Figure 2

Optogenetic stimulation to fine-tune responsivity of lc-LysM GEPII 1.0. (A) Schematic illustration of the setup for optogenetic stimulation of ChrimsonR during live cell imaging (created with BioRender.com). Cells are illuminated with a LED with an emission peak at 617 nm. Cells were co-transduced with AAV-Syn-GCaMP8m and AAV-Syn-ChrimsonR-tdT (left) or AAV-CAG-lc-LysM GEPII 1.0 and AAV-Syn-Chrimson-tdT (right) to allow imaging of calcium in neurons or potassium in neurons and astrocytes during neuronal stimulation respectively. Scale bar represents 25 µm. (B) Cells were stimulated with 10 ms pulses at a stimulation rate of 1 Hz. Representative traces of GCaMP8m [left, vertical scale: 0.2 (dF/F)] and lc-LysM GEPII 1.0 [right, vertical scale: 0.05 (normalized FRET ratio)] in response to different amounts of pulses. Each pulse elicited a calcium spike indicating successful and robust stimulation. Potassium changes were only visible after a train of 30 or more stimulations. (C) Quantification of the amplitudes of the normalized FRET ratio of lc-LysM GEPII 1.0 expressed in neurons in response to an increasing number of stimuli. A drop of neuronal potassium levels was observed starting at 30 stimulations and increased with the number of additional stimulations. Amplitudes were quantified as the difference of the FRET ratios between the baseline before the stimulation and the mean of the last two frames of each train of stimulation. Data is represented as mean ± SD (N = 46 neurons in n = 6 experiments). (D) Quantification of the amplitudes of the normalized FRET ratio of lc-LysM-GEPII1.0 targeted to the plasma lemmal space of neurons. Data is represented as mean ± SD (N = 25 neurons in n = 5 experiments).