Neocortical Projection Neurons Instruct Inhibitory Interneuron Circuit Development in a Lineage-Dependent Manner

Abstract

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development.

Keywords: circuits; cortex; development; embryonic lineage; interneuron; projection neuron; radial migration; single-cell RNA-sequencing; synaptic physiology.

Figure 1.. Reprogramming IT-Type Projection Neurons by Conditional Loss of Satb2

(A) Experimental strategy. (B) Loss of callosal axons (arrows) in Satb2 cKO mice. Myelin basic protein (MBP). (C) Ectopic superficial expression of Ctip2 and CRYM in Satb2 cKO mice. (D) Changes in cortical and layer 1 thickness in Satb2 cKO. *p < 0.001, Wilcoxon rank tests. (E) Responses to hyperpolarizing current pulses for control (black) and Satb2 KO (red) PNs. (F) Intrinsic membrane properties. Resting Vm: control (n = 12 cells) versus cKO (n = 14 cells), t test. Rin: control (n = 22 cells) versus cKO (n = 29 cells), Wilcoxon rank test. Voltage sag: control (n = 22 cells) versus cKO (n = 29 cells), Wilcoxon rank test. *p < 0.001. (G) DAPs in Satb2 cKO (red) but not control (black) PNs (arrows). (H) Initial doublet spikes in Satb2 cKO (red) but not control (black) PNs. (I) Doublet spikes as a function of the presence of DAP. Control (n = 22 cells) versus cKO (DAP) (n = 15 cells) versus cKO (No DAP) (n = 13 cells), *p < 0.001, Kruskal-Wallis test, Dunn-Holland-Wolfe multiple comparisons test. (J) Input resistance as a function of the presence of DAP. Control (n = 22 cells) versus cKO (DAP) (n = 16 cells) versus cKO (No DAP) (n = 13 cells). *p < 0.001, Kruskal-Wallis test, Dunn-Holland-Wolfe multiple comparisons test. (K) Voltage sag as a function of the presence of DAP. Control (n = 22 cells) versus cKO (DAP) (n = 16 cells) versus cKO (No DAP) (n = 13 cells). *p < 0.001, Kruskal-Wallis test, Dunn-Holland-Wolfe multiple comparisons test. (L) Resting Vm as a function of the presence of DAP. Control (n = 12 cells) versus cKO (DAP) (n = 9 cells) versus cKO (No DAP) (n = 13 cells). *p < 0.001, Kruskal-Wallis test, Dunn-Holland-Wolfe multiple comparisons test. Control (black), cKO (red). Error bars represent ± SEM.

Figure 2.. Reprogramming IT-Type PNs Disrupts the Lamination of CGE-but Not MGE-Derived INs

(A) CGE IN cell body positions. (B) (Left) Percentage of CGE INs as a function of normalized cortical depth. (Right) Cumulative percentage. (C) Total percentage of CGE INs in the top 50% (first 10 bins: Superficial) versus bottom 50% (last 10 bins: Deep) of cortex. *p < 0.01, χ² test. (D) PV⁺ IN cell body positions. (E) (Left) Percentage of PV⁺ INs as a function of normalized cortical depth. (Right) Cumulative percentage. (F) Total percentage of PV⁺ INs in the top 50% (Superficial) versus bottom 50% (Deep) of cortex. p = 0.29, χ². (G) SST⁺(GFP⁻) IN cell body positions. (H) (Left) Percentage of SST⁺(GFP⁻) INs as a function of normalized cortical depth. (Right) Cumulative percentage. (I) Total percentage of SST⁺(GFP⁻) INs in the top 50% (Superficial) versus bottom 50% (Deep) of cortex. p = 0.37, χ². (J) Overlap of SST (IHC) and 5HT3A-GFP signal. p = 0.47, χ² test. (K) Cell densities. 5HT3A-GFP⁺ INs: control (n = 23 sections) versus cKO (n = 24 sections), p = 0.62. PV⁺ INs: control (n = 36 sections) versus cKO (n = 33 sections), p = 0.29. SST⁺(GFP⁻) INs: control (n = 35 sections) versus cKO (n = 36 sections), p = 0.57. t tests. Error bars represent ± SEM.

Figure 3.. Loss of IT-Type PNs Disrupts VIP⁺ but Not reelin⁺ IN Lamination and Results in Aberrant CCK⁺ INs

(A) VIP IN cell body positions. (B) Percentage of VIP INs as a function of normalized cortical depth. Inset, total percentage of VIP INs in the top 50% (first 10 bins: Superficial) versus bottom 50% (last 10 bins: Deep) of cortex. *p < 0.02, χ² test. (C) VIP IN densities. Control (n = 35 sections) versus cKO (n = 33 sections). p = 0.7, Wilcoxon rank test. (D) Reelin⁺(GFP⁺) IN cell body positions. (E) Percentage of reelin⁺(GFP⁺) INs as a function of normalized cortical depth. (F) Reelin⁺(GFP⁺) IN densities. Densities: control (n = 35 sections) versus cKO (n = 33 sections). p < 0.05, t test. (G) CCK⁺(GFP⁺) IN cell body positions. (H) Percentage of CCK⁺(GFP⁺) INs as a function of normalized cortical depth. (I) CCK⁺(GFP⁺) IN densities. Densities: control (n = 35 sections) versus cKO (n = 34 sections). p < 0.001, Wilcoxon Rank test. (J) (Left) Total overlap of VIP and reelin(GFP⁺). Control versus cKO: p = 0.29, χ² test. (Right) Within-section overlap. Overlap/VIP total: control (n = 35 sections) versus cKO (n = 33 sections), p = 0.15; overlap/reelin total: control (n = 35 sections) versus cKO (n = 33 sections), p = 0.1. Wilcoxon rank tests. (K) (Left) Total overlap of CCK(GFP⁺) and reelin(GFP⁺). Control versus cKO: *p < 0.05, χ² test. (Right) Within-section overlap. Overlap/CCK total: control (n = 35 sections) versus cKO (n = 34 sections), p = 0.28, t test; overlap/reelin total: control (n = 35 sections) versus cKO (n = 34 sections), *p < 0.001, Wilcoxon rank test. Error bars represent ± SEM.

Figure 4.. Loss of IT-Type Identity Disrupts Synaptic Connectivity between PNs and CGE-Derived INs in the Superficial Cortex

(A) CGE INs in the superficial cortex of cKO mice: late spiking (LS) neurogliaform, irregular spiking (IS) and fast adapting (fAD) bi-tufted. LS cell red trace: depolarizing Vm ramp. Dendrites and soma in black, axon in red. (B) Percentage of IN firing types. Control (n = 38 cells) versus cKO mice (n = 31 cells). bNA2, burst nonadapting 2. (C) Paired whole-cell recordings of synaptic connections in control mice. (1) Recording configuration. (2) PN-to-IN; postsynaptic LS IN. (3) IS-type IN-to-PN. Arrow: asynchronous transmitter release. (4) LS-type IN-to-PN synaptic connection. Gray traces: first 5 trials; pink traces: subsequent failures. Scale bars correspond to (2) unless otherwise noted. 10 overlaid trials in each. (D) Paired whole-cell recordings of synaptic connections in Satb2 cKO mice. (1) Recording configuration. iPT = induced-PT. (2) PN-to-IN; postsynaptic IS IN. (3) IS-type IN-to-PN. Arrow: asynchronous transmitter release. (4) LS-type IN-to-PN synaptic connections lack failures after many trials (cf. Figure 4C4). Scale bars correspond to (2) unless otherwise noted. 10 overlaid trials in each. (E) Connection probabilities (reciprocal in middle). Satb2 cKO PNs denoted iPT. *p < 0.05, χ² test. (F) Connection probabilities by IN firing type. (G) Reconstructed Satb2 KO PN. Dendrites and soma in black, axon in red.

Figure 5.. IT-Type but Not PT-Type PNs Target CGE-Derived INs in Deep Cortical Layers

(A) Experimental design. SC, superior colliculus. (B) IT PN to CGE IN synaptic connection and connection probabilities (reciprocal in middle). *p < 0.01, χ² test. (C) Connection probabilities by IN firing type. (D) Putative VIP⁺ INs with a linear a current-voltage relationship receive synaptic connections at a significantly higher rate than those with inward rectification. *p < 0.01, χ² test.

Figure 6.. Population Input from PNs to CGE INs

(A) Experimental design. Vc, voltage clamp; Ic, current clamp. (B) Light stimuli (blue bars) evoked action potentials in infected PT PNs, without postsynaptic responses in INs. Depolarizing afterpotential (arrow). 10 overlaid trials. (C) Long-duration (10 ms), high-power (26 mW) light stimuli (blue bars) were required to evoke rare, low amplitude postsynaptic responses in 5HT3A-GFP⁺ INs. Note doublet spiking in PT PN. 10 overlaid trials. (D) Experimental design and example of 1.5 mW, 2 ms light stimuli evoked spikes. Lack of a depolarizing afterpotential (arrow). 10 overlaid trials. (E) Amplitude of postsynaptic responses in CGE INs increase as a function of light power. 10 overlaid trials. Plotted are amplitudes of the first postsynaptic response evoked during a stimulus train, normalized to the lowest intensity (0.6 mW) (n = 5 cells). Error bars represent ± SEM.

Figure 7.. Single-Cell RNA Sequencing of CGE INs after Loss of IT-Type PNs

(A) Experimental design. (B) t-distributed stochastic neighbor embedding (t-SNE) plots of CGE INs from control and cKO mice after integration and alignment of transcriptomic datasets. (C) t-SNE plot (overlaid control and cKO datasets) with color-coded clusters identified by unbiased analysis. IN cluster names were manually curated based on prevalence of major molecular markers. Small contaminant clusters also identified (Glutamatergic, Astrocytes, Oligodendrocytes, Immature). (D) Heatmaps of transcription for major molecular markers across IN clusters. Colors represent log normalized average expression values relative to each gene across clusters. (E) Differential expression of selected genes across IN clusters from control and cKO mice. Colors are average expression values within a cluster; dot size is percentage of cells within a cluster expressing the gene.

Figure 8.. CGE INs Upregulate Many Genes Relevant to Migration and Synaptic Connectivity in Mice Lacking IT-Type PNs

Selected genes with significant differential expression after loss of IT-type PNs in manually curated categories: (A) chemokine ligands and receptors, (B) adhesion molecules and receptors, (C) growth factors, and (D) molecules involved in axon guidance and synapse formation. Green arrowheads indicate genes mentioned in the text. Genes and clusters sorted by Euclidean distance. Colors denote log fold change of average expression within each cluster.