Inhibition tunes prefrontal circuit dynamics to promote sociosexual behavior in female mice

Reviewer #1 (Public review):

Summary:

Amadei et al investigate how excitation/inhibition balance in the prefrontal cortex plays a role in social behavior. To address this question, they developed a behavioral task where adult female mice can choose between a social reward (e.g., an adult male for sociosexual choice, or an adolescent female mouse) and a non-social reward (e.g., milk). They found that optogenetic inhibition of inhibitory neurons expressing oxytocin receptors (OXTR neurons) in the prefrontal cortex (PFC) reduces choice for sociosexual interaction compared to non-social reward and to a greater extent in sexually receptive females. They also found that this manipulation increases pyramidal neuron activity. Specifically, the authors identified a neuronal ensemble which represent the male option. Inhibition of OXTR disrupts the ability of the neuronal ensemble to represent the male option during decision-making in the behavioral task. Thus, using computational modeling, the authors proposed that OXTR neurons promote male choice by letting a male-representing pyramidal ensemble outcompete other pyramidal populations in the mPFC.

Strengths:

The study addresses an important topic in social behaviour and reward neuroscience with a focused hypothesis. The combination of behavioral testing and circuit manipulation combined with calcium imaging is a clear strength, and the work has the potential to make a solid contribution.

Weaknesses:

The main weaknesses are limited methodological clarity and details.

Reviewer #2 (Public review):

The authors aim to understand how inhibitory circuitry within the medial prefrontal cortex regulates the selection of sociosexual behaviour. Rather than studying social interaction in isolation, they develop an elegant behavioural paradigm in which female mice repeatedly choose between interacting with a male and obtaining an appetitive non-social reward. This task allows the authors to examine behavioural choice under conditions that more closely resemble natural decision-making. They combine optogenetic inhibition of oxytocin receptor-expressing interneurons, large-scale calcium imaging of pyramidal neurons, slice electrophysiology, and computational modelling to investigate how inhibition shapes cortical representations that ultimately bias behavioural choice.

The study has several notable strengths. The behavioural paradigm is novel and well-designed, allowing repeated choice measurements while controlling for general social motivation by including both male and juvenile female stimuli. The integration of multiple experimental approaches is particularly impressive. The behavioural effects of optogenetic inhibition are complemented by population imaging demonstrating elevated pyramidal activity, electrophysiological recordings confirming monosynaptic regulation of pyramidal neurons, and a computational model that provides a mechanistic interpretation of the observed circuit dynamics. The work therefore spans multiple levels of analysis, from synaptic interactions to behaviour, and the individual datasets are generally of high technical quality.

The imaging analyses identifying a putative "MALE" ensemble are particularly interesting. The observation that a relatively small subset of pyramidal neurons preferentially represents the male option before behavioural commitment provides an attractive framework for understanding how inhibition can stabilise specific behavioural representations. The temporal analysis suggesting that disruption of this representation precedes impaired behavioural choice is especially compelling, as it moves beyond simple correlations between neural activity and behaviour.

Several aspects of the mechanistic interpretation remain somewhat speculative. The central conclusion relies heavily on the computational competition model, which assumes an asymmetric competition between a relatively small male-selective ensemble and a much larger default pyramidal population. While the model successfully reproduces several experimental observations, many of its architectural assumptions are inferred rather than experimentally demonstrated. In particular, the designation of the remaining pyramidal neurons as a functional "OTHER" population representing the non-social alternative is not directly established experimentally. Alternative circuit architectures may be capable of producing similar behavioural and population-level effects, and the current data do not fully distinguish among these possibilities.

Similarly, although the identification of MALE cells is thoughtfully performed, the classification depends on an operational threshold derived from ROC analysis and correlated activity. It remains uncertain whether these neurons constitute a stable functional ensemble across sessions or merely reflect one end of a continuous representational spectrum. Longitudinal analyses examining the stability of these ensembles across days or across changes in behavioural state would strengthen the claim that they represent a dedicated neuronal population.

An additional limitation concerns the specificity of the behavioural interpretation. The reduction in male choice is interpreted primarily as impaired sociosexual decision-making. While the inclusion of juvenile female stimuli substantially improves the experimental design, it remains difficult to completely separate altered sociosexual motivation from broader changes in motivational salience, valuation, or action selection. The observed changes could reflect alterations in multiple components of the decision-making process, and this distinction deserves a somewhat more balanced discussion.

The interaction with the oestrous state is a very interesting aspect of the work and is consistent with previous studies of oxytocin-dependent sociosexual behaviour. However, this analysis is based on relatively modest numbers of animals and sessions, making it difficult to judge the robustness of these effects. The conclusions regarding hormonal modulation would therefore benefit from a more cautious interpretation.

Overall, the authors achieve their primary objective of demonstrating that oxytocin receptor-expressing interneuron-mediated inhibition contributes to the selection of sociosexual behaviour while regulating pyramidal population dynamics in the medial prefrontal cortex. The behavioural, imaging, and electrophysiological datasets provide convincing evidence that inhibition shapes cortical activity during decision-making. The computational model offers a plausible mechanistic framework linking these observations, although some aspects of this framework remain hypothetical and await further experimental testing.

The work is likely to have a significant impact on the fields of cortical circuit function, social neuroscience, and decision-making. Beyond its specific findings, the study introduces a behavioural paradigm that should prove broadly useful for investigating how competing behavioural options are represented within prefrontal circuits. The combination of behavioural neuroscience, population imaging, and computational modelling represents a valuable resource for the community and provides an important foundation for future studies examining how excitation-inhibition balance shapes flexible social behaviour.

Reviewer #3 (Public review):

Summary:

Using a combination of Miniscope imaging and optogenetic manipulation, Amadei et al. reveal how oxytocin receptor neurons in the prefrontal cortex of mice control pyramidal subpopulations and socio-sexual behavior. This work was planned and executed carefully and provides a novel and important angle to study the oxytocin system in the cortex. According to their results, oxytocin receptor neurons help discriminate between sexual and non-sexual stimuli, most likely by controlling different pyramidal subpopulations that are either most active during trials that include a sexual stimulus or that include non-sexual stimuli. I highly appreciate this article; however, I have one major concern related to the modeling part.

Strengths:

(1) Well-designed experiments.

(2) Rigorous analysis.

(3) Generates a new avenue to study socio-sexual decision making and creates a hypothesis about the connectivity of oxytocin-sensitive circuits.

Weaknesses:

(1) Major

In their last figure (Figure 4), the authors generated a computational model that, according to the authors, reveals a potential network mechanism in which oxytocin receptor (OXTR) neurons are connected to both pyramidal populations with certain connectivity rules. Although the model seems to reproduce the experimental results, some assumptions of the model seem to be poorly supported. If I understood correctly, the authors simply assumed that the strength of the connections between OXTR neurons and MALE neurons is the same as the strength of the connections between OXTR neurons and OTHER neurons. The authors neither discuss literature supporting such connectivity nor provide experimental evidence for this. I also could not find information about the magnitude of the synaptic weights to each of these populations. I guess these parameters are critical for the outcome of the simulation, and it may be worth exploring the outcome of simulating the different combinations of connectivity and synaptic weights between OXTR neurons and pyramids, as well as the degree of recurrent connectivity within the pyramidal subpopulations. Further, the authors should at least discuss in depth how inhibitory OXTR neuronal subtypes (they have different properties that could potentially be implemented in the modeling) may match their computational model best. If the current model remains the most promising, the authors should clearly discuss which experimental trajectory should be taken next to actually provide proof for its correctness (e.g. whether and how it would be possible to determine the predicted connectivity experimentally).

(2) Minor

The authors state regarding counterbalancing in Figure 1 and Figure S4C: "The social presentation order (male or female first), as well as the locations of the social and milk options (left or right relative to start arm), were fixed over sessions within a given subject, but varied over subjects (Figure S4C)". In Figure S4C, it looks as if there are fewer animals in which the male was always presented first than animals in which the female was always presented first (~ 16 vs. 20). While the difference is not very big, it may be influential. To fully exclude a sequence effect, I would suggest adding male-first animals until both groups are the same size.