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  1. Evaluation Summary:

    Manipulations of sex hormones in animals in ecologically relevant environments usually involve long-term manipulations using chronic implants or injections of esterified steroids with longer half-lives than the endogenous hormones. This has been done in line with the prevailing idea of the long-lasting effects of steroids mediated by the transcritpional actions of their liganded receptors. The specific novelty of this study lies in the transiency of hormone availability (testosterone's half-life is about 2 hours). This might suggest that the observed effects depend on a mode of action different from the mode of action during chronic sex hormone exposure. It should also be noted that any study in natural settings is significantly more difficult to perform than in the lab. However, as all brain/hormonal functions evolved in natural environments, these studies are absolutely crucial to understand the function of the respective systems.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    This study investigated the biological relevance of transient fluctuations of circulating testosterone (T) experienced by males during social encounters. The authors propose that T pulses have rewarding properties that influence social interactions and place preference. As California mice are monogamous and bi-parental, one prediction was that T pulses influence the attendance of males to the nest. To test this possibility, the movements and vocalizations of pairs of wild California mice identified as mates were followed in the field by telemetry as well as thermal and auditory recordings for 3 nights following the administration to males of three daily short live testosterone injections. The results show that males indeed spent more time in the nest, and even more so if pups were present, suggesting that one function of T pulses is to increase paternal care. T pulses administered to males also reduced time spent by their mate in the nest, regardless of the presence of pups, suggesting that male attendance to the nest allows another use of the female's time. T pulses also resulted in increased production of ultrasonic vocalizations (USVs) by the pair, in a smaller bandwith in treated males and in a change in the types of USV predominantly produced. Together these data thus provide important new information on the significance of acute and short lived changes in testosterone availability to reproductive behavior and fitness in this species. The impact of this work is expected to extend beyond this specific species.

    Overall, the methods are transparent, sufficiently detailed and clear. The conclusions are for the most part supported by the data.

    Strengths :

    Sex steroids are chemical messengers producing slow and lasting responses that prime the circuits underlying behaviors. These actions are mediated by transcriptional changes and their effects are typically studied in response to long-term (seasonal) changes in hormonal circulating concentrations. Yet, the concentration of sex steroid hormones, including testosterone, can rapidly vary in response to social encounters or stressful events and induce rapid responses that are mediated by membrane-initiated actions. Much less is known about these rapid effects of testosterone and even less about their impact on behavior and reproductive success. The demonstration that repeated T pulses induce changes in time allocation and communication, which translate a change in behavioral activities, in a complex and natural environment is thus an important achievement. Moreover, because these effects are thought to reflect a form of conditional place preference, they also provide an illustration of how short lived hormonal secretion can exert long term effects on behavior and reproductive fitness independently of a direct action on gene transcription.

    Weaknesses :

    The authors present this experiment as a modification of classical conditioned place preference (CPP). If this notion is important to understand the frawework of this study, whether the present experimental design constitutes real CPP is debatable, as the authors cannot provide evidence that conditioning truly occured. What occured prior or immediately after the injection is not known. And, the stimuli an individual was exposed to prior to or immediately after an injection may have been different between the three injections and between the different subjects of this experiment. This is of course not detracting from the main findings and conclusions of this study whose results clearly highlight an effect of treatment on place preference but it seems inaccurate to consider the experimental design as a conditioned place preference, even if the underlying mechanism may involve reinforcing properties of testosterone.

    The description of the different types of USVs and what was analyzed here is actually not totally clear such that it is not always easy to interpret the results. The method section about audio recordings describes a variety of ultrasonic vocalizations broken down into sonic vocalizations (SVs), which are further categorized based on the number of calls they are composed of (1SV, 2SV, etc), bark calls as well as simple and complex sweeps. Although SV, barks and complex sweeps are defined, the difference between simple and complexe sweeps is not clear. Moreover, the results only report effects on SVs and barks, but the discussion concludes on sweeps (line 263) which is confusing.

    The results presented in table S3 are confusing. Tables S1 and S2 clearly report information relative to the amount of time males and females, respectively, spent in the nest (which is tehn broken down following different covariates). What Table S3 represents is less clear. The legend indicates that it represents pair time at the nest (but this is broken into male and female data) while the main text mentions within pair comparisons which would suggest that a difference between the time spent by each pair member was computed. How these data were obtained is not described. This is important because this information is key to establish a relationship between the time spent by the two pair members.

  3. Reviewer #2 (Public Review):

    In this manuscript by Petric et al, male California mice in a natural habitat with and without pups received 3 SQ injections of testosterone over 5 nights and the researchers used remote sensing equipment to record behaviors over 3 nights. They found that males administered testosterone spent more time at the nest than males given a control injection. Between males that were given testosterone, those with pups spent more time at the nest than those without pups. However, in control mice, males also spent more time at the nest if they had pups.

    Interestingly, females pair-bonded with males who received testosterone injections spent less time at the nest. The authors then showed that in pairs where the male received Testosterone injections, there was also an increased amount of total USVs at the nest. In addition, pairs who spent more time together produced less USVs. Interestingly, males who received testosterone injections produced calls that had a smaller bandwidth.

    Altogether, this paper provides behavioral evidence that testosterone has an effect on conditioned place preference in a natural, complex setting that is consistent with what has been seen in the laboratory setting. However, the paper is largely descriptive in nature and does not offer any novel mechanistic insights as to how testosterone influences place preference. The data show that having pups has a significant influence on male time at the nest regardless of testosterone. It is unclear from the data if having pups also significantly increases USVs in a manner that is distinct from testosterone pulses. Thus, the specific role of testosterone in driving these behaviors is unclear. For instance, would other rewards or pharmacological agents known to produce CPP, administered at the site of the nest, produce a different behavioral profile than what was observed with testosterone? Thus, while the authors provide evidence in a natural setting that confirms findings seen in the laboratory setting, the mechanism of testosterone in driving the observed behaviors remains unclear.

  4. Reviewer #3 (Public Review):

    While multiple laboratory studies - including some by the authors of this manuscript - have found that giving mice testosterone pulses induces a conditioned place preference, the role of such pulses in nature have not been studied before. In an ambitious field experiment, the authors administer testosterone to wild California mice to study its function in a more ecologically relevant setting. They then track the behavior of the injected male mice and of their pair bonded female partners to study physical movement and ultrasonic vocalizations. While these efforts are commendable, there are limitations to the results and the interpretations.

    1. While the authors find that mice injected with testosterone spend more time at the nest, it is not clear that the effect is specific to the nest or if testosterone would induce a conditioned place preference to other parts spatial locations as well. Moreover, it is not clear from these analyses if animals are spending more time at the nest simply because testosterone made them sick, for example.

    2. What is the evidence that the experimental manipulations are mimicking natural testosterone pulses at the nest?

    3. While it is clear that testing large numbers of animals in an experiment such as this is not trivial, the sample sizes are too small to support many of the results, especially when the authors attempt to estimate the effect of so many covariates. For example, there are only 4 male mice injected with testosterone that have pups. Fitting so many variables when overall sample size is small is problematic and can lead to false positive results.