Light-activated postsynaptic currents. (A) (Left) Acetylcholine postsynaptic current evoked by a 3-ms light pulse (Punc-17::ChR2::mCherry). (Middle) Electrically evoked current from the WT. (Right) A 3-ms light pulse failed to evoke postsynaptic response in unc-13(s69) Punc-17::ChR2::mCherry. (B) GABA postsynaptic current evoked by a 3-ms light pulse (Punc-47::ChR2::mCherry). (C) Comparison of amplitude (Left) and decay time (Right) between light-evoked (gray column, n = 9) and electrically evoked (white column, n = 16) postsynaptic currents. (Electrical: 2.01 nA ± 0.11, τ_decay = 5.6 ms ± 0.4; light: 1.96 nA ± 0.18, τ_decay = 7.4 ms ± 0.5; *P < 0.01, 2-sample t test). (D) UNC-13 is required for light-evoked postsynaptic currents (0.03 nA ± 0.01, n = 5, **P < 0.00001, 2-sample t test).

Repetitive stimulation of postsynaptic currents. (A) Acetylcholine currents evoked by 10 Hz (Left), or a 5-s continuous (Right) light pulse in Punc-17::ChR2::mCherry. (B) GABA postsynaptic currents evoked by 10 Hz (Left) or a 5-s continuous (Right) light pulse in Punc-47::ChR2::mCherry. (C) Recovery of acetylcholine current 10 s after a single light pulse (Left) or after a 5-s train of 10 Hz light pulses (Right). (D) Recovery 10 s after a single light pulse (91.5% ± 2.2%, white column, n = 15) or after a 10-Hz train (52.2% ± 3.2%, gray column, n = 23; *P < 0.00001). (E) Normalized amplitudes (to the first peak amplitude in a 10-Hz train) of evoked acetylcholine currents were plotted with time. Red spheres: light-evoked currents were corrected to the relative muscle photocurrents recorded at 10 Hz (n = 10). Black squares: electrically evoked currents (n = 4). (F) An example of acetylcholine currents electrically evoked by a 10-Hz train from the WT.

Channelrhodopsin-induced photocurrents in muscle. (A) Photocurrents activated by a 1 s (Left) or 3 ms (Right) light pulse. The downward deflection of the blue line indicates that blue light is on. (B) Paired-pulse analysis of channelrhodopsin recovery from the steady state. (Top) Seven overlaid photocurrent traces activated by pairs of 3-ms light pulses with intervals of 50 ms, 100 ms, 200 ms, 500 ms, 1 s, 2 s, and 5 s. All traces were aligned to the first light pulse (black arrow). The second pulses are indicated by colored arrows. (Bottom) Ratio of the second to the first peak was plotted against paired-pulse intervals. The recovery curve was best fit by a double-exponential function (τ_fast = 0.6 s ± 0.5, τ_slow = 6.4 s ± 3.2, n = 6).

Graded acetylcholine release. (A) Photocurrents from muscle and postsynaptic currents from acetylcholine synapses evoked by blue light at variable intensities (Punc-17::ChR2::mCherry). Light intensities (mW/mm²): red, 9.9; orange, 4.9; green, 2.3; blue, 1.4; black, 0.6. (B) Muscle photocurrents (square)) and acetylcholine postsynaptic currents (circle) were normalized to their maximum amplitudes and plotted with light intensity. The normalized muscle and postsynaptic curves each were fitted with a single exponent and overlapped. Color arrows are corresponding to light intensities used in A. (C) Normalized postsynaptic currents are directly proportional to normalized muscle photocurrents. (D) Endogenous acetylcholine release recorded from Punc-17::Halo::GFP before, during, and after illumination. The downward deflection of the yellow line indicates that the yellow light is on. (E) Statistical comparison of mini frequency and amplitude before, during, and after light pulse. Mini frequencies: before (white column), 26.3 ± 5.4; during (yellow column), 10.2 ± 3.1; and after (gray column), 35.3 ± 5.5 (events/s). Mini amplitudes: before, 17.6 pA ± 0.9; during, 16.5 pA ± 1.7; after, 17.1 pA ± 0.9. *P < 0.01, 1-way ANOVA, n = 5.

Synaptic plasticity of acetylcholine and GABA synapses. (A) Corrected light-evoked acetylcholine currents were plotted with time at variable stimulation frequencies. Depression rates at low stimuli frequencies were best fit by single-exponential functions (0.2 Hz: τ = 4.71 s ± 1.24, n = 7; 1 Hz: τ = 0.58 s ± 0.11, n = 9; 5 Hz: τ = 0.09 s ± 0.02, n = 9). Depression rates at high stimuli frequencies were best fit by double-exponential functions (10 Hz: τ_fast = 0.02 s ± 0.03, τ_slow = 3.05 s ± 1.84, n = 10; 20 Hz: τ_fast = 0.02 s ± 0.01, τ_slow = 2.12 s ± 0.84, n = 13). (B) Corrected light-evoked GABA currents were plotted with time at variable stimulation frequencies. Stimuli at 1 Hz did not cause depression (dark blue line, linear fit, n = 8). Depression caused by 5 Hz light stimuli was best fit by a single-exponential function (τ = 1.13 s ± 0.47, n = 8). Depressions following the initial facilitation at 10 Hz and 20 Hz light stimuli were fit by single-exponential decay (10 Hz: τ = 0.83 s ± 0.14, n = 13; 20 Hz: τ = 0.62 s ± 0.08, n = 9). (C) Exogenous acetylcholine or GABA evoked currents. (D) Corrected light-evoked acetylcholine currents depress differently in unc-38(-) (n = 18) or acr-16(-) animals (n = 14).