Diversified expression of Pcdhg isoforms in neocortical neurons.

(A) UMAP analysis displaying 17,438 cells obtained through 5’-end single-cell sequencing after data cleanup and doublet removal. Neurons are depicted as green dots, while non-neuronal cells are marked in red. (B) UMAP clusters of all neurons categorized by the UMI cutoff. Red dots denote cells with fewer than 10 total UMIs of Pcdhg (n=3,671), and green dots denote cells with more than 10 UMIs (n=2,834). (C) Fractions of neurons with more than 10 UMIs in different clusters. (D) Distribution of neurons expressing different Pcdhg isoforms in the neocortex. (E) Fraction distribution illustrating similarity levels in the combinatorial expression of Pcdhg variable isoforms among neurons. Similarity levels were calculated as 𝐴𝐴∩𝐵𝐵. (F) Observed distribution (black) of the fraction of cells expressing varying numbers of isoforms across all neurons. The red curve represents the shuffled distribution generated under the hypothesis of stochastic isoform expression. (G) The variance difference between the observed and shuffled fraction distribution of cells from all clusters.

Increased Synaptic Connectivity in the Absence of γ-PCDH

(A) Representative traces (red/green) from multiple electrode whole-cell patch-clamp recordings conducted on six neurons in layer 2/3 of the barrel cortex. Average traces are shown in red and green, with ten original traces in gray. Positive evoked postsynaptic responses are indicated by arrows. Orange and green arrows denote unidirectional and bidirectional synaptic connections, respectively. Scale bars: 100 mV (green), 50 pA (red), and 50 ms (black). (B, D) Connectivity probability among nearby pyramidal cells in the layer 2/3 of the barrel cortex in Pcdhg cKO (B), Pcdha cKO mice (D) and their littermate WT controls. γCKO: Pcdhg flox/flox::nex-cre mice; αCKO: Pcdha flox/flox::nex-cre mice; Ctrl: Pcdhg+/+::nex-cre or Pcdha+/+::nex-cre mice. (C) Connectivity probability among vertically or horizontally aligned neurons in the layer 2/3 of the barrel cortex in Pcdhg cKO mice and their littermate WT controls. (E) Connectivity probability among vertically aligned pyramidal cells as a function of the distance between recorded pairs in Pcdhg cKO mice and WT mice. (F) Developmental profiling of connectivity probability among vertically aligned neurons in Pcdhg cKO mice and WT mice. Chi-square tests were used in B-F to calculate statistical differences.

Overexpressing identical variables, but not C4, γ-PCDHs in neurons decreased their synaptic connectivity.

(A) Schematic illustrating the brain regions selected for qRT-PCR in both experimental and control groups. (B) qRT-PCR results showing overexpression levels in electroporated regions. Electroporated isoforms are indicated in red, control isoforms in blue, and contralateral sides used as controls are in black. Statistical analysis was conducted using Student’s t-test, where * indicates p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. (C) Diagram illustrating the process of cell extraction for single-cell RT-PCR assays. (D) Results of single-cell RT-PCR for γ-PCDH isoforms after electroporation. Neurons with fluorescence are highlighted in green, while nearby neurons without fluorescence are in black. Negative controls are labeled as N1-N6. Electroporated isoforms are shown in green, with red stars indicating faint signals in negative controls. (E) Impact of overexpressing one or six γ-PCDH isoforms on synaptic connectivity in WT mice. “1 isoform” represents Pcdhga2, and “6 isoforms” denote Pcdhga2, Pcdhga8, Pcdhga10, Pcdhgb1, Pcdhgb2, and Pcdhgb6. “gC4” stands for PcdhgC4, and “Control” indicates plasmid vector without Pcdhg insertion. (F) The influence of overexpressing 6 γ-PCDH isoforms on synaptic connectivity in Pcdhg cKO mice. The same 6 isoforms used as in (E) were employed. Pcdhg cKO: Pcdhg conditional knockout mice; Control: WT littermates. Statistical differences between groups in (E) and (F) were determined using the chi-square test and false discovery rate (FDR, Benjamini-Hochberg method) correction.

Diversified γ-PCDHs are critical for synapse formation in cortical neurons.

(A) Diagram illustrating the sequential in utero electroporation process at E14.5 and E15.5. (B) Overview of the overexpressed γ-PCDH isoforms in different experiments, resulting in varying similarity levels between neurons. S.l.: Similarity level; EP: Electroporation; P.: plasmids mixture; s./d., Same or different isoforms in two electroporations. (C) Sample image of recorded neurons after two rounds of electroporation at E14.5 (mNeongreen) and E15.5 (mRuby3). Neurons labeled as positive for mNeongreen (cells 3 and 4), mRuby3 (cell 6), and negative without fluorescence (cells 1, 2, and 5). Neurobiotin was used in the internal solution to label recorded neurons. The translucent arrows show the positions of the electrodes. Scale bar, left: 100 μm, right: 25 μm. (D) Connectivity probability for neuron pairs overexpressing different sets of γ-PCDH isoforms (labeled with different fluorescence) following sequential in utero electroporation. Statistical differences were determined using the Chi-square test with FDR (Benjamini-Hochberg method) correction. (E) Correlation between the similarity level of overexpressed γ-PCDH combinations and the probability of synaptic connections. Each data point corresponds to the outcome of 100 bootstrapped samples derived from the source data presented in panel D. Error bars indicate the standard deviation (S.D) for each data point. The gray shaded area represents the 95% confidence interval of the curve fitting. (F) Graph summarizing the effect of γ-PCDHs on synapse formation.