Supplementary Figure 2: CoChR-Na_V1.2(II–III)-GFP expression and photostimulation in cultured hippocampal neurons

(a-d) Images are presented for cultured hippocampal neurons expressing CoChR-Na_V1.2(II-III)-GFP, along with the countermarker mCherry. (a) A hippocampal neuron in culture expressing CoChR-Na_V1.2(II-III)-GFP and mCherry, seen in the GFP channel (scale bar: 100 µm). (b) Zoomed-in image from the yellow rectangle of a. (c) The neuron of b, seen in the mCherry channel (magenta), along with other nearby neurons. (d) Merge of b and c (scale bar for b-d: 20 µm). (e) Whole cell current clamp recording of a cultured hippocampal neuron expressing CoChR-Na_V1.2(II-III)-GFP, under current injection (10 ms duration; gray rectangle) and optical stimulation (480nm, 34.84mW/mm², 1 ms duration; blue rectangle). Rectangles not to scale. (f) Box-and-whiskers plot of GFP brightness versus position along a neurite, normalized to GFP brightness at the soma, extracted from neurites of cultured hippocampal neurons expressing CoChR-Na_V1.2(II-III)-GFP (n = 5 neurites taken from 5 cells from 4 cultures). Red line denotes the median. Top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Top and bottom whiskers indicate the highest and lowest values respectively.