Mapping visual functions onto molecular cell types in the mouse superior colliculus

Abstract

The superficial superior colliculus (sSC) carries out diverse roles in visual processing and behaviors, but how these functions are delegated among collicular neurons remains unclear. Here, using single-cell transcriptomics, we identified 28 neuron subtypes and subtype-enriched marker genes from tens of thousands of adult mouse sSC neurons. We then asked whether the sSC's molecular subtypes are tuned to different visual stimuli. Specifically, we imaged calcium dynamics in single sSC neurons in vivo during visual stimulation and then mapped marker gene transcripts onto the same neurons ex vivo. Our results identify a molecular subtype of inhibitory neuron accounting for ∼50% of the sSC's direction-selective cells, suggesting a genetic logic for the functional organization of the sSC. In addition, our studies provide a comprehensive molecular atlas of sSC neuron subtypes and a multimodal mapping method that will facilitate investigation of their respective functions, connectivity, and development.

Keywords: cell types; direction selectivity; inhibitory neurons; single-cell sequencing; superior colliculus; two-photon calcium imaging.

Figure 1.. Molecular identification of superficial superior colliculus (sSC) neuron subtypes

A. Schematic of single-nuclei RNA-sequencing (snRNA-seq) workflow. B. UMAP (uniform manifold approximation and projection) plot of 26,186 sSC neurons clustered by expression of highly variable genes and colored based on cluster identity. Each dot represents a neuron. C. Dendrogram illustrating relatedness of 28 sSC neuron populations, followed by the cluster name, annotated based on selected marker gene(s); number of cells per cluster; number of genes detected per cluster; and plot showing average expression and percent detection of the marker gene selectively enriched for each cluster. See also Figure S1 and S2.

Figure 2.. Neurotransmitter properties of sSC neuron subtypes

A. Expression of excitatory markers (vGluT1, vGluT2, and vGluT3 in magenta) and inhibitory markers (vGAT, Gad1 and Gad2 in cyan) for each cluster. B. UMAP plot color coded based on vGluT2 and vGAT co-expression (gray, low co-expression; magenta/cyan, high co-expression). C. Pie chart showing the percentage of excitatory and inhibitory neurons in the sSC snRNA-seq dataset. D. Fluorescence in situ hybridization (FISH) of vGluT2 and vGAT expression in the SC. An 800 μm × 250 μm yellow rectangle is used to quantify expression and further divided into 16 sub-regions along the SC depth. Staining within the area were shown to the right: DAPI, gray; vGluT2, magenta; vGAT, cyan. Scale bars, 200 μm (left) and 100 μm (right). E. Bar plot showing the percentage of vGluT2+ and vGAT+ neurons within the quantified area as illustrated in D. Each dot is from one image. Mean ± SEM. N = 16 images from 3 mice. F. Plot of excitatory (magenta) and inhibitory (cyan) neuron density along SC depth. G. Bar plot of excitatory (magenta) and inhibitory (cyan) percentage along depth. H. FISH of Vip, vGluT2, and vGAT transcripts in SC and cortex. The areas in the white squares are shown at higher magnification to the right. Scale bar, 200 μm (left) and 50 μm (right). I. Percentage of Vip co-localization with vGluT2 and vGAT. Mean ± SEM. N = 3 animals. See also Figure S3.

Figure 3.. Layer-specific distribution of neuron subtypes in the sSC

A - D. FISH showing spatial expression of marker genes that are restricted to a sublayer (A, B) or spanning multiple sublayers (C, D). Inset: UMAP plot where cells are colored based on their expression of marker genes. Corresponding clusters are highlighted with the dashed line. The laminar organization of SC is indicated below each image according to Allen Mouse Brain reference atlas. DpG: deep gray layer; InWh: intermediate white layer; InG: intermediate gray layer; SO: stratum opticum; and SGS: stratum griseum superficiale. Scale bar, 200 μm. E. Summary table of spatial distribution of sSC neuron subtypes. Individual subtypes are restricted to upper SGS (uSGS), lower SGS (lSGS), SO, or span entire SGS (uSGS and lSGS) or entire SC. See also Figure S4 and S5.

Figure 4.. Mapping visual responses to neurons expressing identified molecular markers.

A. Measurement of visual responses in the sSC. 1. Imaging of the sSC in awake head-fixed mice using two-photon calcium imaging 2. Left panel, average imaging plane of in vivo functional characterization; right panel, sparse labelling by a red fluorescent protein. 3. Selection of regions of interest (ROIs) for the quantification of visual responses. B. FISH of selected marker genes identified by snRNA-seq. 1. Horizontal sectioning and multiplexed FISH following in vivo imaging. 2. Acquisition of individual round of FISH for each marker gene. 3. Identification of ROIs expressing marker genes. C. Registration of in vivo and ex vivo photomicrographs. Selected ROIs in the functional imaging are overlayed with the histological results, allowing the mapping of visual response properties onto molecularly defined cell types based on probe staining. D. Changes in GCaMP6f fluorescence intensity obtained by 2-photon calcium imaging. Shaded areas represent the duration of stimulus presentation, with arrows representing stimulus direction. E. Overlayed response time courses for all registered neurons, grouped by the corresponding marker genes for which they are positive. The response was at the preferred direction of each neuron, i.e., the direction that elicited the highest change from baseline. F. Corresponding tuning curves, representing the average response for each direction aligned to the neuron’s preferred direction (vertical bar). G. Average peak response magnitude to the preferred direction of each registered neuron as a function of marker gene identity. For non-DS neurons, the direction that caused the largest response was selected and the corresponding magnitude was plotted. For suppressed neurons, the minimum was used as the “peak response” in this plot. Mean ± SEM. H. Distribution of gDSI of all registered neurons as a function of marker gene identity. Neurons are classified as responsive if their strongest responses were above 20% ΔF/F0 (shaded in light and dark green) and classified as direction selective (DS) if their gDSI was above 0.2 (dark green). Suppressed neurons are those that showed a decrease in response during stimulus presentation (purple shading). I. Percentage of DS, non-DS, suppressed and non-responsive neurons in each group. J. Venn diagram showing the overlap of neurons positive for a particular candidate marker gene and the total DS population. (total Vip: n=79; Chrnb3: n=12; Cbln4: n=63; and Itga7: n=36, see Figure S4 for animals and replicates per probe). See also Figure S5 and S6.