Fig. 3: Two molecular subtypes of bushy cells.

a UMAP visualization of Hhip⁺ and Atoh7⁺ clusters and distribution of Patch-seq bushy cells in UMAP space, colored by cluster. Individual dots represent each Patch-seq cell. b UMAP visualization of two bushy cell clusters and normalized expression of Hhip, Atoh7, and Sst. Top: snRNA-seq, Bottom: Patch-seq. c FISH co-staining for Atoh7 and Hhip (left), Atoh7 and Sst (middle), or Hhip and Sst (right) in CN sagittal sections. Lines along the images indicate density of single-labeled or double-labeled cells along two axes of CN. Insets show the total counts of single-labeled and double-labeled cells across eight consecutive sagittal sections for each FISH experiment. d Left: A representative morphology of Hhip⁺ or Atoh7⁺ bushy cell. Axon (shown in red) is truncated. Right: Sholl analysis of Hhip⁺ or Atoh7⁺ bushy cell dendrites. Data are presented as mean ± SEM. n = 20 for Hhip⁺ cells from 15 mice, n = 51 for Atoh7⁺ cells from 20 mice, Two-way mixed model ANOVA. e Heat map displaying normalized expression of the top 20 DEGs for two subtypes. Scale bar: Expression level. Cell number in each subcluster is indicated below the figure. f Top: Example responses of Atoh7⁺ (left) or Hhip⁺ (right) bushy cell to a hyperpolarized current step and a near-threshold depolarizing step. Bottom-left: Individual APs from the Hhip⁺ and Atoh7⁺ cells shown above, aligned with onset of the depolarizing current. Bottom-right: Two most-discriminating features for Hhip⁺ and Atoh7⁺ cells, spike delay and spike duration (half-width). n = 67 for Hhip⁺ cells from 25 mice, n = 110 for Atoh7⁺ cells from 29 mice, Two-sided t-test. Data are presented as mean ± SEM. The dots above the bars represent the original values. g Volcano plot showing the log2 fold change (log2FC) and −log10(p-value) of detected genes comparing the two subtypes. The p-values were adjusted by Benjamini–Hochberg correction. Among DEGs are 15 genes encoding voltage-gated ion channels. See Fig. S9 for more analysis. Source data are provided in the Source Data file.