Short-term social isolation acts on hypothalamic neurons to promote social behavior in a sex- and context-dependent manner

Reviewer #1 (Public Review):

Summary:

Zhao et al. perform a series of experiments aimed at identifying the role of the preoptic area (POA) in controlling the impact of social isolation on same-sex female social behavior. They focus their manuscript on the effects of short-term (3d) isolation and females, both of which have been relatively understudied, making the overall topic of the manuscript exciting and important.

Strengths:

The work highlighted is well designed, the experiments original, and the manuscript is elegant and clearly written. The strengths of the manuscript lie in the attention to multiple facets of social behavior (investigation, mounting, USVs), sex differences, and the use of multiple loss- and gain-of-function approaches.

Weaknesses:

The main weaknesses of the paper are a lack of significance in key findings, and relatedly, concluding effects from insignificant findings. Additional elements could be improved to help strengthen this overall well-rounded and intriguing set of results.

Reviewer #2 (Public Review):

Summary:

This study reveals that short-term social isolation increases social behavior at a reunion, and a population of hypothalamic preoptic area neurons become active after social interaction following short-term isolation (POAiso neurons). Effectively utilizing a TRAP activity-dependent labeling method, the authors inhibit or activate the POAiso neurons and find that these neurons are involved in controlling various social behaviors, including ultrasonic vocalization, investigation, and mounting in both male and female mice. This work suggests a complex role for the POA in regulating multiple aspects of social behavior, beyond solely controlling male sexual behaviors.

Strengths:

A few studies have shown that optogenetic activation of the POA in females promotes vocalization and mounting behavior, similar to the effects observed in males. However, those were the results of artificially stimulating POA neurons, and it was unknown whether POA neurons play a role in naturally occurring female social behaviors. This paper clearly demonstrates that there exists a population of POA neurons that are necessary for naturally evoked female social vocalizations and mounting behaviors.

Weaknesses:

The authors conclude that "In the current study, we identify and characterize a population of preoptic hypothalamic neurons that contribute to the effects of short-term social isolation on the social behaviors of mice." This is an interesting hypothesis, but in my opinion, critical control experiments are missing to support this claim.

All the activity-dependent labeling experiments with TRAP mice, including the subsequent neural activity manipulation experiments (Figures 2, 3, 4, 5E-F), were conducted by labeling neurons only in socially isolated animals, not group-housed animals. The authors labeled neurons after 30-minute social interactions, raising the possibility that the labeled neurons simply represent a "social interaction/behavior population" (mediating mounting and USVs in females and males) rather than a set of neurons specific to social isolation.

I strongly recommend including experimental groups that involve labeling neurons after 30-minute social interactions in group-housed female or male mice and inhibit TRAPed neurons after social isolation or activate TRAPed neurons after group housing. If manipulating the group-housed TRAP neurons has similar effects to manipulating the isolated TRAP neurons, it would suggest the current labeling paradigm is not isolating neurons specific to the effect of social isolation per se. Rather, the neurons may mediate more general social interaction or motivation-related activities. Given the known role of POA in male mating behavior, a group-housed TRAP experiment in males with a female visitor is especially important for understanding the selectivity of the labeled cells.

Without proper controls, referring to the labeled neurons as "POAiso" neurons is potentially misleading. The data thus far suggests these neurons may predominantly reflect a "POA social behavior" population rather than a set of cells distinctly responsive to isolated housing.

Overall, this paper is well-written and provides valuable new data on the neural circuit for female social behaviors and the potentially complex role of POA in social behavior control.

Reviewer #3 (Public Review):

Summary:

How short-term isolation acts on the brain to promote social behavior remains incompletely understood. The authors found that social interactions after a period of acute isolation increased investigation promoted mounting, and increased the production of ultrasonic vocalizations (USVs). This was true for females during same-sex interactions as well as for males interacting with females. Concomitant with these increased behavioral readouts, cFos expression in the preoptic area of the hypothalamus (POA) was found to increase selectively in single-housed females. Chemogenetic silencing of these POA neurons attenuated all three behavioral measures in socially isolated females. Surprisingly, ablation of the same POA neurons decreased mounting duration without impacting social investigation or USV production. While optogenetic activation was sufficient to evoke USV production, it did not affect either mounting or social investigation. In males, chemogenetic silencing of POA neurons decreased mounting but not other behaviors. Together, these data point towards a role of POA neurons in mediating social behaviors after acute isolation but the exact nature of that control appears to depend on the choice of perturbation method, sex, and social context in complex ways that are hard to parse. This study is an essential first step; additional experiments will be needed to explain the apparent discrepancy between the various circuit perturbation results and to gain a more comprehensive understanding of the role of POA in social isolation.

Strengths:

The goal of understanding the neural circuit mechanisms underlying acute social isolation is clearly important and topical. Using a state-of-the-art technique to tag specific neurons that were active during certain behavioral epochs, the authors managed to identify the POA as a critical circuit locus for the effects of social isolation. The experimental design is perfectly reasonable and the quality of the data is good. The control experiments (Figures 2B-D) showing that chemogenetic inactivation of other hypothalamic regions (AH and VMH) do not affect social behavior is indeed quite satisfying and points towards a specific role of POA within the hypothalamus. Using a combination of behavioral assays, activity-dependent neural tagging, and circuit manipulation techniques, the authors present convincing evidence for the role of the preoptic area of the hypothalamus in mediating certain behaviors following social isolation. These data are likely to be a valuable resource for understanding how hypothalamic circuits adjust to the challenges of social isolation.

Weaknesses:

While the authors should be commended for performing and reporting multiple circuit perturbation experiments (e.g., chemogenetics, ablation), the conflicting effects on behavior are hard to interpret without additional experiments. For example, chemogenetic silencing of the POA neurons (using DREADDs) attenuated all three behavioral measures but the ablation of the same POA neurons (using CASPACE) decreased mounting duration without impacting social investigation or USV production. Similarly, optogenetic activation of POA neurons was sufficient to generate USV production as reported in earlier studies but mounting or social investigation remained unaffected. Do these discrepancies arise due to the efficiency differences between DREADD-mediated silencing vs. Casp3 ablation? Or does the chemogenetic result reflect off-manifold effects on downstream circuitry whereas a more permanent ablation strategy allows other brain regions to compensate due to redundancy? It is important to resolve whether these arise due to technical reasons or whether these reflect the underlying (perhaps messy) logic of neural circuitry. Therefore, while it is clear that POA neurons likely contribute to multiple behavioral readouts of social isolation, understanding their exact roles in any greater detail will require further experiments.