Is there a reason you also wouldn't look at an astrocyte-specific Cx-43 knockout? In brain, this is the major connexin that forms gap junctions across the astrocyte syncytium. I'm also wondering whether it would be informative to do the immunostaining above (Fig 4) for Cx-43 since good antibodies exist.

Thanks for the interesting study about a naturally derived molecule and potential for addressing an unmet clinical need. For this experiment implicating inhibition of glial activation in after boldine treatment, I think the results would be more robust with the addition of 1) immuno-staining for control glial or other histological markers that would not be expected to change with differential levels of glial activation, and 2) quantification of the fluorescence across conditions for the markers shown here (and those controls) for all animals in each group, to allow the readers to see the variance in the data across animals.

How do these metrics (and stability) differ between the hippocampus and cortex recordings?

How would you predict that pharmacological or genetic manipulation of gap junctions affect the connectivity networks? Have you tested this experimentally?

Regarding co-activation on short (second-long) timescales, I'm wondering how you square this with our recent work (https://doi.org/10.1038/s41586-024-07311-5) showing that subcellular activity in astrocytes percolates through the gap junctionally coupled network on the order of minutes, and other astrocyte research showing much longer timescales of activity.

What layers of cortex were you recording from, and how might you expect layers to influence these area-level differences you describe?

Note typo

For this analysis, it seems that all the calcium activity in an individual astrocyte is grouped together to generate the time-series data. If this is the case, how is the subcellular activity that characterizes these astrocytes accounted for, and how might that affect the findings here?

Very cool section! A couple of questions: 1. Would you expect that astrocyte calcium activity in deeper layers in mPFC—either in somata or microdomains—would be similar to deep layers in primary sensory areas, which I believe you cite in your comparisons. If they are indeed similar in the features you describe here, does that give specific hints about functions of astrocyte across cortical areas with different architecture.

1. There are several indications recently that rather than number/amplitude/duration of astrocyte calcium events being the most salient, their propagation features can distinguish differential astrocyte activity. Have you looked at that for this comparison? I might temper the last sentence of this section more without a propagative analysis included.

This is such an exciting overall result. I'm wondering if you've tested/identified any other bioactive compounds from the same material in addition to HC-HA/PTX3, and/or whether you think there may be other significant contributors to pain inhibition from human birth tissues.

Even though CD44 is expressed at lower levels in satellite glia than DRGs (hard to tell magnitude without supplemental figures), do you expect that CD44-dependent mechanisms you describe below in DRGs, like cytoskeletal rearrangement) are also occurring in these glial cells and are contributing to the in vivo effects?

It would be helpful to readers to clarify whether individual scratches were quantified by observers or detected automatically, i.e. more experimental details, please!

Clarify in figure that n = mouse number?

I think some zoomed in images from Fig 3H showing the apposed puncta would really help convince readers that these are indeed closely apposed. In addition, adding a quantifiable metric for distance between the puncta would be very helpful.

Likely outside the scope of the current study, but it would be interesting to see whether any aspect of this described circuit is specifically dependent on chloroquine-induced itch, or whether administration of other pruritogens would have differing outcomes.

Do you have DCZ-only and saline controls in animals with an AAV-mCherry control virus?

I'd be interested to see a quantification of this overlap of both populations.

Very exciting finding!

Minor point, but I believe this comma should be removed for meaning. Otherwise, the main findings are differently stated than what the data show.

Congrats on the fascinating paper on an organism without a conventional nervous system. I'm interested in the functional analogies you propose to astrocytes (vs neurons) based on the described ATP and glutamate dynamics you describe. Do you think that a comparative approach in terms of other features of nervous system cells—expression profiles, cell morphological features and connectivity (i.e. gap junctions)—would be useful for distinguishing and understanding the function of these sponge cells? Or help us think about how nervous system cells like neurons and astrocytes became more specialized in animals with conventional nervous systems?

It looks like other waves features were correlated between neurons and astrocytes beyond amplitude, including duration. Was that indeed the case? May be other features to explore below as you look at correlations across brain regions.

This is a great way to visualize the waves. I'm wondering how much gap junctions contribute mechanistically to wave propagation within the developing astrocyte networks.

Congrats on a beautiful, technologically impressive piece of work!

This finding, and the one described in the results showing that a single microglia could form 17 plugs made me wonder whether there was anything specific to the microglial morphology itself that could be used to predict these outliers. Would an unbiased analysis of all 3D reconstructed microglia cluster those that formed particularly large or numerous plugs together? Might these cells have a specific morphology? The potentially transient nature of these plugs/contacts might preclude interesting or interpretable findings, but it could lead you to subgroups of microglia to pay attention to.

This question of differences in morphology depending on the type of fixation was lingering as I read the paper, so I'm happy you addressed it directly here. I wonder whether there are any publicly available cryo-fixed datasets (either the original eLife study that you cite or others) where microglia could be ID'ed, to compare with the results in this study.

I'm a fan of using publicly available datasets to explore new science. Kudos on this interesting project. One thing I was wondering as I read the paper was how easy it was for you to use and navigate this dataset, since that can be a significant hurdle depending on the data type. Any comment on challenges, or what was made easy, by the organization of this dataset for your purposes could be really useful for others looking to analyze it.

Congrats on this very cool (lol) study and unexpected result! When reading the Therapeutic Implications section of the discussion, I wondered whether any of the side effects of topical RAP application for current patients could be clues to its future therapeutic potential, especially with regards to changes (in either direction) in pain, somatosensation, and pruritus.

This is a very exciting study overall, and I'm really interested in the mechanistic interaction between the TRESK channel and MrgprA3+ prurireceptors. For the cheek itch assay, it would be great to know whether scratching bouts were counted manually from videos, or whether you used another technique. Thanks!

This summary is really interesting, and I'd love in the discussion for you to speculate on the cellular and circuit function of correlated activity between these two populations of cells? How does this shed light on how astrocytes and neurons are affecting each other? What extracellular signals do these activity patterns make you think might be driving them?

Minor comment: it would be helpful to the reader to have panel titles for Astrocytes and Neurons in C and D, as well as in subsequent figures.

I may be misunderstanding the panels in 2K–L, but could you explain how the Neutral measure of %dF/F in 2L is around 2, while in K it looks much lower than the shock or the eYFP peaks?

Do you think you're getting similar expression levels (% of total cell population) of neurons and astrocytes with the calcium indicators based on histology? Do you think differences here would affect your findings?

For pain vs itch specifically, it would be interesting to hear how your predictions about cross-modality placebo effects were adjusted based on the results of the current study.

How do you think the findings of this paper would be similar or different if the placebo were a different modality than a cream, say something injected, or would that not have been possible because of the study design?

This is a really interesting paper, and as someone outside of the field, I appreciate the clear and compelling background section.

I agree that this interpretation makes sense, but the test with TTX might also mean that non-neuronal cells are playing a role in maintaining oscillatory activity, as they can do in the CNS. The pharmacological inhibition of intracellular calcium you use would also block these widely-described mechanisms in astrocytes, for example.

Did you try application of GRP for shorter than 3 minutes, and if so, did you see less of a persistent response?

Similar to the comment above, would you expect to see similar results with a pruritogen other than 48/40, in particular one that does not involve histaminergic itch?

Could you use the amount of activity in each cell type, i.e. a few GCaMP spikes vs ongoing oscillations, to enrich this analysis beyond a binary response vs non-response? For example, are more active cells to one peptide more or less likely to be activated by other peptides?

Minor note that "theoretical" is misspelled in panels D and E

What a cool finding and nice control!

I see the n's in the legend for Fig. 2, but for clarity, I would recommend putting the n's in the manuscript text for mouse and slice number.

Overall, this paper consists of an elegantly structured set of experiments which are technically challenging and really timely. Thank you for the exciting work! One question here: is it possible here to use a blocker of synaptic transmission (but not spiking) to isolate direct vs indirect responses? Similarly, would widespread activation of neurons via high K+ and sucrose allow you to see what proportion of cells are activatable at all in your prep, to better estimate the denominator of these percentages that you present?

This figure would be helped by labeling the species, and using scale bars to show that they aren't to scale.

Exciting! And a premise many of us that work in mouse models are depending on. I also wonder whether different astrocytic subtypes that have been described as primate-specific may be differentiated in your future assays.

As you're analyzing several glial subtypes together here, this finding brings up several questions, including whether these relationships are driven by a particular glial cell type and whether/how different glial cell type number are determined developmentally relative to each other.

typo alert!

Another small note for several figures is that the resolution is low and sometimes it is difficult to read labels (here, especially the species names).

A small comment on the figure design here is that using the same colors for type of stain (d) and then layer but not stain (e), is a bit confusing.

I find the language a little confusing here, especially as it differs between the intro and the results/discussion. Here, I think you're using "circuit" to mean the same thing as brain region, but elsewhere, you use "region". I think consistency would help with clarity.

In several mouse brain regions, my experience with patching astrocytes and neurons in ex vivo brain slices (as well as DIC images in ex vivo slices from many other groups) shows that astrocytes have smaller cell bodies than neurons, and in fact, can be identified solely based on their smaller somata. I bet that the Nissl staining here likely reflects real neuron-astrocyte size differences, as observed in unfixed tissue.

It's very cool that you were able to obtain tissue from and analyze so many mammalian species, including humans. If you could speculate, do you think you'd find that the same principles you uncovered also hold for non-mammalian vertebrates?

In ex vivo (acute slice) astrocyte calcium imaging work, several groups have reported astrocyte calcium events that propagate both toward and away from the soma. How do you square that with these findings? Do you think that those may be smaller events that are less detectable in vivo, with more light-scattering, etc? How do you think they may be involved in the integration described here?

I'm curious to know whether "propagation" and "integration" are interchangeable here, or differently defined.

I've interpreted the word "global" to mean "within the recorded astrocytes" in the paper. But here, where you cite a review that includes astrocyte calcium dynamics from other brain regions that differ in some cases, and potentially other definitions of the word "global", it feels slightly misleading.

I think there may be accidental problems with this sentence as it transitions from page 4–5, because I don't understand the meaning.

This is exciting because it's very similar to our finding of high correlation between pupil diameter and astrocyte calcium in cortex (Reitman et al, 2023). I'm also interested in whether you think there are differences in this relationship in hippocampus vs cortex.

I'm interested in how the ROIs don't seem to encompass the outer regions of the astrocyte branches, and thus don't tile the field-of-view. Is that because the fluorescence isn't detectable in these regions?

One question I had throughout the paper is whether you observed changes in microglia motility in your experiments, both because of any biological significance of motility changes, as well as because of how motility could have affected your calcium dynamics results. I'd love to hear more!

What a dramatic response of extracellular NE! Do you know if CNO causes this kind of response in animals that don't express the DREADD?

It's so cool that you saw in vivo calcium changes in the microglia processes with this manipulation. I can see from your analyses that there were significant changes in the processes compared to the soma, but I'm wondering whether you observed changes in the processes somewhat evenly distributed across cells, or whether there were particular microglia that had active processes while other cells did not. I know I might be able to answer this question if I could see the videos, but it could be an interesting descriptive analysis to show in addition to what's already in the figure. In addition, whether there's a change in other features of the calcium signal, like propagation, would also be interesting, and might necessitate an non-ROI-based analysis approach.

I'm curious whether delta power during NREM sleep changed in addition to duration and % time. Especially since you later show that Gi and Gq activation in microglia have similar effects on NREM sleep, it might be a differentiating feature. For Gi vs Gq activation in astrocytes, we previously observed that Gi affected sleep depth while Gq changed sleep duration.

I think this is an important point to make, and I'm glad you did! You may want to include some citations here, especially because it may not be widely known that astrocytes can express high levels of adrenergic receptors, and that subtypes of astrocytic adrenergic receptors can be activated by dopamine, as well as norepinephrine, in the prefrontal cortex.

I'd be curious to see how the Gi and the Gq activation differ in their activation of calcium, for example, to be able to compare any summary statistics here to those presented in Fig. 2g–k.

It's very exciting to see the transgenic mouse available, and to compare activity between NE and neurons/astrocytes. A question about crossing to the GFAP-Cre line: Did you confirm that expression was limited to astrocytes, or is it a mixed cell-type population? In others' hands, GFAP-Cre has lead to expression in both astrocytes and neurons, and the inducible Ald1h1-CreER line may be a cleaner option.

This integration of ASTRA to determine morphological features and AQuA to quantify event-based fluorescence features seems particularly useful and exciting. It would be really interesting to hear more about cases you've observed where this does and doesn't work well, and your hypotheses for why; this could help others in understanding the best use-cases for analytical tool integration.

My reading of this is that excitatory neuronal activity in these circuits decreases when astrocytic Gabbr1 is knocked out of astrocytes, but inhibitory activity seems to be less affected. Do you think this points to a homeostatic mechanism by which astrocytes may help regulate excitatory/inhibitory balance in developing cortical circuits? It would be interesting to see if this sort of effect would also occur in adult animals, after circuit maturation. Another interesting follow-up may be to think about how much this sort of homeostatic mechanism is driven by physiology or morphology of the astrocytes.

It's very exciting that you found a mechanism downstream of Gabbr1 that has known cytoskeletal function. Maybe I missed this, but do you have an idea about how activation of Gabbr1 might lead to Ednrb upregulation? (Or why Gabbr1 KO leads to down-regulation?)

Did you observe any changes in intracellular calcium besides frequency or delta F? In some of our experiments, we find that most salient changes in calcium are related to the propagation or area of the calcium signal. The negative result in calcium here implies that the spontaneous calcium activity in developing cortical astrocytes does not arise from spontaneous inhibitory (GABAergic) signal, which is interesting in light of previous studies. Do you think there might be a developmental timecourse over which astrocytes become responsive to spontaneous release of GABA?

Exciting stuff! In this experiment, since the manipulation is neuronal, it's very cool that you see astrocyte-specific morphological effects. I wonder if there also may be neuronal morphology changes at the subcellular level that could precede or accompany the astrocytic changes.