PREreview of Sex-Dependent Vulnerability to PTSD-Like Behaviors in iNOS Knockout Mice

This article investigates the effects of biological sex and iNOS knockout (KO) on Post-Traumatic Stress Disorder (PTSD)-like behaviors related to anxiety, depression, and cognition. The authors provide thorough context of relevant literature about nitric oxide, sex differences in iNOS models, and the behavioral assays used. They clearly illustrate that they are addressing an important knowledge gap by conducting the first systematic behavioral study of female iNOS KO mice, which is significant given that women are twice as affected by neuropsychiatric disorders compared to men yet are underrepresented in preclinical research. The authors conduct a thorough evaluation of how iNOS KO affects behavior in males and females by using multiple assays within each behavioral domain. Their results show that female iNOS KO mice exhibit increased anxiety-like behavior compared to wildtype (WT) mice, whereas male iNOS KO mice show antidepressant behavior and deficits in contextual and cued fear memory extinction.

I would recommend that the authors improve the presentation of their statistical findings to increase clarity and add more discussion about the impact of their findings in the context of studying PTSD. Additionally, the authors could consider conducting additional experiments to probe the mechanistic underpinnings of the behavioral sex differences observed. Overall, I think this study is of high importance given that sex differences are not well characterized in this field, and the authors provide strong support for their claims via a thorough behavioral analysis.

Major Points

• When discussing figure 2, the authors mention that the sex differences in fear conditioning may be related to sex differences in the behavioral expression of fear, yet only freezing is assessed. The conclusions of the conditioning experiment would be greatly strengthened if other defensive behaviors that are common in female mice, such as darting or flight, were assessed as well.

• In the discussion section, the authors should consider discussing the implications of their findings on the future use of iNOS KO as a model of PTSD. Especially given that female iNOS KO animals don’t show differences in fear conditioning, it would be helpful to have the authors’ perspective on whether iNOS KO is a translationally relevant PTSD model in female animals.

• The authors should consider providing a discussion of the limitations of conducting a purely behavioral study and assessment of why the sex differences they found might be seen. While the authors do mention that their results “may be influenced by brain sexual dimorphism”, they don’t elaborate upon this point and explain what, if anything, is known about sexual dimorphisms in the iNOS system. Furthermore, they could indicate future experiments that could be conducted to probe mechanistic differences.

Minor Points

• I found the use of different symbols for different comparisons confusing, especially when lines weren’t used to show which bars were being compared. The authors should consider using color coding (ex. a specific color line for sex comparisons and another for group comparisons). Additionally, the authors should consider including a table that summarizes the main effects and interaction effects to allow the reader to better glean which behaviors yielded significant differences. Some specific examples are detailed below:

  • Panels 1b and 1c have different use of lines but the same symbols, which makes it confusing to know which comparisons are being made. Including lines between the bars for all comparisons would make it easier to read the graphs.

  • Lines are used to show an explicit comparison in panel 2d but not 2c.

  • Panels 2b and 2f show results for several comparisons. A table would summarize the differences well and improve comprehension.

• In the third paragraph of the discussion section, it is unclear whether the authors are comparing the iNOS KO male mice to WT male mice or iNOS KO female mice. In general, I would advise making it very explicit throughout the discussion which comparisons are being discussed since there are several groups.

• While the authors mention that sex differences are not always hormonally based, evaluating estrous cycle effects would provide evidence as to whether hormonal signaling or circuit-level brain sexual dimorphisms are more likely to be underlying the sex differences seen. This would be impactful, as there is evidence from both animal and human studies that sex steroid hormones influence PTSD symptomology.

• To expand upon the scope of the present paper or as future directions, the authors could consider conducting additional assays to improve understanding of the mechanisms underlying the behavioral sex differences shown. For example, one could use immunohistochemistry or in vivo calcium imaging to profile activity levels in relevant brain regions, such as the hippocampus and mPFC, during the behavioral assays. Gene expression profiling or electrophysiology could also be used to understand general cellular differences between male and female iNOS KO mice and WT mice.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.