Combinatorial expression of gamma-protocadherins regulates synaptic specificity in the mouse neocortex

Reviewer #1 (Public Review):

The manuscript by Zhu and colleagues aimed to clarify the importance of isoform diversity of PCDHg in establishing cortical synapse specificity. The authors optimized 5' single-cell sequencing to detect cPCDHg isoforms and showed that the pyramidal cells express distinct combinations of PCDHg isoforms. Then, the authors conducted patch-clamp recordings from cortical neurons whose PCDHg diversity was disrupted. In the elegant experiment in Figure 3, the authors demonstrated that the neurons expressing the same sets of cPCDHg isoforms are less likely to form synapses with each other, suggesting that identical cPCDHg isoforms may have a repulsive effect on synapse formation. Importantly, this phenomenon was dependent on the similarity of the isoforms present in neurons but not on the amount of proteins expressed.

One of the major concerns in an earlier version was whether PCDHg isoforms, which are expressed at a much lower level than C-type isoforms, have true physiological significance. The authors conducted additional experiments to address this point by using PCDHg cKO and provided convincing data supporting their conclusion. The results from PCDHg C4 overexpression, showing no impact on synaptic connectivity, further clarified the importance of isoforms. I have no further concerns, however, I would like to point out that the evidence for the necessity of the PCDHg isoform is still lacking because most experiments were done by overexpression. It would be helpful for the readers if the authors could add this point to the discussion.

Reviewer #2 (Public Review):

This short manuscript by Zhu et al. describes an investigation into the role of gamma protocadherins in synaptic connectivity in the mouse cerebral cortex. First, the authors conduct a single-cell RNA-seq survey of postnatal day 11 mouse cortical neurons, using an adapted 10X Genomics method to capture the 5' sequences that are necessary to identify individual gamma protocadherin isoforms (all 22 transcripts share the same three 3' "constant" exons, so standard 3'-biased methods can't distinguish them). This method adaptation is an advance for examining individual gamma transcripts, and it is helpful to publish the method, the characterization of which is improved in this revised manuscript. The results largely confirm what was known from other approaches, which is that a few of the 19 A and B subtype gamma protocadherins are expressed in an apparently stochastic and combinatorial fashion in each cortical neuron, while the 3 C subtype genes are expressed ubiquitously. Second, using elegant paired electrophysiological recordings, the authors show that in gamma protocadherin cortical slices, the likelihood of two neurons on layers 2/3 being synaptically connected is increased. That suggests that gamma protocadherins generally inhibit synaptic connectivity in the cortex; again, this has been reported previously using morphological assays, but it is important to see it confirmed here with physiology. Finally, the authors use an impressive sequential in utero electroporation method to provide evidence that the degree of isoform matching between two neurons negatively regulates their reciprocal synaptic connectivity. These are difficult experiments to do, and while some caveats remain, the main result is consistent. Strengths include the impressive methodology and improved demonstration of the previously-reported finding that gamma protocadherins work via homophilic matching to put a brake on synapse formation in the cortex. Weaknesses include the writing, which even in the revision fails to completely put the new results in context with prior work, which together has largely shown similar results; a still-incomplete characterization of a new alpha protocadherin KO mouse (a minor point but it should still be addressed); and a lack of demonstration of protein levels in electroporated brains. Because of the unique organization and expression pattern of the gamma protocadherins, it is unlikely that these results will be directly applicable to the broader understanding of the role of cell adhesion molecules in synapse development. However, the methodology, which is now better described, should be applicable more broadly and the improved demonstration of the role of gamma protocadherin's negative role in cortical synaptogenesis is helpful.

Reviewer #3 (Public Review):

In this study, Zhu and authors investigate the expression and function of the clustered Protocadherins (cPcdhs) in synaptic connectivity in the mouse cortex. The cPcdhs encode a large family of cadherin-related transmembrane molecules hypothesized to regulate synaptic specificity through combinatorial expression and homophilic binding between neurons expressing matching cPcdh isoforms. But the evidence for combinatorial expression has been limited to a few cell types, and causal functions between cPcdh diversity and wiring specificity have been difficult to test experimentally. This study addresses two important but technically challenging questions in the mouse cortex: 1) Do single neurons in the cortex express different cPcdh isoform combinations? and 2) Does Pcdh isoform diversity or particular combinations among pyramidal neurons influence their connectivity patterns? Focusing on the Pcdh-gamma subcluster of 22 isoforms, the group performed 5'end-directed single-cell RNA sequencing from dissociated postnatal (P11) cortex. To address the functional role of Pcdhg diversity in cortical connectivity, they asked whether the Pcdhgs and isoform matching influence the likelihood of synaptic pairing between 2 nearby pyramidal neurons. They performed simultaneous whole-cell recordings of 6 pyramidal neurons in cortical slices, and measured paired connections by evoked monosynaptic responses. In these experiments, they measured synaptic connectivity between pyramidal neurons lacking the Pcdhgs, or overexpressing dissimilar or matching sets of Pcdhg isoforms introduced by electroporation of plasmids encoding Pcdhg cDNAs.

Overall, the study applies elegant methods that demonstrate that single cortical neurons express different combinations of Pcdh-gamma isoforms, including the upper layer Pyramidal cells that are assayed in paired recordings. The electrophysiology data demonstrate that nearby Pyramidal neurons lacking the entire Pcdhg cluster are more likely to be synaptically connected compared to the control neurons, and that overexpression of matching isoforms between pairs decreases the likelihood to be synaptically connected. These are important and compelling findings that advance the idea that the Pcdhgs are important for cortical synaptic connectivity, and that the repertoire of isoforms expressed by neurons influence their connectivity patterns potentially through a self/non-self discrimination mechanism. However, the findings are limited to probability in connectivity and do they do not support the authors' conclusions that Pcdhg isoforms regulate synaptic specificity, 'by preventing synapse formation with specific cells' or to 'unwanted partners'. Characterizations of the cellular basis of these defects are needed to determine whether they are secondary to other roles in cell positioning, axon/dendrite branching and synaptic pruning, and overall synaptic formation. Claims that Pcdh-alpha and Pcdhg C-type isoforms are not functionally required are premature, due to limitations of the experiments. Moreover, claims that 'similarity level of γ-PCDH isoforms between neurons regulate the synaptic formation' are not supported due to weak statistical analyses presented in Fig4. The overstatements should be corrected. There was also missed opportunity to clearly discuss these results in the context of other published work, including recent publications focused on the cortex.

Strengths:

- The 5' end sequencing with a Pcdhg-amplified library is a technical feat and addresses the pitfall of conventional scRNA-Seq methods due to the identical 3'sequences shared by all Pcdhg isoform and the low abundance of the variable exons. New figures with annotated cell types confirm that several pyramidal and inhibitory cortical subpopulations were captured.

-Statistical assessment of co-occurrence of isoform expression within clusters is also a strength.

- By establishing the combinatorial expression of Pcdhgs by maturing pyramidal cells, the study further substantiates the 'single neuron combinatorial code for cPcdhs' model. Although combinatorial expression is not universal (ie. serotonergic neurons), there was limited evidence. The findings that individual pyramidal neurons express ~1-3 variable Pcdhg transcripts plus the C-type transcripts aligns with single RT-PCR studies of single Purkinje cells (Esumi et al 2005; Toyoda et al 2014). They differ from the findings by Lv et al 2022, where C-type expression was lower among pyramidal neurons. OSNs also do not substantially express C-type isoforms (Mountoufaris et al 2017; Kiefer et al 2023). Differences, and the advantages of the 5'end -directed sequencing (vs. SmartSeq) could be raised in the discussion.

- Simultaneous whole-cell recordings and pairwise comparisons of pyramidal neurons is a technically outstanding approach. They assess the effects of Pcdhg OE isoform on the probability of paired connections.

- The connectivity assay between nearby pairs proved to be sensitive to quantify differences in probability in Pcdhg-cKO and overexpression mutants. The comparisons of connectivity across vertical vs lateral arrangement are also strengths. Overexpressing identical Pcdhg isoform (whether 1 or 6) reduces the probability of connectivity, but there are caveats to the interpretations (see below).

Weaknesses:

-The experiments support a role for the Pcdhgs in influencing the probability of synaptic connectivity between nearby pairs but are not sufficient evidence for synapse specificity. The cPcdhs play multiple roles in neurite arborization, synaptic density, and cell positioning. Kostadinov 2015 also showed that starburst cells lacking the Pcdhgs maintained increased % connectivity at maturity, suggesting a lack of refinement in the absence of Pcdhgs. The known roles raise questions on how these manipulations might have primary effects in these processes and then subsequently impact the probability of connectivity. Investigations of morphological aspects of pyramidal development would strengthen the study and potentially refine the findings. The authors should more clearly relate their findings to the body of cPcdh studies in the discussion.

- Pcdhg cKO-dependent effects on connectivity occur between closely spaced soma (50-100um - Figure 2E), highlighting the importance of spatial arrangement to connectivity (also noted by Tarusawa 2016). Was distance considered for the overexpression (OE) assays, and did the authors note changes in cell distribution which might diminish the connectivity? Recent work by Lv et al 2022 reported that manipulating Pcdhgs influences the dispersion of clonally-related pyramidal neurons, which also impacts the likelihood of connections. Overexpression of Pcdhgc3 increased cell dispersion and decreased the rate of connectivity between pairs. Though these papers are mentioned, they should be discussed in more detail and related to this work.

- Though the authors added suggested citations and improved the contextualization of the study, several statements do not accurately represent the cited literature. It is at the expense of crystalizing the novelty and importance of this present work. For instance, Garrett et al 2012 PMID: 22542181 was the first to describe roles for Pcdhgs in cortical pyramidal cells and dendrite arborization, and that pyramidal cell migration and survival are intact. Line 52 cited Wang et al 2002, but this was limited to gross inspection. Garrett et al is the correct citation for: 'The absence of γ-PCDH does not cause general abnormality in the development of the cerebral cortex, such as cell differentiation, migration, and survival (Wang et al., 2002).' Second, single cell cPcdh diversity is introduced very generally, as though all neuron types are expected to show combinatorial variable expression with ubiquitous C-Type expression. But those initial studies were limited to Purkinje cells (Esumi 2005 and Toyoda 2014). Profiling of serotonergic neurons and OSN reveals different patterns (citations needed for Chen 2017 PMID: 28450636; Mountofaris et al PMID: 2845063; Canzio 2023 PMID: 37347873), raising the idea that cPcdh diversity and ubiquitous C-type expression is not universal. Thus, the authors missed the opportunity to emphasize the gap regarding cPcdh diversity in the cortex.

- They have not shown rigorously and statistically that the rate of connectivity changes with% isoform matching. In Figure 4D, comparisons of % isoform matching in OE assays show a single statistical comparison between the control and 100% groups, but not between the 0%, 11% and 33% groups. Is there a significant difference between the other groups? Significant differences are claimed in the results section, but statistical tests are not provided. The regression analysis in 4E suggests a correlation between % isoform similarity and connectivity probability, but this is not sound as it is based on a mere 4 data points from 4D. The authors previously explained that they cannot evaluate the variance in these recordings as they must pool data together. However, there should be some treatment of variability, especially given the low baseline rate of connectivity. Or at the very least, they should acknowledge the limitations that prevent them from assessing this relationship. Claims in lines 230+ are not supported: ' Overall, our findings demonstrate a negative correlation between the probability of forming synaptic connections and the similarity level of γ-PCDH isoforms expressed in neuron pairs (Fig. 4E)".

-Figure 4 provides connectivity probability, but this result might be affected by overall synapse density. Did connection probability change with directionality (e.g between red to green cells, or green to red cells).

-Generally, the statistical approaches were not sufficiently described in the methods nor in the figure legends, making it difficult to assess the findings. They do not report on how they calculated FDR for connectivity data, when this is typically used for larger multivariate datasets.

- The possibility that the OE effects are driven by total Pcdhg levels, rather isoform matching, should be examined. As shown by qRT-PCR in Fig. 3, expression of individual isoforms can vary. It is reasonable that protein levels cannot be measured by IHC, although epitope tags could be considered as C-terminal tagging of cPcdhs preserves the function in mice (see Lefebvre 2008). Quantification of constant Pcdhg RNA levels by qRT-PCR or sc-RT-PCR would directly address the potential caveat that OE levels vary with isoform combinations.

-A caveat for the relative plasmid expression quantifications in Figure 3-S1 is that IHC was used to amplify the RFP-tagged isoform, and thus does not likely preserve the relationship between quantities and detection.

-Figure 1 didn't change in response to reviews to improve clarity. New panels relating to the scRNA-Seq analyses were added to supplementary data but many are central and should be included in Figure 1 (ie. S1-Fig6D). In the Results, the authors state that neuronal subpopulations generally show a combinatorial expression of some variable RNA isoforms and near ubiquitous C-type expression. But they only show data for the Layer 2/3 neuron-specific cluster in S1-Fig-6D, and so it is not clear if this pattern applies to other clusters. Fig. S1-5 show a low number of expressed isoforms per cell, but specific descriptions on whether these include C-type isoforms would be helpful. Figure 1F showing isoform profile in all neurons is not particularly meaningful. There is a lot of interest in neuron-type specific differences in cPcdh diversity, and the authors could highlight their data from S1-5 accordingly.

-The concept of co-occurrence and results should be explained within the results section, to more clearly relate this concept to data and interpretations. Explanations are now found in the methods, but this did not improve the clarity of this otherwise very interesting aspect of the study.

- The claim that C-type Pcdhgs do not functionally influence connectivity is premature. Tests were limited to PcdhgC4, which has unique properties compared to the other 2 C-type isoforms (Garrett et al 2019 PMID: 31877124; Mancia et al PMID: 36778455). The text should be corrected to limit the conclusion to PcdhgC4, and not generally to C-type. The authors should test PcdhgC3 and PcdhgC5 isoforms.

-The group generated a novel conditional Pcdh-alpha mouse allele using CRISPR methods, and state that there were no changes in synaptic connectivity in these Pcdh-alpha mutants. But this claim is premature. The Southern blots validate the targeting of the allele. But further validations are required to establish that this floxed allele can be efficiently recombined, disrupting Pcdha protein levels and function. Pcdha alleles have been validated by western blots and by demonstration of the prominent serotonergic axonal phenotype of Pcdha-KO (ie. Chen 2017 PMID: 28450636; Ing-Esteves 2018 PMID: 29439167).

-The Discussion would be strengthened by a deeper discussion of the findings to other cPcdh roles and studies, and of the limitations of the study. The idea that the Pcdhgs are influencing the rate of connectivity through a repulsion mechanism or synaptic formation (ie through negative interactions with synaptic organizers such as Nlgn - Molumby 2018, Steffen 2022) could be presented in a model, and supported by other literature.