RESULTS

Low and medium doses of chronic intranasal oxytocin (OT) reduce quantity of OT cells in PVN of male prairie vole

Caleigh D. Guoynes, Catherine Yun, David Patron, Louiza Livschitz, Griffin Downing, Crystal Vardakis, Chathurika Peiris, Allison Perkeybile & Karen L. Bales

ABSTRACT: For voles given chronic intranasal OT, behavioral assays have shown that low and medium of doses of OT caused adult male prairie voles to lose partner preference and low doses caused adult female prairie voles to attack pups more during alloparental care tests. Our goal was to determine if low, medium, and high doses of chronic intranasal OT change the number of cells containing OT in the PVN and SON in prairie voles. This study used non-behavior tested animals to establish a baseline for OT in brains of voles given one of three OT doses or saline. Using oxytocin immunohistochemistry, we found that voles given the low and medium doses of OT had significantly fewer cells (F=5.55, p=.0274 and F=3.00, p=.0966, respectively) in the PVN. There were no significant changes in the SON and no changes in OT plasma levels across groups. These results suggest that the decreased number of cells containing OT in the PVN largely contributed to the observed behavioral abnormalities.

BACKGROUND & INTRODUCTION

•Oxytocin (OT) is a naturally occurring neuropeptide that has a significant impact on the formation and maintenance of social bonds. •Our study aims to confirm the short-term benefits of intranasal OT and explore possible long-term side effects. •The PVN contains parvocellular cells that make OT available to the brain. The SON contains both parvocellular and magnocellular cells, making OT available to both the brain and body. •Our previous study indicates that low and medium doses of OT cause long-term behavioral deficits, so we predict those doses will cause a decrease in cell number in the brain (Fig. 1). •Quantifying OT antigens may make it possible to understand how various doses of chronic OT affect brain development and the consequent adult social behavior.

•Clinical researchers are using intranasal OT for patients with autism, schizophrenia, and post-traumatic stress in conjunct with behavioral therapy to maximize patient benefit.

METHODS At post-natal day (PND) 21 through PND 42 (sexual maturity) voles were given daily treatments between 8 a.m. and 12 p.m. Each vole was given either a low (0.08 IU/kg), medium (0.8 IU/kg), or high (8.0 IU/kg) dose of OT or saline. Days 42-50 served as a washout period and PND 50-55, brains were removed. After all brains were sliced, immunohistochemistry was performed using anti-rabbit primary and secondary antibodies, and all cells in PVN and SON were counted by C.D.G. The assay for the blood plasma was performed by K.L.B. and T.W. Below, Fig. 2 shows the timeline of these events.

ACKNOWLEDGMENTS Thanks to Julie Vanwesterhuyzen, Meredith Lee, Fontaine Ma, Caryn Covella, and John Helmy. Thanks to Allison Perkeybile and Tamara Weinstein for their help with assays. Special thanks to Dr. Karen Bales for designing the project and giving me this incredible opportunity. This work was supported NIH grant HD071998 to K. Bales.

DISCUSSION •There was a significant decrease in cell number in the PVN of both low and medium dose treated animals.

•It follows that these animals were not producing as much of their own OT as control animals.

•There were no significant changes in the SON, so this suggests the behavioral abnormalities observed in the previous study were mediated by PVN fibers extending into the brain rather than SON fibers extending to the posterior pituitary.

•A previous autoradiography study showed the medium dose in males had significantly higher OT receptor binding in the posterior

cingulate cortex (F=5.85, p=0.0421) and lower binding in the BNST (F=4.62, p=0.0842). There were no significant changes with the low dose in males.

•This suggests that the abnormal social behaviors in the low dose were caused by a decrease in OT cells and the abnormal social behaviors in the medium dose were caused by a combination of changes in receptors and a decrease in OT cells.

•We have plans to quantify the data on female brains and run a vasopressin assay to look for AVP expression in these cells.

Figure 5 Males given the low dose had significantly fewer cells producing OT in the PVN when compared to saline controls. Two-tailed probability shows F=5.55, p=0.0274. For male prairie voles given the medium dose, the one-tailed probability is significant (p=0.0483). **significant for two-tailed probability, *significant for one-tailed probability

•Stained brains were sorted into one of seven levels of PVN (Fig. 2) and averaged for total cell counts throughout the various levels of PVN (Fig. 4). Same methods were used for quantification of SON. •There were fewer OT-expressing cells in the PVN of voles given low and medium doses of OT. Two-tailed probabilities were significant for voles given the low dose (p=0.0274) and showed a trend medium doses (p=0.0965). (Fig. 5) •The one-tailed probability for the medium dose is significant (p=0.0483) as seen in Figure 5.

Figure 4 The averaged number of cells expressing OT in the PVN. Standard errors (STE) were used the calculate the variability of cell number in the PVN between different test subjects.

Dosage Low Medium High Saline

Cell number ± STE 37.97 ± 4.82 40.29 ± 4.81 44.67 ± 4.64 46.71 ± 1.25

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PND 50-55 (adulthood): brain

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Immunohistochemistry assay for OT

Brains sorted from anterior to posterior

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PND 21-42 (juvenile period): daily intranasal

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PND 42-50 (adulthood): washout period, no

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Juvenile prairie vole Adult prairie vole

PVN from anterior to posterior: above, pictures A -G show how the shape of the PVN changes significantly from anterior to posterior. In this study, we averaged the PVN and SON cells for the sum of all brain areas. However, we plan to examine possible changes in PVN density throughout the different areas.

Figure 3 40 micron slice with PVN, PVN fibers, and SON labeled. These areas were stained using a rabbit OT primary and secondary antibody with DAB staining. Slices were mounted within one week of the assay.

PVN fibers

SON

PVN

Figure 1 Results from the behavioral assay (as published in Biological Psychiatry). Short term, OT increased social behavior for all doses. Long-term, low and medium doses caused deficits in social bond while high and saline produced typical social bonds.

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Low dose vs. saline Medium dose vs. saline

High dose vs. saline Ave

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Effects of OT on cell number in PVN of males

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Figure 2 Timeline of study events.

•The PVN and SON were stained and then quantified as shown in Figure 3.

•There was a trend showing that the medium dose had fewer cells (average of 8.988 ) in the SON compared to the saline control (average of 13.532). However, the one-tailed probability of the medium dose versus saline is p=0.05. •There were no significant changes in the SON for the low or high doses compared to the saline dose. •No significant differences were found in OT plasma levels across groups.