Instructing Perisomatic Inhibition by Direct Lineage Reprogramming of Neocortical Projection Neurons

Abstract

During development of the cerebral cortex, local GABAergic interneurons recognize and pair with excitatory projection neurons to ensure the fine excitatory-inhibitory balance essential for proper circuit function. Whether the class-specific identity of projection neurons has a role in the establishment of afferent inhibitory synapses is debated. Here, we report that direct in vivo lineage reprogramming of layer 2/3 (L2/3) callosal projection neurons (CPNs) into induced corticofugal projection neurons (iCFuPNs) increases inhibitory input onto the converted neurons to levels similar to that of endogenous CFuPNs normally found in layer 5 (L5). iCFuPNs recruit increased numbers of inhibitory perisomatic synapses from parvalbumin (PV)-positive interneurons, with single-cell precision and despite their ectopic location in L2/3. The data show that individual reprogrammed excitatory projection neurons extrinsically modulate afferent input by local PV(+) interneurons, suggesting that projection neuron class-specific identity can actively control the wiring of the cortical microcircuit.

Figure 1. iCFuPNs acquire molecular identity of CFuPNs

(A) Timeline of experiments performed. In utero electroporation at E14.5; single-cell gene-expression profiling at P15; electrophysiology at P22-26; histological analysis at P28. (B-C) mRNA expression analysis of CPN and CFuPN markers in single Fezf2-OE PNs and GFP-CPNs at P15 shows that a subset of Fezf2-OE cells acquire a CFuPN-like molecular identity. Fezf2-OE n = 70, GFP-CPN n= 57. (B) Gene expression heatmap of 88 genes assayed by high-density qPCR demonstrates acquisition of a global CFuPN-like signature in a subset of Fezf2-OE neurons. Sample clustering by PAM identified 2 clusters (green/orange and chartreuse/red bars). Top row of blue bars: item consensus (IC) values, a metric of cluster assignment stability. Correlated gene expression modules were identified by WGCNA (M1-M4: Module 1 through Module 4, N: not assigned to a module). Bolding indicates genes with significant expression differences (>2-fold and p<0.05); arrows indicate direction of change in Cluster 2. Also see Table S1. Gene color-coding: red, CFuPN marker; green, CPN marker; purple, synaptic; blue, normalization control; orange, other control. (C) PCA of gene expression data demonstrates that Clusters 1 and 2 separate along PC1. Also see Figure S1 and Table S1.

Figure 2. iCFuPNs in L2/3 acquire electrophysiological properties of L5 CFuPNs

(A) Representative current steps at -100, 50, and 350 pA. iCFuPNs show a firing pattern similar to L5 CFuPNs, whereas Fezf2-NR neurons resemble endogenous L2/3 CPNs and L2/3 GFP-CPNs. Scale bar: 500 ms (horizontal), 20 mV (vertical). (B-C) PCA on 12 intrinsic electrophysiological properties separates algorithmically identified iCFuPNs from Fezf2-NR neurons. Also see Figure S2. (B) Histogram of cell distribution along PC1 shows bimodal distribution of Fezf2-OE cells. Dotted lines: fits to single (L2/3 CPN, L5 CFuPN) or mixed (Fezf2-OE) Gaussian distributions. PDF: probability density function. (C) PCA analysis clusters the Fezf2-NR and iCFuPN populations with L2/3 CPNs and L5 CFuPNs, respectively. Color-coding as in A. Dotted circles: threshold where PDF=0.02 after fitting to 2D Gaussian distribution. (D-K) Intrinsic properties of algorithmically identified iCFuPNs and Fezf2-NRs resemble L5 CFuPNs and L2/3 CPNs, respectively. Endogenous L2/3 CPN n=8, GFP-CPN n=24, L5 CFuPN n=11, iCFuPN n=14, Fezf2-NR n=21. Error bars: mean ± SEM. (D) Firing-current curves (spike numbers in 2 seconds vs. current injection). Asterisks indicate p values (Black, L5 CFuPN vs. L2/3 CPN; Red, iCFuPN vs. L2/3 CPN; Gold, L5 CFuPN vs. iCFuPN): * p<0.05, ** p<0.01, *** p<0.001. Color-coding as in A. (E-G) Quantification of passive membrane properties: (E) Vm, (F) Vsag, and (G) Tau. (H-K) Quantification of action potentials: (H) Sample traces of action potentials, (I) CThr, (J) VThr and (K) fAHP.

Figure 3. iCFuPNs receive increased inhibition

(A-G) mIPSC events demonstrate inhibition in iCFuPNs similar to L5 CFuPNs and higher than L2/3 CPNs. GFP-CPN n=30, Fezf2-OE n=33, L5 CFuPN n=16. Error bars: mean ± SEM. (A) Representative mIPSCs traces. (B) Average mIPSC frequency and (C) cumulative inter-event interval (IEI) from all cells. (D) Average mIPSC amplitude and (E) cumulative amplitude from all cells. (F) Average mIPSC weighted decay (τ_w and (G) cumulative τ_w from all cells. (H-M) Increased inhibition is specific to iCFuPNs within Fezf2-OE brains. (H) Representative responses to hyperpolarizing current steps (-100 to -500 pA), mIPSC traces and image reconstruction. GFP-CPNs n=12, Fezf2-NR n=15, iCFuPN n=6; from 5 Fezf2-OE and 3 GFP-OE brains. Dotted lines: pial surface. (I) iCFuPNs were identified by Vsag higher than the maximal GFP-CPN Vsag value. (J) mIPSC frequency is increased in iCFuPNs compared to Fezf2-NR and GFP-CPNs. (K) mIPSCs amplitude remains constant between all three groups. (L) Comparison of average mIPSC frequency in iCFuPNs and Fezf2-NR cells from the same brains (connected pairs) shows that increased inhibition is specific to iCFuPNs. (M) Fezf2-OE cells increase their apical tuft width regardless of their reprogrammed state.

Figure 4. iCFuPNs receive greater PV⁺ IN input

(A-C) iCFuPNs have increased PV⁺ puncta density. GFP-CPNs n=100, iCFuPNs n=200, L5 CFuPNs n=80. Error bars: mean ± SEM. (A) Representative immunofluorescence images for GFP, Crym and PV at P28. (B) iCFuPNs have greater soma size and (C) PV⁺ puncta density. (D-G) Optogenetic excitation of PV⁺ INs in reprogrammed cortices shows two patterns of response in Fezf2-OE neurons, resembling L2/3 CPNs and L5 CFuPNs, respectively. L2/3 CPNs n=21, Fezf2-OE cells n=21, L5 CFuPNs n=18. (D) Representative max-IPSC_PV (main) and min-IPSC_PV (inset) light-evoked response traces. (E) Amplitude of min-IPSC_PV responses. (F) Amplitude of max-IPSC_PV responses. (G) Fits of max-IPSC_PV response data to single (L2/3 CPNs and L5 CFuPNs) or mixed (Fezf2-OE) Gaussian curves: Fezf2-OE cells show bimodal distribution (R²: L2/3 CPN = 0.499, L5 CFuPN = 0.596, Fezf2-OE = 0.740).