Clustered synapses develop in distinct dendritic domains in visual cortex before eye opening

Reviewer #1 (Public Review):

Summary:
Using concurrent in vivo whole-cell patch clamp and dendritic calcium imaging, the authors characterized how functional synaptic inputs across dendritic arborizations of mouse primary visual cortex layer 2/3 neurons emerge during the second postnatal week. They were able to identify spatially and functionally separated domains of clustered synapses in these neurons even before eye-opening and characterize how the clustering changes from P8 to P13.

Strengths:
The work is technically challenging and the findings are novel. The results support previous EM and immunostaining studies but provide in vivo evidence on the time course and the trajectory of how functional synaptic input develops.

Weaknesses:
There are some missing details about how the experiments were performed, and I also have some questions about the analyses.

Reviewer #2 (Public Review):

In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that has led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite the full control of membrane voltage in-vivo not being realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enables measurements of the best-level precision.

On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

1. The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of the dendrite is a spine on top of (or below) it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability of being just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.

2. The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. Not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggests that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.

3. Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already shows that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.

4. The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.

Reviewer #3 (Public Review):

Summary:
There is a growing body of literature on the clustering of co-active synapses in adult mice, which has important implications for understanding dendritic integration and sensory processing more broadly. However, it has been unclear when this spatial organization of co-active synapses arises during development. In this manuscript, Leighton et al. investigate the emergence of spatially organized, co-active synapses on pyramidal dendrites in the mouse visual cortex before eye-opening. They find that some dendrite segments contain highly active synapses that are co-active with their neighbors as early as postnatal day (P) 8-10, and that these domains of co-active synapses increase their coverage of the dendritic arbor by P12-13. Interestingly, Leighton et al. demonstrate that synapses co-active with their neighbors are more likely to increase their activity across a single recording session, compared to synapses that are not co-active with their neighbors, suggesting local plasticity driven by coincident activity before eye-opening.

The current manuscript includes some replication of earlier results from the same research group (Winnubst et al., 2015), including the presence of clustered, co-active synapses in the visual cortex of mouse pups, and the finding that synapses co-active with their neighbors show an increase in transmission frequency during a recording session. The main novelty in the current study compared to Winnubst et al. (2015) is the inclusion of younger animals (P8-13 in the current study compared to P10-15 in Winnubst et al., 2015). The current manuscript is the first demonstration that active synapses are clustered on specific dendrite segments as early as P8-10 in the mouse visual cortex, and the first to show the progression in active synapse distribution along the dendrite during the 2nd postnatal week. These results from the visual cortex may help inform our understanding of sensory development more broadly.

Strengths:
The authors ask a novel question about the emergence of synaptic spatial organization, and they use well-chosen techniques that directly address their questions despite the challenging nature of these techniques. To capture both structural and functional information from dendrites simultaneously, the authors performed a whole-cell voltage clamp to record synaptic currents arriving at the soma while imaging calcium influx at individual synaptic sites on dendrites. The simultaneous voltage clamp and calcium imaging allowed the authors to isolate individual synaptic inputs without their occlusion by widespread calcium influx from back-propagating action potentials. Achieving in vivo dendrite imaging in live mice that are as young as P8 is challenging, and the resulting data provides a unique view of synaptic activity along individual dendrites in the visual cortex at an early stage in development that is otherwise difficult to assess.

The authors provide convincing evidence that synapses are more likely to be co-active with their neighbors compared to synapses located farther away (Fig. 6F-H), and that synapses co-active with their neighbors increase their transmission frequency during a recording session (Figure 7C). These findings are particularly interesting given that the recordings occur before eye-opening, suggesting a relationship between co-activity and local synaptic plasticity even before the onset of detailed visual input. These results replicate previously published findings from P10-15 pups (Winnubst et al., 2015), increasing confidence in the reproducibility of the data.

The authors also provide novel data documenting for the first time spatially organized, co-active synapses in pups as young as P8. Comparing the younger (P8-10) and older (P12-13) pups, provides insight into how clusters of co-active synapses might emerge during development.

Weaknesses:
This manuscript provides insufficient detail for assessing the rigor and reproducibility of the methods, particularly for age comparisons. The P8-10 vs P12-13 age comparisons are the primary novel finding in this manuscript, and it is, therefore, critical to avoid systematic age differences in the methods and analysis whenever possible. Specific concerns related to the age comparisons are listed below:

• Given that the same research group previously published P12-13 data (Winnubst et al., 2015), it is unclear whether both age groups in the current study were imaged/analyzed in parallel by the same researcher(s), or whether previous data was used for the P12-13 group.

• The authors mention that they used 2 different microscopes, and used a fairly wide range of imaging frame rates (5-15 Hz). It is unclear from the current manuscript whether the same imaging parameters were used across the two age groups. If data for the two experimental groups was collected separately, perhaps at different times, by a different person, or on a different microscope, there is a concern that some differences between the groups may not necessarily be due to age.

• It is unclear whether the image analysis was performed blind to age. Blinding to age during analysis is particularly important for this study, in which it was not possible to blind to age during imaging due to visible differences in size and developmental stage between younger and older pups.

• The relatively low N (where N is the number of dendrites or the number of mice) in this study is acceptable due to the challenging nature of the techniques used, but unintentional sampling bias is a concern. For example, if higher-order dendrites from the apical tuft were imaged at P12-13, while more segments of the apical trunk were imaged at P8-10, this could inadvertently create apparent age differences that were in fact due to dendrite location on the arbor or dendrite depth.

Additional general methodological concerns, which are not specifically related to the age comparisons, are listed below:

• The authors assert that clustered, co-active synapses emerge in the visual cortex before eye-opening, which is an important finding in that it suggests this phenomenon is driven by spontaneous activity rather than visual input. However, this finding hinges on the imaged cells being reliably located in the visual cortex, which is difficult to identify with certainty in animals that have not yet opened their eyes and therefore cannot undergo intrinsic signal imaging to demarcate the boundaries of the visual cortex. If the imaged cells were in, for example, nearby somatosensory cortex, then the observed spatial organization could be due to sensory input rather than spontaneous activity.

• It is unclear how the authors defined a synaptic transmission event in the GCaMP signal (e.g. whether there was a quantitative deltaF/F threshold).

• The authors' division of synapses into spine vs shaft is unconvincing due to the difficulty of identifying Z-projecting spines in images from 2-photon microscopy, where the Z resolution is insufficient to definitively identify Z-projecting spines, and the fact that spines in young animals may be thin and dim. The authors' examples of spine synapses (e.g. in Fig. 2A) are convincing, but some of the putative shaft synapses may in fact be on spines.