Figure 5
iChloCCA, PhobosCA and AuroraCA in CA1 pyramidal cells in organotypic hippocampal slice cultures. (A) Representative photocurrent traces of a PhobosCA expressing CA1 cell evoked with different activation wavelengths and shutoff with 595 nm light. (B) Photocurrent traces in the same cell evoked with 460 nm light and shutoff with indicated wavelengths (10 mW/mm2). (C) Activation spectra (dashed lines) and inactivation spectra (solid lines) of PhobosCA, iChloCCA and AuroraCA in CA1 pyramidal neurons. Lines are interpolations of data points and shaded areas represent SEM (n = 2 to 9). (D) CA1 pyramidal neuron expressing PhobosCA-Citrine 5 days after electroporation (stitched maximum intensity projections of two-photon images, fluorescence intensity shown as inverted gray values). Citrine fluorescence was mainly localized to the plasma membrane across the entire cell. Inset shows magnified view of the apical dendrite. (E) Membrane voltage trace shows reversible suppression of depolarization-induced spiking by photoswitching PhobosCA between open and closed state. (F) Quantification of the spike rate during current injection at indicated time intervals before opening light pulse, after opening light pulse and after closing light pulse in CA1 neurons expressing iChloCCA-Citrine (n = 12 neurons in 12 slice cultures), PhobosCA-Citrine (n = 7) or AuroraCA-Citrine (n = 5). Gray symbols indicate individual experiments. Mean values are shown as rectangular symbols with SEM. ****p < 0.0001, repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test. (G) Quantification of the rheobase shift generated by PhobosCA or AuroraCA and iChloCCA during illumination with increasing light intensities. Bar plots show mean ± SEM. Single measurement data points are shown as open squares.
