Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility

Reviewer #1 (Public Review):

In this study, the authors examined the putative functions of hypothalamic groups identifiable through Foxb1 expression, namely the parvofox Foxb1 of the LHA and the PMd Foxb1, with emphasis on innate defensive responses. First, they reported that chemogenetic activation of Foxb1hypothalamic cell groups led to tachypnea. The authors tend to attribute this effect to the activation of hM3Dq expressed in the parvofox Foxb1 but did not rule out the participation of the PMd Foxb1 cell group which may as well have expressed hM3Dq, particularly considering the large volume (200 nl) of the viral construct injected. It is also noteworthy that the activation of the Foxb1hypothalamic cell groups in this experiment did not alter the gross locomotor activity, such as time spent immobile state. Thus, contrasts with the authors finding on the optogenetic activation of the Foxb1hypothalamic fibers projecting to the dorsolateral PAG. In the second experiment, the authors applied optogenetic ChR2-mediated excitation of the Foxb1+ cell bodies' axonal endings in the dlPAG leading to freezing and, in a few cases, bradycardia as well. The effective site to evoke freezing was the rostral PAGdl, and fibers positioned either ventral or caudal to this target had no response. Considering the pattern of Foxb1hypothalamic cell groups projection to the PAG, the fibers projecting to the rostral PAGdl are likely to arise from the PMd Foxb1 cell group, and not from the parvofox Foxb1 of the LHA. Here it is important to consider that optogenetic ChR2-mediated excitation of the axonal endings is likely to have activated the cell bodies originating these fibers, and one cannot ascertain whether the behavioral effects are related to the activation of the terminals in the PAGdl or the cell bodies originating the projection. Moreover, activation of PMd CCK cell group, which consists of around 90% of the PMd cells, evokes escape, and not freezing. According to the present findings, a specific population of PMd Foxb1 cells may be involved in producing freezing. In addition, only a small number of the animals with correct fiber placement presented sudden onset of bradycardia in response to the photostimulation. Considering the authors' findings, the Foxb1+ hypothalamic groups are likely to mediate behavioral responses related to innate defensive responses, where the parvofox Foxb1 of the LHA would be involved in promoting tachypnea and the PMd Foxb1group in mediating freezing and bradycardia. These findings are very interesting, and, at this point, they need to be tested in a scenario of real exposure to a natural predator.

Reviewer #2 (Public Review):

The authors aimed to examine the role of a group of neurons expressing Foxb1 in behaviors through projections to the dlPAG. Standard chemogenetic activation or inhibition and optogentic terminal activation or inhibition at local PAG were used and results suggested that, while activation led to reduced locomotion and breathing, inhibition led to a small degree of increased locomotion.

The observed effects on breathing are evident and dramatic. However, this study needs significant improvements in terms of data analysis and presentation and some of studies seem incomplete; and therefore the data may not yet support the conclusion.

1. Fig.1 has no experimental data and needs to be replaced with detailed pictures from the viral injected mice showing the projections diagrammed.

2. Fig. 3 needs control pictures and statistical comparison with different conditions in c-Fos. Also expression in other nearby regions needs to be presented to demonstrate the specificity of the expression.

3. Fig. 5, a great effort has been made to illustrate the point that CCK and Foxb1 are differentially expressed. Why not just perform a double in situ experiment to directly illustrate the point?

4. Fig. 7 data on optogenetic stimulation on immobility and breathing, since not all mice showed the same phenotype, what is the criterion for allocating these mice to hit or no hit groups? Given the dramatically reduced breathing and locomotion, what is the temperature response? More data needs to be gathered to support that this is a defense behavior.

5. The authors claim to target dlPAG. However, in the picture shown in Fig. 8C, almost all PAG contains ChR2 fibers and it is likely all the fibers will be activated by light. Thus, as presented, the data does not support the claim of the specificity on dlPAG. Also c-Fos data needs to be presented on the degree of activation of downstream PAG neurons after light exposure.

6. Fig. 9 only showed one case. A statistical comparison needs to be presented.

7. Optogentic terminal activation in the PAG will likely elicit back-propagation and subsequent activation of additional downstream brain sites of Foxb1 neurons. More experiments need to be done to assess this and as presented, the data does not support the role of PAG necessarily.

8. The authors claim negative data from PVH-Cre mice. More data need to be presented to make this case.

The conclusion, even as presented, adds to the known evidence of the PAG in the defense behavior.

Author Response:

We would like to thank the reviewers for their thorough evaluation of the presented manuscript and herewith would like to address their comments and suggestions.

This study was funded by a NSF-grant awarded to Prof. Celio. The animal experimentation license (including animal husbandry, breeding and experiments) that is required by law to perform animal experiments was also issued to Prof. Celio. Therefore, with the retirement of Prof. Celio, the funding for the project was discontinued and the animal license was terminated. We are thus unable to answer the reviewers’ open questions with follow-up experiments. We would however like to discuss some of the reviewers’ open questions or concerns and hope this might be insightful to the interested reader.

Reviewer #1 (Public Review):

“First, they reported that chemogenetic activation of Foxb1 hypothalamic cell groups led to tachypnea. The authors tend to attribute this effect to the activation of hM3Dq expressed in the parvofox Foxb1 but did not rule out the participation of the PMd Foxb1 cell group which may as well have expressed hM3Dq, particularly considering the large volume (200 nl) of the viral construct injected. It is also noteworthy that the activation of the Foxb1hypothalamic cell groups in this experiment did not alter the gross locomotor activity, such as time spent immobile state.”

Because an AAV2 serotype was used for expression of the chemogenetic tools, the spread of viral infection was much more restricted to the injection site in chemogenetic animals than was observed the AAV5-based expression of optogenetic tools. The more restricted spread of viral infection with AAV2 serotypes has previously been shown by a range of other groups (e.g. see https://doi.org/10.3389/fnana.2019.00093). This limited spread of the AAV2 serotype in our chemogenetic animals, together with the absence of the very strong locomotor phenotype observed during optogenetic stimulation experiments makes us hypothesize, that the respiratory phenotype is largely attributable to the ParvafoxFoxb1 neurons.

“In the second experiment, the authors applied optogenetic ChR2-mediated excitation of the Foxb1+ cell bodies' axonal endings in the dlPAG leading to freezing […]. Here it is important to consider that optogenetic ChR2-mediated excitation of the axonal endings is likely to have activated the cell bodies originating these fibers, and one cannot ascertain whether the behavioral effects are related to the activation of the terminals in the PAGdl or the cell bodies originating the projection.”

We did not consider the possibility of backpropagation induced by optogenetic axon terminal stimulation at the time of experiments. We acknowledge that this is the major limitation of our optogenetic experiments that would have to be investigated with further animal experiments.

Reviewer #2 (Public Review):

“3) Fig. 5, a great effort has been made to illustrate the point that CCK and Foxb1 are differentially expressed. Why not just perform a double in situ experiment to directly illustrate the point?”

We came across the publication in which the Cck-expressing PMd neurons’ control escape behaviors, only when we were drafting the manuscript. Because this was already after the retirement of Prof. Celio and we were not able to conduct further experiments involving animals, we leveraged on in silico methods and the publicly available high-quality dataset on the gene expression of the posterior hypothalamic area. The applied in silico method of dimensionality reduction and cluster assignment is well established and widely accepted. We believe in the quality of the dataset and the reliability of these in silico results but we agree with the reviewer that an alternative would have been to illustrate the expression patterns of Cck and Foxb1 by in-situ hybridisation.

“4) Fig. 7 data on optogenetic stimulation on immobility and breathing, since not all mice showed the same phenotype, what is the criterion for allocating these mice to hit or no hit groups?"

We defined the group allocation criteria in the section titled “Optogenetic modulation of Foxb1 terminal in the dlPAG induces immobility” as follows:

“OnTarget_antPAG animals had the tip of the optic fiber implant located above the dlPAG at an anterior-posterior level AP-4.04mm (from bregma) or proxymal. The OffTarget group contains animals with fiber tips located below (i.e. ventral to) the dlPAG and/or located more distal than AP -4.04mm.”