A comparison of propagated action potentials from tropical and temperate squid axons: different durations and conduction velocities correlate with ionic conductance levels
Departments of Physiology and Anesthesiology, UCLA School of Medicine, Los Angeles, CA 90095, USA
* Author for correspondence (e-mail: fbezanil{at}ucla.edu )
Accepted 5 April 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: squid, giant axon, Loligo pealei, Loligo opalescens, Loligo plei, Sepioteuthis sepioidea, temperature adaptation, action potential, conduction velocity, K+ conductance, Na+ conductance, K+ current, Na+ current, conduction velocity, action potential broadening
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Those studies that have examined the temperature adaptation of the action
potential have focused mainly on conduction velocity. For example, compared
with temperate taxa at an equivalent recording temperature, Antarctic teleosts
have relatively rapid conduction velocities
(Macdonald, 1981;
MacDonald et al., 1988
). The
same is true for certain leg nerves in Arctic seagulls
(Chatfield et al., 1953
). In a
few cases, other parameters have been examined (e.g. action potential
duration; Lagerspetz and Talo,
1967
). No reports, however, have identified the underlying
physiological mechanisms for an adaptive change. For example, do
species-dependent conduction velocities result from changes in the magnitude
of gNa, in INa kinetics or in the fiber's cable
properties? These questions are hindered by several factors. First, most axons
are too small to permit the use of voltage-clamp methods to study ionic
conductances. In many preparations, individual neurons cannot be easily
identified. Finally, apparent adaptive differences between dissimilar
organisms may be a consequence of evolutionary distance.
The squid giant axon, long used to examine basic mechanisms of
excitability, is also an ideal model for temperature adaptation. Its
dimensions permit the measurement of both action potentials and the underlying
ionic conductances. Although giant neurons are present in other molluscs, most
consist of large somata, not giant axons useful for studying impulse
propagation. As a result of the isolation of mRNAs for a Na+
channel and a delayed rectifier K+ channel, molecular reagents are
available for the giant axon system
(Rosenthal and Gilly, 1993;
Rosenthal et al., 1996
). The
ionic conductances in this preparation have been investigated intensely for
almost 50 years and are very well described. Finally, the giant axon can be
easily identified in many squid taxa whose representatives inhabit a wide
range of habitats. This study takes advantage of four species within the
family Loliginidae whose collective temperature range spans 20°C.
Our questions address how their propagated action potentials compare and
whether differences correlate with a species' thermal environment. We also
investigate the ionic basis for these differences.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Giant axons were isolated from adult specimens using standard methods. Squid were rapidly decapitated, and the hindmost stellar nerves were removed from the mantle and bathed in filtered sea water. Small nerve fibers and other connective tissue were manually removed from the giant fiber. In general, axons were used for experimentation immediately; however, in some cases, they were stored at 4°C for 4-5 h. All experiments used 10K artificial sea water (10K ASW; in mmol l-1: 430 NaCl, 10 KCl, 50 MgCl2, 10 CaCl2, 10 Hepes, pH 7.5, 970-980 mosmol l-1).
Temperature control and measurements
For all experiments, temperature was controlled using Peltier units, placed
immediately under the recording chamber, driven by a standard
temperature-control circuit. Temperatures were measured directly adjacent to
the axon with a hand-held thermocouple and varied by no more than
±0.5°C. Field temperature measurements were made with a Thermochron
IButton data logger (Dallas Semiconductor, Dallas, TX, USA). The reported
accuracy of this device is ±0.5°C.
Propagated action potential measurements
The details of propagated action potential measurements have been described
previously (Rosenthal and Bezanilla,
2000b). In brief, 4-6 cm lengths of axon were mounted in a
rectangular glass chamber. Action potentials were stimulated at one end of the
axon by brief current pulses (2-20 µA for 200-400 µs) delivered through
an intracellular micropipette (0.4-0.6 M
) connected to the ±10 V
output of the on-line D/A converter. Recordings were made at two points along
the axon with intracellular glass microelectrodes (1-3 M
) connected to
capacity-compensated, high-impedance electrometers. The chamber was grounded
through two Ag/AgCl coils embedded in 10K ASW containing 3% agarose and
connected to the amplifier's virtual ground. Signals were digitized at rates
between 200 kHz and 1 MHz (depending on the chamber temperature) using a PC44
signal processor board (Innovative Integration, Simi Valley, CA, USA) and a
PC-compatible computer. Data were filtered at 1/10th the sampling rate and
analyzed using software written in our laboratory. A calibrated eyepiece
micrometer was used to measure each axon's diameter and the distance between
electrodes. Only electrode impalements that displayed resting potentials more
hyperpolarized than -55 mV were considered for analysis. In all experiments,
axons were bathed in 10K ASW.
Voltage-clamp measurements
The voltage-clamp apparatus utilized in these studies has been described
previously in greater detail (Bezanilla et al.,
1982a,
b
;
Rosenthal and Bezanilla,
2000b
). In general, transmembrane voltage was measured with two
reference electrodes. The external reference, a wide-bore glass capillary, was
positioned adjacent to the axon's mid-point. The internal reference, a
capillary approximately 50-70 µm in diameter, containing a floating 25
µm platinum wire threaded along its entire length, was inserted into the
axon from one end and advanced until its tip reached the mid-point. Voltage
was clamped by passing currents through a platinized platinum wire inserted
down the axon's entire length. The diameter of this wire was normally 75
µm, but for many small axons (<325 µm in diameter) a 50 µm wire
was used. Data were collected as described in the previous section. As before,
10K ASW was used as external solution and native axoplasm served as the
internal solution.
For voltage-clamp experiments that did not involve instantaneous
interruptions of the action potential, signals were leak-subtracted using a
standard P/-4 procedure (Bezanilla and
Armstrong, 1977). Holding potentials were either -65 or -70 mV.
K+ currents were studied by applying tetrodotoxin (TTX) to the
bath. Na+ currents were isolated by subtracting the K+
current (IK) from total membrane currents. To block all inward
current activated by a brief pulse to 0 mV, 200 nmol l-1 TTX was
added. In our hands, TTX subtraction was adequate for studying time points
prior to and including peak Na+ current (INa), a time
range when very little IK was activated. This permitted the
measurement of peak Na+ conductance (gNa) and its
activation kinetics. At later time points, after substantial IK had
been activated, this method became less reliable because of subtraction
artifacts. Accordingly, the inactivation kinetics of INa was not
studied.
Our experimental apparatus was modified slightly for action potential interruptions. To generate the action potential, the axial wire was connected to the output of the current generator and to the output of the control amplifier of the voltage-clamp through an electronic switch (FET switch with TTL input). To switch the voltage-clamp on suddenly, a TTL pulse activated this switch connecting the output of the control amplifier to the axial wire, while the same pulse opened another electronic switch removing an auxiliary feedback loop across the control amplifier. Membrane action potentials were triggered on-line in current-clamp mode by passing brief (approximately 200 µs) current pulses through the axial wire using a battery-powered stimulator. At timed intervals, the current-clamp was changed rapidly to voltage-clamp by activating the switches with TTL pulses generated under computer control. The speed of this transition was assessed by monitoring the voltage signal, and voltages were clamped to their command values within approximately 10 µs. For INa experiments, action potentials were interrupted every 20-50 µs by clamping to the K+ equilibrium potential (EK). For IK experiments, action potentials were interrupted every 100 or every 200 µs by clamping to the Na+ equilibrium potential (ENa). Instantaneous current values were measured immediately after the clamp had settled. Axons were held at -60 mV.
Data analysis
For propagated action potentials, rise times and fall times were measured
between 10 and 90% of the action potential's peak value. In experiments using
two microelectrodes, conduction velocities were computed by dividing the
distance separating the two electrodes by the time between the peak of the
action potential passing each point. Measurements were then normalized by the
square root of the axon's diameter
(Hodgkin and Huxley, 1952;
Taylor, 1963
). For
voltageclamped axons, the time taken for either IK or
INa to reach half its maximal value after a voltage step was used
as an index of activation kinetics. K+ conductance (gK)
was computed by dividing the instantaneous current measured between the end of
a voltage pulse (where IK was fully activated) and the return to
rest by the magnitude of the voltage difference at the same points. For
gNa, peak INa was measured and divided by the magnitude of
the voltage step minus ENa. For action potential
interruption experiments, both gK and gNa were measured
using the second method. Leak subtraction for interruption experiments was
performed manually. For IK interruptions, current was measured
following an instantaneous voltage jump to ENa in the
absence of a stimulated action potential. This current was then subtracted
from experimental values. For INa interruptions, the same strategy
was employed except that voltages were jumped to EK. For
all experiments, gK and gNa were normalized to the axon's
surface area. Surface areas were estimated by visually measuring the axon's
average diameter and assuming the geometry to be that of a cylinder. This
approach assumes that membrane infoldings, if present, were equivalent for the
axons of each species.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Propagated action potential measurements
Like most physiological processes, propagated action potentials are
temperature-sensitive. For example, in squid axons, conduction velocities have
a Q10 of less than 2, and the temperature-sensitivities of the rise
times and fall times are significantly steeper, i.e. Q10 values are
greater than 2 (Chapman, 1967;
Hodgkin and Katz, 1949
;
Rosenthal and Bezanilla,
2000b
). To investigate whether any of these properties correlate
with the squid's thermal environment, recordings were made from axons of each
of the four species identified above. Fig.
1A shows examples of these recordings taken at 15 °C. In each
case, action potentials are characterized by rapid voltage changes of more
than 100 mV, followed by a typical undershoot. A casual inspection of the
recordings indicates that the action potential duration is variable, being
greater in S. sepioidea than in L. opalescens. In
Fig. 1B, the action potentials
have been superimposed by aligning their peaks. Clearly, the duration of the
falling phase differs among all four recordings and correlates with each
species' thermal environment: warmer environments are associated with slower
fall times. The rise time, in contrast, shows a different pattern: the rise
times for all species of Loligo are equivalent and are faster than
those for S. sepioidea.
|
By averaging many experiments, each recorded over a large temperature range, we examined in detail the species-dependent features of the action potentials (Fig. 2). The three parameters investigated are depicted in Fig. 2A. The rise times for all species of Loligo are equivalent (Fig. 2B). Those for S. sepioidea, however, are on average 44% slower at each temperature. The fall times (Fig. 2C) are different for all species and can be ranked from fastest to slowest as follows: L. opalescens < L. pealei < L. plei < S. sepioidea. The relative differences tend to increase at lower temperatures and are particularly apparent below approximately 7.5°C. As with rise times, conduction velocities are equivalent for all Loligo species but different from that of S. sepioidea. At temperatures below 10°C, Loligo conduction velocities are up to 100% faster, whereas above 10°C they are approximately 50% faster. The temperature-sensitivities (Q10) of these processes also vary (Table 1). For each species, the Q10 values of the fall times are large and those of the conduction velocities are relatively small. In general, Q10 values are highest over the lowest temperature range (0-10°C). This feature is particularly exaggerated for S. sepioidea, in which the Q10 for the fall time is 7.9 compared with approximately 4 for the two temperate species of Loligo. That for L. plei, at 5.25, is intermediate. Over higher temperature ranges, the Q10 values for these processes are more similar.
|
|
The temperature limits of the propagated action potential were also studied. No low-temperature block was encountered: all species were able to generate action potentials down to 0°C. However, action potential failure was readily apparent at high temperatures, and the temperature varied among species. For L. pealei and L. opalescens, action potentials failed at 29.5±0.8°C (N=9) and 29.0±0.2°C (N=3) respectively. For L. plei and S. sepioidea, failure temperatures were significantly higher: 37.8±1.3°C (N=4) and 34.9±0.6°C (N=6; means ± S.E.M.) respectively. In all experiments, action potentials could be recovered by lowering the temperature. Interestingly, the failure temperature is reflected in the ratio of the fall time to the rise time (Fig. 2E). Because of the relatively high Q10 of the fall time, this ratio decreases as the temperature increases. As this ratio reaches unity, it is increasingly likely that the action potential will fail. In the case of S. sepioidea, action potentials were measured in which fall times were slightly faster than rise times, but in axons of Loligo failure occurred prior to this point. As with the measurements of the absolute failure temperatures, the fall time:rise time ratio in S. sepioidea and L. plei axons is shifted to the right on the temperature axis.
The preceding analysis of propagated action potentials indicated that both rise times and conduction velocities were similar among Loligo species, but slower in S. sepioidea. By contrast, all species exhibited different fall times. These results suggest that some specific property of the Na+ conductance may differ between S. sepioidea and the Loligo species, and that the K+ conductance may differ among all species. However, the analysis cannot predict which physiological property of a particular conductance will vary. For example, changes in kinetics, voltage-dependence or absolute conductance level could bring about similar results.
Voltage-clamp measurements
To examine gK and gNa directly,
voltage-clamp experiments were performed. Axons from all species except L.
opalescens were compared under identical conditions. Specimens of L.
opalescens were excluded because the axons of most specimens were too
small for this technique. Fig.
3A shows a typical voltage-clamp current recording following a 3.5
ms pulse to 0 mV from an S. sepioidea axon. The general features of
this current were shared by all species. In squid axons, a rapidly activating
inward current is carried by Na+ (INa), and a delayed
outward current is carried by K+ (IK)
(Hodgkin and Huxley, 1952).
IK can be isolated by adding TTX to the bath. In
Fig. 3B, the same axon has been
treated with TTX, and a family of classic `delayed rectifier' K+
currents is shown. Their characteristic shape is derived from rapid activation
following a pronounced delay. INa was more complicated to study.
Fig. 3Ci shows two currents
traces from an L. plei axon after a voltage step to -10 mV before and
after the addition of TTX. The TTX-sensitive current, visualized by
subtracting these two recordings, was considered to be INa
(Fig. 3Cii). INa of
squid axons activates very rapidly, with a much shorter delay than
IK, and then inactivates with a quasi-exponential time course. It
is noteworthy that, when INa is at its peak, virtually no
IK has activated (Fig.
3Ci). This relationship held irrespective of the voltage,
temperature or species (data not shown). At later times, increasingly larger
proportions of IK had to be subtracted. In some axons, this process
introduced artifacts in the time course of INa inactivation. For
this reason, analysis focused on activation kinetics and maximal conductance.
A family of subtracted Na+ currents, recorded at various voltages,
is shown in Fig. 3Ciii. In this
case, currents activate rapidly even after modest depolarizations, and their
direction reverses between approximately +30 and +50 mV. The following
analysis is based on IK and INa recorded from all three
species in an analogous manner.
|
Steady-state and kinetic parameters of gNa and gK were analyzed for all species, and these results are presented in Fig. 4. Clearly, the most striking difference rests in the absolute levels of gNa and gK. For both species of Loligo, gNa reaches a maximum of approximately 75 mS cm-2 at voltages greater than +10 mV (Fig. 4A). In S. sepioidea axons, gNa reaches only 47 mS cm-2 (approximately 63% of the value for the Loligo species). Maximal gK, however, is different for all three species: for L. pealei, L. plei and S. sepioidea, the corresponding values are 73 mS cm-2, 54 mS cm-2 (approximately 74% of the value for L. pealei) and 38 mS cm-2 (approximately 52%), respectively. These differences are highly significant (see figure legend). In Fig. 4C,D, values of gNa and gK have been normalized to their maximum values and then plotted against voltage. In both cases, the conductances activate steeply between approximately -30 mV and +20 mV. The gNa/voltage relationship is particularly steep. In neither case, however, is there a significant inter-species difference in voltage-dependence. Activation kinetics for both currents were also equivalent among species. The half-time (t1/2) for activation versus voltage relationships for both INa and IK are shown in Fig. 4E,F. In general, INa activated approximately 10 times faster than IK. For both INa and IK, half-times for activation were not statistically different among species. All results in Fig. 4 were taken from axons at 10°C. Similar experiments conducted at 20°C yielded the same relative results (not shown).
|
Conductance measurements during the action potential
Data from voltage-clamped axons suggest that differences in absolute
conductance levels are consistent with species-specific differences in the
propagated action potential. Accordingly, slower action potential rise times
for S. sepioidea are due to a smaller gNa;
differences in fall times between all three species are due to different
levels of gK. These hypotheses were tested directly by
reconstructing the time course and magnitude of gNa and
gK during membrance action potentials. The strategy was to
stimulate action potentials in current-clamped axons and then to `interrupt'
them at specific times by rapidly switching to voltage-clamp mode. If the
voltage is clamped to the K+ equilibrium potential
(EK), then the resulting instantaneous current jump is due
to INa. Conversely, if the voltage is clamped to the Na+
equilibrium potential (ENa), then the current is due to
IK. Thus, by interrupting the action potential at many points, the
time course of both gNa and gK can be
measured. The method of action potential interruption, along with the
underlying assumptions, has been described in greater detail previously
(Bezanilla et al., 1970;
Rojas et al., 1970
).
It is assumed that, early during an action potential, the reversal potential (Erev) corresponds to ENa, and later to EK (when INa or IK is large, small leak conductances have a minimal contribution; see above references). Therefore, to determine ENa and EK, Erev was measured at different times during an action potential. Fig. 5 demonstrates an example of this type of experiment. Fig. 5A shows a typical membrane action potential, in this case recorded from a L. pealei axon at 15°C. In Fig. 5Bi, seven action potentials, from the same axon, have been superimposed. In each case, the voltage-clamp was activated after 700 µs and the membrane was clamped to a different potential (-70 mV to +50 mV in increments of 20 mV). Fig. 5Bii shows the resulting current traces. All the following results focus on the magnitude of the instantaneous current jump after the voltage-clamp has been activated and the voltage has settled to its new value. The relationship between this current and voltage (I/V) was used to determine Erev. In this case, Erev was -27 mV (due to activation of both gNa and gK). Fig. 5C shows a time series of Erev determinations plotted on top of an action potential. In this case, Erev begins at approximately +55 mV and, by the end of the action potential, reaches approximately -70 mV. The maximum and minimum values are taken to be ENa and EK, respectively. Similar experiments were repeated for each species, and no significant differences were found among species for either ENa or EK.
|
Values of ENa and EK were then used to examine the time course of gNa and gK during an action potential (Fig. 6). Fig. 6Ai shows an `interrupted' action potential from an L. pealei axon at 10°C. In this case, the action potential is clamped after 700 µs (arrow) to -70mV (EK). The resultant INa is shown in Fig. 6Aii. At the time the clamp is activated, the current jumps almost instantaneously from near 0 µA to approximately 375 µA, after which INa rapidly deactivates back to 0 µA. Thus, the instantaneous current jump is proportional to gNa at 700 µs. Here, gNa is 3.0 mS. A similar experiment, designed to measure gK, is illustrated in Fig. 6Bi. In this case, the action potential is interrupted after 2 ms and clamped to +55 mV (ENa). Upon clamping, the current (Fig. 6Bii) jumps from near zero to approximately 173 µA. Thus, gK at 2 ms is 1.4 mS.
|
These procedures, conducted at many time points, were extended to each species, and the complete time courses for both gNa and gK were determined (Fig. 6C-E). In each case, gNa activates rapidly and reaches its peak simultaneously with that of the action potential. gK activates less steeply and with a pronounced delay, reaching its peak after the action potential has almost fully repolarized. Several features, however, differ between the data presented in Fig. 6C-E. First, peak gK is greatest in L. pealei (approximately 29 mS cm-2), intermediate in L. plei (approximately 23 mS cm-2) and smallest in S. sepioidea (approximately 19 mS cm-2). Peak gNa, in contrast, is greatest in L. plei (approximately 65 mS cm-2), intermediate in L. pealei (approximately 50 mS cm-2) and smallest in S. sepioidea (approximately 44 mS cm-2). In addition, gK appears to turn on and off faster in L. pealei than in the other two species. gNa, in contrast, activates extremely rapidly in each case. It turns off, however, more rapidly in L. pealei than in the other two species. These findings are based on data from a single representative axon for each species. Because of variation among axons, these studies were extended to multiple axons from each species.
Fig. 7 shows mean
gNa and gK for each species. Because
the stimulus duration and action potential latency varied slightly among
axons, all time points were normalized using the action potential's peak as
time zero. Fig. 7A demonstrates
that, contrary to the results presented in
Fig. 6, absolute
gNa is equivalent for both Loligo species,
reaching approximately 55 mS cm-2 as the action potential peaks. In
S. sepioidea, peak gNa is significantly smaller
(approximately 37 mS cm-2; 67% of the value for the Loligo
species; P0.05). In Fig.
7B, gNa measurements have been normalized to
their peak values to compare kinetics. Although gNa
appears to peak slightly more slowly in S. sepioidea, there are no
significant differences in activation. The time course of the falling phase
does differ among species, being fastest in L. pealei, intermediate
in L. plei and slowest in S. sepioidea. This is expected
because, during the action potential, the turning off of
gNa is driven mainly by the turning on of
gK, which is fastest in L. pealei and slowest in
S. sepiodea. Unlike gNa, maximum
gK differs among species. It is greatest in L.
pealei (approximately 36 mS cm-2), intermediate in L.
plei (approximately 24 mS cm-2; 66% of the value for L.
plei) and smallest in S. sepioidea (approximately 17 mS
cm-2; 47%). The kinetics of gK
(Fig. 7D) is indistinguishable
between L. plei and S. sepioidea, but for L. pealei
both the on and off rates are relatively fast. Thus, as with voltage-clamp
experiments, absolute conductances show dramatic interspecies differences.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
L. opalescens is commonly found off the Pacific coast of North
America and is particularly abundant off California. In the winter,
populations are found in southern California, whereas for the rest of the year
they are found off northern and central California. In both cases, surface
temperatures rarely exceed 13°C and are often several degrees lower. These
squid have also been observed below the thermocline at temperatures as low as
6-7°C (Neumeister et al.,
2000). Specimens of L. pealei are commonly encountered
off the Atlantic coast of North America from the Gulf of Maine to the Gulf of
Mexico. Their temperature range has been discussed in greater detail elsewhere
(Boyle, 1983
;
Rosenthal and Bezanilla,
2000b
). In Woods Hole, this squid is most commonly encountered
between approximately 10 and 20°C, although their abundance is greatest
when water temperatures are at the low end of this range. They are reported
actively to avoid temperatures below 8°C
(Summers, 1969
). Specimens of
L. plei are commonly found in the Gulf of Mexico and in the Caribbean
Sea off northern South America. In Mochima, Venezuela, where the specimens for
this study were collected, water temperatures at the capture site (and depth)
were between 18 and 20.5°C. In the Gulf of Mexico, these squid are often
encountered in the same areas as L. pealei; however, the abundance of
each species is stratified with depth. L. plei prefer warmer surface
waters (<50 m), whereas L. pealei prefer cooler, deeper waters
(Hixon et al., 1980
; Whitaker,
1978
,
1980
). S. sepioidea
are normally found adjacent to shallow reefs in the Caribbean Sea and the Gulf
of Mexico (Voss, 1956
). In
Mochima, where specimens were collected, data loggers indicated that daily
temperatures fluctuated between 23.5 and 27°C in April and May. In July
and August, temperatures were significantly higher (26-29°C). It should be
noted that both L. plei and S. sepioidea were maintained at
29-30°C in flowing seawater tanks for several days before use with no
obvious detrimental effects.
The data presented in this report identify three properties of the propagated action potential that vary. First, the duration of the action potential's falling phase differs among all species tested and correlates with the thermal environment of the species; when measured at equivalent temperatures, action potentials from species that inhabit colder environments are shorter. Second, the action potential's rising phase is relatively slow in S. sepioidea, but equivalent in all three species of Loligo. Third, the same relationship holds true for the conduction velocity.
The absolute levels of gK and gNa are consistent with all
three changes in the propagated action potential. It should be stressed that
species-dependent conductances were measured using two independent sets of
experiments (voltage-clamped axons and action potential interruptions) and
both yielded comparable results. Clearly, an increase in gK will
decrease the duration of the action potential's falling phase, an assertion
supported by the Hodgkin and Huxley model (data not shown). In fact, for a
number of systems, the action potential's duration is regulated by changing
the level of gK (Chen et al.,
1996; Kaab et al.,
1996
; Thuringer et al.,
1996
; Zhang and McBain,
1995
). The relatively slow action potential rise time and
conduction velocity in S. sepioidea axons are consistent with its low
levels of gNa. On the basis of the Hodgkin and Huxley model axon, the
level of gNa correlates well with conduction velocity and rise time
(Adrian, 1975
;
Hodgkin, 1975
;
Huxley, 1959
). However, more
quantitative extrapolations of conduction velocity and rise time, using our
measured conductance values, were not attempted. Years of scrutiny have
indicated that several aspects of the Hodgkin and Huxley model require further
refinement to predict properties of conduction with sufficient accuracy for
the present purposes (Armstrong and
Bezanilla, 1977
; Armstrong et
al., 1973
; Armstrong and Hille,
1998
; Vandenberg and
Bezanilla, 1991
; Bezanilla,
2000
; Bezanilla and Armstrong,
1977
). For example, as seen in
Fig. 6, at the action
potential's peak, there is virtually no gK, a result not predicted by
the Hodgkin and Huxley equations using the original parameters
(Hodgkin and Huxley,
1952
).
There are many plausible mechanisms for regulating ionic conductances in
the giant axon. The most straightforward would be to modify the surface
expression of Na+ or K+ channels by transcriptional
mechanisms, by translational mechanisms or by changing turnover rates.
Conceivably, the unitary conductance of Na+ and K+
channels could also be regulated. No evidence supports or refutes any of these
possibilities. Species-dependent differences in slow inactivation could also
influence the available levels of ionic conductances. However, at the holding
potentials used for these studies (-65 mV), no significant slow
inactivation was apparent for any species. For gK, regulation may
also be at the level of subunit assembly. Recent evidence suggests that
SqKv1.1A, a K+ channel mRNA that is thought to underlie
some or all of delayed rectifier gK in the giant axon
(Rosenthal et al., 1996
),
contains several anomalous amino acid residues in a channel domain (T1)
responsible for subunit tetramerization
(Liu et al., 2001
). The amino
acids at these positions, which are regulated by RNA editing
(Rosenthal and Bezanilla,
2000a
), strongly influence the functional expression of
heterologously expressed channels in Xenopus oocytes. Further
evidence suggests that the pattern of RNA editing in the T1 domain of
SqKv1.1A varies among different species of squid
(Rosenthal and Bezanilla,
1998
).
The conductance level changes identified in this study are a plausible
regulatory mechanism for the changes observed in the propagated action
potentials. However, other possibilities exist. For example, inactivation and
deactivation of gNa could contribute to the action potential's rate
of decline. The axon's cable properties (e.g. resistance or capacitance) could
also vary among species. Previous work has demonstrated that, within a
species, seasonal changes in the giant axon's internal resistance can
influence conduction velocity (Rosenthal
and Bezanilla, 2000b).
Do these data describe temperature adaptations? To answer this question,
several factors should first be considered. Does the giant axon system mediate
an equivalent function in all the species tested? On the basis of similarities
in anatomy and behavior, it is assumed that for each species the giant axon
system regulates the jet-propelled escape response
(Otis and Gilly, 1990;
Young, 1938
) and plays a role
in feeding behavior (Preuss and Gilly,
2000
). Are these species sufficiently close on a phylogenetic
level to permit a meaningful comparison? Although there are no established
standards, all four species are members of the family Loliginidae. On the
basis of recent phylogenetic studies using mitochondrial DNA sequences, the
three Loligo species are more closely related to each other than to
other members of the genus. The species S. sepioidea is the outlier
(Anderson, 2000
). Therefore,
when considering our data, more weight should be given to physiological
differences that varied within the genus Loligo (e.g. the level of
gK). All three species of Loligo are active pelagic
predators. Although S. sepioidea are more domercile, it is probable
that the giant axon system mediates the same basic function for all the squid
species tested.
It is also important to consider why a physiological difference would be
adaptive. First, the action potential's duration will be considered in terms
of simple rate compensation. Is it necessary for species that inhabit
different thermal environments to maintain a similar action potential duration
at their native temperatures? If so, modifications of the underlying ionic
conductances are required. Data from this study only partially support this
view. Action potentials from L. opalescens giant axons are relatively
fast and those from S. sepioidea are slow. However, the rate
compensation is not complete. On average, there is an approximately 5 °C
shift in the fall times between L. opalescens and S.
sepioidea and an overall shift of approximately 7.5 °C in the total
action potential duration (fall time plus rise time). The temperature
difference between these two species' environment is approximately double
this. Clearly, there is not perfect rate compensation. When considering the
purpose of the giant axon's action potential, to stimulate an escape jet, the
necessity to compensate the duration in the first place is not immediately
apparent. A single action potential in the giant axon produces an all-or-none
contraction of the mantle musculature
(Prosser and Young, 1937;
Young, 1938
). Does the
duration of this action potential matter? A better understanding of the
relationship between temperature, action potential duration and mantle
contraction could help shed light on this issue.
An alternative hypothesis is that the action potential broadening, seen in the species from warmer habitats (L. plei and S. sepioidea), is merely a consequence of an adaptation to avoid action potential failure at high temperatures. As discussed in the Results section, action potentials in the two species from colder habitats fail at approximately 29 °C. The temperature range for both L. plei and S. sepioidea can approach this level. In fact, in our hands, both species survive in tanks maintained at this temperature. Using the Hodgkin and Huxley paradigm, decreases in gK lead to increases in the failure temperature because less gK competes with gNa following stimulation. Action potentials from both `warm' species exhibited a reduced gK and elevated failure temperature. Other factors, however, could also contribute to differences in failure temperature (e.g. differences in Na+ channel inactivation). Clearly, avoiding action potential failure would be an adaptational advantage.
The reported changes in normalized conduction velocity can also be viewed
in terms of compensatory adaptations. The conduction velocity of the giant
axon of S. sepioidea, at all temperatures, is slower than that of
Loligo species. In fact, the shift of approximately 10 °C between
the conduction velocity versus temperature relationships is in
reasonable agreement with the temperature ranges for these two species. Thus,
to make the escape response as fast as possible, Loligo species have
more gNa in their axons to compensate for a relatively cold
environment. It is important to note, however, that there is no apparent rate
compensation within the genus Loligo, even though the species
utilized in this study span a considerable range of habitat temperature. A
previous study, using two Loligo species, hypothesized that a change
in the slope of the conduction velocity versus temperature
relationship was a temperature adaptation
(Easton and Swenberg, 1975).
In our study, at temperatures above 7 °C, there is no evidence for a
change in the slope of the conduction velocity versus temperature
realtionship. Members of the genus Loligo are not commonly found
below this temperature.
At present, we consider the physiological properties identified in this
study as reasonable candidates for temperature adaptations. Similar
investigations utilizing more representatives within the family Loliginidae
would help clarify the issue. These findings are interesting in light of the
considerable attention that has been paid to the theory of homeoviscous
adaptation (Macdonald, 1988;
Sinensky, 1974
). No
comparative data exist for membrane lipid viscosity in the giant axon;
however, homeoviscous adaptation would be expected to affect the rates of ion
channel gating. These data suggest that absolute conductance levels are more
likely to be targets of adaptation. However, Na+ channel
inactivation and deactivation kinetics need to be measured. For these and
other studies, the squid giant axon remains an excellent system in which to
study temperature adaptation in nerve.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adrian, R. H. (1975). Conduction velocity and gating current in the squid giant axon. Proc. R. Soc. Lond. B 189,81 -86.[Medline]
Anderson, F. E. (2000). Phylogeny and historical biogeography of the loliginid squids (Mollusca: Cephalopoda) based on mitochondrial DNA sequence data. Mol. Phylog. Evol. 15,191 -214.[Medline]
Armstrong, C. M. and Bezanilla, F. (1977). Inactivation of the sodium channel. II. Gating current experiments. J. Gen. Physiol. 70,567 -590.[Abstract]
Armstrong, C. M., Bezanilla, F. and Rojas, E.
(1973). Destruction of sodium conductance inactivation in squid
axons perfused with pronase. J. Gen. Physiol.
62,375
-391.
Armstrong, C. M. and Hille, B. (1998). Voltage-gated ion channels and electrical excitability [see comments]. Neuron 20,371 -380.[Medline]
Bezanilla, F. (2000). The voltage sensor in
voltage-dependent ion channels. Physiol. Rev.
80,555
-592.
Bezanilla, F. and Armstrong, C. M. (1977).
Inactivation of the sodium channel. I. Sodium current experiments.
J. Gen. Physiol. 70,549
-566.
Bezanilla, F., Rojas, E. and Taylor, R. E. (1970). Sodium and potassium conductance changes during a membrane action potential. J. Physiol., Lond. 211,729 -751.[Medline]
Bezanilla, F., Taylor, R. E. and Fernández, J. M. (1982a). Distribution and kinetics of membrane dielectric polarization. I. Long-term inactivation of gating currents. J. Gen. Physiol. 79,21 -40.[Abstract]
Bezanilla, F., Vergara, J. and Taylor, R. E. (1982b). Voltage clamping of excitable membranes. Meth. Exp. Physiol. 20,445 -511.
Boyle, P. R. (1983). Cephalopod Life Cycles. London: Academic Press.
Chapman, R. A. (1967). Dependence on temperature of the conduction velocity of the action potential of the squid giant axon. Nature 213,1143 -1144.[Medline]
Chatfield, P. O., Lyman, C. P. and Irving, L.
(1953). Physiological adaptation to cold of peripheral nerve in
the leg of the herring gull (Laras argeniatus). Am. J.
Physiol. 172,639
-644.
Chen, Y., Sun, X. D. and Herness, S. (1996).
Characteristics of action potentials and their underlying outward currents in
rat taste receptor cells. J. Neurophysiol.
75,820
-831.
Easton, D. M. and Swenberg, C. E. (1975).
Temperature and impulse velocity in giant axon of squid Loligo pealei.Am. J. Physiol. 229,1249
-1253.
Hixon, R. F., Hanlon, R. T., Gillespie, S. M. and Griffin, W. L. (1980). Squid fishery in Texas: biological, economic and market considerations. Mar. Fish. Rev. 42, 44-50.
Hodgkin, A. (1975). The optimum density of sodium channels in an unmyelinated nerve. Phil. Trans. R. Soc. Lond. B 270,297 -300.[Medline]
Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol., Lond. 117,500 -541.[Medline]
Hodgkin, A. L. and Katz, B. (1949). The effect of temperature on the electrical activity of the giant axon of the squid. J. Physiol., Lond. 109,240 -249.
Huxley, A. F. (1959). Ion movements during nerve activity. Ann. N.Y. Acad. Sci. 81,221 -246.
Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.
H., Kass, D. A., Marban, E. and Tomaselli, G. F. (1996).
Ionic mechanism of action potential prolongation in ventricular myocytes from
dogs with pacing-induced heart failure. Circ. Res.
78,262
-273.
Lagerspetz, K. Y. and Talo, A. (1967). Temperature acclimation of the functional parameters of the giant nerve fibres in Lumbricus terrestris. I. Conduction velocity and the duration of the rising and falling phase of the action potential. J. Exp. Biol. 47,471 -480.[Medline]
Liu, T. I., Lebaric, Z. N., Rosenthal, J. J. C. and Gilly, W.
F. (2001). Natural substitutions at highly conserved
T1-domain residues perturb processing and functional expression of squid Kv1
channels. J. Neurophysiol.
85, 61-71.
Macdonald, A. G. (1988). Application of the theory of homeoviscous adaptation to excitable membranes: pre-synaptic processes. Biochem. J. 256,313 -327.[Medline]
Macdonald, J. A. (1981). Temperature compensation in the peripheral nervous system: Antarctic vs. temperate poikilotherms. J. Comp. Physiol. A 142,411 -418.
MacDonald, J. A., Montgomery, J. C. and Wells, R. M. G. (1988). The physiology of McMurdo Sound fishes: current New Zealand research. Comp. Biochem. Physiol. 90B,567 -578.
Neumeister, H., Ripley, B., Preuss, T. and Gilly, W. F.
(2000). Effects of temperature on escape jetting in the squid
Loligo opalescens. J. Exp. Biol.
203,547
-557.
Otis, T. S. and Gilly, W. F. (1990). Jet-propelled escape in the squid Loligo opalescens: concerted control by giant and non-giant motor axon pathways. Proc. Natl. Acad. Sci. USA 87,2911 -2915.[Abstract]
Preuss, T. and Gilly, W. F. (2000). Role of
prey-capture experience in the development of the escape response in the squid
Loligo opalescens: a physiological correlate in an identified neuron.
J. Exp. Biol. 203,559
-565.
Prosser, C. L. and Nelson, D. O. (1981). The role of nervous systems in temperature adaptation of poikilotherms. Annu. Rev. Physiol. 43,281 -300.[Medline]
Prosser, C. L. and Young, J. Z. (1937). Responses of muscles of the squid to repetitive stimulation of the giant nerve fibers. Biol. Bull. 73,237 -241.
Rojas, E., Bezanilla, F. and Taylor, R. E. (1970). Demonstration of sodium and potassium conductance changes during a nerve action potential. Nature 225,747 -748.[Medline]
Rosenthal, J. J. C. and Bezanilla, F. (1998). Evidence for RNA editing in squid giant axon. Am. Zool. 37,190A .
Rosenthal, J. J. C. and Bezanilla, F. (2000a). Editing of delayed rectifier K+ channel mRNA in squid giant axon. Biophys. J. 78,214A .
Rosenthal, J. J. C. and Bezanilla, F. (2000b).
Seasonal variation in conduction velocity of action potentials in squid giant
axons. Biol. Bull. 199,135
-143.
Rosenthal, J. J. and Gilly, W. F. (1993). Amino acid sequence of a putative sodium channel expressed in the giant axon of the squid Loligo opalescens. Proc. Natl. Acad. Sci. USA 90,10026 -10030.[Abstract]
Rosenthal, J. J., Vickery, R. G. and Gilly, W. F. (1996). Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon. J. Gen. Physiol. 108,207 -219.[Abstract]
Sinensky, M. (1974). Homeoviscous adaptation - a homestatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc. Natl. Acad. Sci. USA 71,522 -525.[Abstract]
Summers, W. C. (1969). Winter population of L. pealei in the mid-Atlantic Bight. Biol. Bull. 137,202 -216.
Taylor, R. E. (1963). Cable theory. In Physical Techniques in Biological Research, vol.6 (ed. W. L. Nastuk), pp.219 -262. New York: Academic Press.
Thuringer, D., Deroubaix, E., Coulombe, A., Coraboeuf, E. and Mercadier, J. J. (1996). Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy. Cardiovasc. Res. 31,747 -757.[Medline]
Vandenberg, C. A. and Bezanilla, F. (1991). A sodium channel gating model based on single channel, macroscopic ionic and gating currents in the squid giant axon. Biophys. J. 60,1511 -1533.[Abstract]
Voss, G. L. (1956). A review of the cephalopods of the Gulf of Mexico. Bull. Mar. Sci. Gulf Caribb. 6, 85-119.
Weight, F. F. and Erulkar, S. D. (1976). Synaptic transmission and effects of temperature at the squid giant synapse. Nature 261,720 -722.[Medline]
Whitaker, J. D. (1978). A contribution to the biology of Loligo pealei and Loligo plei (Cephalopoda, Myopsida) off the southeastern coast of the United States. MS thesis, pp. 186. College of Charleston.
Whitaker, J. D. (1980). Squid catches resulting from trawl surveys of the southeastern United States. Mar. Fish. Rev. 42,39 -43.
Young, J. Z. (1938). The functioning of the giant nerve fibres of the squid. J. Exp. Biol. 15,170 -185.
Zhang, L. and McBain, C. J. (1995). Potassium conductances underlying repolarization and after-hyperpolarization in rat CA1 hippocampal interneurones. J. Physiol., Lond. 488,661 -672.[Abstract]
Related articles in JEB: