Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
In this remarkable study, the authors use some of their recently-developed oxytocin receptor knockout voles (Oxtr1-/- KOs) to re-examine how oxytocin might influence partner preference. They show that shorter cohabitation times lead to decreased huddling time and partner preference in the KO voles, but with longer periods preference is still established, i.e., the KO animals have a slower rate of forming preference or are less sensitive to whatever cues or experiences lead to the formation of the pair bond as measured by this assay. This helps relate the authors' recent study to the rest of the literature on oxytocin and partner preference in prairie voles. To better understand what might lead to slower partner preference, they quantified changes to the durations and frequency of huddling. In separate assays, they also found that Oxtr1-/- KOs interacted more with stranger males than wild-type females. In a partner choice assay, they found that wild-type males prefer wild-type females more than Oxtr1-/- KO females. They then performed bulk RNA-Seq profiling of nucleus accumbens of both wild-type and Oxtr1-/- KO males and females, either housed with animals of the same sex or paired with a wild-type of the opposite sex. 13 differentially expressed genes were identified, mostly due to downregulation in wild-type females. These genes were also identified in a module lost in the Oxtr1-/- voles by correlated expression profiling. They also compared results of transcriptional profiling in female and male wild-type vs Oxtr1-/- voles (independently of bonding state) and found hundreds of differentially expressed genes in nucleus accumbens, mostly in females and often with some relation to neural development and/or autism. Some of the reduction in the transcript was confirmed with in-situs, as well as compared to changes in transcription in the lateral septum and paraventricular nucleus (PVN) of the hypothalamus. Finally, they find fewer oxytocin+ and AVP+ neurons in the anterior PVN.
Strengths:
This is an important study helping to reveal the effects of oxytocin receptor knockout on behavior and gene expression. The experiments are thorough and reveal a surprising number of genetic and anatomical differences, with some sexual dimorphism as well, and the authors have more carefully examined the behavioral changes after shorter and longer periods of partner preference formation.
We thank Reviewer #1 for the positive assessment of the study’s significance and for recognizing the value of our behavioral and transcriptional analyses in refining the role of oxytocin signaling in pair bonding.
Weaknesses:
It is surprising that given all the genetic changes identified by the authors, the behavioral phenotypes are fairly mild. The extent of gene changes also might be underreported given the variability in the behavior and relatively low number of animals profiled.
Pair bonding is a robust behavior composed of distinct modules that are supported by redundant and compensatory neural pathways. Our findings support a model in which Oxtr functions in parallel with other mechanisms to modulate specific components of social attachment. We have addressed this point in the discussion. We have also updated our result and method section to more clearly reflect our cohort size which is comparable to similar studies.
Reviewer #1 (Recommendations for the authors):
How do the wild-type males 'know' which animal is which during the three-chamber assay test of Figure 4B? Do the Oxtr1-/- KO females act in some way different from the wild types in this experiment?
We thank the reviewer for this question. During follow-up analyses prompted by reviewer requests to characterize the behaviors underlying the apparent bias in WT male choice, we discovered a labeling error in the metadata used to analyze these assays. The error flipped the genotypes of the tethered stimulus animals at the ends of the chamber. After correcting this error and reanalyzing the data, we find that naïve WT males do not show a significant preference for naïve WT females over naïve Oxtr1-/- females. We have reconfirmed the metadata used in all assays in this study; no other datasets or conclusions are affected.
While overall choice frequency is equivalent for males and females, our revised analyses demonstrate that Oxtr loss nonetheless alters the dynamics of social interactions in a sex-specific manner. In particular, the presence of an Oxtr1-/- male significantly alters WT females’ social behavior—enhancing prosocial engagement and reducing aggression—independent of which male is ultimately chosen. These findings support the conclusion that Oxtr function modulates early reciprocal social interactions rather than categorical choice outcomes.
MOAT and LOAT seem like cumbersome acronyms, more so than something simpler like vole 1 vs vole 2.
We have replaced these acronyms throughout the manuscript with the simpler, descriptive terminology; winner (MOAT) and loser (LOAT).
Only three animals per condition seemed to have been used for RNA-Seq studies in Figure 5. Given the high behavioral variability in the earlier figures, did the authors screen for animals with exemplar or similar behavior within groups? The lack of significance of other genes or across other groups might just be due to a low-powered experiment given the high behavioral and genetic variability.
We thank the reviewer for raising the important point regarding behavioral preselection, which has been performed in some similar studies. For our study, animals were not preselected based on exemplar or matched behavioral performance prior to tissue collection, as doing so would risk introducing variation in gene expression patterns due to the experience of complex social interactions. Instead, given that our prairie vole lines are maintained on an outbred background, tissue from three animals was pooled for each RNA-seq sample to reduce inter-individual variability and to capture representative transcriptional states within each experimental group. While this approach increases robustness to individual variability, we acknowledge that it may limit sensitivity to detect low expression behavior linked gene transcripts.
On lines 426-429, the authors state that "While there was no significant difference in Oxtr transcript levels by genotype (padj = 0.753)-consistent with minimal nonsensemediated decay despite a premature stop codon-we have previously shown that no functional protein is produced in Oxtr1-/- animals (52)." This assertion could use strengthening, even if just to explain how this was verified in their previous publication. What is the evidence for nonsense decay and a full knockout of functional receptors at the protein level?
We agree that this point benefits from clarification. Although Oxtr transcript levels were not significantly different by genotype (padj = 0.753), consistent with minimal nonsense-mediated decay, transcript abundance alone does not reflect receptor functionality. In our prior study, we directly assessed Oxtr protein function using receptor autoradiography and found a complete absence of specific ligand binding in Oxtr1-/- animals across brain regions that show robust Oxtr binding in wild-type voles, demonstrating a full loss of functional receptor protein. We have clarified this in our manuscript.
Reviewer #2 (Public review):
Summary:
This manuscript uses a recently published oxytocin receptor null prairie vole line to examine the effects of this mutation on pair bonding behavior and PVN gene expression. Results reveal that Oxtr sex specifically influences early courtship behavior and partner preference formation as well as suppressing promiscuity toward novel potential mates. PVN gene expression varies between Oxtr null and WT prairie voles.
Strengths:
Behavioral analyses extend beyond the typical reporting of frequency and duration. The gene expression models and analyses are well-done and convincing. The experimental designs and approaches are strong.
We thank Reviewer #2 for highlighting the strengths of the gene expression modeling and behavioral analyses.
Weaknesses:
More details and background literature explaining the role of the Oxt system in pair bonding behaviors is necessary, particularly for the Introduction. The authors overstate several times that Oxtr expression is not necessary for partner preference formation, based on their previous findings. However, it does appear, particularly, in the short cohabitation that it is necessary. Thus, the nuanced answer may be that Oxt may accelerate partner preference formation. Improving the presentation of the statistics and figures will make the manuscript more reader-friendly.
We thank the reviewer for this thoughtful feedback and agree that additional background on the oxytocin (Oxt) system’s role in pair bonding will strengthen the manuscript. We have revised the introduction to expand our discussion of prior pharmacological and comparative studies suggesting that Oxt signaling modulates multiple components of pair bonding.
Finally, in response to the reviewer’s suggestion, we have improved the presentation of figures and statistical reporting by interlacing figures with figure legends and updating the supplementary statistics table.
Reviewer #2 (Recommendations for the authors):
Major concerns
(1) The Introduction provides a "broad strokes" approach to link the oxytocin and vasopressin systems as neuromodulators of social attachment processes. This study is a follow-up to a recent publication by the senior authors' groups which reported that the Oxtr null prairie voles were able to form typical pair bonds. Now, the authors are revisiting the same question by developing a series of behavioral assays to probe distinct aspects of pair bonding behavior. However, the Introduction lacks a nuanced examination of how the oxytocin system has been shown to regulate an array of social behaviors in prairie voles and other social species.
We thank the reviewer for this observation and agree that the original Introduction did not capture the breadth and nuance of oxytocin system involvement in social behavior. We have substantially revised the Introduction in response to the reviewer’s suggestion to include a more detailed discussion of the role played by oxytocin signaling in social behaviors displayed across multiple phyla, including during the early stages of pair bonding.
(2) In addition, there seems to be relevant viral Oxtr KD and KO studies in prairie voles which could be referenced to reflect differences between acute pharmacological Oxtr inhibition and prolonged viral KD of Oxtr on behavioral outcomes. This could also be put into context with the authors' first paper in prairie voles and others' work with mice showing how congenital Oxtr null rodent models may result in behavioral changes that are not reflected in the pharmacological or viral manipulation research. This could help justify the approach of the current study.
We thank the reviewer for suggesting this comparison and have included a section in the discussion comparing pharmacological manipulations and global knock outs as well as the discrepancy in phenotypes that arise due to these methods. This expanded discussion clarifies why a congenital genetic model provides complementary insights: it allows us to identify which components of pair bonding are robust to developmental loss of Oxtr and which remain sensitive, thereby distinguishing between Oxtr-dependent behavioral modules and those supported by parallel mechanisms. Additionally, we have included viral manipulations of Oxtr in prairie voles during the early phase of interactions between the sexes in the introduction, to contextualize our study in the broader field.
(3) On lines 129-130: The authors state, "We previously found that Oxtr is not required for the display of partner preference following 1 week of cohabitation". While this is the general conclusion of their previous publication, this seems like a rather larger overgeneralization. There are many studies that have documented the functional regulation and necessity of the Oxt system for partner preference behavior in prairie voles. Therefore, it would be more appropriate to state that their previous study demonstrated that "Oxtr null prairie voles are able to develop a partner preference", but not that Oxtr is not necessary for partner preference formation. This may be a question about when the KO occurs, whether it be congenital or conditional.
(4) This statement is repeated in Lines 350-352. However, the authors can now qualify this statement at this point in the manuscript with their new data which suggests that Oxtr null voles fail to form a partner preference after short cohabitation, but WT still form such preferences. This would suggest the qualification of this statement should be on the onset of partner preference formation as Oxtr is necessary for partner preference formation after a "short" cohabitation. Therefore, both findings are more in line with previous results which suggest that Oxt signaling accelerates partner preference formation.
We have revised this language throughout the manuscript to state that our prior work demonstrated that Oxtr null voles are capable of forming a partner preference after extended cohabitation.
(5) It appears Supplementary Table 1 is not scaled to the page size, so not all statistical results are clear. This limits the accuracy of my review.
This table has been reformatted to ensure all statistical results are properly scaled to page size.
(6) It is not always clear what statistical analyses are being performed. For example, how were the data in Figures 4G-H analyzed? What statistics were used and the output should be more readily available.
During follow-up behavioral analyses prompted by Reviewer #1 requests to characterize the basis of the apparent WT male bias, we discovered a labeling error in the metadata associated with a subset of naïve three-chamber choice assays. In these cases, the genotypes of the tethered stimulus animals had been inadvertently flipped. After correcting this error and reanalyzing the data, we find that naïve WT males do not show a significant preference for naïve WT females over naïve Oxtr1-/- females. We have rechecked the metadata for all assays included in this study and confirmed that this was the only instance in which such an error occurred. We further analyzed the temporal dynamics of naive choice to find that Oxtr function modulates early reciprocal social interactions but does not affect the genotype ultimately chosen.
To improve the clarity of the statistical analyses performed, we have reformatted our presentation of figure legends and our statistics table. All statistical tests, sample sizes, and relevant parameters (including exact tests used, correction methods where applicable, and definitions of units of analysis) are explicitly stated in the figure legends and compiled in the supplementary statistical summary table, in accordance with eLife reporting guidelines.
(7) Oxytocin plays a critical role in development as early as embryogenesis. It may be useful to frame some of the Introduction and Discussion recognizing the congenital deletion of Oxtr may affect much of development. With that in mind, it is not surprising to see changes in gene expression associated with neurodevelopmental disorders.
We now explicitly acknowledge in both the Introduction and Discussion that congenital Oxtr deletion likely impacts neural development which provides context for the observed enrichment of neurodevelopmental gene expression changes.
Minor concerns
(1) It was not clear why vasopressin was referenced in the Introduction. Specifically, the study documents that Oxtr null prairie voles have a reduction in Avp neurons in the PVN, which would suggest some aspects of Oxt signaling regulate Avp expression. However, the Introduction is not focused on how Oxt regulates the Avp system but rather on how each is a modulator of social attachment. It would improve the justification of this study to focus on Avp expression if the Introduction presented this concept.
We thank the reviewer for pointing out the need for greater clarity around our reference to vasopressin (Avp) in the Introduction. We have simply stated that the potential for pair bonding is correlated with the patterns of expression of Oxtr and V1ar in the introduction. The goal of this study was to find evidence of behavior and gene expression changes due to the chronic loss of Oxtr which lead to our finding that a population of Avp neurons is lost in the animals lacking Oxtr. As we did not intend to justify our study on this basis, we have clarified our discussion to include previous studies where OT manipulation affects Avp neurons.
(2) Figures and supplemental figures need figure legends.
We have re-arranged the figure legends for each figure (including the supplementary figures) to follow the figures for easier readability and accessibility.
(3) Figure 1 Timeline is focused more on the male timeline with "bond formation" and "bond maintenance" reflecting the days required to form a partner preference for males. The figure should be revised to reflect similar time points for female pair bonding.
Figures have been revised to reflect each sex's bonding timeline.
(4) Figure 1 has a color theme with females represented by red/pink and males represented by dark/light blue. However, this is not true for Figures 1C and 1D. Please revise these color schemes.
Color schemes have been standardized across all figures.
(5) It is not clear what is being graphed in Figures 2 and 3. The duration graphs have many more data points than the frequency graphs. Can this be explained?
We thank the reviewer for pointing out this lack of clarity. The difference in the number of data points reflects how these measures are defined. Duration plots are generated at the level of individual huddle events, specifically pooling all huddles whose duration falls within the top quartile for a given animal, whereas frequency plots are generated at the level of individual animals and therefore contain one data point per subject. As a result, duration graphs necessarily include more data points than frequency graphs. The figure legends and Methods section explicitly state the unit of analysis for each metric and to clarify why the number of data points differs between duration and frequency plots.
(6) What are the black bars in Figure 4H meant to represent?
We thank the reviewer for this question. In the original submission, the black bars in Figure 4H were intended to indicate time periods showing statistically significant convergence in the chooser’s preference for the MOAT (More Of Assay Time, now winner) animal, based on the sliding preference index analysis. However, as mentioned during revision we identified a metadata error affecting the dataset used to generate this figure. After correcting the error, the figure was fully reanalyzed and regenerated. As a result, Figure 4H now presents a different analysis and no longer includes these black bars, and the conclusions drawn from this panel have been revised accordingly. The updated figure, legend, Results text and statistics table now accurately reflect the new analysis.
