Freezing suppression by oxytocin in central amygdala allows alternate defensive behaviours and mother-pup interactions

Thank you for submitting your article "Freezing Suppression by Oxytocin in Central Amygdala Allows Alternate Defensive Behaviors and Mother-Pup Interactions" for consideration by eLife. Your article has been favorably evaluated by four reviewers, one of whom, Peggy Mason (Senior Editor), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Josh Neunuebel (Reviewer #4).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This is an exciting piece of work that shows modulation of fear immobility by the presence of pups. The phenomenon is robust with dams showing freezing in the absence of pups, orientation toward the conditioned fear stimulus or toward the pups in the presence of young and old pups, respectively. The age-dependent pup effect is dependent on mom's stage initially and then on pup's stage. Injection of an OXT-R antagonist into Ce-Amygdala blocks the pups' modulation of mom's fear response, so that the moms freeze now even with the pups around. Finally, the pups learn to fear the odor not from the moms' freezing but from the moms' modulated reaction (not freezing).

Essential revisions:

There are detailed reviews below that are there to help you prepare your revision. The changes that must be addressed are:

Better justify that the modulated behavior is pup protection or soften the conclusions;

Change language to acknowledge that OXT-R involvement is tested for NOT OXT release;

Calculation of the% : what are the bin sizes?

Are the responses really starting before the stimulus at t=0? The lines between pre and post are steep and straight and give this impression. if there is a break in data collection to introduce the stimulus, please illustrate this. In any case, please clarify;

Figure 2 does not match the text;

Neutral stimulus for mom to pup transmission experiment is needed to rule out overall increase in anxiety/fear or tone down discussion;

Improve or omit videos.

Reviewer #1:

This is an exciting piece of work that shows modulation of fear immobility by the presence of pups. The phenomenon is robust with dams showing freezing in the absence of pups, orientation toward the conditioned fear stimulus or toward the pups in the presence of young and old pups, respectively. The age-dependent pup effect is dependent on mom's stage initially and then on pup's stage (see below for a concern with the reporting of this result). Injection of an OXT-R antagonist into Ce-Amygdala blocks the pups' modulation of mom's fear response, so that the moms freeze now even with the pups around. Finally, the pups learn to fear the odor not from the moms' freezing but from the moms' modulated reaction (not freezing). The discussion of this latter result should be augmented a bit to mention the lack of pup copying of mom's behavior.

Figure 2 does not match the text. As the text reads dams of young pups (matched) orient toward the threat when tested with young pups and yet Figure 2B has them orienting toward the pups. And in 2C, it appears that the dams of older pups (matched) orient toward the threat when the text says that they orient toward the pups. Please explain.

Subsection “Regulation of dams’ defensive behavior by the presence of pups”, first paragraph: doesn't maternal aggression increase between birth and weaning?

Please comment on how the sample sizes were determined. The numbers in the groups are greatly different. E.g. 17 controls and 7 OTA.

The videos appeared inconsistently sized on my browser. Moreover, they are simply not useful as is. Please label the time and location of the smell. Annotate the videos (in the videos) so that a viewer can easily glean the point of each video.

From the videos that I could see, it appears that the dams of older pups interact with them closely through taction and one would guess calls as well as odors. The possibility that either taction or audition plays a role in communicating threat to the pups should be at least mentioned.

Reviewer #2:

While the results presented in the manuscript by Rickenbacher et al. are novel and interesting, I have some concerns that should be addressed before the paper is published.

1) I have a concern about the interpretation of the results. It is not clear whether increased social contact of the dams with older pups (PP19-21) is a switch from self-defense to offspring protection. The data do not show if the suppression of freezing is limited to the presence of pups or if this suppression occurs in the presence of any conspecific. It is conceivable that the dams engage in social contact with their pups just as they would do with unrelated adult animals (which could result in social buffering effects). Therefore, the assumption should be better justified, either by an additional control group or by a reference to already available results.

2) The sentence "Next, we tested our hypothesis that pup presence leads to release of oxytocin by PVN neurons projecting to the CeL, which suppress freezing and permits expression of maternal defensive behaviors." is an overstatement. There is no data here directly showing that oxytocin is actually released in the CeA when the CS is presented to the dams with pups. Similarly, the specific projections as well as the specific region of the CeA were not targeted in the presented experiments. Furthermore, the abbreviations for the central amygdala are inconsistent throughout the text. In the Introduction, there are references to the CeL (centro-lateral amygdala), in Materiasl and methods, Surgery Protocol the central amygdala is abbreviated to the CeM (which is a common abbreviation for the medial part of the central amygdala). It should be clearly stated that the extent of infusion site is not verified here – so the authors can say that they target the central amygdala, but not one of its parts. Since it is known from other works that the receptors for oxytocin are in the lateral and capsular subdivisions of the central amygdala (Huber et al., 2005), the results suggest that these parts are crucial for oxytocin effects.

3) In the description of the methods:

A) The dose of the oxytocin antagonist is not given. It is particularly important in the context of mixed results on the role of oxytocin in the CeA in maternal aggression. This issue should be addressed.

B) In the Materials and methods section, the ITIs between CSs during odor test are described as 5min, 3 min and 3 min. In figures all ITIs are 3 min. Moreover, the response to the third stimulus is not shown.

C) It should be clearly stated how the freezing scores shown in the results were obtained. It is not clear as it is described now.

D) "The final behavioral values are the mean of multiple blind and independent scorings." – it should be clearly stated how many.

E) It is not clear for which periods of time (3-min bins?) the δ scores showing change of behavior upon odor presentation are calculated.

4) There is a difference in the amount of time "toward pups" before the presentation of the CS (in the baseline) between dams of younger and older pups (Figure 1G); this should be discussed.

5) In the last experiment on transmission of fear from mother to pup, there is some variability in the freezing response. Is there any correlation between freezing and intensity of social contact with the dam during the test? Are there differences between pups tested with different dams?

6) The data shown in Figure 2 are inconsistent with the description in the text, probably graphs should be in different order.

7) Why are different behavioral measures shown in Figures 1 and 2 – δ time and time during odor, respectively?

Reviewer #3:

There is a lot to like in this paper. The finding that dams express conditioned fear responses that depend on both the presence of pups and the pups' age is novel and provocative. The identification of oxytocin as a permissive factor for the expression of the defensive response demonstrates a potential mechanism. I also really like the raster plots showing behavior for each animal as well.

One thing that I'm finding unclear is exactly how the data for all of the% time graphs (e.g. Figure 1C-G) were calculated. I don't know if I'm missing something somewhere but what are these values a percentage of? Is it across the entire session? If there is a "binning" of time blocks somewhere I can't find it. Normally in cued conditioning experiments there is a time block (e.g. a 30s tone) and freezing% is calculated by what% of those 30s the animals spend freezing. However, since we have continuous line graphs here across time, this doesn't seem to be the approach. Along the same lines, I don't see where the authors describe how the change in% is calculated. Is it the change from before the first presentation of the odor to some kind of aggregate of the behavior during the odor presentation? More detail is needed in exactly where all of these% values come from (again, unless I'm missing where this information is in the manuscript).

Reviewer #4:

Rickenbacher et al. conducted a series of elegant experiments to address how self-defense behaviors are suppressed when switching to offspring protection. The authors reported that dams condition to fear an odor froze when tested alone, but when pups were present, they remained in close contact with them or targeted the threat. Oxytocin (OT) signaling in the centro-lateral amygdala (CLA) appear to be crucial for this behavior because blocking OT by injecting oxytocin antagonous into the (CLA) prevented suppression of maternal freezing. Moreover, pups exposed to the odor in the presence of the conditioned dam later froze when re-exposed alone. However, if oxytocin signaling in the dam had been blocked, pups failed to learn. The authors concluded that OT in the CLA plays a role in suppressing self-defense behaviors and allows mothers to protect their pups and transmit information about danger to their offspring. Although the results are striking and fill a gap in knowledge about the circuitry underlying maternal behaviors during innate behavior, a component that needs to be emphasized more in the paper, there are three major, yet addressable concerns. One of which, requires additional experiments.

First, the reported behavioral changes of the dams in Figures 1–4 all start before the delivery of the odor. The timing of this response is odd because behavioral responses to odor delivery typically occur with a slight delay and not before the odor is delivered. There is clearly a difference in the behaviors of the dams with pups and without pups. The question is what are they mothers really responding to the environment. It doesn't appear to be the odor.

Second, the text for Figure 2 does not match data in the figure. For Figure 2, shouldn't PP4-6 matched replicate your results in Figure 1? Shouldn't PP19-21 matched replicate your results in Figure 1? This is not the case. For Figure 2, it looks like the plots were mislabel and this is concerning. Therefore, the raw data needs to be shown like Figure 1B.

Third, you claim that the mothers are transmitting the learned fear response to the pups, but you haven't rule out that the pups could just be over anxious and more sensitive to any stimulus. A vital control was omitted. You needed to also show that animals are not freezing to a neutral odor. If the pups from the control dams don't show freezing to a neutral odor, then you say that they learned the association between peppermint and threat from the mom and make the claim that this learning was transmitted from the moms. Without doing this experiment, then you need to tone down the conclusions you make in the Discussion.