We recruited 103 prairie vole subjects (52 males, 51 females) from our breeding colony located in the Department of Psychology at the University of California, Davis. We maintained the animals on a 14:10 h light cycle at approximately 21°C and provided food (Purina High Fiber Rabbit Chow, PMI Nutrition International, Brentwood, MO, USA) and water ad libitum. Animals were housed in large polycarbonate cages (44 cm × 22 cm × 16 cm) with their parents and marked with non-toxic Nyanzol D dye (American Color and Chemical Corporation, Charlotte, NC, USA) for identification purposes until weaning at postnatal day (P) 20. We then separated all subjects from their parents, gave them ear clip markings, and placed them with a same-sex sibling in smaller cages (27 cm × 16 cm × 13 cm) until sacrifice. Subjects that were treated with IN-AVP were housed with untreated siblings. To help control for potential litter effects, each litter had at least one AVP-treated animal and one saline-treated animal within sex.

Each test subject was randomly assigned to one out of four treatment groups, including saline control, low-dose AVP (0.05 IU/kg), medium-dose AVP (0.5 IU/kg), and high-dose AVP (5.0 IU/kg). The medium dose was specifically calculated to represent the same dose given in some clinical trials (https://ClinicalTrials.gov Identifier: NCT01962870), only controlled for weight. AVP solutions were purchased from Sigma-Aldrich (V0377 SIGMA), already mixed in NaCl. The solution was then diluted to provide the necessary concentrations for the treatment groups and aliquoted into 200 µL test tubes. The tubes were stored in a refrigerator at 4°C until use.

From P15 to 21 (early juvenile period), voles were given intranasal treatments twice daily. Each day, the first treatment was given between 0900 and 1100 hours, while the second treatment was given between 1500 and 1800 hours. Treatments were administered through cannula tubing, which was attached to a blunt cannula needle (33 gauge, 2.8 mm length; Plastics One, Roanoke, VA, USA) secured to an airtight Hamilton syringe (Fisher Scientific, Pittsburgh, PA, USA). The animal was held still while 25 µL of solution was expelled slowly through the cannula system and allowed to absorb into the nasal mucosa (divided evenly between the two nostrils). Following administration, the animal was returned to its home cage while the Hamilton syringes and cannula system were cleaned with 70% isopropyl alcohol solution and de-ionized water. Treatment order was randomized each day and administration was rapid (less than 30 s) making handling consistent across treatment groups.

All animals were subjected to a series of testing paradigms from weaning to adulthood. These tests were digitally recorded and manually scored using Behavior Tracker 1.5 (www.behaviortracker.com). Each scorer was blind to subject group assignment. See Figure 1 for a summary of all experimental procedures.

As direct treatment comparisons across the sexes were confounded by the difference in behavioral baselines, we decided to analyze males and females separately. Thus, we examined the effects of developmental AVP exposure in both sexes but not between sexes. We also controlled for the potentially confounding effect of litter on our results by assigning a unique identifier to all pups from the same litter and including this variable in our analyses.

All analyses were conducted using R version 3.3.3 (27). We began by fitting two models for each dependent variable, one including the litter variable as a random effect and one without it; both models included treatment group as a fixed effect. These two models were compared using an exact likelihood ratio test from the RLRsim package (28) to determine whether the presence of the variance component provided a better fit for the model. The test statistic from this likelihood ratio test is based on simulated values from the exact sample distribution as derived by Crainiceanu and Ruppert (29). When the statistic of the observed likelihood ratio was significant (α = 0.05), we chose the mixed model over the linear model, having found evidence for a significant effect of litter on the dependent variable. Each model that included the random effect was fit using the lme4 package (30), while all other models were fit using base R functionality.

After selecting the best model, we conducted a series of follow-up tests to confirm that our model met the assumptions for ANOVA testing. For the normality assumption, we prioritized visual inspection of Q–Q plots (31), but confirmed our observations using a combination of the Shapiro–Wilk test and measures of skewness and kurtosis. Despite the Shapiro–Wilk test having the best power for a given significance when compared to other normality tests (32), it is biased by sample size (33). Therefore, normality was assumed when the Shapiro–Wilk test was statistically insignificant (α = 0.05), the Shapiro–Wilk test statistic was high (W > 0.95), or when values for skewness and kurtosis fell between −2 and +2 (34, 35). We also utilized Levene’s test to determine whether group variances were homogenous. When models contained outliers or heteroscedastic data, we refit the model using robust techniques (36, 37). Robust linear models were fit using the MASS package (38), while robust mixed linear models were fit using the robustlmm package (39).

After selecting the best model for each dependent variable and satisfying the assumptions for one-way ANOVA testing, we passed the models to the car package (40) to produce the ANOVA tables. For mixed ANOVA models, F-test statistics were calculated using Kenward–Roger’s approximation for degrees of freedom. Using the lsmeans package (41), we conducted post hoc analyses on all models that contained a statistically significant effect of treatment (α = 0.05). We only considered direct comparisons between each treatment group and control, warranting the use of Dunnett’s test to control for Type I errors (42).

To determine whether overall parental handling differed between the groups, we combined the data from the dam with the sire and then compared total parental handling on individual observation days. Preliminary analyses confirmed no treatment differences within each observation day, so we then summed all parental handling behaviors across both days and reanalyzed the data. For partner preference data, we standardized the contact scores by subtracting the time spent with the stranger from the time spent with the partner, depicting the magnitude of the preference for the partner over the stranger. We tested whether our difference scores were significantly greater than 0, indicating a preference for the partner over the stranger. Then, we compared these scores across treatment groups to see if the magnitude of partner preference was affected by AVP treatment.

Intranasal AVP administration had no effect on acute parental handling; Table 1. For the open field test, IN-AVP altered fecal boli production in males, F (3, 51) = 2.839, p < 0.05. Specifically, males treated with the medium dose produced more fecal boli than control, z = 2.801, p < 0.05. IN-AVP also altered fecal boli production in females, F (3, 50) = 4.497, p < 0.01; high-dose exposure increased fecal boli production relative to control, z = 3.650, p < 0.001. We did not find effects of IN-AVP on any other recorded behavior in either sex; Table 2.

During both phases of the novel object recognition test, the time spent in each object’s interaction zone was similar across treatment groups regardless of sex. In addition, none of the treatment groups, including control, preferentially maintained proximity with the novel object over the familiar object. Like with open field testing, we found no treatment group differences in anxiety or exploratory measures across both phases of recognition testing; Table 2. We decided to combine fecal boli production across the three paradigms to confirm an overall effect of treatment. For males, IN-AVP altered the total fecal boli production across testing days [F (3, 51) = 3.656, p < 0.05], confirming an increase for medium-dose males (M = 11.70, SEM = 3.13) compared to control (M = 5.00, SEM = 0.99), z = 3.036, p < 0.01; Figure 2. AVP also impacted the total fecal boli produced across testing days in females, F (3, 50) = 3.069, p < 0.05; Figure 2. While the high dose (M = 9.44, SEM = 2.71) tended to increase [t = 2.39, p = 0.056], the medium dose (M = 9.50, SEM = 2.49) significantly increased fecal boli production relative to control (M = 3.54, SEM = 0.95), t = 2.502, p < 0.05.

For juvenile affiliation, we found no effect of IN-AVP on social- or anxiety-related behaviors in males. But, IN-AVP did impact play behavior in females [F (3, 50) = 2.750; p = 0.05]; the medium dose increased bouts of play compared to control, t = 2.729, p < 0.05; Figure 2. No other behaviors were altered by juvenile IN-AVP treatment; Table 3.

As with juvenile affiliation, we found no effect of IN-AVP across all recorded behaviors during the first intrasexual aggression test in males; Table 3. However, IN-AVP did have an effect on male aggression during the second intrasexual aggression test, F (3, 45) = 4.735, p < 0.01. Males treated with the high dose engaged in more bouts of aggressive behavior than control males, z = 3.031, p < 0.01; Figure 3. For intrasexual aggression testing in females, we found no effect for IN-AVP in females across all recorded behaviors regardless of testing day.

For the first partner preference test, we found no evidence of mate preference for any of the treatment groups; Table 4. However, IN-AVP did have an effect on partner preference in males during the second partner preference test, F (3, 50) = 5.847, p < 0.01 (Figure 4; Figure S1 in Supplementary Material). Further analyses revealed that while the control and low dose groups significantly preferred their partners over strangers (t = 6.096, p < 0.00001 and t = 4.329, p < 0.0001, respectively), such preference was not seen in both the medium- and high-dose groups. In addition, male medium- and high-dose groups spent significantly less time in preferential contact with their partner than control, t = 2.856, p < 0.05 and t = 3.055, p < 0.05, respectively. For these two groups, the reduction in time spent in contact with the partner could not be explained by increases in time spent in the neutral compartment, F (3, 47) = 0.378, p = 0.769.

Intranasal AVP did not affect partner contact in females during the first partner preference test and no group preferred the partner over the stranger. As for the second test, we did not find a significant effect of treatment on preferential mate choice. But while no differences existed between treatment groups, we did conduct post hoc analyses to confirm partner preference within each group. Unlike males in the second test, all female treatment groups did demonstrate a significant partner preference (control: t = 7.347, p < 0.00001; low: t = 4.755, p < 0.0001; med: t = 5.914, p < 0.00001, high: t = 4.127, p < 0.01) (Figure 4; Figure S1 in Supplementary Material). See Table 4 for all partner preference testing descriptive statistics.

Finally, each animal was weighed on the first day of treatment and on the day of sacrifice. We found an effect of IN-AVP on weight change across life in males [F (3, 32.481) = 5.234, p < 0.01], but not in females; Figure 5. Further analysis revealed an increase in weight for low-dose males compared to control, t = 3.672, p < 0.01.

We hypothesized that the effects of IN-AVP would (1) vary by dose, (2) be more prominent in males, and (3) exhibit contrasting effects depending on context. Our results confirm that IN-AVP modulates behavior in dose-specific ways. For example, the low dose increased weight gain, the medium dose increased fecal boli production, and the high dose increased aggression. We also found the most profound effects of IN-AVP administration in males, who exhibited both behavioral and physiological changes across life. Specifically, we found no effects of IN-AVP on adult female behavior, but males experienced impairments in partner preference formation and increases to aggression depending on the dose. We also detected context-specific contrasts in behavior. While IN-AVP did increase aggression during competitive encounters, we did not find increases in social behavior during non-threatening encounters. IN-AVP impaired sociability in males during partner preference testing without impacting sociability in juvenile affiliation or the first intrasexual aggression test (prior to partner preference). The changes to aggression experienced after partner preference testing also confirm that the effects of IN-AVP depend on context. This is further supported by the IN-AVP-stimulated increases in anxiety experienced by females in non-social contexts and increases to play behavior during social contexts (e.g., juvenile affiliation).

In the present study, we found increases in fecal boli production across open field and novel object recognition testing for both males and females treated with the medium dose. Context-specific increases in fecal boli production have been associated with increased anxiety (43, 44); habituated animals produce successively fewer fecal boli with repeated testing (45). But, previous studies have also demonstrated a role for AVP in the regulation of gastric motility; systemic AVP injections increase gastric motility (46, 47) while AVP microinjections within the rat vagal nerve inhibit gastric motility (48). Given the fact that we did not find increases in other anxiety-related behaviors (e.g., freezing, autogrooming) alongside, the increases to fecal boli production, we suspect that peripheral AVP receptor effects on gastric motility may provide a stronger explanation for our findings. It would have been interesting to see if a more anxiogenic paradigm would have elicited a stronger response or if our results could be blocked by antidepressants.

The effects of IN-AVP during the juvenile period also appear to differ by context. While the treatment may potentially increase anxiety in both males and females during non-social novelty, we found evidence for increases in sociality with a novel social partner. Specifically, the medium dose selectively increased play bouts in females (with no increase in males). This may reflect slight differences in the quantity of play exhibited by male and female prairie voles; male control voles engaged in marginally higher bouts of play than female control voles. Alternatively, it is also possible that females may be more susceptible to the effects of AVP in play behavior since male prairie voles have more AVP-containing neurons than females in several neural regions, such as the medial amygdala and bed nucleus of the stria terminalis (22).

In rodents, the AVP system seems to regulate social play differently between males and females. For example, intracerebroventricular (ICV) administration of AVPR1a antagonists in rats increased social play in females and decreased it in males (49). However, site-specific injections of AVPR1a into the lateral septum produced the opposite results, increasing play in males while reducing it in females (6). Without measuring the effect of chronic IN-AVP on regulation of AVP receptors and peptide throughout the brain, it is difficult to determine the neural mechanism for our findings that medium-dose IN-AVP increased play bouts in females. Repeated activation of AVPR1a has been shown to cause internalization, decreasing the membrane density of AVPR1a (50). It is possible that the administration period and frequency implemented in this study was sufficient to decrease AVPR1a densities across the brain, approximating the effect of ICV AVPR1a antagonist administration as shown by Veenema et al. (49). But, if downregulation of AVPR1a is the cause of our observed effects, it is curious that we did not see a simultaneous decrease in play activity for males treated with IN-AVP. Regardless, we might expect the effects of intranasally administered AVP in prairie voles to differ from other more direct delivery routes (e.g., microinjections) and from other rodent species, especially, since the mechanism for behavioral effects of intranasal delivery has not yet been confirmed (51).

Early studies suggested that OT activity on the OTR was more important for pair-bond formation in females, while AVP activity on the AVPR1a was more important in males (52–55). More recent studies have produced more subtlety than this strict dichotomy, suggesting instead that OT and AVP are involved in partner preference formation in both sexes (56–59). Cho et al. (57) found that ICV administration of OT or AVP could facilitate pair-bond formation in both males and females while concurrent administration of either OT receptor or AVPR1a antagonists blocked this effect. In our study, we demonstrated that a single week of twice daily exposure to IN-AVP during the early juvenile stage can disrupt pair-bond formation later in life. These effects were sex-dependent, occurring only in the male medium- and high-dose groups, and did not reflect changes in the amount of time spent in the neutral cage. On the contrary, all experimental groups in females successfully demonstrated a partner preference by the time of the second test.

Previous studies have demonstrated that pair-bond behavior in prairie voles is highly susceptible to manipulation of the AVP system. In adult prairie voles, site-specific AVPR1a antagonists within the ventral pallidum prevented pair-bond formation (60), while selectively increasing AVPR1a densities in the ventral forebrain facilitated pair-bond formation (61). Overexpression of AVPR1a within the ventral pallidum (60) and ventral forebrain (62) of the promiscuous meadow vole substantially increased partner preference behavior. As mentioned before, repeated activation of AVPR1a can lead to physiological tolerance (50). Since the observed chronic effects of IN-AVP administration in our study approximate the effects of acute AVPR1a antagonist administration in other studies, we suspect that a single week of twice daily exposure to AVP should be sufficient to decrease AVPR1a densities in specific neural regions, which are critical for pair-bond formation in males. Complementary work from our lab has shown that chronic OT exposure also impairs partner preference formation in prairie voles (63) with subsequent changes to OTR receptor and AVP peptide concentrations (unpublished data).

Given our results, we suspect that the physiological mechanisms behind the disrupted partner preference in males may differ between the medium- and high-dose groups. Specifically, the high dose also resulted in a substantial increase in aggressive behavior during the second intrasexual aggression test, which was conducted approximately 24 h following partner preference testing. These findings are similar to those found by Stribley and Carter (7); early postnatal exposure to the high dose of AVP increased aggression in sexually naïve prairie voles compared to control. In prairie voles, aggressive tendencies naturally increase following induction of the pair-bond. Gobrogge et al. (64) showed that 2 weeks of cohabitation with a female intensely increased male aggression toward both novel male and female conspecifics; these males maintained elevated levels of social affiliation with their female partners during this time. But, the increase in aggression experienced by high-dose males in our study occurred in the absence of a preference for a female partner.

Outside of the pair-bond, AVP modulates aggressive behavior in sex-specific ways. AVP injections within the anterior hypothalamus stimulate aggression in male Syrian hamsters but inhibits it in females (65, 66). Also, male prairie voles who received low amounts of parental handling early in life engaged in more aggression in adulthood (67). Given the stimulatory effects of AVP in males on aggression and the lack of pair-bond formation in high-dose males, the increases in aggression is likely unaffiliated with mate-guarding. On the contrary, there is also the potential for delayed pair-bond formation in the high-dose males. While this group may not have formed a partner preference at 24 h, they may have formed it at some point between the end of the second partner preference and the second intrasexual aggression test (which was an additional 24 h). Thus, it is possible that high-dose males experienced a delay in partner preference formation, but had an exacerbated mate-guarding response once the pair-bond occurred. Regardless, the medium and high doses may have different effects on AVPR1a in brain regions involved in pair bonding versus aggression.

We also found that the low-dose males gained significantly more weight than control. Inappropriate AVP secretion to the periphery (68) and the use of synthetic AVP (desmopressin) has been linked to weight gain in humans (69). Though we reported only the results of weight gain across life, we also measured the difference in weight gain across the dosing period. No significant difference in weight gain was found for any of the treatment groups during this time, but there was a suggestive increase in weight gain for the low-dose group in males (Cohen’s d = 0.66). As supported by this experiment, early life manipulations can change adult behavior (and likely physiology). Therefore, it is possible that the slight (statistically insignificant) changes in weight gain for males treated with the low dose of IN-AVP across the dosing period were subsequently exacerbated across life.

Caveats in interpretation of these results are the lack of any animal model for autism with clear constructive validity (70, 71), and the variability of OT and AVP receptors across different taxa and species. This variability may lead to differences in responses to these neuropeptides across species. In a previous study, we found that chronic OT impaired pair bonding in male prairie voles at certain doses (63). However, the same dose did not change mouse social behavior, either in BTBR mice (a rodent model of reduced sociability) or in their strain control (72). In our quest to translate neuropeptide results from animal models, we should consider the neurobiology and natural history of the animal model, as well as the dosage, sex, context of administration, and other testing conditions. Future studies in animal models and in humans will reveal which model is most predictive.

Another limitation is the potential for the confounding effects of repeated behavioral testing on the results. Our study employs several behavioral paradigms in the early juvenile period as well as adulthood. It is possible that this combination of testing could have obscured or attenuated treatment and sex effects of IN-AVP, particularly given the density of testing and the frequency of experimental handling early in life. But as mentioned previously, the increases in aggression experienced by the high-dose group in adulthood do replicate the results of Stribley and Carter (7) who did not employ the same intensity of behavioral testing.