IL-6 mRNA Expression in WHHL Rabbits

62. In Vivo OT infusion in ApoE Knockout Mice (Nation et al., Psychosom Med, In Press) Recently, we demonstrated that chronic OT infusions significantly attenuated atherosclerosis in a transgenic murine model, the ApoE knockout mouse OT infused mice exhibited less disease in the thoracic aorta than vehicle infused animals Ex vivo IL-6 release from adipocytes was attenuated in OT infused mice.

63. Conclusions Stable social environment, characterized by increased affiliative behavior & less agonistic behavior, slows the progression of atherosclerosis in animals genetically predisposed to disease Vascular cells, macrophages, and adipocytes express oxytocin receptors Oxytocin inhibits oxidative stress in cultured vascular cells, and inflammatory cytokine release in macrophages and adipocytes

64. Conclusions In vivo infusion of OT slows the progression of atherosclerosis in a site-specific manner It is proposed that the beneficial effects of a prosocial environment on disease progression may be mediated through the direct effect of peripheral oxytocin on pathophysiological mechanisms occuring in vascular wall

65. Collaborators University of Miami Department of Psychology: Angela Szeto, Daniel Nation, Larry Brooks, Crystal Noller, Maria Rossetti, Julie Gonzales, Jim Paredes, Maria Llabre, & Neil Schneiderman University of Miami Medical School: Armando Mendez, Julie Zaias, Ron Goldberg Eehscience LLC, Pickerington, OH Edward Herderick