Physiological responses of mechanosensory systems in the head of larval zebrafish (Danio rerio)

Abstract

The lateral line system of zebrafish consists of the anterior lateral line, with neuromasts distributed on the head, and the posterior lateral line, with neuromasts distributed on the trunk. The sensory afferent neurons are contained in the anterior and posterior lateral line ganglia, respectively. So far, the vast majority of physiological and developmental studies have focused on the posterior lateral line. However, studies that focus on the anterior lateral line, especially on its physiology, are very rare. The anterior lateral line involves different neuromast patterning processes, specific distribution of synapses, and a unique role in behavior. Here, we report our observations regarding the development of the lateral line and analyze the physiological responses of the anterior lateral line to mechanical and water jet stimuli. Sensing in the fish head may be crucial to avoid obstacles, catch prey, and orient in water current, especially in the absence of visual cues. Alongside the lateral line, the trigeminal system, with its fine nerve endings innervating the skin, could contribute to perceiving mechanosensory stimulation. Therefore, we compare the physiological responses of the lateral line afferent neurons to responses of trigeminal neurons and responsiveness of auditory neurons. We show that anterior lateral line neurons are tuned to the velocity of mechanosensory ramp stimulation, while trigeminal neurons either only respond to mechanical step stimuli or fast ramp and step stimuli. Auditory neurons did not respond to mechanical or water jet stimuli. These results may prove to be essential in designing underwater robots and artificial lateral lines, with respect to the spectra of stimuli that the different mechanosensory systems in the larval head are tuned to, and underline the importance and functionality of the anterior lateral line system in the larval fish head.

Keywords: lateral line; mechanosensation; statoacoustic; trigeminal; zebrafish.

FIGURE 1

Overview of the lateral line and trigeminal system in zebrafish larvae. (A) Schematic representation of the lateral line system and its subsystems, the anterior lateral line (blue), and the posterior lateral line (green). The blue and green circles represent the anterior lateral line and posterior lateral line ganglia, respectively. All afferent neurons are located in the ganglia and receive their sensory inputs from neuromasts while sending signals via ascending projections into the hindbrain (cyan). For the sake of simplicity, only one neuromast (red circle) is shown. Left inset: maximum intensity z-projection of a larva expressing mCherry (magenta) driven by islet2B and SILL:GFP (green). Anterior lateral line ganglion (ALLG), posterior lateral line ganglion (PLLG), and trigeminal ganglion (TG). Shown are a transverse plane (bottom) and a sagittal plane (top). Right inset (top): maximum intensity z-projection of a larva expressing GFP (Brn3c:GFP) in hair cells. The left side shows a lateral view of a neuromast. The right side shows a dorsal view. Right inset (bottom): schematic representation of a neuromast. For convenience, only two hair cells are depicted (in yellow). Afferent connections from the ganglia neurons are shown in blue. Innervation by different efferent systems is illustrated in red. (B) Schematic overview of the trigeminal system in the zebrafish larvae. Shown are three representative trigeminal afferent neurons with their arborizations and fine nerve endings covering the skin of the head. (C) Schematic demonstration of the tactile stimulation procedure. The black solid line depicts the platinum wire used for touching the front head. The default starting position of the wire is 100 µm away from the skin. It then approaches the front head with a preset speed, touches, and slightly impresses the skin for a preset of time and then returns back to the starting position. The corresponding time points are shown on the right as red dots.

FIGURE 2

Layout and temporal development of anterior neuromasts in larval zebrafish. (A) Micrograph of the head of a 7dpf larva expressing GFP under the Brn3c promotor in hair cells, tectum (TEC), and lens with superimposed schematic representation of the neuromast layout and innervation derived from imaging of afferent neurons, as shown in (B). The supraorbital (SO1–3) and otic neuromasts (O1–2), as well as infraorbital neuromasts (IO1–4) and nasal neuromasts, comprise the anterodorsal lateral line (magenta). They are innervated by four nerve branches arising from the anterodorsal ganglion. The anteroventral lateral line system (red) comprises the mandibular (MD1), hyomandibular (HM1–2), and opercular (OP1–2) neuromasts. These neuromasts are innervated by nerve branches originating from the anteroventral ganglion. When viewed from the side, the trigeminal ganglion (TG) is located between the anterior lateral line ganglia (cyan). Neuromasts also located on or in proximity of the head which are part of the posterior lateral line are the medial neuromasts (M1–3) innervated by neurons arising from the medial ganglion (light blue). The dorsal neuromasts (D1–3) are innervated by the dorsal branch of the posterior lateral line arising from the posterior lateral line ganglion (blue). Also labeled with GFP are hair cells of the sensory epithelia of the statoacoustic system, the anterior and posterior cristae (AC and PC), the anterior macula (MC), utricle (UTR), and sacculus (SAC). (B) Maximum intensity projection of confocal stack of the lateral view of the region around the lateral line ganglia with neurons of the cranial ganglia expressing YFP driven by islet2B in a 7 dpf larva. Innervation of the otic neuromasts, IO4, HM1, and opercular neuromasts is visible. In addition to the neuromasts visible in (A), the opercular dorsal (OPd) neuromast can also be observed (asterisk). Nerve branches of the posterior lateral line, the posterior lateral line nerve (P), and innervation of the skin by trigeminal neurons and the trigeminal nerve projecting caudally (T) are also visible. (C) Micrographs depicting the temporal patterning of neuromasts in the anterior lateral line system in larvae expressing GFP in hair cells driven by Brn3c. Age of imaged larvae is given in the bottom left corner in hours post fertilization (hpf) and finally at 4dpf. Abbreviations as in (A). Larva imaged at 45.5 hpf also expressed GFP driven by sill in afferent neurons of the anterior and posterior lateral line ganglia (ALLG and PLLG). Later stages are still represented with head pointing down for comparison although head position relative to body axis has changed. Outline of the eye and otic capsule is indicated by dashed white lines up to 54.5 and 60.5 hpf for orientation.

FIGURE 3

Afferent trigeminal and anterior lateral line neurons respond to tactile stimulation. (A) Schematic representation of the experimental setup and the stimulus shapes (step and ramp) used in the experiments. (B) Representative calcium traces (dF/F) of selected trigeminal neurons (TG), anterior lateral line neurons (ALLG), posterior lateral line neurons (PLLG), and auditory ganglion neurons (SAG). Stimulus trace (stim) is shown in red with ramp (r) and step (s). (C, E, G, I) Mean activity (z-score) of individual neurons to ramp and step stimulation for TG neurons (C), ALLG neurons (E), PLLG neurons (G), and auditory neurons in the SAG (I). Stimulus onset at t = 0 s indicated by a pink vertical line. Z-score values over time are color-coded, as represented in the scale bar to the left. The y-axis shows the IDs of the individual neurons, sorted from top to bottom starting with the highest activity during ramp stimulation (mean activity from stimulus onset to 10 s after stimulus onset). The two subtypes of trigeminal neurons in (C) are indicated by the orange (subtype 1) and cyan (subtype 2) bars between the stimulus-type blocks and separated by a black dotted line. (D, F, H, J) Mean activity (z-score) of all neurons of a given cell type (solid lines). The shaded area represents the standard error of the mean (SEM). The pink vertical line indicates the stimulus onset. Trigeminal subtype 1 (n = 13), trigeminal subtype 2 (n = 34), anterior lateral line (n = 10), posterior lateral line (n = 29), and auditory ganglion (n = 54).

FIGURE 4

Tuning of trigeminal and anterior lateral line neurons to the velocity and duration of tactile stimulation. Boxplots for responses (z-score) of trigeminal subtype 1 neurons (A), trigeminal subtypes 2 neurons (B), and anterior lateral line neurons (C) to ramp and step stimulation. Blue and green dots indicate the mean activity of individual neurons between the stimulus onset and 15 s after stimulus onset. Red lines represent the median values. Horizontal dotted lines indicate z = 0. R² values are for a linear model including only the ramp velocities. Ramp stimuli are given in velocity [µm/s] increasing from left to right and step stimuli in duration [s] increasing from right to left, as further indicated by the blue (velocity) and green (duration) arrows. Trigeminal subtype 1 (from left to right): n_62.5 = 7; n₁₂₅ = 3; n₂₅₀ = 7; n₅₀₀ = 3; n₁₀₀₀ = 7; n_0.4 = 1; n_0.1 = 7. Trigeminal subtype 2 (from left to right): n_62.5 = 40; n₁₂₅ = 27; n₂₅₀ = 40; n₅₀₀ = 27; n₁₀₀₀ = 40; n_0.4 = 16; n_0.1 = 40. Anterior lateral line for all: n = 10.

FIGURE 5

Only auditory neurons respond to acoustic stimulation. (A) Schematic representation of the experimental setup and the stimulus used in the experiments. A 2-kHz carrier sine wave was modulated with a step function to turn sound stimulation on and off, thereby generating pulsed tones with a duration of 2 s. (B) Calcium traces (dF/F) of selected trigeminal neurons (TG), anterior lateral line neurons (ALLG), and auditory ganglion neurons (SAG). The stimulus trace is shown in red with single pulses (s) with a duration of 2 s and pulse trains with 15 pulses with a pulse duration of 2 s and an interval of either 5 s or 10 s between the pulses. (C, D) Left in each panel: mean activity (z-score) of individual trigeminal (C) and anterior lateral line (D) neurons to single sound pulses. The y-axis shows the IDs of the individual neurons, which are sorted from top to bottom starting with the highest activity during stimulation (10 s after the stimulus onset). Right in each panel: mean activity (z-score) of all trigeminal (C) and all anterior lateral line (D) neurons (n: 4 and 16, respectively). (E, F) Top in each panel: Mean activity (z-score) of individual auditory ganglion neurons (n = 14) to single pulses [(E), left], train pulses [(E), right], and entire trains with inter-pulse intervals (ISI) of 5 s [(F), left] and 10 s [(F), right]. Bottom in each panel: mean activity (z-score) of all auditory ganglion neurons (SAG). The solid black line in [(C, D), right] and [(E, F), bottom] represents the mean response of all neurons; shaded region represents SEM. The pink vertical bar indicates stimulus onset in all panels. Activity (z-scores) in [(C, D), left] and [(E, F), top] is color-coded, as shown in the scale bars.

FIGURE 6

Afferent anterior lateral line neurons respond to water flow stimulation. (A) Schematic representation of the experimental setup and the stimulus used in the experiments. Two glass pipettes (diameter 30 µm) deliver laminar water jets in either head-to-tail (HT) or tail-to-head direction (TH). (B) Standard deviation of intensity projection of calcium imaging recording showing the neurons of the anterior lateral line ganglion (ALLG) and the trigeminal ganglion (TG). The red line indicates the border between those two ganglia. ROI numbers 1 and 2 (yellow circles) correspond to recordings shown in (C). (C) Calcium traces (z-score) of two anterior lateral line ganglion neurons responding to head-to-tail (HT: green bars) stimulation but not to tail-to-head (TH: red bars) stimulation.