Department of Physiology and Biophysics, Dalhousie University,
Halifax, Nova Scotia B3H 4H7, Canada
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INTRODUCTION |
Voltage-activated
INa is the main current responsible
for the rising phase of the action potential in many neurons. Some
neurons have two components of INa,
one inactivating in the millisecond range and the other being more
persistent. The slower component has been found in neurons that adapt
slowly, where it recovers from inactivation more rapidly than the fast
component (e.g., Elliott and Elliott 1993
), and it was
suggested to have a role in allowing high-frequency firing. However, a
different role for slow INa
inactivation has also been presented, where it was assumed to cause
adaptation of action potential firing (Gestrelius and Grampp
1983
).
The lyriform slit-sense organ VS-3 in the patella of the spider,
Cupiennius salei (nomenclature of Barth and Libera
1970
) has seven to eight slits, which are each innervated by a
pair of bipolar mechanosensory neurons. Both neurons in each pair
respond to a sustained mechanical or electrical stimulus with a rapidly adapting burst of action potentials. However, adaptation to silence in
type A neurons occurs in <20 ms, often producing only one or two
action potentials, while type B neurons respond with a burst of action
potentials lasting several hundred milliseconds (Seyfarth and
French 1994
). Part of the difference in adaptation to
mechanical stimuli can be explained by differences in the dynamic
properties in the transduction step from mechanical stimulus to
receptor potential (Juusola and French 1998
), but a
similar adaptation difference is present even when the neurons are
stimulated electrically, bypassing the mechanical stage. We have
studied the active membrane conductances and their contributions to
overall dynamic behavior of the VS-3 neurons. We began by examining the
kinetics of voltage-activated IK
(Sekizawa et al. 1999
) but found only small differences
between type A and type B neurons, which may contribute to the
differences in their adaptation properties but are unlikely to be major
factors. Subsequently, we found a large transient
low-voltage-activated (LVA) ICa in
both types of VS-3 neurons (Sekizawa et al. 2000
), but
this also seemed very similar in the two neuron types. We have also
found that these neurons do not have
Ca2+-activated K+ currents
[IK(Ca)] or a classical A-type
current (IK) (Sekizawa et al.
1999
), which have both been associated with spike frequency adaptation in some insect mechanosensory neurons (Torkkeli and French 1994
, 1995
) and other neurons (e.g.,
Crest and Gola 1993
; Rogawski 1985
).
The action potentials in VS-3 neurons are normally driven by the
voltage-activated INa. Therefore any
differences in the kinetics of INa
between type A and type B neurons could have a significant impact on
their adaptation properties. Research into the properties of
INa in VS-3 neurons is limited by the
fact that these neurons are too small for two-electrode voltage clamp,
and it has not been possible to voltage clamp this propagating current,
with rapid activation and inactivation kinetics, using single-electrode voltage-clamp methods. Here, we reduced the bath
Na+ concentration to about one-half that in
normal saline, which reduced the amplitude of
INa to a level where it could be well clamped. This approach allowed us to investigate the kinetic
differences between type A and type B neurons to determine the role of
this current in action potential frequency modulation.
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METHODS |
Preparation
Experiments were carried out using autotomized legs from adult
Central American Wandering Spiders, Cupiennius salei, Keys. The spiders were kept in a laboratory colony at room temperature (22 ± 2°C, mean ± SD). A piece of patellar cuticle
containing slit-sense organ VS-3 was dissected free in spider saline,
which contained (in mM) 223 NaCl, 6.8 KCl, 8 CaCl2, 5.1 MgCl2, 5 sucrose, and 10 HEPES, pH 7.8 (Höger et al. 1997
).
The hypodermis preparation of VS-3 neurons described in detail by
Sekizawa et al. (1999)
was used in all experiments and
is shown in Fig. 1. Briefly, the neurons
were detached from the cuticle but remained embedded in an internal
membrane (hypodermis), which was spread onto a small coverslip. The
axons and dendrites were crushed at ~100 µm from the somata to
improve the voltage-clamp conditions. The coverslip was placed on a
preparation holder where the neurons could be superfused with different
solutions during recordings.

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Fig. 1.
Hypodermis preparation of VS-3 neurons. Intracellular recording
electrodes are shown impaling both neurons in pair 4.
Type A neurons usually fire only one action potential, while type B
neurons fire bursts of up to several hundred milliseconds. Methods of
measuring the threshold depolarization (a), action potential amplitude
(b), and action potential duration (c) are shown in the recording from
the type A neuron. In each case the letter indicates the distance
between the 2 lines where it is placed.
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Recording and stimulation
Recordings were performed using the discontinuous
single-electrode current- and voltage-clamp methods (Finkel and
Redman 1984
) with an SEC-10 l amplifier (NPI Electronic, Tamm,
Germany). We have previously described the conditions for successful
single-electrode voltage- and current-clamp in detail (Torkkeli
and French 1994
), and the same methods have been used in VS-3
neurons to study voltage-activated IK
(Sekizawa et al. 1999
) and
ICa (Sekizawa et al.
2000
). A horizontal puller (P-2000, Sutter Instrument, Novato,
CA) was used to pull microelectrodes from borosilicate glass (1 mm OD
and 0.5 mm ID). Electrodes were filled with 3 M CsCl, and their
resistances were 40-90 M
. Electrodes were coated with petroleum
jelly to reduce stray capacitance (Juusola et al. 1997
),
and they had time constants of 1-3 µs in solution. Switching
frequencies of 20-40 kHz and a duty cycle of 1/4 (current
passing/voltage recording) were used in all experiments. All
voltage-clamp experiments were performed by averaging three current
recordings with 5-s intervals.
The neurons were observed under bright-field optics (Olympus, Tokyo,
Japan). They were impaled with high-frequency oscillation ("buzzing") followed by a 15-min stabilizing period before
recordings. All current- and voltage-clamp experiments were controlled
by an IBM compatible computer with custom written software (ASF
Software, Halifax, Nova Scotia, Canada). Voltage and current stimuli
were provided by the computer via a 12-bit D/A converter. The membrane potential recording was low-pass filtered at 33.3 kHz and the current
at 3.3 kHz by the voltage-clamp amplifier. Membrane resistance was
calculated from the linear part of the current-voltage curve at
hyperpolarizing potentials, and this value was used for leakage subtraction.
Statistical analyses were performed using Prophet 6.0 software (AbTech
Corporation, Charlottesville, VA). Data were analyzed using a two-sided
unpaired t-test for significantly different means or one-way
unblocked ANOVA.
Chemicals
To record INa, extracellular
Na+ concentration was reduced from 223 to 100 mM,
because normal Na+ concentration did not allow
stable voltage clamp. Voltage-activated ICa was blocked by a combination of 50 µM NiCl2 and 100 µM
CdCl2, and voltage-activated
IK were blocked by a combination of 25 mM tetraethylammonium chloride (TEA, Kodak) and 25 mM 4-aminopyridine (4-AP) in the bath. CsCl (3 M) in the electrode further reduced IK, but with sharp electrode only a
small amount of the pipette solution can reach the cell interior, and
therefore the blockade of IK was never
complete at strong depolarizations as seen in Fig.
2A. INa was
completely blocked by bath application of 1 µM TTX, and the
subtracted currents (Fig. 2B) could be used for kinetic analysis of INa. Sucrose was used to
adjust the osmolarity.

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Fig. 2.
INa in VS-3 neurons. A:
currents elicited from a holding potential of 100 mV to potentials
from 90 to 30 mV at 10-mV intervals. Top panel
shows the control recording, and the bottom panel shows
identical recording after INa was blocked
with 1 µM TTX. B: an example of subtraction of the
current trace after TTX from a trace in a control recording to
eliminate the outward currents. This recording was obtained with a
voltage pulse of 20 mV.
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 |
RESULTS |
Voltage response
Na+ is responsible for producing action
potentials in the VS-3 neurons (Sekizawa et al. 2000
;
Seyfarth and French 1994
). We examined the voltage
response of both types of neurons to learn whether there were any
differences between their action potential firing, other than the
well-known difference in the number of action potentials. Figure 1
shows typical responses of both types of neurons, and Table
1 lists the electrophysiological
parameters and P values for statistical tests between type A
and type B neurons. While the action potential amplitudes and durations
were very similar in both neuron types, there were statistically
significant differences in the action potential thresholds and membrane
potentials. In type A neurons the level of depolarization needed to
produce an action potential was about 7 mV more positive than in the
type B neurons.
Voltage-activated Na+
current (INa)
Figure 2A shows a typical voltage-clamp recording from
a type A neuron in response to positive voltage stimuli when
the extracellular solution contained blockers for
IK and
ICa and had low
Na+ concentration (see METHODS). A
significant part of the outward IK
remained at high depolarizing voltages. However,
INa could be completely blocked by 1 µM TTX, and the residual outward currents subtracted to uncover
INa (Fig. 2B).
The time courses of activation and inactivation of
INa at different test potentials were
fitted by the Hodgkin-Huxley equation for an inactivating current
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(1)
|
where I
is the current level
expected in the absence of inactivation,
t1 is the time constant of activation,
t2 is the time constant of
inactivation, and m is an integer exponent (Hodgkin
and Huxley 1952
). m values of 3 or 4 gave the best
fit for all of the current traces. The inset in Fig.
3A shows an example of this
fit. The mean (±SD) activation and inactivation time constants of
INa for 6 type A and 11 type B neurons
are shown in Fig. 3. Both time constants were voltage dependent, being
slightly faster at more depolarizing potentials, but there were no
statistically significant differences in either parameter between the
two neuron types.

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Fig. 3.
Activation and inactivation time constants of
INa. A, inset:
an example of a current trace following a stimulus to 20 mV fitted
with an exponential power equation (Eq. 1) with a time
constant of 1.5 ms for activation and 16.9 ms for inactivation. Mean
activation time constants (±SD) from 6 type A ( ) and 11 type B
( ) neurons are plotted against test voltage.
B: mean inactivation time constants (±SD) from the same
neurons as in A at different test voltages.
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INa activated at about
50 mV (Fig.
4A) and reached its maximum
amplitude at potentials close to
20 mV in both types of neurons. There were no statistically significant differences in
INa amplitudes between the two neuron
types (Table 1). The experimental activation data were fitted by
a Boltzmann distribution of the form
|
(2)
|
where I is the current at test potential V,
Imax is the maximum current,
V50 is the potential giving the
half-maximum current, and s is the slope factor. Fitting was
performed by the Levenberg-Marquardt general nonlinear fitting
algorithm (Press et al. 1990
). Boltzmann fits for 11 neurons of both types are shown in Fig. 4B, and the mean
(±SD) values for V50 and s
from fits of the same neurons are given in the Table 1. There were no
statistically significant differences in these values between the two
neuron types.

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Fig. 4.
Voltage dependence of activation of INa.
A: points show the normalized mean (±SD) peak
INa from 11 type A ( ) and 11 type B ( ) neurons plotted against test potentials.
B: steady-state activation was determined by fitting the
peak currents from each experiment with a Boltzmann distribution
(Eq. 2). Data from the same neurons as in
A.
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INa inactivation was also voltage
dependent. We determined the steady-state inactivation using 20-ms
conditioning pulses followed by a test pulse to a fixed potential (Fig.
5A). Currents were then
normalized by the maximum current, plotted as functions of test
potential, and fitted by Eq. 2 with appropriate change in the exponential sign (Fig. 5B). The fitted parameters from
seven experiments with type A neurons and eight experiments with type B
neurons are given in Table 1. While the s values were not
different for the two neuron types, the
V50 values were significantly more negative for type B than for type A neurons, indicating that
INa inactivation occurred at more
positive potentials in type A than type B neurons. In fact, when the
conditioning potential was greater than or equal to
20.0 ± 8.2 mV (n = 7) in the type A neurons and greater than or
equal to
31.3 ± 11.3 mV (n = 8) in the type B
neurons, INa was completely suppressed. Maximum
current could be produced when the conditioning potential was less than
or equal to
78.6 ± 19.5 mV (n = 7) in type A
neurons and less than or equal to
98.8 ± 8.3 mV
(n = 8) in type B neurons. Therefore the ability of
type B neurons to fire a burst of action potentials, in contrast to the
one or two action potentials produced by type A neurons, does not arise
from INa being more easily inactivated in type A neurons.

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Fig. 5.
Steady-state inactivation of INa.
A: example of a recording where holding potentials from
80 to 20 mV for 20 ms were used before a test pulse to a constant
potential of 20 mV. B: steady-state inactivation was
determined from experiments such as A by fitting the
peak currents from each experiment at different voltages with a
Boltzmann distribution (Eq. 2). Points show the mean
(±SD) for 7 type A neurons ( ) and 8 type B neurons
( ).
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Inactivation at more depolarized potentials could allow a faster return
to potentials from which the neuron can fire a new action potential. To
examine the time course of INa
recovery from inactivation, we activated the current from a series of
holding potentials (
40,
60,
80, and
100 mV) to the voltage
where a peak current for each cell was recorded (
30 to 0 mV) for 10 and 20 ms and then applied a similar test pulse after varying periods at the same holding potential (Fig.
6A). Peak
INa activated by the second test pulse
was normalized to the peak INa
elicited by the first test pulse, and the normalized current was
plotted against time between the two test pulses, and fitted by a
single exponential decay. The data revealed a difference in time course of recovery from inactivation between the two neuron types. Type B
neurons recovered significantly faster than type A neurons, and the
difference between the time constants of the two neuron types was
significant at all holding potentials and stimulus durations that were
tested (Figs. 6 and 7 and Table
2). However, the recovery from
inactivation was also significantly dependent on the holding potential,
being fastest at the lowest and slowest at the highest holding
potential (Table 2). Interestingly, the duration of the test pulse only
caused a statistically significant difference in the type B neurons
being faster when the test pulse lasted 10 ms and slower with 20-ms
test pulses (Table 3). These results indicate that the ability of INa in
type B neurons to inactivate at more depolarized potentials allows a
more rapid recovery from inactivation and contributes to the production
of more action potentials.

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Fig. 6.
INa recovery from inactivation.
A: recovery from inactivation was studied
by varying the duration (1-50 ms) at a constant holding
potential of 40, 60, 80, and 100 mV between test pulses
to fixed voltage, in this example 0 mV. Each experiment was
performed using test pulse durations of 10 and 20 ms.
B-D: experiments with 10-ms test pulses with holding
potentials 40 mV (B), 60 mV (C), and 80 mV
(D). Normalized data from both types of neurons were fitted
with single exponential decay time constants. The mean values and
statistical comparisons are shown in Table 2.
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Fig. 7.
INa recovery from inactivation. Similar
pulse protocols were used as described in Fig. 6A. In these
experiments a test pulse duration of 20 ms was used from holding
potentials of 40 mV (A), 60 mV (B), 80 mV
(C), and 100 mV (D). Normalized mean values of
experimental data were fitted using single exponential decay time
constants, and the statistical comparison is shown in Table 2.
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DISCUSSION |
This is our third investigation into the properties of
voltage-activated currents in the two types of spider VS-3 neurons. We
showed previously that the difference between the adaptation properties
of the two neuron types is not determined by LVA
ICa (Sekizawa et al.
2000
), even though the calcium current can produce large enough
depolarizations to bring the neurons to threshold, and even produce
large action potentials when INa and
IK are blocked. LVA
ICa is not significantly different
between the two neuron types, and its ultimate functional significance
has yet to be established (Sekizawa et al. 2000
). It is
clear that the normal action potentials in both types of neurons are
produced by an inactivating INa and
their repolarization is driven by IK
(Sekizawa et al. 1999
).
IK activation occurs closer to the
resting potential in type B neurons than in the type A neurons
(Sekizawa et al. 1999
), and here we found that the same
is true for the INa inactivation. In
addition, we showed here that INa
recovers faster from inactivation in type B than type A neurons. Are
the differences in INa inactivation properties responsible for the difference in the adaptation properties of these two neuron types?
INa
The extracellular Na+ concentration was
reduced to allow successful voltage-clamp experiments, so the amplitude
of INa was also decreased from its
normal value at the physiological Na+
concentration. The maximum INa
recorded here was small (530 pA in type A neurons and 510 pA in type B
neurons) when compared with the INa
amplitudes in some other neurons that use Na+
action potentials, where it is measured in tens or even hundreds of
nanoamps (Baker and Bostock 1997
; Benoit et al.
1985
; Lapied et al. 1990
; Purali and
Rydqvist 1998
). Another factor affecting INa amplitude here was that the axons
and dendrites of the VS-3 neurons were crushed, and the recordings
restricted to the somata and initial axons.
INa amplitudes from dissociated
neurons (Elliott and Elliott 1993
; Herness and
Sun 1995
; Honmou et al. 1994
) are closer to the
values recorded here. However, the resting somatic resistance of these
neurons typically exceeds 100 M
(Sekizawa et al.
1999
) so INa recorded here are
adequate to produce normal action potentials, as we have previously
found (e.g., Höger et al. 1997
; Sekizawa et
al. 2000
).
The rates of activation and inactivation of
INa in both types of VS-3 neurons were
voltage dependent (Fig. 3), but the dependence was not very strong. At
20 mV, where INa reached its maximum amplitude, the current activated with a time constant of about 2-3 ms
and inactivated with a time constant of about 5-10 ms. Significantly
faster inactivation time constants have been reported for
INa in other neurons, where they are
usually close to or less than 1 ms (Baker and Bostock
1997
; Benoit et al. 1985
; Bezanilla and
Armstrong 1977
; Herness and Sun 1995
;
Lapied et al. 1990
; Purali and Rydqvist
1998
). However, in many of these neurons, INa inactivation follows two
exponential decay time constants, and the slower component varies from
3 ms (Benoit et al. 1985
) to persistent (Baker
and Bostock 1997
; Elliott and Elliott 1993
). Slowly inactivating INa has been
suggested to be part of the adaptation process in the slowly adapting
lobster stretch receptor neuron (Gestrelius and Grampp
1983
) and to decrease the adaptation in rat dorsal root
ganglion neurons (Elliott and Elliott 1993
). In VS-3
neurons we only found one type of INa,
with no indication of a more slowly inactivating component. These
results suggest that INa inactivation
in the VS-3 neurons may account for their relatively rapid adaptation
properties, compared with more slowly adapting neurons such as the
crustacean stretch receptor neurons, but it may not be able to account
for the smaller difference between type A and type B neurons.
Peak INa in both types of VS-3 neurons
was produced close to
20 mV (Fig. 4A), and the
steady-state activation curves followed similar courses (Fig.
4B). The V50 values for
INa activation in type A and type B
neurons were
30.6 and
32.7 mV, respectively, both close to the
values reported in other invertebrate (Lapied et al.
1990
) and vertebrate (Elliott and Elliott 1993
;
Herness and Sun 1995
) neurons. These results show that
INa activates at similar potentials in
both types of neurons, but when the actual threshold voltages (Resting
potential-Threshold in Table 1) are applied to the activation curves
of Fig. 4B, they indicate that type A neurons need 67% of
total INa at threshold, in contrast to
49% in type B neurons.
The major finding here was the significant difference in the
steady-state inactivation properties of VS-3 neurons. Type A neurons
inactivated at significantly more positive potentials than type B
neurons, with the V50 values being
40.1 mV for type A neurons and
58.1 mV for type B neurons. These
values are within the range observed for other sensory neurons in
vertebrates (Baker and Bostock 1997
; Benoit et
al. 1985
; Elliott and Elliott 1993
; Herness and Sun 1995
) and invertebrates (Lapied
et al. 1990
; Purali and Rydqvist 1998
), where
V50 varied between
40 and
65 mV.
Interestingly, the rapidly and slowly adapting crayfish stretch
receptor neurons have V50 values of
45 and
41 mV, respectively (Purali and Rydqvist 1998
), which was not a statistically significant difference.
However, it was in the opposite direction to that found here for VS-3
neurons, indicating that there is no simple relationship between this
parameter and the firing patterns of sensory neurons.
We found that type B neurons recovered significantly faster from
inactivation than type A neurons. It is well known that recovery of
INa from inactivation is strongly
dependent on the amplitude of the conditioning voltage, and this is
also the case with VS-3 neurons; the more negative the holding voltage,
the faster was the recovery of INa
(Fig. 8 and Table 2). The time constant
of INa recovery from inactivation in
the squid giant axon was 2.7 ms when a
70-mV prepulse was used and
accelerated to 0.6 ms when the prepulse was
130 mV (Bezanilla
and Armstrong 1977
). Similar differences were seen in cockroach
dorsal unpaired median neurons, where a prepulse of
60 mV
produced a recovery with two time constants of 2.8 and 31.4 ms, and a
100-mV prepulse reduced these values to 0.8 and 8.4 ms (Lapied
et al. 1990
). Our results with both types of VS-3 neurons are
similar to above-mentioned observations (Fig. 8 and Table 2), and a
clear difference between the two neuron types is seen at all holding
potentials. These results indicate that the faster recovery from
inactivation of the type B neurons may be the factor that explains why
these neurons have different adaptation properties.

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Fig. 8.
INa recovery from inactivation at different
holding potentials in type A and type B neurons. Pulse protocols were
the same as described in Fig. 6. Normalized mean values of experimental
data were fitted using single exponential decay time constant, and the
statistical comparison is shown in Table 2.
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Comparison with the crustacean stretch receptor neuron
The crustacean stretch receptor organ has been subject of a number
of studies regarding the different adaptation properties of the slowly
and rapidly adapting mechanosensory neurons (Swerup and Rydqvist
1992
), with recent work concentrating on the action potentials
and Na+ current in the crayfish stretch receptor
neurons (Lin and Rydqvist 1999
; Purali and
Rydqvist 1998
). In these studies the rapidly adapting neurons
were shown to produce action potentials of lower amplitude and shorter
duration than slowly adapting neurons and having smaller inward
INa (Purali and Rydqvist
1998
). Both type A and type B spider VS-3 neurons adapt
significantly more rapidly in response to a constant stimulus than the
crayfish stretch receptor neurons, and they produce action potentials
with smaller amplitudes and longer durations. However, we found no
significant differences in the action potential amplitudes or durations
between type A and type B neurons, and the amplitude of
INa was also similar in both neurons
(Table 1). Purali and Rydqvist (1998)
suggested that the
large difference in the action potential and
INa amplitudes in the rapidly and
slowly adapting stretch receptor neurons were due to differences in the
densities of Na+ channels close to the somata
(Lin and Rydqvist 1999
). The VS-3 neuron
Na+-channel distribution has been studied using
immunocytochemistry, and no differences between the two neuron types
were found (Seyfarth et al. 1995
).
Concluding remarks
The most plausible explanation for the different adaptation
properties of type A and type B neurons based on our results is that
the Na+ channels have different inactivation
properties: INa inactivated closer to
the resting potential in the more rapidly adapting type A neurons, and
it recovered from inactivation significantly slower in type A than type
B neurons. The tendency of type A neurons to remain inactivated for a
longer period than type B neurons could explain why they adapt so much
more rapidly. These assumptions may be tested in the future by
single-channel analysis.
Address for reprint requests: P. H. Torkkeli (E-mail:
Paivi.Torkkeli{at}dal.ca).