Figure 1.
The myo6b promoter drives ChR2-YFP expression in hair cells of the ear and lateral line.
(A) The myo6b:ChR2-YFP meganuclease construct was inserted into the zebrafish genome by plasmid injection along with the I-SceI enzyme into the 1-cell stage embryo. (B) Hair cells of a 5 days post fertilization (dpf) zebrafish larva express YFP (pseudo-colored yellow) in the lateral line and ear (arrow). The lateral line is composed of neuromasts, clusters of hair cells, at regular intervals around the head and the length of the fish (examples at arrowheads, scale bar 0.05 mm). B′ ChR2-YFP larval eye at 5 dpf. B″ Wild-type larval eye at 5 dpf. (C) YFP expression in neuromast hair cells. C′ DIC image of the neuromast C″ composite of YFP and DIC images (scale bar 0.0125 mm) (D) Adult transgenic fish continue to express ChR2-YFP (background removed for clarity, scale bar 2 mm). D′ YFP expression in an adult neuromast (scale bar 0.05 mm).
Figure 2.
Comparison of spike encoding following mechanical and optical activation of hair cells.
(A) Mechanical stimulation of neuromast hair cells with a waterjet and optical stimulation with ∼470-nm light produced similar patterns of spiking in the afferent neuron. Stimulus protocol is below the traces (scale bar 100 ms). (B) Plots of all spikes in response to 60 repeated sweeps for the cell recorded in A, using either a mechanical (upper) or optical (lower) 100-ms stimulus. Each successive spike is represented by circles of incrementing color: first spike of each sweep is dark blue, the second light blue, the third green, the fourth brown, with subsequent spikes fading from brown to white. (C) The mean number of spikes per sweep for cells (n = 9) recorded with both mechanical (black) and optical (grey) stimuli. The overall mean plus SEM across all recorded cells are represented by the bars. The cell labeled red in C, D, and E corresponds to the cell shown in A and B. (D) The mean first spike latency for all cells recorded with either mechanical or optical stimuli. Symbols and bars are as in C. (E) The coefficient of variation (C.V.) of the first spike latency for all cells recorded with both mechanical and optical stimuli. Symbols and bars are as in C and D.
Figure 3.
Mechanical stimulation is required for the temporal fidelity of phase-locked spiking.
(A) Top: Phase-locked spiking for a mechanical 20-Hz stimulus. Middle: Phase-locked spiking for an optical 20-Hz stimulus. Bottom: 20-Hz stimulus protocol (scale bar 25 ms). Note that stimuli were 25-ms in length and delivered at a 20-Hz rate. (B) Polar plots from 60 sweeps of mechanical (upper) and optical (lower) stimulation. Plots constructed from all spikes elicited by 20-Hz stimulation of the cell shown in A. (C) The vector strength of phase-locked spiking for multiple cells recorded during 60 sweeps of 10, 20, and 40 Hz mechanical and optical stimulation. The bars represent the mean of the vector strength from all cells.
Figure 4.
Escape responses from touch and optical stimulation of wild type and myo6b:ChR2 transgenic larvae.
(A) Diagram depicting the Mauthner cells (M), a pair of neurons in the hindbrain of teleost fish. The axons of the M-cells project into the spinal cord where they synapse on primary motor neurons and elements of the central pattern generator responsible for left-right tail motions. (B, C) Diagrams of the setup for field recordings of M-cell potentials from larval zebrafish. (B) A waterjet was used to stimulate touch receptors on the head of a larva embedded in low melt agarose. (C) For optical stimuli, field potentials were collected from free-swimming transgenic larvae. (D) In both wild type and myo6b:ChR2 transgenic larvae, the M-cell was activated in response to a 100-ms touch stimulus (onset at arrowhead). (E) In wild-type larvae, the M-cell was not activated by flashes of ∼470-nm light (n = 18). Transgenic myo6b:ChR2 larva responded to ∼470-nm light with both a field potential (n = 55; scale bar 2 ms for D and E) and an escape response (not shown). (F) Increasing the duration of optical flashes increased the percentage of observed field potentials (seen in E) and escape responses in transgenic larvae (n = 12). Note that flashes that were 100-ms or greater resulted in 100% success rate for observed escape responses and field potentials.