Fig. 6: Electrophysiological characterization of CapChRs in organotypic hippocampal slice culture.
From: Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling
A Construct design and fluorescent confocal micrographs showing organotypic hippocampal cultures (DIV 20) transduced with AAVs-expressing mScarlet-tagged ChRs. All three constructs C2-LC, CapChR1 and CapChR2 were homogeneously expressed throughout the hippocampal tissue after 3–4 days post-transduction (example shown for CapChR2). B Representative whole cell current traces depicting the effects of 500 ms of 490 nm (1–2 mW/mm2, saturating) light exposure at different calcium concentrations. C Charge transfer (normalized to values at 0 mM [CaCl2]e) between 1 and 2 s of illumination (n = 4 for C2-LC; n = 5 for CapChR1; n = 4 for CapChR2). D Photocurrents from CapChR2 in presence of blockers and the total charge transfer in % of the control. Apamin (Apam; 10 µM) Paxilline (PAX; 10 µM) (n = 3), Ryanodine (Ry, 50 µM) (n = 4) and Niflumic Acid (NFA, 0.5 mM, n = 4). E Effects of NFA application on photocurrents of C2-LC (n = 7), CapChR1 (n = 6) and CapChR2 (n = 7). F Exemplary whole cell voltage traces of CapChR2-expressing neurons, depicting the effects of 1 s of 490 nm light exposure in current clamp mode in the presence or absence of NFA in the bath solution. G Number of spikes elicited during illumination in (F) (mean, ±SEM; n = 4 cells). H Summary of the effect of NFA on pyramidal cell depolarization (mean, ±SEM; n = 4). I Schematic representation of the physiological response in CapChR2-expressing slices. Calcium entry via CapChR2 elicits calcium store release and activates calcium-activated chloride channels (TMEM16). Graphs depict the mean ± SEM. n = X biologically independent cells. AU arbitrary units, EC extracellular, IC intracellular, ER endoplasmic reticulum.
