In vivo physiological recording from the lateral line of juvenile zebrafish

Abstract

Key points: Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish.

Abstract: Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish.

Figure 1. Effect of MS‐222 on the K⁺ currents recorded from young‐adult lateral line hair cells

A–C, K⁺ current recordings from hair cell before (A), during (B) and after (C) local superfusion of 0.017% MS‐222. For this recording, the decapitated zebrafish (19 dpf) was maintained in a normal extracellular solution without MS‐222, which was locally superfused onto the patched hair cell. Currents were obtained by applying a series of voltage steps in nominal 10 mV increments from –124 mV, starting from the holding potential at –84 mV. The presence of MS‐222 largely reduced both the peak and steady‐state of the outward K⁺ current (B). D, peak current–voltage relation obtained from panels A–C above.

Figure 2. Benzocaine did not block the K⁺ currents in hair cells from young‐adult zebrafish

A and B, characteristic K⁺ current recordings from lateral line hair cells positioned in the edge (left panels; 25 dpf) and centre (right panels; 25 dpf) of the neuromast before (top panels) and during (bottom panels) the local application of 100 mg l^–1 benzocaine. Voltage protocol is as in Fig. 1. Note that hair cells recorded from the edge and centre of a neuromast show a different current profile, which has previously been characterized (Olt et al. 2014). All cells expressed a delayed rectifier K⁺ current (I _K,D). However, most cells in the edge region expressed a Ca²⁺‐activated K⁺ current (I _K,Ca), a small A‐type current (I _A(s)) and an h‐type current (I _h), whilst those in the centre expressed a large I _A. C–E, average peak and steady‐state current measured near 0 mV before (black bars) and during the superfusion of different concentrations of benzocaine (grey bars) from 24–26 dpf zebrafish: C: control: peak 400 ± 32 pA, steady 317 ± 43 pA, n = 2; benzocaine (50 mg l^–1): peak 388 ± 65 pA, steady 349 ± 58 pA, n = 2; D: control: peak 435 ± 94 pA, steady 265 ± 66 pA, n = 5; benzocaine (100 mg l^–1): peak 399 ± 93 pA, steady 273 ± 105 pA, n = 5; E: control: peak 426 ± 67 pA, steady 261 ± 45 pA, n = 7; benzocaine (300 mg l^–1): peak 393 ± 62 pA, steady 224 ± 43 pA, n = 7.

Figure 3. Benzocaine does not affect the MET current in hair cells

A and B, fluorescence images from 14 dpf zebrafish neuromasts (primI) showing that hair cells take up FM1‐43 when applied alone (A) or together with 50 mg l^–1 benzocaine (B). CB indicates the cell body of the hair cells within the neuromast. C, fluorescence images with the DIC image superimposed from 5 dpf zebrafish neuromasts treated for 5 min with 5 mm BAPTA before the application of FM1‐43. Note that FM1‐43 labelled the stereociliary hair bundle, most likely due to the partitioning of the dye in to the outer leaflet of the hair bundle plasma membrane (HC: left panel), but not the cell body (CB: right panel) of hair cells. Scale bars in A–D: 10 μm. D and E, mechanoelectrical transducer (MET) currents recorded from a P6 mouse OHC before (black trace) and during (grey trace) the superfusion of 50 mg l^–1 benzocaine (D) and before (black trace) and during (grey trace) the superfusion of 100 μm dihydrostreptomycin (DHS) (E). MET currents were obtained by applying saturating sinusoidal force stimuli of 50 Hz to the hair bundles at −81 mV. The driver voltage (DV) signal of ±40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The arrow indicates the inward MET current and the arrowhead the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements. Dashed lines indicate the holding current, which is the current at the holding membrane potential. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Ca²⁺ currents and neurotransmitter release in lateral line hair cells is not affected by benzocaine

A and B, Ca²⁺ current (I _Ca) recorded from hair cells of a 35 dpf zebrafish lateral line before (A) and during (B) the local superfusion of 100 mg l^–1 benzocaine. Currents were elicited by a depolarizing voltage step to –31 mV (200 ms in duration) from the holding potential of –79 mV. C, average I _Ca from seven hair cells from 27–35 dpf decapitated zebrafish before (black) and during (grey) benzocaine. Control I _Ca: 6.1 ± 1.1 pA, n = 7; I _Ca in benzocaine: 5.2 ± 0.8 pA, n = 7. D and E, changes in membrane capacitance (ΔC _m) recorded from a hair cell of 35 dpf zebrafish. Recordings were obtained in response to 1 s voltage steps from the holding potential of −79 mV to near the peak of I _Ca (−31 mV). F, average ΔC _m recorded from four hair cells of 27–31 dpf zebrafish before (black) and during (grey) benzocaine. Control ΔC _m: 5.3 ± 0.7 fF, n = 4; ΔC _m in benzocaine: 6.7 ± 1.6 pA, n = 4.

Figure 5. Benzocaine affects the afferent activity in the zebrafish lateral line

A and B, spontaneous firing recorded with loose‐patch voltage clamp from the posterior lateral line ganglion (PLLg) of a 5 dpf zebrafish in the presence of normal extracellular solution (A) and during the application of 50 mg l^–1 benzocaine (B). C and D, coefficient of variation (C, CV) and mean firing rate (D) as a function of recordings time. E, CV from each recording against the respective firing rate (single recording: open symbols; averages: filled symbols). The total duration of the recoding time and spike number for each experimental condition was: control 70.4 min and 14604 spikes, 50 mg l^–1 benzocaine 89 min and 8753 spikes, wash 85.4 min and 17767 spikes. F–H, spontaneous firing from a PPLg of a 4.5 dpf zebrafish in normal extracellular solution (F), during the application of 500 mg l^–1 benzocaine (G) and the final washout (H). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. In vivo recordings from lateral line hair cells of young adult zebrafish

A, image showing a 31 dpf intubated zebrafish under anaesthesia (using 50 mg l^–1 benzocaine) at the bottom of the microscope recording chamber. Scale bar: 0.4 mm. B, images showing the cannula inserted in the mouth of a 21 dpf zebrafish. Scale bar: 1 mm (top) and 0.5 mm (bottom). The cannula delivered extracellular solution that was not oxygenated. C and D, example of K⁺ currents recorded from hair cells of 34 dpf (A) and 48 dpf (B) zebrafish elicited using the same voltage protocol as in Fig. 1. E, comparison of the average size of the peak (black columns) and steady‐state (white columns) outward K⁺ current at 0 mV recorded in vivo from larval (from Olt et al. 2014) and juvenile zebrafish hair cells. Larval (3–5.2 dpf) hair cells: peak 468 ± 16 pA, steady 335 ± 18 pA, n = 41; juvenile (34–38) hair cells: peak 287 ± 32 pA, steady 194 ± 31 pA, n = 8). [Colour figure can be viewed at wileyonlinelibrary.com]