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  1. Author Response:

    Reviewer #1 (Public Review):

    Mulholland et al show that there is a very close relationship between the development of excitatory and inhibitory networks in the developing cortex. This paper makes an important contribution to our understanding of the structure of inhibition during an early stage in cortical development. The work has been carefully performed and analysed. The changes I suggest are principally to improve clarity in some places.

    Lines 48-55: express these possibilities more didactically as individual items in a list, rather than grouping the first two together.

    We thank the reviewer for this suggestion and have revised our manuscript accordingly.

    Fig 2: Show a raw image of the imaging window, with a scale bar.

    We thank the reviewer for the suggestion, and we have added a raw image of the imaging window to Figure 2.

    Line 81: clarify the type of imaging used (both wide-field and 2-photon are mentioned in the Introduction).

    The experiments in Figure 2 were performed using wide-field calcium imaging. We have now clarified this point in the main text and figure legend.

    Line 89: This is presumably in a different set of animals: make this clearer. n value?

    The reviewer is correct that the animals in Figure 3 are a different experiment from those in Figure 2. We have clarified the text, and now more clearly state the number of animals in these experiments.

    Fig 3b: "Example mean trace…"

    We have edited the figure legend to incorporate this suggestion.

    Fig 3e: Is this for one animal or averaged over all? Why not show negative correlations as well?

    Figure 3e shows the values of correlation maxima for a single representative experiment shown in panels (b-d, f). We quantified the spatial extent of correlations by measuring the amplitude of positively correlated peaks in the correlation pattern as a function of distance from the seed point, and therefore only plotted the values of these peaks in Figure 3e.

    We have revised the axis label for this panel to more clearly indicate that we are plotting the values of correlations at local maxima (New label: “Correlation at maxima”).

    Fig 3f: This appears to be the same image as S1c?

    The reviewer is correct. Our original Figure S1 (now Figure S2) aims to elaborate on the description of correlation fractures shown in Figure3f, and thus reproduces the fracture image shown previously with a more detailed explanation. We have revised the legend for Figure S2c to clearly indicate that this panel is reproduced from Figure 3f.

    Line 119-120: Suggest you explain more clearly that this is a preliminary step towards the later simultaneous E-I imaging, and why it's still useful given that it's somewhat indirect compared to the later simultaneous imaging.

    We thank the reviewer for the suggestion, and have revised the manuscript to better convey the rationale behind imaging spontaneous inhibitory and excitatory activity in separate animals.

    Line 124: Why tenth of maximum? Can you confirm that the main results are not highly sensitive to this choice?

    We selected the tenth of the maximum of the fitted two-dimensional gaussian as a way to estimate the diameter of the active pixels within an event domain. When using an alternative threshold, such as the full-width at half maximum, the values for inhibitory and excitatory domain size scale together, indicating that the relationship between the relative size of inhibitory and excitatory active domains is not sensitive to this threshold. However, we found that full-width half max underestimated the diameter of the active region, leaving out active pixels. We have now included the values for full-width half max in the text of the results, to show that the threshold does not impact the relationship between inhibitory and excitatory domain size.

    Fig 4: It would be clearer to give the graph parts of a and d their own panel labels. Is it worth explicitly flagging that none of the E-I distributions are significantly different? It would be useful to explicitly define pink and blue in the caption. Does panel e have units? In panel a I'm confused about what is being referred to as "circles" and what as "lines". Panel f caption: "principal components"

    We have incorporated these suggestions into a revised figure. We have included bars to indicate non-significance of E-I distributions, updated panel e and f figure axes, and edited the figure legend to provide more explicitly define inhibitory and excitatory data color scheme.

    Line 143: suggest "similarity in the statistical structure", to make it clearer what you mean by "structure".

    We have revised the text to explicitly refer to “similarity in the spatial structure” in this paragraph.

    Line 159: state the n value here.

    We have updated the text to include the number of animals used in this particular experiment.

    Fig 5: It would be interesting to speculate about the significance of the correlation fractures. In the rhs of panel b the red seed points are almost impossible to see, since they occur on top of a red patch (perhaps include a black or white border to these circles?). a caption: "-2-2 z-score" is confusing; perhaps say "-2 to +2 z-score". g caption: Is this correlation similarity for E, I or both?

    Fractures show areas in the network where there are sharp discontinuities in the spatial patterns of the correlations. As discussed in the results, mature functional maps in the visual cortex also exhibit smooth variation punctuated by abrupt discontinuities, such as pinwheels and fractures in orientation preference maps (Bonhoeffer and Grinvald, 1991), or direction reversals in direction preference maps (Okai et al., 2005). Previous work demonstrated that correlation fractures derived from spontaneous activity after eye opening precisely coincided with the highrate-of-change regions in the orientation preference map (Smith et al., 2018). Therefore, the correlation fractures observed from spontaneous inhibitory activity prior to eye opening reveal that the underlying network is already precisely organized, and while the fractures are refined over development (Smith et al 2018), they may provide insight into the future structure of the mature orientation preference map, although this remains to be directly tested.

    We thank the reviewer for the suggestions to improve the clarity of this figure. We have changed the color of the seed points, to make them easier to see, and have adjusted the figure legends for Panel a. In Panel g, the correlation similarity is comparing E vs I networks as a function of distance, and the figure legend has been updated to more explicitly state this.

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  2. Evaluation Summary:

    Mulholland et al show that there is a very close relationship between the development of excitatory and inhibitory networks in the developing cortex. This paper makes an important contribution to our understanding of the structure of inhibition during an early stage in cortical development. It is therefore of great interest to scientists interested in development, and in computation in cortical circuits. The work has been carefully performed and analysed.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Mulholland et al show that there is a very close relationship between the development of excitatory and inhibitory networks in the developing cortex. This paper makes an important contribution to our understanding of the structure of inhibition during an early stage in cortical development. The work has been carefully performed and analysed. The changes I suggest are principally to improve clarity in some places.

    Lines 48-55: express these possibilities more didactically as individual items in a list, rather than grouping the first two together.

    Fig 2: Show a raw image of the imaging window, with a scale bar.

    Line 81: clarify the type of imaging used (both wide-field and 2-photon are mentioned in the Introduction).

    Line 89: This is presumably in a different set of animals: make this clearer. n value?

    Fig 3b: "Example mean trace..."

    Fig 3e: Is this for one animal or averaged over all? Why not show negative correlations as well?

    Fig 3f: This appears to be the same image as S1c?

    Line 119-120: Suggest you explain more clearly that this is a preliminary step towards the later simultaneous E-I imaging, and why it's still useful given that it's somewhat indirect compared to the later simultaneous imaging.

    Line 124: Why tenth of maximum? Can you confirm that the main results are not highly sensitive to this choice?

    Fig 4: It would be clearer to give the graph parts of a and d their own panel labels. Is it worth explicitly flagging that none of the E-I distributions are significantly different? It would be useful to explicitly define pink and blue in the caption. Does panel e have units? In panel a I'm confused about what is being referred to as "circles" and what as "lines". Panel f caption: "principal components"

    Line 143: suggest "similarity in the statistical structure", to make it clearer what you mean by "structure".

    Line 159: state the n value here.

    Fig 5: It would be interesting to speculate about the significance of the correlation fractures. In the rhs of panel b the red seed points are almost impossible to see, since they occur on top of a red patch (perhaps include a black or white border to these circles?). a caption: "-2-2 z-score" is confusing; perhaps say "-2 to +2 z-score". g caption: Is this correlation similarity for E, I or both?

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  4. Reviewer #2 (Public Review):

    The paper by Mullholland and colleagues investigates the spatial structure of spontaneous events in inhibitory neurons in ferret primary visual cortex before eye opening. This work is a logical and important extension of previous studies by the senior author which investigated the same issue from the perspective of excitatory neurons. Two major findings result from this work: First, spontaneous events are tightly coupled in inhibitory and excitatory networks. Neurons of both kinds participate in the same event, and the strength of inhibitory and excitatory activation in each event is well matched, presumably to maintain a somewhat constant overall activation level. Second, the spontaneous activity reveals spatial structure in excitatory and inhibitory networks already at this relatively early developmental stage, with very similar spatial scales for both kinds of networks. Both findings are well supported by careful experimental work and matching quantitative analyses. At the same time, the conclusion that spatial scales for inhibitory and excitatory networks match could be further strengthened. A particular concern in this respect are the potentially quite different firing rates of inhibitory and excitatory neurons. In combination with the non-linearities of calcium imaging, they introduce potential confounds when comparing measurements between inhibitory and excitatory networks. The two calcium sensors used for some of the work similarly might introduce systematic differences between the 2 types of cells. Finally, the paper could be strengthened by a more thorough analysis confirming that differences in calcium indicator expression levels are not limiting the detectable spatial scale of spontaneous events.

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  5. Reviewer #3 (Public Review):

    The manuscript provides key information about the state of cortical networks at around the time that feed-forward input can begin to provide external drive. The data show convincingly that early activity in inhibitory networks exhibits a modular structure that overlaps with the same modular activity of immediately neighboring excitatory neurons. This early architecture contrasts strongly with the non-specific pooling that has been shown in rodent visual cortex. The paper also contains a small related gem: evidence that GABAergic signaling has a net inhibitory effect on cortical activity at P21 in the ferret, showing that the transition from GABAergic signaling from depolarizing to hyperpolarizing has already occurred during this period. The paper borrows analysis techniques from a prior publication that examined excitatory activity (Smith et al. 2018), so the methods and techniques are already proven. The paper is highly polished.

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