RESEARCH Pharmacological Characterization Hillary Ihrig, McKenna Rich, and Sara M Freeman, PhD of the Receptor Antagonist Figure 3- L-368,889 chemical structure. L-368,899 for Coyote Receptors Introduction Results - Oxytocin is a hormone that mediates - Slides exposed to OXTR radioligand multiple social behaviors, social recognition, exhibited the expected decline in binding as maternal behaviors, and pair-bonding. the concentration of L-368,899 increased. - Coyotes display monogamy. In other - Slides exposed to AVPR1a radioligand did mammalian models, oxytocin has been exhibit some binding density reduction with shown to influence this behavior. increased L-368,899, but these slides did not - Oxytocin and ’s structural develop as we expected; many were too similarities result in binding promiscuity -- faint for quantification. both molecules bind to both receptors. - Based on Ki values, OXTR 12.38 nM and - We determined the binding affinity of the AVPR1a 870.7 nM, L-368,899 showed a 70x antagonist, L-368,899 greater binding affinity for oxytocin. -7.5 using competitive binding autoradiography. - ½ of max binding was identified at ~ 10 - If this antagonist effectively blocks the for OXTR. At this same concentration, the coyote oxytocin receptor without antagonist was incapable of competing off effectively blocking the vasopressin 1a the radioligand binding to AVPR1a. receptor, it can be used in live coyotes to Conclusion study oxytocin-dependent social - Increasing concentrations of the behaviors. antagonist, L-368,899 increasingly inhibits Methods binding of OXTR and AVPR1a radioligands. L-368,899 is more selective for oxytocin - 6 Frozen coyote brains were blocked into receptors than vasopressin 1a receptors. slabs and stored at -80C until sectioning. - Lower concentrations of L-368,899 out- - Brain sections were sliced using a cryostat compete binding of the oxytocin at 20 micron thickness, and mounted on radioligand, effectively blocking these microscope slides. receptors while leaving vasopressin 1a - Competitive binding autoradiography receptor binding undisturbed. was performed with increasing - We believe variability in AVPR1a selectivity concentrations of the oxytocin receptor is due to an error in our experimental (OXTR) antagonist L-368,899 in design. Replication of our experiment is competition with a consistent needed to have full confidence in L- concentration of the commercially

AVPR1a OXTRAVPR1a vs. 368,899’s selectivity for future in vivo available radioligands: 1) OXTR experiments. radioligand 125I-ornithine vasotocin analog 125 REFERENCES ( I-OVTA) and 2) vasopressin 1a receptor -12 -11 -10 -9 -8 -7 -6 -5 125 10 10 10 10 10 10 10 10 Boccia, M. L., Goursaud, A. -. S., Bachevalier, J., Anderson, K. D., & Pedersen, C. A. (2007). (AVPR1a) radioligand I-linear Peripherally administered non-peptide oxytocin antagonist, L368,899®, accumulates in limbic brain 125 areas: A new pharmacological tool for the study of social motivation in non-human primates. vasopressin antagonist ( I-LVA). Concentration of L-368,899 (M) Hormones and Behavior, 52(3), 344-351. Freeman, S. M., Walum, H., Inoue, K., Smith, A. L., Goodman, M. M., Bales, K. L., & Young, L. J. - After quantifying the binding densities, (2014). Neuroanatomical distribution of oxytocin and vasopressin 1a receptors in the socially Figure 1 (top)- Binding selectivity competition curves. monogamous coppery titi monkey (callicebus cupreus). Neuroscience, 273, 12-23. we generated a competition curve, which Smith, A. S., Ågmo, A., Birnie, A. K., & French, J. A. (2010). Manipulation of the oxytocin system Figure 2 (bottom)- Radioligand binding with increasing concentrations of antagonist L-368,899. alters social behavior and attraction in pair-bonding primates, callithrix penicillata. Hormones and calculated binding affinity (Ki). Behavior, 57(2), 255-262.