Fig. 5 |. CellREADR-enabled targeting and recording of human cortical neuron types.

a, Schematic of binary AAV vectors for targeting human neuron types. ClipF, sequence coding for an engineered enzymatic tag here used as a spacer in the READR vector; smV5, sequence coding for spaghetti monster fluorescent protein fused with the epitope tag V5. b, Genomic structure of the human FOXP2 gene and the design of two sesRNAs targeting FOXP2 mRNA. c, READR targeting of human FOXP2 cells. Left, upper layer FOXP2 cells (labelled with mNeon, native fluorescence) targeted with READR^FOXP2(1) AAVs in organotypic slices from temporal neocortex. Right, deeper layer FOXP2 cells targeted with READR^FOXP2(2) from parietal neocortex. Insets show magnified views of the boxed regions. Wt. matter, white matter. d, Immunostaining of FOXP2 in neurons labelled with READR^FOXP2(2). Arrowheads indicate cells labelled with both mNeon (left) and FOXP2 immunofluorescence (middle); the arrow indicates a cell labelled with mNeon only. e, The specificity of READR^FOXP2(2) assayed by immunofluorescence. f, Current-clamp recording traces of three mNeon-labelled neurons (top). Input–output curves (bottom) for cells depict similar spiking behaviours. g, Morphology of cell 3 recorded in f. The cell was filled with biocytin and was visualized by silver staining on a bright field. Brown, cyan and purple arrowheads indicate soma, axons and dendrites, respectively. h, SesRNA designed to target exon 2 of human VGAT mRNA. i, An organotypic slice from temporal neocortex infected with AAV-READR^VGAT and TRE3g-mNeon and visualized with native fluorescence 8 days after infection. j,k, Morphologies of interneurons labelled by AAV-READR^VGAT. l, Colocalization of mNeon native fluorescence (left) with VGAT mRNA detected by in situ hybridization (middle). Arrowheads indicate neurons labelled with both mNeon and in situ hybridization; the arrow indicates a cell labelled with mNeon only. m, The specificity of READR^VGAT assayed by VGAT mRNA in situ hybridization. n, Current-clamp recording traces (top) of three labelled neurons, filled with biocytin and visualized with streptavidin. Input–output curves (bottom) depict distinct spiking behaviours: accommodating (blue), fast (green) and delayed onset (pink). o, The morphologies of cells labelled by READR^VGAT and recorded in n. Scale bars, 50 μm. Data in e,m are mean values. n = 2 biological replicates.