Sodium and potassium currents of larval zebrafish muscle fibres
Department of Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton, Alberta, T6G 2E9, Canada
* Author for correspondence (e-mail: declan.ali{at}ualberta.ca)
Accepted 15 December 1003
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Summary |
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Key words: zebrafish, Danio rerio, muscle, sodium current, potassium current, action potential
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Introduction |
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K+ channels associated with muscle fibres have been described
from rat (Duval and Leoty,
1980a,b
)
and frog (Camacho et al.,
1996
). An inactivating, outward K+ current exists in
both rat and frog skeletal fibres, while a noninactivating outward
K+ current is associated with rat slow-twitch muscle (Duval and
Leoty,
1980a
,b
).
The zebrafish Danio rerio offers many advantages for investigating
the relationship between ion channel expression and muscle fibre activity
in vivo. In particular, the ready accessibility of the developing
motor system to currently available electrophysiological techniques, the
availability of a large number of viable mutants, and the ease of recording
from both red (outer) and white (inner) muscle fibres, make this preparation
particularly attractive for investigating differences in ion channel
expression on physiologically different fibre types in developing animals
(Drapeau et al., 2002).
As in most other teleosts, zebrafish axial muscle comprises both red and
white fibre types (Greer-Walker and Pull,
1975). In the embryo and larva, these have been shown to perform
different roles in swimming (Buss and
Drapeau, 2002
), with red fibres being derecruited at faster
fictive swimming rates and white fibres probably being inactive during slow
swimming (Buss and Drapeau,
2002
). Although the development of two cell types (red and white
fibres) with distinct behavioural roles in the developing zebrafish axial
muscle system offers a convenient model in which to examine the
differentiation of electrical phenotypes, to date the electrophysiology of
larval zebrafish muscle has received surprisingly little attention.
Based on voltage recordings of embryonic and larval red and white fibres,
an initial study (Buss and Drapeau,
2000) concluded that the development of both red and white fibres
was similar, but not identical. Neither cell type was reported to exhibit
action potentials, and both types were found to be able to follow trains of
pulsatile electrical stimulation at up to 30 Hz
(Buss and Drapeau, 2000
). This
raises the question of how excitationcontraction coupling occurs in the
absence of muscle action potentials.
Here we use whole-cell, patch-clamp (under voltage-clamp and current-clamp
modes) to determine the kinetic and steady state properties of voltage-gated
ion currents of larval axial red and white muscle. Embryos undergo a
characteristic developmental sequence of motor behaviours that starts with
spontaneous alternating trunk contractions (1730 h post fertilization;
h.p.f.), followed by the emergence of coiling in response to touch
(2127 h.p.f.), and finally by active swimming in response to touch
(after 27 h.p.f.) (Saint-Amant and
Drapeau, 1998). We investigated trunk muscles in zebrafish
46 days post fertilization (d.p.f.), since at this age the larvae
exhibit mature locomotor behaviours
(Plaut, 2000
;
Saint-Amant and Drapeau,
1998
). This is the first step in a larger study aimed at
determining how developmental changes in ion channel expression affect spiking
parameters of maturing muscle cells. Here we report for the first time in this
species, that inner (white) muscles support action potentials while the outer
(red) muscles do not. Interestingly, inner white muscles were found to be
capable of generating only one action potential in response to sustained
depolarizations beyond threshold. We propose that this characteristic serves
as a mechanism whereby inner muscle sustains once-only firing during swimming
whilst preserving the capacity for high frequency firing in response to
periodic neuronal stimulation.
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Materials and methods |
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Electrophysiology
Glass pipettes were prepared from borosilicate glass (GC150T, World
Precision Instruments, Sarasota, FL, USA), pulled on a P-97 pipette puller
(Sutter Instrument Co., Novato, CA, USA) and fire polished (Micro-Forge
MF-830; Narishige, Japan) to a resistance of 0.52 M. Signals
were amplified using an Axopatch 200B (Axon Instruments, Foster City, CA, USA)
and displayed on an IBM-compatible PC using pClamp 8.02 software (Axon
Instruments) and on a digital oscilloscope (TDS 220; Tektronix, Beaverton, OR,
USA). Immediately after the establishment of the whole-cell mode
(Hamill et al., 1981
), series
resistance was compensated by at least 80%, and usually by 90%, using the
amplifier's compensation circuitry. Experiments were aborted if the series
resistance changed by more than 15%. Data were sampled at 50 kHz using a
Digidata 1322A (Axon Instruments) analogue to digital converter, or at 250 kHz
when recording Na+ currents. Data were analyzed using Clampfit 8.0
software (Axon Instruments) and plotted using SigmaPlot 7.0 (SPSS).
Capacitative and leak currents were subtracted on-line using the P/N protocol
provided with pClamp 8.02, in which we used 4 depolarizing pulses.
Salines and pipette-filling media
Salines and pipette-filling media were prepared as in
Table 1. The pH of all
extracellular solutions was adjusted to 7.8 with NaOH or KOH depending upon
the experiment, while the pH of all intracellular solutions was adjusted to
7.6 with CsOH or KOH. The final osmolarity of all solutions was adjusted to
290±2 mOsm l1. Pipette filling solutions were
supplemented with Na2ATP (4 mmol l1) and LiGTP
(0.4 mmol l1). The pipette-filling solutions for the
current-clamp experiments consisted of either the `Normal' solution
(Table 1) or a low
Cl solution composed of the following: 140 mmol
l1 D-gluconic acid K+ salt, 6 mmol
l1 KCl, 4 mmol l1 MgCl2, 10
mmol l1 EGTA, 10 mmol l1 Hepes, 4 mmol
l1 Na2ATP and 0.4 mmol l1
LiGTP. 1-Heptanol (2 mmol l1) was added to the extracellular
saline for the voltage-clamp experiments in order to block gap junctions and
reduce electrical coupling between cells
(Nguyen et al., 1999;
Saint-Amant and Drapeau,
2000
).
All solutions and drugs were bath-applied at a flow rate of 2 ml min1. All drugs were acquired from Sigma, unless otherwise indicated.
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Results |
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Fire-polished glass patch pipettes readily formed high-impedance (G)
seals with both inner and outer muscle fibres, and the whole-cell patch-clamp
configuration was achieved either by application of negative pressure or by a
combination of negative pressure and a brief depolarizing current. Most cells
recorded in the whole-cell patch clamp mode had input resistances of less than
80 M
, although many cells had markedly higher input resistances (more
than 300 M
; Table 2).
Because many of the currents recorded in these experiments had amplitudes to
the order of 10 nA for white fibres and 4 nA for red fibres, this study
included only those cells in which the ratio of the input resistance to the
access resistance (both estimated electronically by the data acquisition
software) exceeded 10 both before and after recordings. The large size of the
muscle fibres coupled with series resistance will introduce errors in voltage
control of the fibres (Penner,
1995
). We calculated that the compensated series resistance
results in a maximum voltage error for red fibres on the order of
approximately 12 mV, while for white fibres the error is approximately
35 mV. In addition, series resistances ranging from 2.7 to 3.1 M
for recordings from red fibres, and membrane capacitance values of 2837
pF (Table 2) results in
membrane-charging time constants (
=RsxCm) to
the order of 1215 µs after
85% compensation, whereas for white
fibres the time constant ranges between 20 and 26 µs.
|
To ensure that we recorded from only individual muscle fibres, we included
the gap junction blocker 1-heptanol (2 mmol l1) in the
exctracellular saline in order to uncouple the muscle fibres
(Nguyen et al., 1999). When
Lucifer Yellow (0.1%) was included in the pipette, only individual fibres were
stained in the presence of 1-heptanol (Fig.
1C,D), suggesting that the presence of the alcohol effectively
isolated the cells.
Currents of outer and inner muscle
When all major ions (Na+, K+, Ca2+ and
Cl) were present in the saline and in the pipette-filling
medium, depolarizations of voltage-clamped fibres evoked outward currents in
outer muscle (Fig. 2A) and both
inward and outward currents in inner muscle
(Fig. 2C). In outer muscles,
stepwise, 100 ms depolarizations from a range of potentials from 85 to
+50 mV (at 5 mV intervals) from a holding potential of 90 mV gave rise
to outwardly directed currents with some evidence of an inactivating component
at greater levels of depolarization (Fig.
2A). Inwardly directed currents were not visible in these
recordings. Plotting the peak amplitudes of the outward currents against the
amplitude of the depolarizing step revealed that currents were evoked at
potentials more positive to around 40 mV, and continued to increase
with more depolarized potentials (Fig.
2B).
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In contrast, similar voltage protocols applied to inner muscle evoked a pronounced, brief inward current followed by outwardly directed currents with a strong inactivating component (Fig. 2C and inset). Currentvoltage plots (Fig. 2D) of the peak amplitudes of the inward and outward currents revealed that the inward current was evoked at membrane potentials more positive to around 40 mV, and the outward at potentials more positive to 20 mV.
Na+ currents of inner muscle
Putative Na+ currents were isolated from inner muscle by
performing voltage-clamp recordings in saline in which all the potassium ions
were replaced by equimolar cesium ions, all the calcium ions replaced with
equimolar cadmium ions, and BAPTA added to the intracellular medium to 10 mmol
l1 to reduce twitching
(Table 1). The addition of
BAPTA to the pipette medium prevented contractions in inner muscle, but not in
outer muscle. Under these recording conditions, stepwise, 5 ms depolarizations
from a holding potential of 90 mV to a range of potentials from
90 to +70 mV evoked rapidly activating and rapidly inactivating,
inwardly directed currents (Fig.
3B, inset) up to 10 nA in amplitude. These currents appeared in
response to potentials more positive than about 40 mV and reversed at
+48±3.7 mV (N=8). When similar protocols were applied to
voltage-clamped outer muscle fibres using the same recording conditions, no
currents greater than 0.5 nA were observed
(Fig. 3A) in any of the five
cells tested.
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Steady state properties of Na+ currents
Steady-state activation and inactivation properties were determined for
Na+ currents of inner muscle fibres
(Fig. 3C). The
voltage-dependence of steady-state activation was determined by first
measuring the reversal potential for each set of IV
traces (such as those summarized in Fig.
3B) and then measuring the ratio of the peak current at each
potential to the driving force (estimated as the difference between the
potential and the reversal potential). This ratio, which is the estimated
conductance, plotted against the amplitude of the depolarizing pulse, yielded
a saturating curve that, when normalized to the maximum values, was fitted to
a Boltzmann equation of the form:
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Steady-state inactivation was determined by measuring the amplitude of depolarization-evoked currents following a preconditioning period. A series of 50 ms depolarizations from a holding potential of 100 mV to a range of potentials from 115 to 10 mV in 5 mV steps was followed by a 5 ms step to 5 mV to evoke the inward current (Fig. 3C, inset). The ratio of the amplitude G of the inward currents to the maximum amplitude Gmax was plotted against the membrane potential during the preconditioning step. The data thus derived were then fitted to a Boltzmann function, which yielded an estimated value of V50 of inactivation of 74.5±1.1 mV and slope of 6.0±0.2 mV/e, N=9.
Inactivation kinetics of Na+ currents of inner muscle
The time constants of inactivation and the voltage-dependence of recovery
from inactivation of Na+ currents of inner muscle were determined.
To derive the time constant of inactivation, the decaying phases of
Na+ currents illustrated in Fig.
3B were each fitted to a single exponential, the time constant
(ms) of which was plotted against the amplitude of the depolarizing
pulse. The rate of inactivation was found to be strongly voltage-dependent
(Fig. 4A) and could be
described by the function:
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The rate of recovery of inactivation was measured using a paired pulse
protocol. A 5 ms depolarizing step to 20 mV was applied to completely
inactivate the Na+ currents, followed by a 0.59 ms recovery
step to allow partial recovery of the currents. A second test pulse, of
identical form to the first, was applied to determine the proportion of
currents that had recovered. The ratio of the amplitudes of currents evoked by
the first and second test pulses was plotted against the duration of the
recovery period. The data thus obtained at different interstimulus potentials
(from 70 to 150 mV in 10 mV steps) could best be fitted to
simple, single exponential functions (Fig.
4B), which serve as estimates of the time-constant of recovery
from inactivation. Plotting these time constants against the interstimulus
potential (Fig. 4C), revealed
that the rate of recovery of inactivation was strongly voltage-dependent, and
could be described by the function
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The inward currents observed in Na+-isolating salines were reversibly blocked by the application of micromolar concentrations of TTX (1 µmol l1; Fig. 4D) and reversed near the equilibrium potential for sodium ions calculated from the sodium ion concentrations using the Nernst equation, suggesting that they are carried by sodium ions.
K+ currents of inner and outer muscle
Isolation of K+ currents of inner and outer muscle of 46
d.p.f. zebrafish larvae was accomplished by applying depolarizing pulses to
voltage-clamped fibres using saline and pipette media in which all sodium ions
were replaced with equimolar choline ions, all calcium ions replaced with
equimolar cadmium ions and BAPTA added to the intracellular medium to 10 mmol
l1. The use of high concentrations of potassium ions in the
pipette appeared to compromise the health of the fibres, as determined by seal
and input resistances and by the rapid deterioration of whole cell recordings,
especially in the case of outer fibres. However, high-quality recordings could
be obtained from these cells in sufficient numbers. K+ currents
recorded in outer muscle in response to a series of 250 ms depolarizations
imposed from a holding potential of 100 mV to a range of potentials
from 95 to 25 mV showed little or no evidence of inactivation
(Fig. 5A). These currents
appeared at membrane potentials more positive to around 40 mV and
increased as the depolarizing potentials were made more positive
(Fig. 5B; N=8).
Similar protocols applied to inner muscle evoked outwardly directed currents
that were of larger amplitude than those of outer muscle, and that inactivated
rapidly and completely (Fig.
5C). Currentvoltage plots of the peak amplitudes of these
currents revealed that the currents were activated at potentials more positive
than around 20 mV, and that there remained some residual inward current
(Fig. 5C,D; N=8).
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Steady-state activation and inactivation of K+ currents
Steady-state activation and inactivation of putative K+ currents
of inner muscle were determined. Steady-state activation was estimated from
isochronal isopotential tails, thereby eliminating errors introduced by
estimating the reversal potential of the putative K+ currents since
the measurements are made at the same potential. 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 (Fig.
6A, right inset) were applied. These pulses were followed
immediately by a step to 130 mV to enable the measurement of tail
currents, which at this potential are slower and therefore easier to measure.
The amplitudes of the tails were normalized to a maximum value of 1 and
plotted against the amplitude of the activating pulse
(Fig. 6A). These data were
fitted to a Boltzmann function giving an estimated V50 of
activation of 1.03±1.02 mV, N=7 and slope factor of
10.82±0.48 mV/e, N=7.
|
Steady state inactivation was determined using similar protocols as for Na+ currents (Fig. 6A, left inset). A 250 ms conditioning pulse was followed immediately by a 20 ms test pulse to 10 mV. The peak amplitude of the response to the test pulse was normalized to a maximum value of 1 and minimum value of 0 and plotted against the conditioning membrane potential (Fig. 6A, open circles). These data could be fitted to a Boltzmann function giving a V50 of inactivation of 30.4±0.9 mV, N=8 and slope factor of 4.4±0.1 mV/e, N=8.
These outwardly directed currents were blocked by continual bath perfusion of 10 µmol l1 4-aminopyridine (4AP) (Fig. 6B) Stepwise, 50 ms depolarizations from a holding potential of 100 mV to a potential of 0 mV evoked outward currents regardless of how long the preparation had been exposed to 4AP, even after other fibres had previously been tested in 4AP. Responses to subsequent depolarizing pulses, however, were progressively smaller (Fig. 6B). The rate of decline in amplitude was independent of the interval between the pulses, and was a function of the number of previous pulses, indicating that the block by 4AP is use-dependent.
Action potentials in inner muscle
Using the patch-clamp amplifier in current-clamp mode, action potentials
were recorded from inner muscle in response to injected current. The Axopatch
200B patch clamp amplifier is not ideally suited for accurately recording the
true kinetics of an action potential, and tends to distort the action
potential waveform (Magistretti et al.,
1996). However, since it was our intention to record the
occurrence of action potentials without regard to precise details of their
waveforms, we used the Axopatch 200B in current-clamp mode accepting that
there would unavoidably be errors associated with the shape of the action
potential.
Two different pipette-filling solutions were used. One was the `Normal'
solution that we used to voltage-clamp the fibres when investigating total
currents. The second solution was a potassium gluconate-based solution
containing only approximately 14 mmol l1
Cl, a concentration that is close to physiological for these
muscle fibres (Bretag, 1987).
The current-clamp results for both solutions were indistinguishable, apart
from a slight slowing of the kinetics when recording with the low
Cl solution. This is likely due to a smaller rate of change
of voltage with time as a consequence of smaller current injections into the
fibres compared with results using the high Cl solutions. In
all experiments, background current was injected to control the `resting'
membrane potential to around 70 mV, a value that has been reported as
the average resting membrane potential of larval inner, muscle fibres
(Buss and Drapeau, 2000
). In
all inner muscle fibres recorded (N=15 in Normal solution and
N=5 in low Cl), the injection of depolarizing
current resulted in one, and only one, action potential if the `resting'
potential was sufficiently negative (Fig.
7A,B). Injection of depolarizing current above a threshold value
(which varied from cell to cell) resulted in an overshooting action potential,
whereas the same depolarizing current applied to a background `resting'
potential of approximately 45 mV evoked a highly attenuated spike. No
action potentials could be evoked by any current applied to outer muscle
fibres (Fig. 7E,F; N=5
in Normal solution; N=6 in low Cl), even when the
background, `resting' potential was set to 100 mV (data not shown).
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Progressively increasing the amplitude of the depolarizing current failed to elicit more than one action potential from inner muscle fibres (Fig. 7C,D; N=10 for Normal solution and N=5 for low Cl). A second action potential could, however, be elicited by applying two depolarizing pulses separated by an interval of at least 25 ms (Fig. 8A,B). When hyperpolarizing current was injected during the interstimulus interval, the minimum interval for which spiking could be evoked was around 0.5 ms (Fig. 8C,D; N=10 in Normal solution; N=5 for low Cl).
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Discussion |
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The use of whole-cell patch in the current-clamp mode suffers from two
experimental disadvantages: (1) the use of these headstages can distort the
action potential waveform significantly, and (2) the intracellular environment
is changed by dialysis with the pipette contents. We cannot therefore draw
confident conclusions on the shape of the action potentials recorded in these
experiments. It seems unlikely, however, that the phenomenon of once-only
firing, which has also been observed in recordings from muscle fibres using
sharp intracellular electrodes (Adrian and
Bryant, 1974; Bryant
1962
), is an artefact of the amplifier's circuitry, and so our
findings provide confirmation that this phenomenon also occurs in zebrafish.
Further, we cannot exclude the possibility that the inclusion of BAPTA in
pipette-filling media may have altered the properties of ion channels through
interference with calcium-dependent signalling pathways. We used the same
pipette solutions in both current-clamp and voltage-clamp experiments, and
since once-only firing is seen in our experiments as well as in those using
sharp microelectrodes, it is unlikely that dialysis has greatly changed the
properties of the channels that contribute to the firing pattern, although we
cannot exclude the possibility that dialysis might have effected subtle
alterations in channel properties.
The steady state and kinetic properties of skeletal muscle Na+
channels have been investigated in a variety of preparations including humans
(Almers et al., 1984), the rat
(Duval and Leoty, 1978
;
Ruff et al., 1987
;
Moczydlowski et al., 1986
),
mouse (Gonoi et al., 1989
),
frog (Campbell and Hille,
1976
) and elasmobranch fish
(Stanfield, 1972
). In
addition, skeletal muscle Na+ channels from rat have been cloned
and sequenced (Trimmer et al.,
1989
; Kallen et al.,
1990
). In comparison to some of these previous studies, our
estimated V50 of activation of the Na+-currents
in zebrafish fast-twitch muscle (of 7 mV) is notably more positive than
that found in these other preparations, although the half-inactivation voltage
in zebrafish white muscle (74 mV) is very similar to previously
published values (O'Leary,
1998
), which range from 70 to 94 mV, with the vast
majority falling between 70 and 76 mV. Many studies also report
an outward K+-current associated with fast-twitch muscle with
properties similar to an A-current (Conor and Stevens, 1971a), in that it
peaks and then inactivates with variable time courses
(Adrian et al., 1970
;
Duval and Leoty, 1978
;
Stanfield, 1972
;
Vázquez, 1998
).
Our finding that inner muscle is capable of supporting action potentials,
is in contrast to the findings of Buss and Drapeau
(2000), who were unable to
evoke spikes from either red (outer) or white (inner) fibres. We show here
that spikes evoked from a more depolarized resting membrane potential are
highly attenuated. Buss and Drapeau
(2000
) report a resting
membrane potential of 78 mV in 1-day embryos, to 71 mV in 6-day
larvae, both values within the range over which we were able to elicit spikes.
In our hands we found the outer, red muscle incapable of supporting action
potentials, similar to larval (Buss and
Drapeau, 2000
) and adult zebrafish preparations
(Westerfield et al., 1986
).
The red muscle in other preparations, however, has been shown to support
action potentials (Takeuchi,
1959
; Stanfield,
1972
), but these instances were rare, and to the best of our
knowledge the majority of red fibres lack the ability to produce spikes.
The kinetic and steady state properties of the Na+ and
K+ currents of inner muscle fibres are adapted to the behavioural
functions of these muscles. Although inner muscle, in response to
depolarization, produces a single, large action potential without the
development of spike trains, it is nonetheless capable of following a train of
depolarizing inputs at around 35 cycles per second
(Buss and Drapeau, 2001). The
inability of these muscles to produce spike trains could possibly serve as a
safeguard against the depolarization accompanying one phase of the swimming
cycle from evoking either tetany or a train of spikes, so ensuring strictly
one spike per swim cycle. Earlier work suggested that a relatively
high-density chloride shunt conductance may be responsible for the once-only
firing in fast-twitch muscle (Adrian and
Bryant, 1974
; Bryant
1962
), which is suggested to maintain the membrane potential at a
hyperpolarized level following a spike. Our results show that white fibres are
able to support once-only firing when filled with either a high (
140 mmol
l1 Cl) or a low (
14 mmol
l1 Cl) Cl solution,
whilst red fibres are unable to support action potentials. We found that when
recording with the low Cl solution we had to inject less
current into both white and red fibres in order to depolarize them, compared
with the high Cl solutions. Even though red fibres were only
injected with
5 nA of current, they depolarized to values approaching
+150 mV, and further stimulation appears unlikely to produce spikes at such
highly unphysiological potentials.
An alternative explanation for once-only firing might lie in the
steady-state inactivation and voltage dependence of recovery from inactivation
of the Na+ current and the presence of the A-type K+
currents. Sharp microelectrode studies of adult white fibres reported an
average resting membrane potential of 81±8 mV
(Westerfield et al., 1986),
and more recent work on larval white fibres indicates a resting potential of
approximately 71 mV, near to the value we assume in our current-clamp
experiments. Our data indicate that at 71 mV, the reported value of the
resting potential in larval white muscle
(Buss and Drapeau, 2000
), some
30% of the Na+ channels are inactivated
(Fig. 3). It is therefore
possible that Na+ channel inactivation is the likely explanation
for the graded attenuation of action potentials upon background membrane
depolarization (Fig. 7A,B).
This in turn may provide a mechanism to prevent spike trains, since the steep
voltage dependence of the rate of recovery from inactivation makes the
relative refractory period dependent upon interspike hyperpolarization. This
would effectively make high frequency firing conditional upon interspike
repolarization. The role of the A current might accordingly be to ensure an
interspike hyperpolarization. At the reported resting membrane potentials for
zebrafish larval muscle, none of the K+ channels would be activated
and almost none inactivated, providing further reason for anticipating that
these currents play a role in hastening repolarization. A role in producing
slow, non-zero firing, as suggested by Connor and Stevens
(1971b
), is unlikely, since
such firing is not expected in this muscle, which is rather associated with
rapid swimming (Buss and Drapeau,
2002
).
Thus, the kinetic and steady-state properties of the Na+ and K+ currents of inner muscle underlie a phenotype that permits high frequency firing in response to pulsatile depolarizing inputs of the kind expected during fast swimming, whilst safeguarding against the danger of tetanic spike trains in response to a single, strong depolarization, so permitting a large safety factor.
These predictions await further testing, either directly using manipulations that alter the ionic current properties, or indirectly using computer modeling. Thus, the larval zebrafish provides a particularly convenient model in which to trace behavioural adaptations in a motor system to molecular properties of ion channels.
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Acknowledgments |
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References |
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