Post-Mating Inhibition of Female Sexual Drive via Heterogeneous Neuronal Ensembles in the Medial Preoptic Area

Reviewer #1 (Public Review):

Summary:
This manuscript by Ishii et al utilizes a classical, but extremely understudied, female self-paced assay to directly address aspects of female sexual motivation independent from the male's behavior. This allowed for a clear separation of appetitive and consummatory events, of which whole brain unbiased activity was mapped. Mating completion in females was then focused on the medial preoptic nucleus where the authors performed a rigorous set of single-cell GCaMP recordings in populations marked by Vglut2 and Vgat, finding the latter display stronger and prolonged activity after the onset of mating completion. Finally, they demonstrate function to these Fos-TRAPPED completion cells demonstrating their capacity to suppress female sexual behavior.

Strengths:
This manuscript sought to explicitly explore the female mating drive as dictated by the female, a very rare angle for those studying mating behavior which almost always is controlled by the male's behavior. To achieve this, the authors went back to old literature and modified a classical paradigm in which a measurable approach and avoidance of male conspecifics can be measured in female mice using a self-paced mating assay. Strengths include a detailed quantification of female behaviors demonstrating a robust attenuated sexual motivation in females after mating completion. To determine the neural basis behind this, a brain-wide analysis of cells responding to mating completion in the female brain was conducted which revealed numerous anatomical regions displaying increased Fos activity, including the MPOA, of which the authors concentrated the remaining of their study. Employing microendoscopic imaging, the authors discovered that this mating completion signal was strongly represented in the MPOA. The single cell data analyses are of very high quality as is the number of individual cells resolved. While they identified both excitatory and inhibitory cell types that were activated by mating completion, they found the latter exhibited stronger and more persistent activity. Segmentation into individual mating behaviors reinforced the importance of GABAergic completion cells, which display prolonged activity late after the onset of mating completion. This information provides a potential mechanism for how female mice suppress further mating activity following completion. The authors then definitively demonstrate this function by TRAP'ping completion cells with chemogenetic actuators and show that CNO-induced activation of these cells specifically and strongly suppresses female sexual behavior. All experiments were extremely well-designed and performed carefully and expertly with the necessary controls solidifying the conclusions.

Weaknesses:
While there are no glaring weaknesses in this study, it should be noted that a great deal of literature has pinpointed the MPOA (and specifically inhibitory cells in this area) as being critical to sexual behavior, including female mating. However, no study to my knowledge has explored self-paced female mating with such fine control over manipulating and monitoring cellular activity in this region. In addition, this study may act to inspire others to further explore the additional brain regions found to show upregulation of neural activity (Fos) during mating completion in the female using the data sets generated here.

Reviewer #2 (Public Review):

Summary:
In this set of studies, the authors identify cFos activation in neurons in female mice that mated with males, and after experiencing male sexual behavior that is either restricted to appetitive behavior or including ejaculation. The medial preoptic nucleus was identified as an area with high cFos induction following ejaculation. Characterization of neurochemical phenotypes of cfos-expressing neurons showed a heterogenous distribution of activated neurons in the MPOA, including both inhibitory and excitatory cell types. Next, in vivo calcium imaging was used to show activation of Vgat and Vglut neurons in female mice MPOA after displaying sniffing of the male, experiencing male appetitive, or male consummatory sexual behavior, demonstrating significantly higher activation and of a greater subpopulation of Vgat neurons than Vglut neurons. Moreover, the greatest activation of Vgat neurons was detected following experiencing ejaculation, and ejaculation activated different subpopulations of MPOA cells than consummatory or appetitive sexual behaviors experienced by the female. Finally, pharmaco-genetic activation of the subpopulation of MPOA neurons that were previously activated following ejaculation resulted in a significant reduction of approach behavior by the female mice towards the male, interpreted as suppression of female sexual motivation. In conclusion, a subpopulation of inhibitory cells in the MPOA is activated in female mice after experiencing ejaculation, in turn contributing to the suppression of sexual approach behavior.

Strengths:
The current set of studies replicates previous findings that ejaculation causes longer latencies to initiate interactions with a male after receiving an ejaculation in a paced mating paradigm, which is widely validated and extensively used to investigate sexual behavior in female rodents. Studies also confirm that ejaculation increases cFos expression in the MPOA while extending prior findings with a careful analysis of the neurochemical phenotype of activated neurons. A major strength of the studies is the use of cell-specific in vivo imaging and pharmaco-genetic activation to reveal a functional role of specific neuronal ensemble within the MPOA for post-ejaculatory female sexual behavior.

Weaknesses:
The authors include an elegant manipulation of ejaculation-activated neurons in the MPOA using DREADD. However, this study was limited to show that activation of previously activated cells was sufficient to reduce approach behavior in a paced mating paradigm and receiving intromissions in a home cage mating paradigm. An inhibition approach using DREADD would have been a great complement to this study as it would have examined if activation of the cells was required. Moreover, additional tests for sexual motivation would have greatly strengthened the overall conclusions.

Reviewer #3 (Public Review):

Summary:
Ishii et al used molecular genetics and behavioral analyses in mice to study the functional role of a subset of MPOA neurons in the regulation of female sexual drive. They first employed a self-paced mating assay during which a female could control the amount of interaction time with a male to assess female sexual drive after completion of mating. The authors observed that after mating completion females spent significantly less time interacting with the mated males, indicating that their sexual drive was reduced. Next, the authors performed a brain-wide analysis of neurons activated during the completion of mating and identified the MPOA as a strong candidate region. However, their activity labeling was not exclusive to neurons activated during mating completion but included all neurons activated before, during, and after the mating encounter. This makes it difficult to interpret these data. Importantly, the authors do provide in vivo calcium imaging data showing that a subset of MPOA neurons responds significantly and specifically to mating completion and not other behaviors during the social encounter. The authors performed these studies in both excitatory and inhibitory populations of the MPOA. Their analysis identified a subpopulation of inhibitory neurons that exhibit sustained increased activity for 90 sec following mating completion. Finally, the authors used chemogenetics to activate MPOA neurons during home cage mating, condition place preference, pup retrieval, and the self-paced mating assay. They found that activation of these neurons significantly reduced mating behaviors and time spent interacting with a male during the self-paced mating assay. The authors suggest that their chemogenetic activation is restricted to neurons activated during mating completion, but their activity-dependent labeling strategy resulted in chemogenetic activation of all MPOA neurons activated either before, during, or after mating.

The authors' experimental execution is rigorous and well-performed. Their data identify inhibitory neurons in the female MPOA as a neural locus that is activated following the completion of mating and potentially a key neural population in the regulation of female sexual motivation. However, the conclusions and interpretation of the data extend beyond what is reasonable given the limitations of the activity-dependent labeling strategy employed.

Strengths:

1. The use of the self-paced mating assay in combination with neural imaging and manipulation to assess female sexual drive is innovative. The authors correctly assert that relatively little is known about how mating completion affects sexual motivation in females as compared to males. Therefore, the data collected from these studies is important and valuable.

2. The authors provide convincing histological data and analyses to verify and validate their brain-wide activity labeling, neural imaging, and chemogenetic studies.

3. The single-cell in vivo calcium imaging data are well performed and analyzed. They provide key insights into the activity profiles of both excitatory and inhibitory neurons in the female MPOA during mating encounters. The authors' identification of an inhibitory subpopulation of female MPOA neurons that are selectively activated following the completion of mating is fundamental for future experiments which could potentially find a molecular marker for this population and specifically manipulate these neurons to understand their role in female sexual motivation in greater detail.

Weaknesses:

1. Their activity-dependent labeling strategy is not exclusive to mating completion but instead includes all neurons active before, during, and after the social encounter. In the manuscript, the authors did not discuss the time course of Fos activation or the timeframe of the FosTRAP labeling strategy. Fos continues to be expressed and is detectable for hours following neural activation. Therefore, the FosTRAP strategy also labels neurons that were activated 3 hours before the injection of 4-OHT. The original FosTRAP2 paper which is cited in this manuscript (DeNardo et al, 2019) performed a detailed analysis of the labeling window in Supplementary Figure 2 of that paper. Here is quoted text from that paper: "Resultant patterns of tdTomato expression revealed that the majority of TRAPing occurred within a 6-hour window centered around the 4-OHT injection." Thus, the FosTRAP "mating completion" groups throughout this manuscript also include neurons activated 3 hours before mating completion, which includes neurons activated during appetitive and consummatory mating behaviors.

This makes all of the FosTRAP data very difficult to interpret. Compounding this is the issue that the two groups the authors compare in their experiments are females administered 4-OHT following appetitive investigation behaviors (with the male removed before mating behaviors occurred) and females administered 4-OHT following mating completion. The "appetitive" group labeled neurons activated only during appetitive investigation, but the "completion" group labeled neurons activated during appetitive investigations, consummatory mating bouts, and mating completion. Therefore, in the brain-wide analysis of Figure 2, it is impossible to identify brain regions that were activated exclusively by mating completion and not by consummatory mating behaviors. This could have been achieved if the "completion" group was compared to a group of females that had commenced consummatory mating behaviors but were separated from the male before mating was completed. Then, any neurons labeled by the "completion" FosTRAP but not the "consummatory" FosTRAP would be neurons specifically activated by mating completion. In the current brain-wide analysis experiments, neurons activated by consummatory behaviors and mating completion can not be disassociated.

This same issue is present in the interpretation of the chemogenetic activation data in Figure 6. In the experiments of Figure 6, the authors are activating neurons naturally activated during consummatory mating behaviors as well as those activated during mating completion.

1. This study does not definitively show that the female mice used in this study display decreased sexual motivation after the completion of mating. The females exhibit reduced interaction with males that had also just completed mating, but it is unclear if the females would continue to show reduced interaction time if given the choice to interact with a male that was not in the post-ejaculatory refractory period. Perhaps, these females have a natural preference to interact more with sexually motivated males compared to recently mated (not sexually motivated) males. To definitively show that these females exhibit decreased sexual motivation the authors should perform two control experiments: 1) provide the females with access to a fully sexually motivated male after the females have completed mating with a different male to see if interaction time changes, and 2) compare interaction time toward mated and non-mated males using the self-paced mating assay. These controls would show that the reduction in the interaction time is because the females have reduced sexual motivation and not because these females just naturally interact with sexually motivated males more than males in the post-ejaculatory refractory period.

2. It is unclear how the transient 90-second response of these MPOA neurons following the completion of mating causes the prolonged reduction in female sexual motivation that is at the minutes to hours timeframe. No molecular or cellular mechanism is discussed.

3. The authors discuss potential cell types and neural population markers within the MPOA and go into some detail in Figure S3. However, their experiments are performed with only the larger excitatory and inhibitory MPOA neural populations.