Department of Physiology and Biophysics, Dalhousie University,
Halifax, Nova Scotia B3H 4H7, Canada
 |
INTRODUCTION |
Slit sensilla are mechanoreceptors characteristic
to spiders. They respond to strains that compress slits on the spider
exoskeleton (Barth 1985
). The lyriform VS-3 organ
(nomenclature of Barth and Libera 1970
) in the patella
of the spider Cupiennius salei consists of seven to eight
sensilla, each of which is innervated by a pair of mechanosensory
neurons. Both neurons of each pair respond to a sustained mechanical or
electrical stimulus with a rapidly adapting burst of action potentials.
In type A neurons this adaptation takes place in <20 ms, often
producing only one or two action potentials, but in type B neurons the
response is a burst lasting several hundred milliseconds
(Seyfarth and French 1994
). Although this difference in
adaptation properties can be partly explained by differences in the
dynamic properties in the transduction step from the mechanical
stimulus to the receptor potential (Juusola and French
1998
), active membrane conductances seem to dominate the
overall dynamic behavior.
Voltage-activated outward K+ currents have been shown to
have an important function in regulating action potential firing
frequency (Connor and Stevens 1971
) as well as
controlling the duration of individual action potentials
(Hodgkin and Huxley 1952
). The contribution of
K+ currents to adaptation has been described in two
different types of invertebrate mechanoreceptors: 1) the
rapidly adapting tactile spine neuron of the cockroach (Torkkeli
and French 1994
, 1995
), where an A current and a
Ca2+-activated K+ current were shown to have
major roles in the adaptation and the delayed rectifier current was the
major action potential repolarizing current, and 2) the
rapidly and slowly adapting stretch receptor neurons of the crayfish
(Purali and Rydqvist 1992
), where the voltage-activated
outward currents were not shown to play major roles in adaptation. We
examined the kinetics and pharmacological sensitivity of
voltage-activated K+ currents in type A and type B neurons
of the spider VS-3 organ to learn if these currents are responsible for
the differences in their adaptation properties.
 |
METHODS |
Preparation
A laboratory colony of Central American wandering spiders,
Cupiennius salei, Keys., was kept at room temperature
(22 ± 2°C). Legs from adult spiders of either sex were
autotomized, and a concave piece of patellar cuticle containing slit
sense organ VS-3 was dissected free in spider saline. The neurons are
directly beneath the cuticle embedded in an internal membrane
(hypodermis). We detached the hypodermis with the neurons attached to
it from the cuticle as described by Höger and Seyfarth (1995)
and
placed them on a small round glass coverslip coated with 5 µg/ml
Collagen IV (Sigma; Oakville, ON). The coverslip was fixed to the
bottom of a 35-mm culture dish (Falcon 3001, Becton Dickinson; Franklin Lakes, NJ) (Fig. 1.). This "hypodermis
preparation" differs from the previously described "cuticular
preparation" of the VS-3 organ (e.g., Juusola and French
1995
; Seyfarth and French 1994
) in two important ways. First, the neurons are facing up, with the hypodermis below them, which makes it easier to impale the neurons with
intracellular electrodes and allows faster exchange of solutions.
Second, the axons and dendrites can be crushed to prevent current
propagation and consequently improve voltage-clamp conditions. In these
experiments the axons and dendrites were crushed at 100-200 µm from
the somata. This preparation does not allow research into the
mechanical properties of the VS-3 neurons because the mechanically
activated ion channels are located on the tips of the dendrites
(Höger et al. 1997
). The action potential
initiation zone obviously remains intact in this preparation because
the neurons retain their typical adaptation patterns in response to
depolarization.

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Fig. 1.
"Hypodermis preparation." Shaded area: hypodermis that covers the
neurons in an intact preparation. Neurons appear in pairs, which are
numbered from 1 to 7 from right to left. Type A and type B neurons
cannot be identified visually because their locations with respect to
each other vary in different preparations. Pair 2 is an exception
because the type A neuron is always smaller than the type B neuron. The
dendritic portion of the neurons is broken off from the cuticle, and
the axons join the main leg nerves, which run to the CNS (proximal).
Before the experiments the axons and dendrites were crushed at 100-200
µm from the somata.
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Recording and stimulation
Current- and voltage-clamp recordings were performed with the
discontinuous single-electrode method (Finkel and Redman
1984
) with an SEC-10 l amplifier (NPI Electronic; Tamm,
Germany). The conditions for successful single-electrode voltage and
current clamp were described in detail by Torkkeli and French (1994)
, and the same methods were used previously in VS-3 neurons
(Höger et al. 1997
; Juusola et al.
1994
). Borosilicate microelectrodes (1 mm OD and 0.5 mm ID)
were pulled with a horizontal puller (P-2000, Sutter Instrument;
Novato, CA). The electrodes were filled with 3 M KCl and coated with
petroleum jelly to decrease stray capacitance (Juusola et al.
1997
). Electrode resistances were 45-80 M
with time
constants of 1-3 µs in solution. Switching frequencies of 20-23 kHz
and a duty cycle of 1/8 (current passing/voltage recording) were used
in all experiments.
The neurons were observed with an inverted microscope with bright field
optics (Olympus; Tokyo), identified, and then penetrated through a thin
layer of spider saline. Penetration was achieved by high-frequency
oscillation ("buzzing"). Impaled cells were allowed to stabilize
for 15 min before the start of experiments. Criteria for reliable
recordings were stable resting potentials of approximately
60 mV,
action potential amplitudes of >40 mV, and thresholds for firing
action potentials of
0.75 nA. With higher thresholds it was not
possible to reliably classify type A and type B behavior because a
small number of action potentials could indicate that the cell was
damaged during penetration or that it normally fires only one spike.
All current- and voltage-clamp experiments were controlled by an
IBM-compatible computer with custom written software (ASF Software;
Halifax, NS). The computer provided voltage or current stimuli via a
12-bit D/A convertor. Membrane potential was low-pass filtered at 33.3 kHz and current at 3.3 kHz by the voltage-clamp amplifier. In
current-clamp experiments the membrane resistance and time constant
were estimated by measuring voltage responses to a hyperpolarizing
current pulses of 0.25 nA. In voltage-clamp experiments the 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 analysis was made with the Student's
two-tailed t-test for significantly different means,
assuming different variances (Press et al. 1990
).
Chemicals
The spider saline used in these experiments contained (in mM)
223 NaCl, 6.8 KCl, 8 CaCl2, 5.1 MgCl2, 5 mM
sucrose, and 10 HEPES, pH 7.8 (Höger et al. 1997
).
Pharmacological agents used to block membrane currents were dissolved
in spider saline and freshly prepared for each experiment or kept
frozen at the same or higher concentrations. Voltage-activated
Na+ currents were blocked with 1 µM TTX. In some
experiments Ca2+ currents were blocked by replacing
CaCl2 with an equal concentration of CoCl2, but
the cells started to deteriorate rapidly in these conditions, and in
most experiments the Ca2+ currents were blocked with 50 µM CdCl2. Because Cd2+ is a more potent
blocker of Ca2+ currents it can be used as an additive
rather than replacement of all bath Ca2+ ions
(Lancaster 1991
). To keep the bath osmolarity unchanged when 5 mM tetraethylammonium chloride (TEA, Kodak) or 5 mM
4-aminopyridine (4-AP) were used, sucrose was removed from the saline.
Ten to 100 nM charybdotoxin (CTX), 10-100 nM apamin, and 100 µM
quinidine were added to the normal saline. The effects of blocking
agents usually took place in 5-10 min. All chemicals were purchased
from Sigma unless otherwise stated.
 |
RESULTS |
Passive membrane properties
Passive membrane properties of VS-3 neurons have been examined
previously with the cuticular preparation (Juusola and French 1998
). Here, we examined the passive membrane properties of
neurons in the hypodermis preparation (Fig. 1) in which the axons and dendrites were crushed at 100- to 200-µm distance from the soma. The
membrane resistance (Rin) and time constant
(
) of both type A and type B neurons were significantly higher in
the hypodermis preparation than reported for the cuticular preparation
(Table 1). Mean values for type A neurons
were Rin = 178.9 M
and
= 16.8 ms
(compared with 61.1 M
and 6.3 ms, respectively, in Juusola
and French 1998
), and mean values for type B neurons were Rin = 114.1 M
and
= 9.9 ms (70.8 M
and
7.6 ms, respectively, in Juusola and French 1998
). The
differences between Rin and
values in type A
and type B neurons were also statistically significant (Table 1).
However, membrane capacitances were not significantly different between
the neuron types (type A: 90.0 pF; type B: 81.9 pF) (Table 1). Because
most of the neurons that we used for experiments had large somata
(radius ~25 µm and length ~100 µm), the contributions of axons
to membrane capacitance were quite small, and we found only a small
difference in the membrane capacitance when compared with the cuticular
preparation (110 pF) (Juusola and French 1998
). Resting
membrane potentials of type A and type B neurons were also close to
each other (Table 1), suggesting similar resting conductances. We also
compared the threshold depolarization, which was defined as the
potential difference between the holding potential and the point where
the action potential rose steeply (Fig.
2) (Nakajima and Onodera
1969
). The mean threshold depolarization of type A neurons was
42.5 mV, and that of type B neurons was 36.5 mV. As shown in Table 1
this difference was statistically significant.

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Fig. 2.
Threshold depolarization was measured as the potential difference
between the holding potential and the inflection point between the
slowly rising potential and the steeply rising action potential.
Stimulus amplitude in this recording was 500 pA. : mean
threshold depolarization of type A neurons, 42.5 ± 8.9 mV
(n = 33); : mean threshold
depolarization of type B neurons, 36.5 ± 8.2 mV
(n = 34). Difference was statistically significant
(Table 1).
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Voltage-activated outward currents
We recorded voltage-activated outward currents after inward
Na+ currents were blocked with 1 µM TTX and
Ca2+ currents were blocked with either Co2+ or
Cd2+. Without Co2+ or Cd2+ in the
bath, addition of K+-channel blockers (TEA, 4-AP, or
quinidine) revealed a rapidly activating and inactivating inward
current at high voltages. Blockade of Ca2+ currents would
also inhibit Ca2+-activated K+ currents, but
because the amplitude of the outward currents did not change
significantly and in a few experiments it actually increased after
administration of Ca2+-channel blockers rather than
decreased we do not believe that a large fraction of outward current
was dependent on Ca2+.
Blockade of inward currents increased the membrane resistance
approximately twofold; type A neurons had a mean membrane resistance of
395.6 M
, and type B neurons had a resistance of 240.3 M
. This
difference in the membrane resistances was statistically significant
(Table 1). Both types of VS-3 neurons had a similar profile of outward
currents (e.g., Fig. 7A, type A, and Fig. 8A, type B) with a rapid activation and slower inactivation at high recording voltages. These currents could be separated into two parts
with the pulse protocols shown in Fig. 3.

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Fig. 3.
Voltage-clamp recordings after inward currents were blocked with TTX
and Cd2+. Actual voltage records are shown below each set
of current recordings (A-C).
A: slightly hyperpolarizing prepulses were used to
elicit transient outward current. B: noninactivating
currents produced with a depolarizing prepulse were subtracted from the
currents in A. C: inactivation kinetics
of the transient outward current were studied with prepulses to
different voltages and by recording the transient at a constant level.
D: transient outward current after subtraction of the
trace at 30 mV in B from the corresponding trace in
A. Current trace was fitted by 2 exponential decay time
constants (Eq. 5).
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Transient outward current
Rapidly activating and inactivating outward current could be
elicited with a stimulus from resting or hyperpolarized potential (Fig.
3A) and the transient component revealed by subtracting a
current that was produced after a depolarizing prepulse (Fig. 3B). The maximum amplitude of the transient outward current
was 0.24 nA in type A neurons and 0.45 nA in type B neurons. Voltage dependencies of activation and inactivation of transient outward current were studied by measuring the peak current during
double-voltage pulse protocols (Fig. 3, A and C).
The experimental activation data were fitted by a Boltzmann
distribution of the form
|
(1)
|
where g is conductance, V is membrane
potential, V50 is the membrane potential at
half-maximal activation, and s is the slope factor.
Conductance was calculated from
|
(2)
|
where I is the current and Erev
is its reversal potential. Fitting was performed by the
Levenberg-Marquardt general nonlinear fitting algorithm (Press
et al. 1990
). The reversal potentials were defined from the
tail currents of the peak transient outward currents (Ritchie
1987
; Torkkeli and French 1995
). The results obtained from fits of 11 type A neurons and 10 type B neurons are shown
in Table 2, and Fig.
4 shows examples of Boltzmann fits in
both types of neurons and bar graphs of V50
values from all recordings. V50 values of type A
neurons were significantly more positive than type B neurons, but all
other parameters were similar in both neuron types.

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Fig. 4.
Voltage dependence of activation of the peak transient outward current.
: fitted Boltzmann distributions to normalized conductances.
: data from type A neuron; : data from
type B neuron. Bar graphs: V50 values for
activation data from 11 type A and 10 type B neurons. Mean
V50 value of type A neurons was 9.6 ± 16.1 mV, and of type B neurons it was 13.1 ± 15.1 mV.
Difference was statistically significant (Table 2).
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The equivalent gating charges (e) for activation was
calculated from
|
(3)
|
where k is the Boltzmann constant, T is
absolute temperature, e is the elementary charge, and
z is the valency of equivalent gating charge. For type A
neurons with s = 12.6 mV (Table 2) and
kT/e = 25.26 mV (Hille 1992
)
the gating charge for activation was 2.0, and for type B neurons with
s value of 14.1 mV (Table 2) it was 1.8. The activation in
both cases was clearly voltage dependent and followed a sigmoidal time
course that indicates multiple gating particles (Hodgkin and
Huxley 1952
).
Equations 1 and 2 were also used to fit the
inactivation data with appropriate change in exponential sign. The
fitted parameters from 8 experiments with type A neurons and from 10 experiments with type B neurons are given in Table 2. One example of a
Boltzmann fit of each neuron type is shown in Fig.
5. A similar shift in the
V50 values was visible in the inactivation as in
the activation, but this difference was not statistically significant.
The amplitude of transient outward current did not increase when
prepulse potentials of less negative than
70 mV were used, indicating
that this current was not the typical A current found in many other
neurons.

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Fig. 5.
Voltage dependence of inactivation of the peak transient outward
current. : fitted Boltzmann distributions to normalized conductance.
: data points from 1 type A neuron; :
data points from 1 type B neuron. Bar graphs:
V50 values for inactivation data from 8 type
A neurons and 10 type B neurons. Mean V50
values were 38.4 ± 12.0 mV for type A neurons and 31.1 ± 9.2 mV for type B neurons. Difference was not statistically
significant (Table 2).
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The time course of the transient outward current was fitted with
exponential power equations with either one or two inactivation time
constants (Eqs. 4 and 5). Two time constants of
inactivation were needed when the current amplitude was larger.
|
(4)
|
|
(5)
|
where I
is the current level expected in
the absence of inactivation,
1 is the time constant of
activation,
2 and
3 are time constants of
inactivation, m is the integer exponent, and
is the
relative amplitude of the two inactivation components (Hodgkin
and Huxley 1952
). An example of a fit with Eq. 5
with an m value of 4 is shown in Fig. 3D. The
activation and inactivation time constants of transient outward current
are given in Table 2, and there were no statistically significant
differences between type A and type B neurons.
Noninactivating outward current
Voltage sensitivity of activation of the noninactivating outward
current was studied with the steady-state currents from recordings such
as Fig. 3B, where a depolarizing prepulse was used to
eliminate the transient outward current. Noninactivating current made
up a major part of the outward current. Its mean maximum amplitude was
1.2 nA in type A neurons and 1.7 nA in type B neurons (Table 3). The steady-state activation was well
fitted by the Boltzmann distribution (Eq. 1; Fig.
6). The reversal potential was defined from the tail currents of the steady-state currents after 100-ms pulses
to several depolarizing voltages (Ritchie 1987
;
Torkkeli and French 1995
). The fitted parameters from 17 sets of data from type A neurons and 18 recordings of type B neurons
are shown in Table 3. The only differences that were statistically
significant between the two neuron types were positive shifts in the
V50 values of the type A neurons when compared
with type B neurons that was also found in the transient outward
current, and also the s value was significantly more
positive in type A neurons than in type B neurons (Table 3). It was not
possible to study the time course of the noninactivating outward
current because the current was already activated at the start of the
recording. The equivalent gating charge (e) for the
activation of noninactivating outward current was calculated with
Eq. 3. For type A neurons it was 2.3, and for type B neurons
it was 3.2.

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Fig. 6.
Voltage dependence of activation of noninactivating outward current.
: fitted Boltzmann distributions to normalized conductances.
: data points from a type A neuron; :
data points from a type B neuron. Bar graphs: mean values of
V50 from 17 type A neurons ( 48.9 ± 4.9 mV) and 18 type B neurons ( 56 ± 4.2). Difference was
statistically significant (Table 3).
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From mean reversal potentials and the value of 6.8 mM for extracellular
K+ concentration we calculated with the Nernst equation
that the intracellular K+ concentration was 199 mM in the
type A neurons and 190 mM in the type B neurons.
Pharmacology
Experiments with the potassium channel blockers TEA and 4-AP are
shown in Figs. 7 and
8. When 5 mM TEA was added to the bath solution it blocked the transient outward current and a small part of
the noninactivating outward current. The experiment shown in Fig. 7 was
done in a type A neuron, and results were very similar with type B
neurons. 4-AP (5 mM) also blocked the transient current and almost
one-half of the noninactivating current (Fig. 8). The blocking effect
of 4-AP was stronger than that of TEA in both neuron types. Larger
concentrations of either blocker (25 and 50 mM) did not produce
stronger block. Similar effects were produced when 100 µM quinidine
was used as a blocker. We also tried two toxins that block
Ca2+-sensitive K+ currents in many neuron
types, apamin and CTX (Hille 1992
), both at 10- to
100-nM concentrations. These experiments were done in normal bath
Ca2+ concentrations and without Co2+ or
Cd2+ in the saline. There were no differences in the
outward current amplitudes before or after applying the blockers.

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Fig. 7.
Tetraethylammonium chloride (TEA) blocked the transient outward current
and a small portion of the steady outward current in this type A
neuron. The pulse protocol below in A is the actual
voltage recording from the amplifier. Leakage currents were subtracted
from both current recordings. B: current recordings from
a 30-mV voltage pulse before (Control) and after TEA in the bath
together with the current that was blocked by TEA (Subtracted).
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Fig. 8.
Effect of 4-aminopyridine (4-AP) on the outward currents in a type B
neuron. Actual voltage from the amplifier is shown below in
A and the current recordings with (4-AP) and without
4-AP (Control) above. B: when current after 4-AP
application was subtracted from the control current it revealed a block
of the transient outward current and ~1/2 the steady current
(Subtracted).
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The effects of K+-channel blockers on the action potentials
were studied in the current-clamp configuration (Fig.
9). The effects of 4-AP and TEA were very
similar; the amplitude and duration of action potentials increased, but
the firing frequency was not affected in either neuron type. Type A
neurons fired usually one large action potential after 4-AP or TEA was
added to the bath solution (Fig. 9A), and type B neurons
produced smaller numbers of action potentials after the blockers (Fig.
9B), but this occurred because of longer-duration spikes
rather than a direct effect on firing frequency. The current that was
needed to induce action potentials in both neuron types was reduced by
both blockers, and in some experiments they produced small spontaneous
depolarizations. For example, in the recording shown in Fig.
9A, the minimum stimulus current amplitude to produce an
action potential fell from 500 to 250 pA after 4-AP application. In the
recording shown in Fig. 9B the current amplitude needed to
cross the threshold decreased from 750 to 250 pA. The effect of
quinidine was stronger than that of 4-AP or TEA. It actually prevented
the neuron from repolarizing after the first action potential (Fig.
9C). Ten to 100 nM apamin or CTX did not affect individual
action potentials or firing patterns.

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Fig. 9.
Effects of K+-channel blockers on action potentials.
Amplitude and duration of action potentials increased when 4-AP was
added to the bath of a type A neuron (A). Stimulus
amplitude in control and 4-AP recordings was 500 pA. When TEA was added
to the bath of a type B neuron (B) the amplitude and
duration of action potentials increased as well as the baseline
depolarization, indicating that the repolarization was partially
inhibited. Stimulus amplitude in control recording was 1.25 nA, and in
TEA recording it was 1 nA. C: quinidine completely
prevented cell repolarization after the first action potential in a
type B neuron. Stimulus amplitude in control and quinidine recordings
was 750 pA.
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DISCUSSION |
Since the cuticular preparation of the spider VS-3 organ was
first developed by Seyfarth and French (1994)
, it has been successfully used to study responses of the sensory neurons to mechanical stimuli (e.g., Höger et al. 1997
; Juusola et al.
1994
). This preparation provides a useful tool for such studies
even in the voltage-clamp configuration because the voltage control is
good enough to examine small amplitude current changes produced by
mechanical stimuli when rapidly changing voltage-activated currents are
blocked (Juusola and French 1998
). However, the control
of voltage-activated currents can be greatly improved when their
propagation into the neural processes is inhibited. Therefore we
utilized here the hypodermis preparation developed by Höger and
Seyfarth (1995)
, which allowed crushing the neurites at a short
distance from the soma. Another benefit of this preparation was
improved access to the neurons with pharmacological agents. The passive
membrane properties of the neurons were significantly different when
the results from the hypodermis preparation were compared with the
cuticular preparation (Juusola and French 1998
), with
higher membrane resistances and longer time constants indicating that
the numbers of open ion channels were smaller in the hypodermis
preparation. This result is expected when the propagation of currents
into the processes is prevented. The differences in membrane
resistances and time constants between type A and type B neurons were
new findings (Table 1). The membrane capacitances in the type A and
type B neurons were not significantly different, indicating that the average sizes of these neurons are not different, although a clear size
difference has been found in one pair (pair 2, Fig. 1), where the type
A neuron is always smaller than the type B neuron (Fabian-Fine et al. 1999
). These results indicate that the resting
conductance of the type B neuron is larger than the type A neuron, at
least on the soma and the parts of neurites close to the soma. The
threshold depolarization was higher and voltage-activated outward
currents activated at more depolarizing voltages in type A neurons than in type B neurons, indicating differences in the relative distributions and densities of both the Na+ and K+ channels
in these neurons.
Although the voltage-activated outward currents in VS-3 neurons could
be separated into two components, these components did not clearly fall
into the two major categories of voltage-activated outward currents
that have been found in many other types of neurons in invertebrates
and vertebrates, namely the A current and delayed rectifier
(Hille 1992
). However, there were similarities. The noninactivating outward current probably has the same function in
repolarizing the action potentials as the delayed rectifier, although
it activated without delay and did not have strong sensitivity to TEA.
The transient outward current in the VS-3 neurons activated maximally
from rest and did not need a hyperpolarizing prepulse to be fully
activated, which is different to the classical A current (Connor
and Stevens 1971
; Rogawski 1985
; Torkkeli
and French 1994
). However, it had a very fast time course, and
it may have functions similar to the A current. From the firing
patterns of VS-3 neurons (Fig. 9) it can be seen that a
hyperpolarization-induced A current cannot be involved because the
action potentials return to a depolarized level as long as the stimulus
is applied and do not hyperpolarize below the resting potential.
However, they return to a level where the transient outward current in
these neurons starts to activate. In both neuron types the number of
action potentials increased with increasing stimulus amplitude,
although in type A neurons even the largest depolarizations did not
produce more than two or three action potentials. The function of
encoding graded depolarization into firing frequency is believed to
depend strongly on A current because some neurons and the axons of many
neurons that do not have an A current fire similar bursts of action
potentials independent of stimulus intensity (Rogawski
1985
). The transient outward current in VS-3 neurons could be
activated at the range of voltages where it can affect frequency
encoding, and, although the K+ channel blockers that we
used were not specific for the transient outward current, they usually
decreased the threshold for firing action potentials as well as
increasing the duration and amplitude of the spikes. None of the
blockers produced an increase in the firing frequency, but if a type A
neuron originally had more than one action potential the blockers
usually reduced the number to one, and they decreased the rate of
repetitive firing in type B neurons. These effects indicate that
neither of these currents could be responsible for action potential
adaptation because removal by blockers would then induce more vigorous
firing. The action potential repolarizing function of the
noninactivating outward current in VS-3 neurons is similar to that of
the delayed rectifier K+ current in many neurons of other
invertebrate species such as molluscan soma (Adams et al.
1980
; Aldrich et al. 1979
), Aplysia bursting pacemaker neurons (Adams and Benson 1985
), and
cockroach tactile spine neurons (Torkkeli and French
1995
). In vertebrates this current is less important in spike
repolarization (Storm 1987). For example, in frog
sympathetic ganglia the Ca2+-activated K+
current is the main repolarizing current (Adams et al.
1980
), and in rat ganglia the A current has an
important function in action potential repolarization (Beluzzi
et al. 1985
).
We found only minor differences between type A and type B neurons. Both
had transient outward currents with small maximum conductances that
activated and inactivated at approximately the same rate in both
neurons (Table 2). Also the membrane conductance responsible for the
noninactivating current was similar in type A and type B neurons (Table
3). The only significant differences were in the activation kinetics of
outward currents because both of them activated at more positive
potentials in type A neurons than in type B neurons (Tables 2 and 3).
We did not find any evidence for the existence of
Ca2+-activated K+ current in the VS-3 neurons.
Experiments with CTX or apamin did not reveal sensitivity to these
blockers in current- or voltage-clamp recordings. However, it is
possible that these neurons have K+ current that is
activated by Ca2+ but not sensitive to these toxins. Purali
and Rydqvist (1992)
did not find a Ca2+-activated
K+ current in the slowly or rapidly adapting stretch
receptor neurons of crayfish using the same blockers, but
single-channel recordings revealed that Ca2+-activated
K+ channels are present in these neurons (Erxleben
et al. 1991
). The density of these channels in some neurons
could be so small that the whole cell currents cannot be easily
detected. Ca2+-activated K+- current was found
to be one of the main components of adaptation in one type of insect
cuticular mechanoreceptor neuron, the cockroach tactile spine
(Torkkeli and French 1995
), where approximately one-half
of the total outward current was activated by Ca2+. The
mechanisms of adaptation of VS-3 neurons are probably very different
from those of the tactile spine because their adaptation behavior is
different. The tactile spine is a phasic receptor, but it can fire
bursts that last several seconds (Torkkeli and French
1995
), the action potentials always return to hyperpolarizing potentials, and their threshold depolarization is substantially lower
than VS-3 neurons (~5 mV, unpublished observation). Consequently, the
tactile spine neuron has a large A current that activates when the
membrane is hyperpolarized below the resting potential (Torkkeli
and French 1994
). The VS-3 neurons do not have the slow afterhyperpolarization that has been shown to be produced by
Ca2+-activated K+ currents in many neurons and
is assumed to slow the rate of firing (Hille 1992
).
In investigations of voltage-activated outward currents of slowly
and rapidly adapting crayfish stretch receptor neurons (Rydqvist and Purali 1991
; Rydqvist and Zhou 1989
) the
activation of outward currents was faster and occurred at more negative
potentials in the rapidly than slowly adapting neurons. These findings
are opposite to our results from VS-3 neurons, but the adaptation
behavior of stretch receptor neurons is also very different from VS-3
neurons because both the rapidly and slowly adapting neurons can fire bursts of action potentials several seconds long, and VS-3 neurons only
fire for a maximum of a few hundred milliseconds. However, Purali and
Rydqvist (1992)
did not regard the differences in the outward current
kinetics as a major contributor to the variations in the adaptation
behavior of stretch receptor neurons. Likewise, we do not believe that
the small differences we found here between the activation kinetics of
type A and type B neurons can have a major effect on the adaptational
differences of VS-3 neurons. It seems more likely that the firing
pattern is defined by other factors, possibly differences in the
inactivation kinetics of the inward currents. Recently, sodium current
inactivation has been shown to begin at more negative potentials, and
it is also faster in the rapidly than in the slowly adapting stretch
receptor neuron of the crayfish (Purali and Rydqvist
1998
), suggesting a major regulatory function. Neither sodium
nor calcium currents of VS-3 neurons have yet been investigated, and
these currents may dominate their dynamic properties.
Address for reprint requests: P. Torkkeli, Dept. of Physiology and
Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
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