Optimization of interneuron function by direct coupling of cell migration and axonal targeting

Abstract

Neural circuit assembly relies on the precise synchronization of developmental processes, such as cell migration and axon targeting, but the cell-autonomous mechanisms coordinating these events remain largely unknown. Here we found that different classes of interneurons use distinct routes of migration to reach the embryonic cerebral cortex. Somatostatin-expressing interneurons that migrate through the marginal zone develop into Martinotti cells, one of the most distinctive classes of cortical interneurons. For these cells, migration through the marginal zone is linked to the development of their characteristic layer 1 axonal arborization. Altering the normal migratory route of Martinotti cells by conditional deletion of Mafb-a gene that is preferentially expressed by these cells-cell-autonomously disrupts axonal development and impairs the function of these cells in vivo. Our results suggest that migration and axon targeting programs are coupled to optimize the assembly of inhibitory circuits in the cerebral cortex.

Figure 1. Different interneurons exhibit migratory route choice biases.

a–c, Coronal sections through the neocortex (NCx) showing immunohistochemistry for GFP in Vgat^Cre;RCE embryos at various stages, repeated with similar results in 3 animals. d, Bar graphs represent mean ± s. e. m for fraction of interneurons in the MZ (n = 3 animals per stage). e,f,h,i, Coronal sections through the E15.5 cortex of Nkx2-1^Cre;RCE (e) and Gad65-Gfp (f), Sst^Cre;Ai9 (h), and Dlx1/2^CreER;Ai9, repeated with similar results in 3 animals (i) showing distribution of different groups of GABAergic interneurons. g, Cumulative frequency of fraction of interneurons in the marginal zone (MZ) for each genotype at all stages examined (n = 12 sections per animal, 3 animals per genotype and stage). j, Bar graphs represent mean ± s. e. m for fraction of interneurons in the MZ for each genotype at all stages examined (n = 3 animals per genotype and stage). Two-way ANOVA, post-hoc Tukey HSD, ***p < 0.001. H, Hippocampus; LGE, lateral ganglionic eminence; SVZ, subventricular zone. Scale bars equal 200 µm.

Figure 2. Different classes of SST+ interneurons migrate through distinct routes.

a, Heatmap representing relative levels (z-scores) of genes differentially expressed in SST+ cells migrating through the marginal zone (MZ) or the subventricular zone (SVZ) at E17.5, with FDR set at < 5% using the Benjamini-Hochberg method (n = 3 litters of embryos). b, Heatmap representing relative levels (z-scores) of MZ-enriched genes (from a) in different classes of SST+ interneurons from the adult visual cortex (data from Ref. 8). c, Bar graph represent mean ± s. e. m. z-scores for all MZ- (b) and SVZ-enriched genes (Supplementary Fig. 5) expressed in SST+ interneurons (n = 41 cells for Chodl, 19 for Cdk6, 68 for Cbln4, 14 for Tascst2d, 41 for Myh8, 33 for Th). The red boxes indicate molecularly distinct classes of SST+ interneurons with positive mean z-scores in MZ-enriched genes. d, Coronal sections through the E17.5 telencephalon illustrating mRNA expression of Mafb, Elfn1 and Neto1, repeated in 2 animals with similar results. e, Violin plot showing individual cell (dot) of z-scores for individual MZ-enriched genes at E17.5. Five genes are highlighted based on expression (n = 41 cells for Chodl, 19 for Cdk6, 68 for Cbln4, 14 for Tascst2d, 41 for Myh8, 33 for Th). Five genes are highlighted based on expression. Three genes show positive mean z-scores uniquely in SST+/Cbln4+ cells (putative Martinotti cells) and negative or zero mean z-scores in all other populations of SST+ interneurons. H, hippocampus; NCx, neocortex; SP, subplate; Str, striatum. Scale bar equals 250 µm.

Figure 3. Martinotti cells and PV+ translaminar interneurons preferentially migrate through the MZ.

a,h, Schematic of experimental design for the labeling of interneuron progenitor cells using conditional retroviruses in Cre-expressing embryos. c,j, Schematic of experimental design for the labeling of interneurons migrating through the marginal zone (MZ) using pial surface electroporation of conditional reporter plasmids in Cre-expressing embryos. b,d,f,g, Representative images of SST+ interneurons at P21 obtained through viral labeling at E14.5, repeated with similar results in 30 cells from 5 animals (b,f) or pial surface electroporation at P0, repeated with similar results in 40 cells from 10 animals (d,g). e, Quantification of the proportion of Martinotti cells found in viral labeling and pial surface electroporation experiments (n = 30 and 40 cells from 5 and 10 animals for viral injections and electroporation, respectively). Error bars represent binomial proportion confidence interval. Two-tailed Fisher's exact test, **p = 0.004. g,i, Representative images of translaminar PV+ interneurons (g, left panel; i, both panels) and intralaminar PV+ interneurons (g, right panel) at P21 obtained through viral labeling at E12.5, repeated with similar results in 60 cells from 7 animals (g) or pial surface electroporation at P0, repeated with similar results in 39 cells from 8 animals (i). l, Quantification of the proportion of translaminar PV+ interneurons found in viral labeling and pial surface electroporation experiments (n = 60 and 39 cells from 7 and 8 animals for viral injections and electroporation, respectively). Fisher's exact test, ***p = 1.108e-05. Scale bar equals 50 µm.

Figure 4. Martinotti cells leave their nascent axon in the MZ while migrating into the cortical plate.

a,c, Time-lapse images of migrating interneurons in coronal slices through the cortex of P0 Dlx1/2^CreER;Ai9 (a) and Nkx2-1^CreER;Ai9 (c) over a period of 60 h. Time-lapse experiments were repeated with similar results in 11 and 8 cells from 8 and 5 animals, respectively. For each time, track lines represent the trajectory of the cell soma (blue) and the tip of the neural process (red) closest to the marginal zone (MZ). Dotted lines represent the boundary between the MZ and cortical plate (CP). b, Box plots represent median, 1^st and 3^rd quartile, and 1.5 IQR of the speed of each cell soma or process tip n = 11 and 8 cells from 8 for Dlx1/2^CreER;Ai9 and 5 Nkx2-1^CreER;Ai9 mice, respectively. Two-tailed Mann Whitney U test with Bonferroni correction, *p = 0.02. d, Bar graphs represent mean ± s. e. m. for distance between the cell soma and the tip of the trailing process after 60 h (n = 11 and 8 cells from 8 for Dlx1/2^CreER;Ai9 and 5 Nkx2-1^CreER;Ai9 mice, respectively). Two-tailed Student t-test, *p = 0.03. e, Schematic of experimental design for the labeling of nascent axons in prospective Martinotti cells via pial surface electroporation at P0. f, Representative image of a prospective Martinotti cell at P3, repeated in 14 cells from 5 animals, of which 11 cells showed similar results. Arrowheads point to Kif5CΔ560 expression in the nascent axon; the open arrowhead indicates lack of Kif5CΔ560 expression in the leading process. Scale bars equal 100 µm (a,c) and 50 µm (f).

Figure 5. Abnormal migration of SST+ interneurons in conditional Mafb mutant mice.

a,b,e,f, Coronal sections through the cortex of E16.5, repeated with similar results in 4 and 5 mice per genotype (a,b) and P21, repeated with similar results in 3 mice per genotype, showing the distribution of SST+ (GFP+) interneurons in Sst^Cre; Mafb^fl/+;RCE (a,e) and Sst^Cre;Mafb^fl/fl;RCE (b,f) mice. c, Cumulative frequency plot of the fraction of cells in the marginal zone (MZ) for each genotype. d, Quantification of the fraction of SST+ cells migrating through the MZ (n = 4 control and 5 mutant mice). Two-tailed Student t-test, **p = 0.004 for MZ, **p = 0.007794 for SVZ. g, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of laminar distribution of SST+ (GFP+) cells in the neocortex (n =12 sections per animal, 3 mice per genotype). Two-way ANOVA, p = 0.90. h,i, Coronal sections through the cortex of P21 Sst^Cre;Mafb^fl/+;RCE (h) and Sst^Cre;Mafb^fl/fl;RCE (i) mice illustrating the distribution of SST+ (GFP+) axons in layers 1 and 2/3 of the somatosensory cortex. j, Line charts representing relative GFP intensity at various distances from the layer I-layer II boundary. Dark lines and light shadings represent means and s.e.m. for both genotypes. n = 10 sections per animal, 5 mice per genotype. k, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of mean GFP intensity in layers I (right) and III (left) of the neocortex; n = 10 sections per animal, 4 mice per group; two-way ANOVA, **p = 0.004. CP, cortical plate; IZ, intermediate zone; sp, subplate; SVZ, subventricular zone. Histograms represent mean ± s. e. m. Scale bars equal 100 µm (a,b,e,f) and 20 µm (h,i).

Figure 6. Abnormal development of Martinotti cell layer I axons in conditional Mafb mutant mice.

a, Schematic of experimental design for the labeling of interneuron progenitor cells using conditional retroviruses in Cre-expressing embryos. b,c, Coronal sections through the neocortex of P21 showing the morphology of Martinotti cells labeled through retroviral infection in Sst^Cre;Mafb^fl/+ (b) and Sst^Cre;Mafb^fl/fl (c), repeated with similar result for n = 29 and 37 cells from 8 and 10 mice, respectively. d, Schematic of experimental design for the labeling of interneurons migrating through the marginal zone (MZ) using pial surface electroporation of conditional reporter plasmids. e,f, Coronal sections through the neocortex of P21 showing the morphology of Martinotti cells labeled by pial electroporation in Sst^Cre;Mafb^fl/+ (e) and Sst^Cre;Mafb^fl/fl (f), repeated with similar result for n = 36 and 32 Martinotti cells from 9 and 8 mice, respectively. g,h, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of length of Martinotti cell axons labeled by retroviral infection in layer 1 (g) and layers 2-6 (h) at P21 (n = 29 and 37 Martinotti cells from 8 and 10 mice in control and mutant respectively). Two-tailed Student t-test with Bonferroni correction, ***p = 0.0005. Cumulative frequency plots for the length of Martinotti cell axons in layer 1 (g) and layers 2-6 (h) are shown next to the corresponding histograms. i, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of length of Martinotti cell axons labeled by pial surface electroporation in layer 1 and layers 2-6 at P21 (n = 36 and 32 Martinotti cells from 9 control and 8 mutant mice, respectively). Two-tailed Student t-test with Bonferroni correction, p = 0.681 and p = 0.911, respectively. j, Schematic of experimental design for the subcellular ChR2-assisted circuit mapping (sCRACM) of SST+ interneuron outputs. Each circle depicts the target of a 70 μm² laser spot used to evoke inhibitory input onto a single recorded pyramidal cell, whose position is indicated by a white triangle. IPSC amplitude from each inhibitory spot is plotted as a heatmap in the overlaying image. R1-R5, rows 1 to 5. k, Representative laminar profiles of mean IPSCs recorded in layer 3 pyramidal cells from control and mutant mice. For these profiles, heatmaps were collapsed into one dimension by averaging evoked IPSC amplitude at each spot per row. The position of the recorded cells is indicated by a triangle. l, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of IPSCs for each input row (n = 21 cells from 10 Sst^Cre;Mafb^fl/+ mice and 19 cells from 11 Sst^Cre;Mafb^fl/fl mice). Two factorial ANOVA mean IPSC versus Genotype-Row, **p = 0.004.

Figure 7. Impaired visual responses of SST+ interneurons in Mafb mutant mice.

a, Schematic of experimental design for the expression of GCaMP6s in SST+ interneurons using conditional AAV plasmids in Cre-expressing mice. b, Experimental set-up for two-photon calcium imaging in V1 of awake head-fixed mice and representative two-photon images of layer 2/3 SST+ interneurons labeled with GCaMP6s in Sst^Cre;Mafb^fl/+ and Sst^Cre;Mafb^fl/fl mice. c, Calcium transients (ΔF/F₀) of representative SST+ interneurons imaged during the presentation of 8 oriented drifting gratings (0 to 315°; arrows and numbers indicate the angle of drift direction). For each genotype, the upper trace shows a single trial with grey regions indicating the periods of visual stimulation. The lower traces show the responses for all trials (grey) and the average response across trials (black). The orientation tuning polar plot is also shown for each neuron. d, Average tuning curve for all SST+ interneurons in Sst^Cre;Mafb^fl/+ and Sst^Cre;Mafb^fl/fl mice, calculated from the average stimulus evoked response (SER) for each of the 8 directions of drifting gratings and normalized to the preferred direction. Light grey shading indicates SEM (n = 61 neurons from 7 Sst^Cre;Mafb^fl/+ mice and 79 neurons from 7 Sst^Cre;Mafb^fl/fl mice). Kruskal-Wallis test, ***p = 0.001, **p = 0.005, orthogonal orientations p = 0.063. e, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of trial-by-trial variability using the average standard error of the mean (SEM) of the stimulus-evoked responses (SER) across trials, for both the preferred orientation, **p = 0.005 and for the orientation orthogonal to the preferred p = 0.314 (n = 61 neurons from 7 Sst^Cre;Mafb^fl/+ mice and 79 neurons from 7 Sst^Cre;Mafb^fl/fl mice). Kruskal-Wallis test. f, Quantification of the distribution of orientation selectivity indices (OSI) of SST+ interneurons for each genotype. g, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR of OSI for all SST+ interneurons for each genotype (n = 61 neurons in Sst^Cre;Mafb^fl/+ mice and 79 neurons in Sst^Cre;Mafb^fl/fl mice). Kruskal-Wallis test, **p=0.009. h, Boxplots represent median, 1^st and 3^rd quartile, and 1.5 IQR for percentage of orientation selective neurons (OSI > 0.25) for each field of view per genotype (n = 7 fields of view for each genotype). Kruskal-Wallis test, *p = 0.015. i, Pairwise correlation (Pearson correlation coefficient) matrices for all SST+ interneurons in one representative field of view for each genotype (n = 9 neurons in Sst^Cre;Mafb^fl/+ mice and 10 neurons in Sst^Cre;Mafb^fl/fl mice). j, The central panel shows the average of all pairwise correlations for each field of view, between SST+ interneurons (left; p = 0.035) and between putative excitatory neurons (pExc) (right; p = 0.016), for each genotype. Red crosses indicate the average across all fields of view (n = 7 fields of view for each genotype for SST+ interneurons and 6 fields of view for each genotype for pExc neurons). Kruskal-Wallis test. The side panels show quantification of the distribution of pairwise correlations between SST+ interneurons (left) (n = 61 neurons from 7 Sst^Cre;Mafb^fl/+ mice and 79 neurons from 7 Sst^Cre;Mafb^fl/fl mice) and pExc neurons (right) (n = 1321 neurons from 7 Sst^Cre;Mafb^fl/+ mice and 1424 neurons from 7 Sst^Cre;Mafb^fl/fl mice). k, Classification accuracy of a template-matching decoder trained on the activity of SST+ interneurons or pExc neurons to decode grating identity. Red crosses indicate the average across all fields of view. Dashed line indicates chance level of decoding accuracy (n = 7 fields of view for each genotype for SST+ interneurons, **p = 0.008 and 6 fields of view for each genotype for pExc neurons, p = 0.032). Kruskal-Wallis test. Scale bar equals 100 µm. All boxplots represent median, 1st and 3rd quartile, and 1.5 IQR.