Intensity-dependent timing and precision of startle response latency in larval zebrafish

Abstract

Key points: Using high-speed videos time-locked with whole-animal electrical recordings, simultaneous measurement of behavioural kinematics and field potential parameters of C-start startle responses allowed for discrimination between short-latency and long-latency C-starts (SLCs vs. LLCs) in larval zebrafish. Apart from their latencies, SLC kinematics and SLC field potential parameters were intensity independent. Increasing stimulus intensity increased the probability of evoking an SLC and decreased mean SLC latencies while increasing their precision; subtraction of field potential latencies from SLC latencies revealed a fixed time delay between the two measurements that was intensity independent. The latency and the precision in the latency of the SLC field potentials were linearly correlated to the latencies and precision of the first evoked action potentials (spikes) in hair-cell afferent neurons of the lateral line. Together, these findings indicate that first spike latency (FSL) is a fast encoding mechanism that can serve to precisely initiate startle responses when speed is critical for survival.

Abstract: Vertebrates rely on fast sensory encoding for rapid and precise initiation of startle responses. In afferent sensory neurons, trains of action potentials (spikes) encode stimulus intensity within the onset time of the first evoked spike (first spike latency; FSL) and the number of evoked spikes. For speed of initiation of startle responses, FSL would be the more advantageous mechanism to encode the intensity of a threat. However, the intensity dependence of FSL and spike number and whether either determines the precision of startle response initiation is not known. Here, we examined short-latency startle responses (SLCs) in larval zebrafish and tested the hypothesis that first spike latencies and their precision (jitter) determine the onset time and precision of SLCs. We evoked startle responses via activation of Channelrhodopsin (ChR2) expressed in ear and lateral line hair cells and acquired high-speed videos of head-fixed larvae while simultaneously recording underlying field potentials. This method allowed for discrimination between primary SLCs and less frequent, long-latency startle responses (LLCs). Quantification of SLC kinematics and field potential parameters revealed that, apart from their latencies, they were intensity independent. We found that increasing stimulus intensity decreased SLC latencies while increasing their precision, which was significantly correlated with corresponding changes in field potential latencies and their precision. Single afferent neuron recordings from the lateral line revealed a similar intensity-dependent decrease in first spike latencies and their jitter, which could account for the intensity-dependent changes in timing and precision of startle response latencies.

Figure 1. Combined field potential recordings and high‐speed videos of C‐starts allow for discrimination between SLCs and LLCs

A, diagram of recording apparatus depicting a transgenic larval zebrafish head‐mounted in agarose (grey square inside light blue Petri dish). C‐starts were evoked by activating ChR2 with a blue LED. Field potentials and videos were recorded simultaneously and were time‐locked to stimulus onset. B, representative field potentials from an SLC (top) and LLC (bottom) response. Blue arrow indicates stimulus onset. Inset: large, initial biphasic peak of the SLC field potential waveform. C, selected frames (1 ms per frame) from videos of C‐starts that occurred with the field potentials in B. First frame is at stimulus onset (Stim on; t = 0 ms). Subsequent frames show initiation of tail movement (C‐start onset), time of maximum angular velocity (Max ang vel), and time of the maximum tail angle (Max tail angle). D and E, change in tail angle (D) and angular velocity (E) over time (1 circle per 1 ms frame) for the SLC (top) and LLC (bottom) responses shown in C with coloured symbols corresponding to coloured times in C. Asterisks in C–E indicate the subtle contraction seen at onset of all LLCs. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. SLCs and LLCs have overlapping latencies with response probabilities that depend on stimulus intensity

A, relative frequency histogram of C‐start startle responses for SLCs (black bars), LLCs (grey bars), and failures (white bars) at each delivered intensity. Failures were defined as responses where the larva did not perform a C‐bend upon optical stimulation. Light intensities are binned at 50 W m⁻² per bin. B, relative frequency histogram of C‐start latencies for the population of 205 SLCs (black bars) and 29 LLCs (grey bars). Startle latencies are binned at 20 ms per bin.

Figure 3. Timing and precision of SLC field potentials and C‐starts are correlated with light intensity

A, left panel: diagram of the features of the field potential (FP): FP latency, FP max amplitude, and FP slope. A, right panel: diagram of three features of the C‐start: C‐start latency, tail angle, and angular velocity. B, representative SLC field potentials (left) and tail angle vs. time plots (right) from a single larva evoked at increasing stimulus intensities. C, box plots of parameters from SLC field potentials and C‐starts (n = 6 animals). Coloured symbol represents mean, box represents 25th to 75th percentiles, and whiskers represent minimum to maximum values. FP max amplitude was normalized for each animal. Numbers in top panel correspond to total trial number (n) from all animals for each intensity bin (50 W m⁻²) across all panels in C–E. D, dose–response plots of mean FP latency (left top) and mean C‐start latency (left bottom) versus stimulus intensity are fitted by single‐phase exponential decay equations. Data are represented as means ± SD (shaded area). The SD at each intensity bin for FP latencies (right top) and C‐start latencies (right bottom) are also fitted by single‐phase exponential decay equations. E, plots of the difference between the mean FP latency and the C‐start latency (left) and between the FP latency SD and the C‐start latency SD (right) at each intensity. The results were fitted by linear equations with zero slope. F, the latencies of C‐starts and underlying field potentials are linearly correlated (dashed line) with a slope of approximately 1. The Y‐intercept (4.9 ± 0.3 ms) of the linear fit corresponds to the mean time delay between field potential onset and initiation of the C‐start. G, the mean delay is also determined by subtraction of FP latency from C‐start latency for every recorded SLC response (n = 205). The resulting values are normally distributed with a histogram fitted by a Gaussian distribution. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Intensity‐dependent changes in evoked spike trains in afferent neurons during optical hair‐cell stimulation

A, cartoon diagram of recording setup for optical stimulation of ChR2‐expressing hair cells within a single neuromast. PLLg = posterior lateral line ganglion of afferent neurons. NM = posterior lateral line neuromast. B, representative responses from a single afferent neuron to 50 ms optical stimuli of different intensity. C, mean spike number (n = 10 repetitions) per stimulus interval from the recording in B for a range of stimulus intensities. D, mean FSL (n = 10 repetitions) from the recording in B as a function of stimulus intensity. E, left panel: mean spike number (n = 5 neurons) within the 50 ms stimulus interval (open circles) and within the FP Latency interval for minimal, moderate and maximal light intensities (see Methods section; filled squares). E, right panel: SD of spike number plotted as a function of stimulus intensity. F, left panel: mean FSL (n = 5 neurons) for each stimulus intensity (filled circles). F, right panel: intensity‐dependent changes in jitter. G, comparison of mean FP Latency (n > 20 repetitions; n = 6 larvae) versus mean FSL at minimal, moderate and maximal light intensities. Data are represented as means ± SD (bars in C, D and G and shaded area in E and F). Data in C were fitted by a Hill equation (dashed line) and data in D–G were fitted by either a single‐phase exponential decay or linear regression equation (dashed lines). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Intensity‐dependent changes in amplitude of microphonic potentials generated by stimulation of neuromast hair cells with a fluid jet mechanical stimulus

A, average traces (n = 200 repetitions) of microphonic potentials displayed for multiple pressure intensities in response to a 50 ms square‐wave stimulation (bottom trace) delivered to a single neuromast. The dashed red line indicates the onset time of the microphonic potential (∼8 ms) after stimulus onset. B, plot of the total microphonic potential (quantified as the area under the evoked portion of the microphonic waveform divided by the duration of stimulus, 50 ms) at each recorded stimulus intensity from the recordings of a single neuromast in A. C, plot of normalized data from multiple neuromast recordings (n = 5 neuromasts; n = 4 larvae). Data in B and C were fitted by Hill equations (dashed line). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Intensity‐dependent changes in evoked spike trains in afferent neurons during mechanical stimulation of hair cells

A, cartoon diagram of recording setup for mechanical stimulation of PLL hair cells using a fluid jet. PLLg = posterior lateral line ganglion of afferent neurons. NM = posterior lateral line neuromast. B, representative responses from a single PLL afferent neuron to 50 ms mechanical stimuli at different pressure intensities (same neuron from Fig. 4 B–D). C, mean spike number (n = 10 repetitions) per stimulus interval from the recording shown in B for a range of stimulus intensities. D, mean FSL (n = 10 repetitions) from the recording in B as a function of stimulus intensity. Ea, mean spike number (n = 5 neurons) within the 50 ms mechanical stimulus interval. Eb, intensity‐dependent changes in SD of spike number. Fa, mean FSL (n = 5 neurons) for each stimulus intensity. Fb, intensity‐dependent changes in jitter. Data are represented as means ± SD (bars in C and D and shaded area in E and F). Data in C–F were fitted by either a single‐phase exponential decay or linear regression equation (dashed lines). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. Cartoon model of the mechanism underlying intensity‐dependent changes in the timing and precision of SLC startle response latencies in larval zebrafish

A, stimuli of increasing intensity generate a greater depolarization of the receptor potential of hair cells (microphonic traces beneath brown hair cells), which leads to faster and larger release of neurotransmitter and more rapid and precise initiation of action potentials in afferent neurons (green traces below grey afferent neurons). The afferent neuron from the lateral line is represented by a dashed line as lateral line inputs have not been shown to evoke startle responses independently from ear afferent neurons. Earlier arrival of first spikes with less jitter at the Mauthner cell (purple) leads to more rapid spiking of the M‐cell with greater precision. Propagation of the M‐cell spike generates excitation of motoneurons (blue) and muscle cells (grey) that together produce the whole‐animal field potential (red traces beneath the M‐cell) and initiate the rapid, C‐shaped contraction of the larval zebrafish (orange traces and images beneath the muscle cells). B, data presented here are consistent with the intensity‐dependent changes in latency of startle responses occurring downstream of hair cell depolarization (blue bars; fixed length) and upstream of spike arrival at the M‐cell (green bars; variable length), which would include presynaptic vesicle fusion, neurotransmitter release, postsynaptic depolarization, and spike initiation. Following a short delay before onset of the field potential (red bar; fixed length), there is a time delay of approximately 4 ms (from Fig. 3 F and G) from onset of the startle circuit until onset of the C‐start contraction (orange bar; fixed length), which probably arises from signal propagation through the M‐cell, motoneurons, and muscle cells that generate the startle response. [Colour figure can be viewed at wileyonlinelibrary.com]