Oxytocin neurons signal state-dependent transitions from rest to thermogenesis and behavioral arousal in social and non-social settings

Reviewer #1 (Public review):

Summary:

The authors identify and investigate a specific population of PVNOT neurons (oxytocin neurons of the paraventricular hypothalamus) that seem to be involved in both behavioral and autonomic thermoregulation. These cells are activated by social thermoregulatory behaviors, but can influence thermoregulation in both social and nonsocial contexts, specifically during transitions and when mice are at low core body temperature (Tb).

Strengths:

The manuscript has many strengths.

This is a novel study, with a clear question that is addressed using an array of well-designed experiments employing integrative methods. Most of the figures are well-developed, and the analysis is generally rigorous and well-detailed. The authors are clearly very experienced in this field, and indeed, their scholarly introduction and discussion sections are to their credit.

The link between thermoregulation and the oxytocin system is well established, as is the link between social behavior and the same broad system. However, the link between these three things is novel, if it can be well substantiated. I am not persuaded that was achieved here, but I do think this manuscript has many novel and useful offerings.

The authors use a cooling floor, and only go down to 10 degrees Celsius. This is fine, but I would like to see the effects using ambient temperature also. This is not a crucial issue, as it is not necessary for the authors' interpretations, but it could improve measurement sensitivity.

Through an elegant behavioral experiment in Figure 1, the authors identify c-Fos patterns in the PVN that are activated by active social huddling, and they show that at the RNA level these cells overlap with oxytocin, indicating that they are oxytocin-producing cells. But this is not well discussed or indeed quantified.

The authors engage in a deep analysis of fiber photometry experiments, first by observing PVNOT neuron overall activity during a variety of different behaviors in the context of three different temperatures. Activity was associated with nesting, quiescence, and both types of huddling (when social opportunities exist). Social situations did not strongly affect this, nor did temperature conditions. These analyses indicate that the PVNOT neurons are involved in mediating specific behavioral outputs.

With more detailed analysis, the authors investigated how PVNOT neuronal activity relates to behavioral state transition. They found that the probability of peak PVNOT neural activity strongly predicts the offset of quiescence or quiescent huddling, and therefore can be argued to signal an increase in physical activity, and as such, increased metabolism. However, the opposite pattern was observed for huddling and nesting (onset being associated with PVNOT activity), again arguing for increased thermogenesis as a function.

What is particularly compelling is that these peaks of activity tend to occur during low Tb, again arguing for the function in increasing body warmth.

The authors then employ an impressive setup where they image brown adipose tissue (BAT) in tandem with DeepLabCut (DLC) based animal tracking. Crucially, BAT activity and surface temperature correlated with the calcium peak of PVNOT neurons.

Lastly, optogenetic activation of PVNOT neurons increased Tb when it was in the lower range, but not when in the higher range. It also affected BAT and rump temperature, again at low Tb. However, there is no real effect on behavior, except a trend in activity.

The authors do some interesting tracing work at the end, though this is not functionally explored. That is not a criticism, as it does seem like this would be a whole follow-up study.

Weaknesses:

While novel and valuable, the manuscript feels incomplete in its current form.

The main evidence lacking is a loss of function of the experiment. Ideally, the authors would chronically and/or acutely inhibit PVNOT neurons to establish their necessity. I know this seems obvious, but I think it is important.

The relative lack of behavioral analysis following optogenetic activation of PVNOT neurons is puzzling. The authors must surely want to study what this intervention does to behavioral state transitions. I feel that the current level of analysis limits the overall conclusions of this study to a large extent.

A broader criticism is that the social dimension of this manuscript seems overplayed. Naturally, oxytocin signalling can be implicated in social behavior based on a large literature. However, the focus on social thermogenesis seems like a crude integration of social behavior and thermogenesis. Given that the authors see their effects in both social and nonsocial cases of thermoregulation, I am not sure the attempts at integrating social functions and thermogenic functions of PVNOT neurons are warranted. That is, unless the authors have further experiments or analysis that can convincingly justify this link.

In addition, the analysis of virgin females and lactating mothers seems out of place in Figure 4.

The c-Fos/oxytocin overlap needs to be quantified.

The methods section could be improved by explaining how the authors exclude animals that exhibit both types of huddling, if they occur within a 90-minute time window. This seems like it could cause significant confounds.

The computer vision model is not well-explained. The authors need to be far more explicit here about how it was validated.

The authors should cite and consider this preprint: https://www.biorxiv.org/content/10.1101/2024.09.17.613378v1

Reviewer #2 (Public review):

Summary:

This is a very interesting study from Vandendoren and colleagues examining the role of PVN oxytocin neurons during thermoregulatory behaviors, in particular during thermoregulatory huddling. The findings are important and compelling, and have implications for the thermoregulation field as well as the social/naturalistic behavior field.

Strengths:

The study is very creative and tackles a challenging task to examine how natural and social behavior influences neural circuits for a homeostatic system such as thermoregulation. The authors use a combination of state-of-the-art tools (photometry, optogenetics, automated behavior tracking, thermal imaging, and core body temperature measurement), often in combination with each other, to produce a rigorous and high-dimensional dataset. Carrying out tightly temperature-controlled experiments and examining natural behavior, neural activity, and body physiology simultaneously is quite a feat. I applaud the authors for taking this on in a rigorous and detailed manner. This paper will be valuable for both the thermoregulation field as well as for researchers interested in naturalistic social behaviors. The conclusions are supported by the data.

Weaknesses:

I have a number of questions and suggestions for clarification that would help improve the interpretation of the findings.

(1) Figure 1D-F: It would be helpful to include representative images of cFos expression in the PVN, LS, and DMH during both quiescent and solo huddling conditions, to better illustrate the reported differences.

(2) Figure 1C: The data suggest a general suppression of neural activity during sleep-associated quiescent huddling, which somewhat complicates the interpretation of what specifically the active huddling cells are responding to. A more informative control might have been a comparison between huddling and a more generic form of social engagement (e.g., dyadic sniffing) to assess whether huddling-responsive neurons are broadly tuned to social stimuli. While it may not be feasible to add this experimentally at this time, a brief discussion of this limitation in the main text would be valuable.

(3) Figure 2H-J vs. Figure 1: The fiber photometry data suggest increased PVN activity during quiescent huddling vs active huddling, which appears to contrast with the cFos results from Figure 1. It would be helpful for the authors to comment on possible reasons for this discrepancy-e.g., methodological differences, temporal resolution, or cell-type specificity.

(4) Figure 2O: A comparable linear regression for active huddling would be informative to assess whether the observed relationships extend across behavioral states.

(5) Temperature manipulation: The use of floor temperature changes presents a distinct physiological and sensory experience from, for example, manipulation of ambient temperature. A discussion of how this choice may affect neural circuit engagement or interpretation of thermoregulatory responses would be beneficial.

(6) Correlations with behavior: Across the manuscript, it would be informative to see correlations between huddle duration and neural activity (e.g., cFos expression, calcium signal magnitude). Similarly, do longer huddles produce greater thermogenic effects?

(7) Lactating vs. virgin mothers: The inclusion of maternal data is intriguing but feels somewhat disconnected from the central huddling-thermoregulation narrative. If these experiments are to remain, additional explanation of their rationale and how they fit into the broader story would help clarify their relevance.

(8) Optogenetic manipulation: Have the authors tested the effect of PVN OT neuron stimulation or inhibition during huddling? Even a negative result would be of interest to the field. If these data exist (main or supplementary), I apologize for missing them. If not, the authors might consider including them or commenting briefly on any attempts or challenges in carrying out these experiments.

Reviewer #3 (Public review):

Summary:

The authors aimed to elucidate the relationship between physiological state (i.e., behavioral status and thermogenic sympathetic activity) and the activity of hypothalamic paraventricular oxytocin (PVNOT) neurons in female mice. They studied this by combining automated classification of mouse behavior via video-based analysis with calcium imaging of PVNOT neuron activity. Sympathetic thermogenesis was inferred from surface temperature changes captured by infrared thermography, and the authors provided their custom analysis scripts in the manuscript. Notably, they found that a strong, pulsatile activation of PVNOT neurons was "occasionally" observed immediately before the animals transitioned from a resting to an active state. This pulsatile activity was observed in both pair-housed and individually housed animals. While PVNOT neurons are often associated with social behaviors, this finding suggests that the oxytocinergic system is also engaged during naturalistic behaviors, even in the absence of social interactions. If experiments were more convincingly performed and presented, the results would point to a broader physiological role of central oxytocin, including in the regulation of fundamental brain states and homeostatic processes, and offer a new perspective on the functional significance of central oxytocin signaling.

Strengths:

The oxytocinergic neural system is believed to subserve a wide range of physiological functions, and elucidating these roles requires monitoring PVNOT neuronal activity under various behavioral contexts, as well as manipulating this activity to establish causal links. In the present study, the authors show a technically sound experimental framework that integrates behavioral tracking in both individually and group-housed mice with the observation and manipulation of PVNOT neuron activity. This experimental setup represents a valuable methodological resource for researchers investigating the physiological functions of oxytocin.

Weaknesses:

While this study successfully established a new experimental setup for simultaneous analyses of behavior and PVNOT neuronal activity, there are several concerns regarding the interpretation of the results and the robustness of the conclusions, which should be more thoroughly addressed.

(1) The study relies on the assumption that calcium imaging and optogenetic manipulation were restricted only to PVNOT neurons. However, the specificity of AAV-mediated gene expression was not verified quantitatively. A fair number of cell bodies in the PVN expressed GCaMP8s, but not OT, indicating potential off-target expression (see Figure S2A, B). The lack of quantitative validation weakens confidence in the causal interpretation of the results.

(2) The study focuses on the transition from rest to active states following pulsatile activity of PVNOT neurons. However, the physiological significance of this pulsatile activity remains unclear. According to the authors, pulsatile activity occurred with an approximately 20% probability within 100 seconds prior to the end of the resting state. This implies that, in the remaining 80% of rest-to-active transitions, pulsatile PVNOT activity did not occur, suggesting that it is not essential for initiating the transition. A comparative analysis of behavioral and thermogenic changes between transitions with and without pulsatile PVNOT activity would help to further clarify the functional relevance of this phenomenon and strengthen the authors' interpretation of the findings.

(3) The study identifies a correlation between pulsatile activity of PVNOT neurons and rest-to-active transitions, and tests for a causal relationship using optogenetic stimulation. However, since PVNOT neurons are known to co-release other neurotransmitters such as glutamate, it remains unclear whether the observed effects are mediated specifically through oxytocin receptor signaling. To address this question, functional intervention experiments using oxytocin receptor antagonists or receptor knockout mice are necessary.

(4) The authors attempted to detect BAT thermogenesis and skin vasomotion using infrared thermography. This technique measures only skin hair temperatures (since the skin was not shaved), but does not measure "BAT temperature" or "vasomotor tone". As seen in Figure 5E, the temperatures of the body surface areas ("BAT", "Rump", and "Dorsal surface") mostly changed in parallel, indicating that these temperatures are strongly affected by body core temperature. Therefore, the thermographic measurements in this study did not provide convincing information on BAT thermogenesis or skin vasomotion. To avoid misleading reports, the authors need to use other techniques to directly measure temperatures, such as telemetry.

(5) Photostimulation of PVNOT neurons increased Tb after 400 sec (6.6 min) (Figure 5). This latency is too long to conclude that the neuronal stimulation elicited BAT thermogenesis. A more reasonable explanation is that the increase in Tb was caused by the induction of physical activity (Figure S4C), which slowly generates heat and contributes to the elevation of Tb. However, this view contradicts the authors' claim. To address this concern, the authors should directly measure BAT thermogenesis and compare it with the rate of Tb elevation. If BAT thermogenesis occurs, the rate at which the BAT temperature increases must exceed the rate at which Tb rises.

Author response:

(1) Maternal lactation assay and PVN oxytocin neuron identity

Reviewers and editors noted that the maternal lactation assay felt out of place (Editors, R1, R2) and asked for clearer validation of AAV specificity in the PVN (R3). These issues are linked: the primary purpose of the lactation assay was to physiologically validate that the recorded neurons are oxytocinergic, as PVNOT neurons exhibit well-established pulsatile activity during lactation.

In response, we will (i) explicitly frame the lactation assay as a validation experiment, (ii) streamline its presentation to sit naturally with our identity-validation rationale, and (iii) clarify our AAV targeting and expression controls; we will also address our oxytocin immunohistochemistry quantification and its limitations (we observed notable intra-individual and technical variability in oxytocin immunoreactivity), which motivated the complementary physiological approach.

(2) Clarifications and analyses.

The reviewers pointed to several analyses, inferences, and conclusions that should be clarified. We will clarify: (i) the oxytocin histology in Figure 1 (marker definitions and quantification), (ii) the roles of floor versus ambient temperature, and (iii) further elucidate some of the quantitative links among behavioral state, neural activity, and body temperature (e.g., behavior bout duration vs. neural responses and Tb), (iv) the computer vision methodology. These additions will address the reviewers’ requests for clearer inferences and presentation.

(3) Optogenetic inhibition.

We appreciate the suggestion to include an inhibition experiment (Editors, R1, R2). While interesting, this is beyond the scope of the current revision. Our stimulation experiments were designed to functionally test a specific observation from calcium imaging, namely, that PVNOT neurons show bursts of heightened activity at transitions from quiescence to arousal/thermogenesis, and to assess causal sufficiency for thermogenic/arousal-related readouts. We will make this rationale explicit, discuss the scope limits of the current dataset, and note inhibition as an important direction for future work.