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First published online January 27, 2004
Journal of Experimental Biology 207, 841-852 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00839

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Sodium and potassium currents of larval zebrafish muscle fibres

Steven D. Buckingham and Declan W. Ali^*

Department of Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton, Alberta, T6G 2E9, Canada

View larger version (90K): [in a new window]   Fig. 1. (A,B) Nomarski Differential Interference Contrast (DIC) images of larval zebrafish outer, red (A) and inner, white (B) muscle fibres. The outer, red fibres constitute a single, superficial cell layer and run parallel to the notochord, while the inner, white fibres are several cell layers thick and run obliquely to the notochord and midline. (C,D) Individual red (C) and white (D) muscle fibres from 5 d.p.f. larvae filled with Lucifer Yellow (0.1%) in the presence of 1-heptanol (2 mmol l–1). Arrows point to the edges of the muscle fibres. Scale bars, 50 µm.

View larger version (33K): [in a new window]   Fig. 2. Voltage-gated currents recorded from outer (A,B) and inner (C,D) muscle of 4–6 d.p.f. zebrafish larvae in normal physiological saline (Na+, K+, Ca2+ and Cl– present). (A) Stepwise, 100 ms depolarizations from a range of potentials –85 to +50 mV (at 5 mV intervals) from a holding potential of –90 mV give rise to almost exclusively outwardly directed currents in voltage-clamped, outer muscle fibres. These currents show some evidence of an inactivating component. (B) Currents are evoked at potentials more positive to around –40 mV, and continue to increase with more depolarized potentials. (C) Similar voltage protocols applied, using the same salines, to inner zebrafish muscle evoke a pronounced, brief inward current (inset) followed by outwardly directed currents with a strong inactivating component. Current–voltage plots (D) of the peak amplitudes of the inward and outward currents reveal that inward currents are evoked at potentials more positive to around –40 mV, and outward currents more positive to around –20 mV. Filled symbols, outward currents; open symbols, inward currents.

View larger version (15K): [in a new window]   Fig. 3. Isolation of Na+ currents of 4–6 d.p.f. zebrafish outer (A) and inner (B,C) muscle. In these experiments, Na+ currents were isolated by performing voltage-clamp recordings in saline in which all the potassium ions were replaced by equimolar cesium ions, and all the calcium ions replaced with equimolar cadmium ions, with BAPTA (10 mmol l–1) supplementing the intracellular medium. (A) Stepwise, 5 ms depolarizations fail to elicit inward, Na+ currents in outer muscle. (B,C) Stepwise, 5 ms depolarizations from a holding potential of –90 mV to a range of potentials from –85 to +30 mV evoked rapidly activating and rapidly inactivating, inwardly directed currents (inset), which appeared at potentials more positive than about –40 mV and reversed around +50 mV. Values are means ± S.E.M. of 8 experiments on separate muscle fibres. (C) Steady state activation and inactivation of Na+ currents recorded from inner muscle. Values are means of 12 (activation) and 9 (inactivation) separate experiments ± S.E.M. The data thus derived for both activation and inactivation were then fitted to a Boltzmann function giving estimated values of V50 of activation of –7.3±1.6 mV and slope 8.4±0.5 mV/e (N=12) and V50 of inactivation of –74.5±1.1 mV and slope of –6.0±0.2 mV/e (N=9). See text for details.

View larger version (25K): [in a new window]   Fig. 4. Time constants of inactivation and voltage-dependence of recovery from inactivation of Na+ currents of inner muscle. (A) The decaying phase of Na+ currents illustrated in Fig. 3B were fitted to single exponentials and the time constants plotted against the amplitude of the depolarizing pulse. The rate of inactivation can be seen to be strongly voltage-dependent. (B) The rate of recovery of inactivation was measured using a paired two-pulse protocol. A 5 ms depolarization to 20 mV was applied to completely inactivate the Na+ currents. This was then followed by a 0.5–9 ms recovery step to one of a range of potentials (from –70 to –150 mV in 10 mV steps). The amplitude of currents evoked by a second test pulse (of identical duration and amplitude to the first) was plotted against the duration of the recovery period. The data thus derived were fitted to a single exponential function, the estimated time constants of which were plotted against the membrane potential of the recovery period (C), revealing a strong voltage dependence of the rate of recovery of inactivation. (D) The inward currents observed in Na+-isolating salines are blocked by the application of micromolar concentrations of TTX, confirming their identity as Na+ currents.

View larger version (29K): [in a new window]   Fig. 5. Isolation of K+ currents from inner and outer muscle of 4–6 d.p.f. zebrafish larvae was performed by applying depolarizing pulses to voltage-clamped fibres in saline in which all sodium ions were replaced with equimolar choline ions and all calcium ions were replaced with equimolar cadmium ions and supplemented with BAPTA (10 mmol l–1). (A) K+ currents of outer muscle. A series of 250 ms depolarizations from a holding potential of –100 mV to a range of potentials from –95 to 25 mV evoked outwardly directed currents with little or no evidence of inactivation. (B) A current–voltage plot of such currents shows that they appear at membrane potentials more positive to around –40 mV and increase with more positive potentials. (C) K+ currents of inner muscle. (D) The current–voltage plot of these currents reveals that they are activated at potentials more positive than around –20 mV. There is still some residual inward current. Values are means ± S.E.M. of at least eight separate experiments

View larger version (22K): [in a new window]   Fig. 6. (A) Steady-state activation and inactivation of putative K+ currents of 4–6 d.p.f. zebrafish inner muscle. Steady-state activation (filled circles; N=7) was determined by applying a series of 5 ms activating pulses from a holding potential of –100 mV to a range of potentials from –45 to +65 mV at 5 mV intervals (inset, right). These pulses were followed immediately by a step to –130 mV to enable the measurement of tail currents. The amplitudes of the tails were normalised to a maximum value of 1 and plotted against the amplitude of the activating pulse. Steady state inactivation (open circles; N=6) was determined as in Fig. 3C (inset, left). (B) These outwardly directed currents are blocked by 10 µmol l–1 4-aminopyridine (4AP) in a use-dependent manner. Stepwise 250 ms depolarizations from a holding potential of 100 mV to a potential of 0 mV were applied as the larva was perfused in saline containing 10 µmol l–1 4AP. Numbers indicate the sequence of depolarizing pulses.

View larger version (35K): [in a new window]   Fig. 7. Action potentials recorded in 4–6 d.p.f. zebrafish inner and outer muscle. (A,B) Action potentials were recorded from inner muscle fibres under current-clamp conditions in response to the injection of depolarizing current when the fibre was previously held at –70 mV (N=15). However, when the same depolarizing current was injected after holding the fibre at –45 mV, the evoked spike was considerably attenuated. Increasing the amplitude of the depolarizing current did not restore spike shape (data not shown). (A) Normal solution, (B) low Cl– in the pipette-filling solution (N=5). (C,D) Applying 10 ms depolarizing pulses of increasing amplitude from a `resting' membrane potential of around –70 mV never evoked more than one spike (N=10) in (C) Normal solution and (D) with low Cl– in the pipette-filling solution (N=5). (E,F) In contrast to inner muscle fibres, depolarization of outer muscle fibres never elicited an action potential (N=5) in (E) Normal solution and (F) with low Cl– in the pipette-filling solution (N=5).

View larger version (43K): [in a new window]   Fig. 8. Action potentials recorded in 4–6 d.p.f. zebrafish inner muscle. (A,B) Although no amount of depolarization of the inner muscle was found to elicit more than one spike, further spiking could be elicited in response to a second depolarization following a brief (>25 ms) return to the `resting' potential in (A) Normal solution and (B) low Cl– solution. (C,D) The minimal interstimulus interval required to elicit a second spike was greatly diminished by hyperpolarizing the membrane between stimuli. The recovery of the action potential was graded rather than all-or-none. (C) Normal solution, (D) in low Cl– solution.