Eye-specific synaptic clustering through activity-dependent stabilization and punishment mechanisms in the developing visual system

Reviewer #1 (Public Review):

Summary:
This publication applies 3D super-resolution STORM imaging to understanding the role of developmental neural activity in the clustering of retinal inputs to the mouse dorsal lateral geniculate nucleus (dLGN). The authors argue that retinal ganglion cell (RGC) synaptic boutons start forming clusters early in postnatal development (P2). They then argue that these clusters contribute to eye-specific segregation of retinal inputs by activity-dependent stabilization of nearby boutons from the same eye. The data provided is N=3 animals for each condition of P2, P4, and P8 animals in wild-type mice and in mice where early patterns of structured retinal activity are blocked.

Strengths:
The 3D storm imaging of pre and postsynaptic elements provides convincing high-resolution localization of synapses.

The experimental design of comparing ipsilateral and contralateral RGC axon boutons in a region of the dLGN that is known to become contralateral is elegant. The design makes it possible to relate fixed time point structural data to a known outcome of activity-dependent remodeling.

Weaknesses:
Based on previous literature, it is known that synapse density, synapse clustering, and synaptic specificity increase during postnatal development. Previous work has also shown that both the changes in synaptic clustering and synaptic specificity are affected by retinal activity. The data and analysis provided by the authors add little unambiguous evidence that advances this understanding.

General problem 1: Most of the statistical analysis is limited to ANOVA comparison of axons from the contralateral and ipsilateral retina in the contralateral dLGN. The hypothesis that ipsilateral and contralateral axons would be statistically identical in the contralateral dLGN is not a plausible hypothesis so rejecting the hypothesis with P < X does not advance the authors' arguments beyond what was already known.

General problem 2: Most of the interpretation of data is qualitative. While error bars are provided, these error bars are not used to draw conclusions. Given the small sample size (N=3), there is a large degree of uncertainty regarding the magnitude of changes (synapse size, number, specificity). The authors base their conclusions on the averages of these values when the likely degree of uncertainty could allow for the opposite interpretation.

General problem 3: Two of the four results sections depend on using the frequency of single active zone vGlut2 clusters near multiple active zone vGlut2 as a proxy for synaptic stabilization of the single active zone vGlut2 clusters by the multiple active zone vGlut2 clusters. The authors argue that the increased frequency of same-eye single active zone clusters relative to opposite-eye single active zone clusters means that multiple active zone vGlut2 clusters are selectively stabilizing single active zone clusters. There are other plausible explanations for this observation that are not eliminated. An increased frequency of nearby single active zone clusters would also occur if RGC axons form more than one synapse in the dLGN. Eye-specific segregation is, by definition, a relative increase in the frequency of nearby boutons from the same eye. The authors were, therefore, guaranteed to observe a non-random relationship between boutons from the same eye. The authors do compare their measures to a random model, but I could not find a description of the model. I would expect that the model would need to account for RGC arbor size, arbor structure, bouton number, and segregation independent of multi-active-zone vGlut2 clusters. The most common randomization for the type of analysis described here, a shift in the positions of single-active zone boutons, would not be adequate.

In discussing the claimed cluster-induced stabilization of nearby boutons, the authors state that the specificity increases with age due to activity-dependent refinement. Their quantification does not support an increase in specificity with age. In fact, the high degree of clustering "specificity" they observe at P2 argues for the trivial same axon explanation.

Analysis of specific claims:

Result Section 1

Most of the figures show mean, error bars, and asterisks, but not the three data points from which these statistics are derived. Large changes in variance from condition to condition suggest that displaying the data points would provide more useful information.

Claim 1: Contralateral density increases more than ipsilateral in the contralateral region over the course of development. This claim is supported by the qualitative comparison of means and error bars in Figure 2D. The argument could be made quantitative by providing a confidence interval for synapse density increase for dominant and non-dominant synapse density. A confidence interval could then be generated for the difference in this change between the two groups. Currently, the most striking effect is a big difference in variance between P4 and P8 for dominant eye complex synapses. Given that N=3, I assume there is one extreme outlier here.

Claim 2: The fraction of multiple-active zone vGlut2 clusters increases with age. This claim is weakly supported by a qualitative reading of panel 1E. The error bars overlap so it is difficult to know what the range of possible increases could be. In the text, the authors report mean differences without confidence intervals (or any other statistics). The reported results should, therefore, be interpreted as a description of their three mice and not as evidence about mice in general.

Figure S1. Panel A makes the point that the study could not be done without STORM by comparing the STORM images to "Conventional" images. The images are over-saturated low-resolution images. A reasonable comparison would be to a high-quality quality confocal image acquired with a high NA objective (~1.4) and low laser power (PSF ~ 0.2 x 0.2 x 0.6 um) that was acquired over the same amount of time it takes to acquire a STORM volume.

Result section 2.

Claim 1: The ipsi/contra (in contra LGN) difference in VGluT2 cluster volume increases with development. While there are many p-values listed, the main point is not directly quantified. A reasonable way to quantify the relative increase in volume could be in the form: the non-dominant volumes were 75%-95%(?) of the dominant volume at P2 and 60%-80% (?) at P8. The difference in change was -5 to 15%(?).

Claim 2: Complex synapses (vGlut2 clusters with multiple active zones) represent clusters of simple synapses and not single large boutons with multiple active zones. The authors argue that because vGlut2 cluster volume scales roughly linearly with active zone number, the vGlut2 clusters are composed of multiple boutons each containing a single active zone. Their analysis does not rule out the (known to be true) possibility that RGC bouton sizes are much larger in boutons with multiple active zones. The correlation of volume and active zone number, by itself, does not resolve the issue. A good argument for multiple boutons might be that the variance is smallest in clusters with 4 active zones (looks like it in the plot) since they would be the average of four active zones to vesicle pool ratios. It is very likely that the multi-active zone vGlut2 clusters represent some clustering and some multi-synaptic boutons. The reference cited by the authors as evidence for the presence of single active zone boutons in young tissue does not rule out the existence of multiple active zone boutons.

Several arguments are made that depend on the interpretation of "not statistically significant" (n.s.) meaning that "two groups are the same" instead of "we don't know if they are different". This interpretation is incorrect and materially impacts the conclusions.

Several arguments are made that interpret statistical significance for one group and a lack of statistical significance for another group meaning that the effect was bigger in the first group. This interpretation is incorrect and materially impacts the conclusions.

Result Section 3.

Claim 1: Complex synapses stabilize simple synapses. There are alternative explanations (mentioned above) for the observed clustering that negate the conclusions. 1) Boutons from the same axon tend to be found near one another. 2) Any form of eye-specific segregation would produce non-random associations in the analysis as performed. The authors compare each observation to a random model, but I cannot determine from the text if the model adequately accounts for alternative explanations.

The authors claim that specificity increases over time. Figure 3b (middle) shows that the number of synapses near complex synapses might increase with time (needs confidence interval for effect size), but does not show that specificity (original relative to randomized) increases with time. The fact that nearby simple synapse density is always (P2) very different from random suggests a primarily non-activity-dependent explanation. The simplest explanation is that same-side boutons could be from the same axon whereas different-side axons could not be.

Claim 2: vGlut2 clusters more than 1.5 um away from multi-active zone vGlut2 clusters are not statistically significantly different in size than vGlut2 clusters within 1.5 um of multi-active zone vGlut2 clusters. Therefore "activity-dependent synapse stabilization mechanisms do not impact simple synapse vesicle pool size". The specific measure of 1.5 um from multi-active zone vGlut2 clusters does not represent all possible synapse stabilization mechanisms.

Result Section 4.

Claim: The proximity of complex synapses with nearby simple synapses to other complex synapses with nearby simple synapses from the same eye is used to argue that activity is responsible for all this clustering.

It is difficult to derive anything from the quantification besides 'not-random'. That is a problem because we already know that axons from the left and right eye segregate during the period being studied. All the measures in Section 4 are influenced by eye-specific segregation. Given this known bias, demonstrating a non-random relationship (P
The results can be stated as: If you are a contralateral complex synapse, contralateral complex synapses that are also close to contralateral simple synapses will, on average, be slightly closer to you than contralateral complex synapses that are not close to contralateral ipsilateral synapses. That would be true if there is any eye-specific segregation (which there is).

It is an overinterpretation of the data to claim that the lack of a clear correlation between vGlut2 cluster volume and distance to vGlut2 clusters with multiple active zones provides support for the claim that "presynaptic protein organization is not influenced by mechanisms governing synaptic clustering".

Reviewer #2 (Public Review):

Summary:
In this manuscript, Zhang and Speer examine changes in the spatial organization of synaptic proteins during eye-specific segregation, a developmental period when axons from the two eyes initially mingle and gradually segregate into eye-specific regions of the dorsal lateral geniculate. The authors use STORM microscopy and immunostain presynaptic (VGluT2, Bassoon) and postsynaptic (Homer) proteins to identify synaptic release sites. Activity-dependent changes in this spatial organization are identified by comparing the β2KO mice to WT mice. They describe two types of presynaptic organization based on Bassoon clustering, the complex and the simple synapse. By analyzing the relative densities and distances between these proteins over age, the authors conclude that the complex synapses promote the clustering of simple synapses nearby to form the future mature glomerular synaptic structure.

Strengths:
The data presented is of good quality and provides an unprecedented view at high resolution of the presynaptic components of the retinogeniculate synapse during active developmental remodeling. This approach offers an advance to the previous mouse EM studies of this synapse because of the CTB label allows identification of the eye from which the presynaptic terminal arises. Using this approach, the authors find that simple synapses cluster close to complex synapses over age, that complex synapse density increases with age.

Weaknesses:
From these data, the authors conclude that the complex synapse serves to "promote clustering of like-eye synapses and prohibit synapse clustering from the opposite eye". However, the authors show no causal data to support these ideas. There are a number of issues that the authors should consider:

1. Clustering of retinal synapses is in part due to the fact that retinal inputs synapse on the proximal dendrites. With increased synaptogenesis, there will be increased density of retinal terminals that are closely localized. And with development, perhaps simple synapses mature into complex synapses. Simple synapses may also represent ones that are in the process of being eliminated as previously described by Campbell and Shatz, JNeurosci 1992 (consider citing). Can the authors distinguish these scenarios from the ones that they conclude?

2. The argument that "complex" synapses are the aggregate of "simple" synapses (Fig 2, S2) is not convincing.

3. The authors use of the β2KO mice to assess changes in the organization of synaptic proteins in retinal terminals that have disrupted retinal waves. However, β2-nAChRs are also expressed in the dLGN and other areas of the brain and glutamatergic synapse development has been reported in the CNS independent of the disruption in retinal waves. This issue should be considered when interpreting the total reduced retinal synapse density in the dLGN of the mutant.

4. Outside of a total synapse density difference between WT and β2KO mice, the changes in the spatial organization of synaptic proteins over development do not seem that different. In fact % simple synapses near complex synapses from the non-dominant eye in the mutant is not that different from WT at P8 (Fig 3C), an age when eye-specific segregation is very different between the genotypes. Can the authors explain this discrepancy?

5. The authors use nomenclature that has been previously used and associated with other aspects of retinogeniculate properties. For example, the phrases "simple" and "complex" synapses have been used to describe single boutons or aggregates of boutons from numerous retinal axons, whereas in this manuscript the phrases are used to describe vesicle clusters/release sites with no knowledge of whether they are from single or multiple boutons. Likewise, the use of the word "glomerulus" has been used in the context of the retinogeniculate synapse to refer to a specific pattern of bouton aggregates that involves inhibitory and neuromodulatory inputs. It is not clear how the release sites described by the authors fit in this picture. Finally the use of the word "punishment" is associated with a body of literature regarding the immune system and retinogeniculate refinement-which is not addressed in this study. This double use of the phrases can lead to confusion in the field and should be clarified by clear definitions of how they are used in the current study.

Reviewer #3 (Public Review):

Summary:
This manuscript is a follow-up to a recent study of synaptic development based on a powerful data set that combines anterograde labeling, immunofluorescence labeling of synaptic proteins, and STORM imaging (Cell Reports 2023). Specifically, they use anti-Vglut2 label to determine the size of the presynaptic structure (which they describe as the vesicle pool size), anti-Bassoon to label a number of active zones, and anti-Homer to identify postsynaptic densities. In their previous study, they compared the detailed synaptic structure across the development of synapses made with contra-projecting vs ipsi-projecting RGCs and compared this developmental profile with a mouse model with reduced retinal waves. In this study, they produce a new analysis on the same data set in which they classify synapses into "complex" vs. "simple" and assess the number and spacing of these synapses. From these measurements, they make conclusions regarding the processes that lead to synapse competition/stabilization.

Strengths:
This is a fantastic data set for describing the structural details of synapse development in a part of the brain undergoing activity-dependent synaptic rearrangements. The fact that they can differentiate eye of origin is also a plus.

Weaknesses:
The lack of details provided for the classification scheme as well as the interpretation of small effect sizes limit the interpretations that can be made based on these findings.

1. The criteria to classify synapses as simple vs. complex is critical for all of the analysis in this study. Therefore this criteria for classification should be much more explicit and tested for robustness. As stated in the methods, it is based on the number of active zones which are designated by the number of Bassoon clusters associated with a Vglut2 cluster (line 697). A second part of the criteria is the size of the presynaptic terminal as assayed by "greater Vglut2 signal" (line 116). So how are these thresholds determined? For Bassoon clusters, is one voxel sufficient? Two? If it's one, how often do they see a Bassoon positive voxel with no Vglut2 cluster and therefore may represent "noise"? There is no distribution of Bassoon volumes that is provided that might be the basis for selecting this number of sites. Unfortunately, the images are not helpful. For example, does P8 WT in Figure 1B have 7 or 2? According to Figure 2C, it appears the numbers are closer to 2-4.

The Vglut volume measurements also do not seem to provide a clear criterion. Figure 2 shows that the distributions of Vglut2 cluster volumes for complex and for simple synapses are significantly overlapping.

The authors need to clarify the quantitative approach used for this classification strategy and test how sensitive the results of the study are to how robust this strategy is

2. Effect sizes are quite small and all comparisons are made on medians of distributions. This leads to an n=3 biological replicates for all comparisons. Hence this small n may lead to significant results based on ANOVAS/t-tests, but the statistical power of these effects is quite weak. To accurately represent the variance in their data, the authors should show all three data points for each category (with a SD error bar when possible). They should also include the number of synapses in each category (e.g. the numerators in Figure 1D and the denominators for Figure 1E). For other figures, there are additional statistical questions described below.

3. The authors need to add a caveat regarding their classification of synapses as "complex" vs. "simple" since this is a terminology that already exists in the field and it is not clear that these STORM images are measuring the same thing. For example, in EM studies, "complex" refers to multiple RGCs converging on the same single postsynaptic site. The authors here acknowledge that they cannot assign different AZs to different RGCs so this comparison is an assumption. In Figure 2 they argue this is a good assumption based on the finding that the Vglut column/active zone is constant and therefore each represents a single RGC. However, the authors should acknowledge that they are actually seeing quite different percentages than those in EM studies. For example, in Monavarfeshani et al, eLife 2018, there were no complex synapses found at P8. (Note this study also found many more complex vs. simple synapses in the adult - 70% vs. the 20% found in the current study - but this difference could be a developmental effect). In the future, the authors may want to take another data set in the adult dLGN to make a direct comparison based on numbers and see if their classification method for complex/simple maps onto the one that currently exists in the literature.

4. Figure 3 assays the relative distribution of simple vs. complex synapses. They found that a larger percentage of simple synapses were within 1.5 microns of complex synapses than you would expect by chance for both ipsi and contra projecting RGCs, and hence conclude that complex synapses are sites of synaptic clustering. In contrast, there was no clustering of ipsi-simple to contra-complex synapses and vice versa. The authors also argue that this clustering decreases between P4 and P8 for ipsi projecting RGCs.

This analysis needs much more rigor before any conclusions can be drawn. First, the authors need to justify the 1.5-micron criteria for clustering and how robust their results are to variations in this distance. Second, these age effects need to be tested for statistical significance with an ANOVA (all the stats presented are pairwise comparisons to means expected by random distributions at each age). Finally, the authors should consider what n's to use here - is it still grouped by biological replicate? Why not use individual synapses across mice? If they do biological replicates, then they should again show error bars for each data point in their biological replicates. And they should include the number of synapses that went into these measurements in the caption.

5. Line 211-212 - the authors conclude that the absence of clustered ipsi-simple synapses indicates a failure to stabilize (Figure 3). Yet, the link between this measurement and synapse stabilization is not clear. In particular, the conclusion that "isolated" synapses are the ones that will be eliminated seems to be countered by their finding in Figure 3D/E which shows that there is no difference in vesicle pool volume between near and far synapses. If isolated synapses are indeed the ones that fail to stabilize by P8, wouldn't you expect them to be weaker/have fewer vesicles? Also, it's hard to tell if there is an age-dependent effect since the data presented in Figures 3D/E are merged across ages.