A genetic model to ablate Nkx2.1-lineage neurons in the lateral septum.

A) Coronal sections through the forebrain of postnatal day 30 (P30) control (labeled as ‘WT’; Nkx2.1-Cre;Ai14, left) and mutant (labeled as ‘cKO’; Nkx2.1-Cre;Prdm16flox/flox;Ai14, right) mice, where cells derived from Nkx2.1-expressing progenitors are labeled by the fluorescent reporter tdTomato (red). Nuclei are counterstained with DAPI (blue). Scale bars, 1 mm. B) Closeup of the septum of WT (left) and cKO (right) samples, as highlighted by white dashed line boxes in A. The main anatomical divisions of the mature septum are indicated by white dashed lines: medial septum (MS), lateral septum (LS), and dorsal (LSd), intermediate (LSi) and ventral (LSv) nuclei within the LS. Scale bar, 500 µm. C) Cartoon representing forebrain coronal sections at the three rostrocaudal positions used in subsequent analyses, labeled as sections ‘I’, ‘II’ and ‘III’ throughout the article. The septal area is highlighted by red ellipses. D) Quantification of the density of cells positive for tdTomato per mm2 in the lateral septum of WT (green circles, n = 9) and cKO (purple squares, n = 9) mice. E) Quantification of the density of tdTomato+ cells per mm2 in the medial septum of WT (green circles, n = 5) and cKO (purple squares, n = 5) mice. F) Quantification of the area of the septum relative to the total area of its corresponding coronal brain section in WT (green circles, n = 6) and cKO (purple squares, n = 5) male mice. Measurements are normalized to the corresponding WT average. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

Loss of Crhr2-expressing neurons in cKO mice.

A) Cartoon illustrating the experimental strategy. The septa of P35 ‘WT’ (Nkx2.1-Cre;Sun1-GFP) or ‘cKO’ (Nkx2.1-Cre;Prdm16flox/flox;Sun1-GFP) animals carrying a Cre-dependent fluorescent reporter inserted into the nuclear membrane were dissected out, sorted into Nkx2.1+ vs. Nkx2.1– populations via Fluorescence-Activated Nucleus Sorting (FANS), and submitted to single-nucleus RNA sequencing using the 10x platform. B) Uniform Manifold Approximation and Projection (UMAP) plot representing a 2D dimensional reduction of the transcriptional similarity among all cells identified as LS neurons within the dataset (4984 cells from a total of 6 WT and 6 cKO animals [3 males and 3 females each]). C) Dot plot showing the expression of select marker genes in each of the LS clusters outlined in B, allowing their identification as distinct cell types. D) UMAP plot as in B, with sample identities highlighted as indicated (WT, green; cKO, purple). E) Bar graph showing the proportion of cells belonging to WT (green) vs. cKO (purple) samples within each of the 17 clusters outlined in B. F) Violin plots showing the expression levels of select marker genes within clusters 7, 8, 9, and 13 (i.e. all clusters where ≥75% of cells belong to WT samples), split by genotype (WT, green; cKO, purple). G) Feature plots showing the gene expression levels within the UMAP representation. H) Overview coronal images of the septum of P30 WT (left) and cKO (right) mice submitted to in situ hybridization for Crhr2 (gray). Scale bars, 250 µm. I) Closeup view of the white dashed line boxes in H, showing the Crhr2 signal (green) combined with immunofluorescence staining for tdTomato (magenta), in the LS of WT (top) and cKO (bottom) mice. Full yellow arrowheads indicate examples of cells positive for both Crhr2 and tdTomato, empty yellow arrowheads show examples of Crhr2–, tdTomato+ cells, and empty cyan arrowheads highlight examples of Crhr2+, tdTomato– cells. Scale bars, 50 µm. J) Bar graph showing the density (per mm2) of all cells positive for Crhr2 (gray squares) and the subset of those cells that are also tdTomato+ (red circles), in section II of WT vs. cKO animals (N = 3 [2 males, 1 female] for WT; N = 4 [2 males, 2 females] for cKO).

Disrupted connectivity in the LS of cKO mice.

A) Overview coronal images of the septum of P30 WT (left) and cKO (right) mice submitted to immunofluorescence staining for urocortin-3 (gray). Scale bars, 250 µm. B) Closeup view of the white dashed line boxes in A, showing the urocortin-3 signal (UCN-3, green) combined with tdTomato (magenta), in the LS of WT (top) and cKO (bottom) mice. Empty orange arrowheads indicate examples of tdTomato+ cells surrounded by urocortin-3 perineuronal baskets, while the blue arrowhead shows a basket formed around a tdTomato– cell. Scale bars, 50 µm. C) Quantification of normalized fluorescence intensity in the different LS subnuclei of P30 WT (green circles) and cKO (purple squares) samples where urocortin-3 was detected by immunofluorescence staining. D) Quantification of the proportion (in %) of all urocortin-3 perineuronal baskets surrounding tdTomato+ cells throughout the entire LS of WT (green circles) and cKO (purple squares) mice at P30. E) Breakdown of the data in D by LS subnucleus. F) Quantification of the density of urocortin-3 baskets per mm2 in the different LS subnuclei of P30 WT (green circles) and cKO (purple squares) mice. C-F: N = 8 (4 males, 4 females) for WT; N = 7 (3 males, 4 females) for cKO. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences. E, F and G show quantifications in rostro-caudal section II, as defined in Figure 1C.

Intrinsic electrophysiological properties of WT and cKO LS neurons.

A) Schematic of the experimental setup: whole-cell recordings were obtained from lateral septal neurons, both tdTomato+ and tdTomato–, on slices from WT and cKO animals at 3 weeks of age. B, C) Representative traces of the firing patterns of tdTomato– (B) and tdTomato+ (C) neurons of WT (top) and cKO (bottom) samples subjected to current injection at the indicated intensities. D-F) Comparison of intrinsic electrophysiological properties of LS neurons of WT and cKO samples: D, resting membrane potential; E, membrane capacitance; F, rheobase. G) Comparison of the firing frequency of tdTomato– (left) and tdTomato+ (right) LS neurons subjected to step-wise current injections at 25 pA intervals, from -200 to 150 pA (data are represented as average +/-SEM). For WT tdTomato+, n = 10 (D, E), n = 9 (F), n = 16 (G); for WT tdTomato–, n = 6 (D), n = 5 (E, F), n = 8 (G); for cKO tdTomato+, n = 16 (D, E), n = 13 (F), n = 7 (G); for cKO tdTomato–, n = 8 (D-F), n = 10 (G). After removal of outliers via Grubbs’ test, 2-way ANOVA tests with multiple comparisons were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

cKO mice display increased exploratory drive.

A-C) Top, cartoons illustrating the outline of each behavior test; bottom, summary of the main anxiety-related readout for both males and females. The behavioral tests performed were: A, open field; B, novel object; C, light-dark box. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences. D) Experimental design: mice were placed in a cylindrical arena with a single odor inlet near the bottom, and exposed to two consecutive phases, where either a neutral (Blank) or an aversive (TMT) smell was pumped into the arena. E) Occupancy plots showing the average proportion of experiment time spent in each location throughout the arena by WT (n = 15 males, 13 females) or cKO (n = 14 males, 10 females) mice, under blank odor (“Blank”) or anxiogenic (“TMT”) conditions, as well as the difference between both plots (“cKO-WT”), separated by sex (top, males; bottom, females). F) Plot displaying the average occupancy of the area within a 40 mm radius around the odor inlet during Blank (gray triangles) and TMT (golden triangles) conditions, separated by sex and genotype. Each line joins the occupancy values of an individual mouse. Bars indicate the average. Note the same range in the y-axis of males and females. After removal of outliers via Grubbs’ test, 2-way ANOVA tests with multiple comparisons were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

Nkx2.1-lineage neurons in the LSd are activated by an acute stressful stimulus.

A) Experimental design: mice were subjected to 30 minutes of forced restraint and sacrificed 1 hour later; neurons firing in response to the anxiogenic stimulus were identified by immunofluorescence staining for c-Fos. Scale bars, 250 µm. B) Examples of coronal sections of the septum of WT (left) and cKO (right) mice after the forced restraint experiment, immunostained for c-Fos (green) and NeuN (blue). C) Closeup view of the white dashed line boxes in B, showing c-Fos (green) and tdTomato (magenta), in the different subnuclei within the LS of WT (left) and cKO (right) mice. Yellow arrowheads indicate examples of cells positive for both c-Fos and tdTomato; cyan arrowheads highlight c-Fos+, tdTomato– cells, and the empty yellow arrowhead shows an example of a c-Fos–, tdTomato+ cell. Scale bars, 50 µm. D) Comparison of the density of c-Fos+ neurons in the LS of untreated controls (‘baseline’, empty symbols; n = 4 WT, green circles; n = 4 cKO, purple squares) and animals subjected to forced restraint (‘restraint’, full symbols; n = 4 WT, green circles; n = 4 cKO, purple squares). E) Proportion of tdTomato+ neurons within the c-Fos+ population in the LS of WT mice, comparing untreated controls (‘baseline’, empty circles, n = 4) and animals subjected to forced restraint (‘restraint’, full circles, n = 4). F) Comparison of the density of c-Fos+ neurons in the LSd of WT (green circles, n = 4) and cKO (purple squares, n = 4) animals subjected to forced restraint. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

related to Figure 1.

A) Coronal sections along the rostral (left) to caudal (right) axis through the forebrain of P30 WT (top) and cKO (bottom) mice, submitted to immunofluorescence staining for tdTomato (magenta) and counterstained with DAPI (blue). Scale bars, 250 µm. B) Quantification of the density of cells positive for tdTomato per mm2 in the different subnuclei within the lateral septum of WT (green circles, n = 9) and cKO (purple squares, n = 9) mice. LSd, dorsal lateral septum; LSi, intermediate lateral septum; LSv, ventral lateral septum. C) Quantification of the density of cells positive for Zic per mm2 in the lateral septum of WT (green circles, n = 4) and cKO (purple squares, n = 4) mice. D) Quantification of the area of the septum relative to the total area of its corresponding coronal brain section in WT (green circles, n = 6) and cKO (purple squares, n = 5) female mice. Measurements are normalized to the corresponding WT average. E) Quantification of the density of cells positive for tdTomato per mm2 in the lateral septum of WT (green symbols, n = 5 males, 4 females) and cKO (purple symbols, n = 5 males, 4 females) mice, as in Figure 1D, separated by sex as indicated. F) Example images of the septa of P30 WT (left) and cKO (right) mice, submitted to immunofluorescence staining for Sox9 (green) and counterstained with DAPI (blue). Scale bars, 250 µm. G) Quantification of the density of cells positive for Sox9 per mm2 in the lateral (left) and medial (right) septum of WT (green circles, n = 4) and cKO (purple squares, n = 4) mice. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

related to Figure 2.

A) Uniform Manifold Approximation and Projection (UMAP) plot representing a 2D dimensional reduction of the transcriptional similarity among all cells identified within the dataset (24323 cells from a total of 6 WT and 6 cKO animals [3 males and 3 females each]). B) Dot plot showing the expression of select marker genes in each of the clusters in A, allowing their identification as distinct cell types. Clusters containing septal neurons are highlighted by dashed red lines. C) UMAP plot of all septal neurons as identified in A and B (8783 cells). D) Dot plot showing the expression of select marker genes in each of the clusters in C, allowing their identification as distinct cell types. Clusters containing lateral septum neurons are highlighted by dashed red lines. E) Bar graph showing the proportion of cells belonging to the Nkx2.1-lineage (green) vs. all other cells (gray) within each of the 17 clusters outlined in Figure 2B. F) Violin plots showing the expression levels of select marker genes within clusters 7, 8, 9, and 13 of Figure 2B (i.e. all clusters where ≥75% of cells belong to WT samples), split by genotype (WT, green; cKO, purple) and lineage (Nkx2.1-lineage: GFP-positive; all other lineages: GFP-negative). G) Bar graphs showing the density (per mm2) of all cells positive for Crhr2 (gray squares) and the subset of those cells that are also tdTomato+ (red circles), in sections I and III of WT vs. cKO animals. H) Quantification of the percentage of Crhr2+ cells that are tdTomato+ throughout the entire LS of WT (green circles) vs. cKO (purple squares) animals. I-K) Detailed quantifications of total Crhr2+ cell density (I); Crhr2+, tdTomato+ cell density (J); and % of tdTomato+ cells within the Crhr2+ population (K), split by sections across the rostro-caudal axis (sections I, II and III) and by LS subdivisions (LSd, LSi, LSv), as shown in Figure 1. G-K: (N = 3 [2 males, 1 female] for WT; N = 4 [2 males, 2 females] for cKO)

related to Figure 3.

A) Overview coronal images of the septum of P30 WT (left) and cKO (right) mice submitted to immunofluorescence staining for enkephalin (gray). Scale bars, 250 µm. B) Closeup view of the white dashed line boxes in A, showing the combined signals for enkephalin (green) and urocortin-3 (magenta), in the LS of WT (top) and cKO (bottom) mice. Scale bars, 50 µm. C) Quantification of fluorescence intensity in the different LS subnuclei of P30 WT (green circles) and cKO (purple squares) brains where enkephalin was detected by immunofluorescence staining, at three levels along the rostro-caudal axis (Sections I through III). D) Quantification of the density of enkephalin+ baskets per mm2 in the different LS subnuclei, at three levels along the rostro-caudal axis (Sections I through III), in WT (green circles) and cKO (purple squares) mice. E) Overview coronal images of the septum of P30 WT (left) and cKO (right) mice submitted to immunofluorescence staining for tyrosine hydroxilase (TH, gray). Scale bars, 250 µm. F) Overview coronal images of the septum of P30 WT (left) and cKO (right) mice submitted to immunofluorescence staining for serotonin (5-HT, gray). Scale bars, 250 µm. C-D: N = 8, N = 5 and N = 5 for WT; N = 5, N = 4 and N = 3 for cKO in Sections I, II, and III, respectively. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.

A-H) Comparison of intrinsic electrophysiological properties of tdTomato+ and tdTomato– neurons in the LS of WT and cKO samples at 3 weeks of age: A, input resistance; B, membrane time constant; C, peak amplitude of the first spike; D, latency to the first spike; E, half-width; F, spike threshold; G, adaptation ratio; H, afterhyperpolarization. After removal of outliers via Grubbs’ test, 2-way ANOVA tests with multiple comparisons were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences. For WT tdTomato+, n = 10 (A, B), n = 9 (C, D, G), n = 8 (E, F, H); for WT tdTomato–, n = 6 (A, B, C, D, E, F, H), n = 5 (G); for cKO tdTomato+, n = 16 (A,) E, G, J, K, Q, R), n = 15 (B), n = 13 (D, E, F), n = 12 (C, H), n = 11 (G); for cKO tdTomato–, n = 8 (A-H).

related to Figure 5.

A) Overview of the timeline used when performing anxiety-related behavior tests. B-D) Left, cartoons illustrating the outline of each behavior test; right, summary of the main anxiety-related readout for males and females. The behavioral tests performed were: B, elevated plus-maze; C, social interaction; D, Y-maze. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences. E-L) Syllable usage in female (E-H) and male (I-L) WT mice during both parts of the experiment, with syllables most enriched in the corresponding portion of the experiment or the genotype as indicated above each graph. Data points in E-L represent the average ± 95% confidence interval of the proportion (in %) of test time spent using the corresponding syllable. Significantly different syllable usage (indicated by asterisks) was determined using a Kruskal-Wallis test, post-hoc Dunn’s two-sided test with permutation, and multiple comparisons correction using the Benjamini-Hochberg procedure with a false discovery rate of 0.05.

related to Figure 4.

A) Examples of coronal sections of the septum of WT (left) and cKO (right) age-matched control mice (i.e., not subjected to the forced restraint experiment), immunostained for c-Fos (green) and counterstained with DAPI (blue). B) Quantification of the density of c-Fos+ neurons per mm2 in the entire LS of WT (green circles, n = 4) and cKO (purple squares, n = 4) control animals. C) Proportion of tdTomato+ neurons within the c-Fos+ population in the different subnuclei within the LS of WT (green circles, n = 4) and cKO (purple squares, n = 4) mice subjected to forced restraint. D) Proportion of tdTomato+ neurons within the c-Fos+ population in the LS of cKO mice, comparing untreated controls (‘baseline’, empty squares) and animals subjected to forced restraint (‘restraint’, full squares). E) Comparison of the density of c-Fos+ neurons in the LSi (top) and the LSv (bottom) of WT (green circles) and cKO (purple squares) animals subjected to forced restraint. Unpaired t-tests with Welch’s correction were performed; the p-values are shown above the corresponding compared sets of data: bold typeface indicates statistically significant (p<0.05) differences.