Figure 4. Electrophysiological recording of photocurrents in primary motor neurons.

(A) Schematics of experimental setup for optogenetic stimulation with in vivo whole-cell patch clamp recordings. Image shows a patched primary motor neuron (pMN) expressing CoChR in a 6 dpf Tg(mnx1:GAL4;UAS:CoChR-tdTomato) larva. Scale bar 5 μm. (B) Membrane resistance was not affected by opsin expression (mean ± SD, across cells). (C) Resting membrane potential was similar between opsin-expressing and control neurons (mean ± SD). (D) Examples of inward photocurrents in response to 5 ms light pulses (20 mW/mm²). (E) Peak photocurrent amplitude. CoChR and ChrimsonR induced the largest photocurrents (mean ± SEM, across cells). Dotted lines show range of pMN rheobase. Data is pooled across stimulus intensity (1–30 mW/mm²) but see Figure 4—figure supplement 1 for currents at varying irradiance. (F) Photocurrent activation time was similar across opsins (mean ± SEM). (G) Chronos photocurrents had the fastest deactivation time constant, while CoChR and ChrimsonR showed similar deactivation kinetics (mean ± SEM). See also Figure 4—figure supplement 1.

Figure 4—figure supplement 1. Wavelengths used in electrophysiological recordings and photocurrent amplitude and kinetics as a function of irradiance.

(A) LED emission wavelength (centre/bandwidth, nm) and irradiance levels used for each opsin line and control cells. (B) Number of cells patched in each group. Numbers and coloured bars indicate included cells while grey bars indicate excluded cells (see Materials and methods for inclusion criteria). (C,D) Access resistance (C) and cell capacitance (D) were comparable between groups (mean ± SD, across cells). (E) Example photocurrents from a CoChR-expressing cell at different irradiance levels (3–20 mW/mm²). F–H Peak photocurrent amplitude (F), activation time (G) and deactivation time constant (H) vs. irradiance (mean ± SEM, across cells). Dotted lines in (F) show range of pMN rheobase. Asterisks indicate a significant non-zero slope.