The rules of sex and strain: deciphering the chemical language of inbred and wild mouse conspecific scents

Reviewer #1 (Public Review):

In this manuscript, Nagel et al. sought to comprehensively characterize the composition of urinary compounds, some of which are putative chemosignals. They used urines from adult males and females in three different strains, including one wild-derived strain. By performing mass spectrometry of two classes of compounds: volatile organic compounds and proteins, they found that urines from inbred strains are qualitatively similar to those of a wild strain. This finding is significant because there is a high degree of genetic diversity in wild mice, with chemosensory receptor genes harboring many polymorphisms.

In the second part of this work, the authors used calcium imaging to monitor the pattern of vomeronasal neuron responses to these urines. By performing pairwise comparisons, the authors found a large degree of strain-specific response and a relatively minor response to sex-specific urinary stimuli. This is a finding generally in agreement with previous calcium imaging work by Ron Yu and colleagues in 2008. The authors extend the previous work by using urines from wild mice. They further report that the concentration diversity of urinary compounds in different urine batches is largely uncorrelated with the activity profiles of these urines. In addition, the authors found that the patterns of vomeronasal neuron response to urinary cues are not identical when measured using different recipient strains. This fascinating finding, however, requires an additional control to exclude the possibility that this is not due to sampling error.

There are several weaknesses in this manuscript, including the lack of analysis of the compositions of sulfated steroids and other steroids, which have been proposed to be the major constituents of vomeronasal ligands in urines and the indirect (correlational) nature of their mass spectrometry data and activity data.

Overall, the major contribution of this work is the identification of specific molecules in mouse urines. This work is likely to be of significant interest to researchers in chemosensory signaling in mammals and provides a systematic avenue to exhaustively identify vomeronasal ligands in the future.

Reviewer #2 (Public Review):

This manuscript by Nagel et al provides a comprehensive examination of the chemical composition of mouse urine (an important source of semiochemicals) across strain and sex, and correlates these differences with functional responses of vomeronasal sensory neurons (an important sensory population for detecting chemical social cues). The strength of the work lies in the careful and comprehensive imaging and chemical analyses, the rigor of quantification of functional responses, and the insight into the relevance of olfactory work on lab-derived vs wild-derived mice.

With regards to the chemical analysis, the reader should keep in mind that a difference in the concentration of a chemical across strain or sex does not necessarily mean that that chemical is used for chemical communication. In the most extreme case, the animals may be completely insensitive to the chemical. Thus, the fact that the repertoire of proteins and volatiles could potentially allow sex and/or strain discrimination, it is unclear to what degree both are used in different situations.

Reviewer #3 (Public Review):

Summary:
The manuscript by Nagel et al. describes studies of mouse vomeronasal sensory neuron (VSN) tuning to mouse urine samples across different sexes and strains, including wild mice, alongside mass spectrometry analysis of the same samples. The authors performed live Ca2+ imaging (CAL520 dye) of VSNs in acute vomeronasal organ (VNO) slices to determine how VSNs are tuned to pairs of stimuli that differ in their origin (e.g. male C57BL/6 versus male BALB/c urine, male C57BL/6 versus female C57BL/6, etc.). For each pair of tested odorants, the results measure the proportion of VSNs that respond to both stimuli ("generalists") or just one of the two ("specialists"), as well as metrics of tuning preference and response reliability. The authors find in most cases that generalists make up a larger proportion of responsive VSNs than specialists, but several pairwise comparisons showed a high degree of strain selectivity. Notably, the authors evaluated VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding strain-dependent differences in the representation of mouse urine. Alongside these measurements of VSN tuning, the authors report results of mass spectrometry analyses of volatiles and proteins in the same urine samples. These analyses indicated a number of molecules in each category that vary across sex and strain, and therefore represent candidate vomeronasal ligands. However, this study did not directly test whether any of these candidate molecules drives VSN activity, limiting the interpretability of these comparisons. Overall, this work provides useful information related to mouse vomeronasal chemosensation, but future work will be necessary to link the physiological measurements to the observed molecular diversity.

Strengths:
A strength of the current study is its focus on characterizing the neural responses of the VNO to urine derived from wild mice. The majority of existing vomeronasal system research has relied on the use of inbred strains for both neural response recordings and investigations of candidate vomeronasal system ligands. Inbreeding in laboratory environments may alter the chemical composition of bodily secretions, thereby potentially changing the information they contain. Moreover, the more homogeneous nature of inbred strains could be critical when studying the AOS mediated social aspects. If there exist noticeable differences in the chemical composition of secretions from wild animals compared to inbred strains, this would suggest that future research must consider natural sources of candidate ligands outside of inbred strains. This work identifies some intriguing differences - worthy of further exploration - between the urine composition of wild mice versus inbred mice, as well as disparities in how the VNO responds to urine from these different sources. However, the molecular composition and VNO responsiveness to wild mouse urine was found to be highly overlapping with inbred mouse urine, supporting the continued investigation of candidate ligands found in inbred mouse urine.

Another positive aspect of this work is its use of the same set of stimuli as a previous study by the same authors (Bansal et al., 2021) in the downstream accessory olfactory bulb. The consistency in stimulus selection facilitates a comparison of information processing of sex and strain information from the sensory periphery to the brain. Although comparisons between the two connected regions are not a focus of this work, and methodological differences (e.g., Ca2+ imaging versus electrophysiology) may introduce caveats into comparisons, the support of "apples to apples" comparisons across connected circuits is critical to progress in the field.

Finally, this study directly measured VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding subtle but important differences in the representation of mouse urine in these two recipient strains. Given that there is a long history of research into strain-specific differences in social behavior, this research paves the way for future studies into how different mouse strains detect and process social chemosignals.

Weaknesses:
One of the primary objectives in this study is to ascertain the extent to which the response profiles of VSNs are specific to sex and strain. The design of these Ca2+ imaging experiments uses a simple stimulus design, using two interleaved bouts of stimulation with pairs of urine (e.g. male versus female C57BL/6, male C57BL/6 versus male BALB/c) at a single dilution factor (1:100). This introduces two significant limitations: (1) the "generalist" versus "specialist" descriptors pertain only to the specific pairwise comparisons made and (2) there is no information about the sensitivity/concentration-dependence of the responses.

The functional measurements of VSN tuning to various pairs of urine stimuli are consistently presented alongside mass spectrometry-based comparisons. Although it is clear from the manuscript text that the mass spectrometry-based analysis was separated from the VSN tuning experiments/analysis, the juxtaposition of VSN tuning measurements with independent molecular diversity measurements gives the appearance to readers that these experiments were integrated (i.e., that the diversity of ligands was underlying the diversity of physiological responses). This is a hypothesis raised by the parallel studies, not a supported conclusion of the work. This data presentation style risks confusing readers.

The impact of mass spectrometry findings is limited by the fact that none of these molecules (in bulk, fractions, or monomolecular candidate ligands) were tested on VSNs. It is possible that only a very small number of these ligands activate the VNO. The list of variably expressed proteins - especially several proteins that are preferentially found in female urine - is compelling, but, again, there is no evidence presented that indicates whether or not these candidate ligands drive VSN activity. It is noteworthy that the largest class of known natural ligands for VSNs are small nonvolatiles that are found at high levels in mouse urine. These molecules were almost certainly involved in driving VSN activity in the physiology assays (both "generalist" and "specialist"), but they are absent from the molecular analysis.