Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na+/K+ ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory. Electronic supplementary material The online version of this article (10.1007/s13238-020-00720-y) contains supplementary material, which is available to authorized users.

(B) Confirmation of the recording neuron. Left panel: neurobiotin staining in brain slice. Red for Texas Red staining; blue for DAPI, scale bar: 50 µm. Right panel: 3-D reconstruction of respective neurons. Scale bar, 30 µm. Figure S2: Basic electrophysiology properties of recorded neurons (A-B) Statistics of membrane capacitance (A) and input resistance (B) of recorded neurons (n=11 for LS and n=11 for NS). Error bar, mean ± s.e.m. The significance was estimated based on the corrected P value using the unpaired t-test, P >0.05 for non-significance.   (B) Visualization of the expression of known marker genes for excitatory neurons (Slc17a7), layer 2/3 (Cux1 and Cux2) and layer 4 (Rorb) via t-SNE. Cells were colored according to the gene expression levels (red, high; grey, low).
(C) Visualization of interneurons from published visual cortex scRNA-seq data via t-SNE. Each dot represents an individual cell. Cells were annotated to 14 subclusters.
(D) Visualization of the expression of known marker gene for interneuron (Gad1), layer 2/3 (Cux1 and Cux2) and layer 4 (Rorb) via t-SNE. Cells were colored according to the gene expression levels (red, high; grey, low).

(E) Integration of our data with layer 2/3 excitatory neurons and interneurons from published data was visualized via t-SNE. Excitatory neurons and interneurons were annotated into 10 and 4 subclusters, respectively.
(F) The distributions of cells from our study were visualized by t-SNE. Dots were colored based on the major cell type annotated in our sequencing dataset and the published dataset, (excitatory neurons in the published data, light grey; interneurons in the published data, grey; LS neurons from our data, orange; NS neurons from our data, blue).  . Error bar, mean ± s.e.m. The significance was estimated based on the corrected P value using the unpaired t-test, P >0.05 for non-significance.
Video S1: Two-photon in vivo calcium imaging of V1 neurons in light-evoked mouse, followed by LS neuron soma extraction.
Cal 520-labeled cells in layer 2/3 of mouse V1 were recorded to measure the calcium response to the 5time repeated light stimulus, under the 'green' channel of the two-photon microscope. A light-responding neuron was subsequently targeted to extract the contents of the soma via a glass cuspidal electrode. The light response cells are indicated by yellow arrows, and the target cell is indicated by the red arrow. The frame of calcium imaging was shifted to the left for a better view of the soma extraction. The cells marked with white arrowheads are coordinate references for locating the target cell. Scale bar, 20 µm.

Video S2: Two-photon in vivo whole-cell configuration.
Left panel: 5-minute movie of whole-cell patch clamp configuration with the cuspidal electrode under the 'red' channel of the two-photon microscope. The shadow of the target cell is visible. Scale bar, 20 µm. Right panel: the corresponding membrane test monitor from the recording software of Clampex. The cells marked with white arrowheads are coordinate references for the target cell. Scale bar, 20 µm.

Video S3: In vivo dual recording of light-evoked calcium response and action potential firing.
Left panel: the video shows the calcium response to light stimulation of a whole-cell patched neuron (Cal-520 labeled), under the 'green' channel of the two-photon microscope. The target neuron is indicted by a yellow arrow, and the position of the glass electrode is marked. Scale bar, 20 µm. Right panel: the calcium response and the action potential firing are dynamically represented in the 'calcium signal' and 'electrophysiological signal' windows, respectively. Video S4: 3-D morphological reconstruction of a neuron in layer 2/3 of V1.