Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
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ABSTRACT |
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Sekizawa, Shin-Ichi, Andrew S. French, and Päivi H. Torkkeli. Low-Voltage-Activated Calcium Current Does Not Regulate the Firing Behavior in Paired Mechanosensory Neurons With Different Adaptation Properties. J. Neurophysiol. 83: 746-753, 2000. Low-voltage-activated Ca2+ currents (LVA-ICa) are believed to perform several roles in neurons such as lowering the threshold for action potentials, promoting burst firing and oscillatory behavior, and enhancing synaptic excitation. They also may allow rapid increases in intracellular Ca2+ concentration. We discovered LVA-ICa in both members of paired mechanoreceptor neurons in a spider, where one neuron adapts rapidly (Type A) and the other slowly (Type B) in response to a step stimulus. To learn if ICa contributed to the difference in adaptation behavior, we studied the kinetics of ICa from isolated somata under single-electrode voltage-clamp and tested its physiological function under current clamp. LVA-ICa was large enough to fire single action potentials when all other voltage-activated currents were blocked, but we found no evidence that it regulated firing behavior. LVA-ICa did not lower the action potential threshold or affect firing frequency. Previous experiments have failed to find Ca2+-activated K+ current (IK(Ca)) in the somata of these neurons, so it is also unlikely that LVA-ICa interacts with IK(Ca) to produce oscillatory behavior. We conclude that LVA-Ca2+ channels in the somata, and possible in the dendrites, of these neurons open in response to the depolarization caused by receptor current and by the voltage-activated Na+ current (INa) that produces action potential(s). However, the role of the increased intracellular Ca2+ concentration in neuronal function remains enigmatic.
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INTRODUCTION |
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Calcium currents
(ICa) that activate in a low voltage
range (LVA), and often transiently (T type), have been observed in a variety of neurons from vertebrate and invertebrate central and peripheral nervous systems. These currents activate at potentials low
enough to gate the activity of other depolarizing voltage-activated ion
channels and therefore their putative functional roles include lowering
the threshold for spike generation (reviewed by Huguenard 1996), contribution to the depolarizing envelope that underlies neuronal burst firing (Llinas and Yarom 1981
) and
promotion of intrinsic oscillatory behavior (Lewis and Hudspeth
1983
; McCormick and Huguenard 1992
).
LVA-Ca2+ channels have been shown to exist in the
dendrites of several central neurons where they can be activated by
synaptic potentials and may therefore enhance the synaptic input
leading to action potentials (Magee and Johnston 1995
).
One important putative function of LVA channels is to increase
intracellular Ca2+ concentration, which could
lead to various Ca2+-dependent secondary
responses (McCobb and Beam 1991
).
The lyriform slit sense organ VS-3 in the patella of the spider,
Cupiennius salei, (nomenclature of Barth and Libera
1970) consists of seven to eight slits each innervated by a
pair of bipolar mechanosensory neurons. Because one neuron (Type A) in each pair usually fires only one or two action potentials after a step
stimulus whereas the other (Type B) can fire a burst of up to several
hundred milliseconds (Seyfarth and French 1994
), the
VS-3 organ provides an excellent model for examining the role of
ICa in regulating excitability. The
difference in spiking behavior can be explained only partially by
differences in the time courses of the receptor potentials
(Juusola and French 1998
), and voltage-activated potassium currents do not contribute to spiking behavior
(Sekizawa et al. 1999
). We have discovered a large
transient ICa in the somata of both
types of VS-3 neurons, which activates in the range previously
described for LVA currents. Here we examine the kinetics and
pharmacological sensitivity of this current with the main focus being
its possible functional significance in the spiking behavior of these
two different types of neurons.
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METHODS |
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Preparation
A laboratory colony of Central American wandering spiders,
C. salei, Keys. was kept at room temperature (22 ± 2°C; mean ± SD). Legs from adult spiders of either sex
were autotomized, and 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 then was spread
onto a small coverslip that was either uncoated or coated with 5 µg/ml Collagen IV (Sigma, Oakville, ON). The coverslip then was
placed on the preparation holder. The axons and dendrites were crushed
at ~100 µm from the somata to improve the voltage-clamp conditions.
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Recording and stimulation
Current- and voltage-clamp recordings were performed by 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 have been described in detail by Torkkeli and
French (1994)
, and the same methods were used previously in
VS-3 neurons to study voltage-activated currents (Sekizawa et
al. 1999
). Borosilicate microelectrodes (1 mm OD and 0.5 mm ID)
were pulled with a horizontal puller (P-2000, Sutter Instument, Novato,
CA). For current-clamp experiments, the electrodes were filled with 3 M
KCl, and for voltage-clamp experiments 3 M CsCl was used in the
pipette, in both cases the electrode resistances were 45-85 M
.
Electrodes were coated with petroleum jelly to decrease 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 the experiments. All voltage-clamp experiments were performed by
averaging three recordings with 5-s intervals.
The neurons were observed by an inverted microscope with bright field
optics (Olympus, Tokyo, Japan). Neurons were impaled with
high-frequency oscillation ("buzzing"), and they were allowed to
stabilize for 15 min before the start of experiments. Only neurons with
resting potentials of approximately 60 mV, action potential
amplitudes of >40 mV and thresholds for firing action potentials of
0.75 nA were used for recording.
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 and current stimuli via a
12-bit D/A converter. 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 input resistances were estimated by
measuring voltage-responses to hyperpolarizing current pulses of 0.25 nA. The mean input resistance of Type-A neurons was 145.6 ± 124.6 M (mean ± SD, n = 34) and of Type-B neurons
113.5 ± 59.6 M
(n = 32). In voltage-clamp
experiments the input resistance was calculated from the linear part of
the current-voltage curve at hyperpolarizing potentials, and this value
was used for leakage subtraction. The mean values of input resistances
after blockade of INa and
IK (see following text) were
259.0 ± 168.0 M
(n = 19) for Type-A neurons
and 208.8 ± 151.1 M
(n = 19) for Type-B
neurons. Similar differences in the input resistances between Type-A
and Type-B VS-3 neurons have been found before (Sekizawa et al.
1999
).
Statistical analyses were performed with the unpaired t-test for significantly different means assuming different variances.
Chemicals
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. To record ICa, voltage-activated
INa were blocked with 1 µM
tetrodotoxin (TTX) and IK with a
combination of 25 mM tetraethylammonium chloride (TEA, Kodak) and 25 mM
4-aminopyridine (4-AP) in the bath (to adjust osmolarity sucrose was
removed and NaCl concentration was reduced to 178 mM) and replacing the
electrode filling solution with 3 M CsCl. With sharp microelectrodes,
only a small amount of the pipette solution can reach the cell
interior, and therefore the blockade of
IK was never complete at strong
depolarization. Amplitude of outward current at higher depolarizations
varied significantly between different neurons, indicating that CsCl dialysis was stronger in some neurons than others. The data used for
analysis were collected from neurons that did not have outward currents
at potentials of 60-80 mV from resting potential even when
ICa was blocked with
Cd2+.
In several experiments, extracellular CaCl2 was
replaced with equal (8 mM) concentrations of
BaCl2 or SrCl2. We also
tested several known blockers of ICa
in current- and voltage-clamp experiments including
CdCl2, NiCl2, nifedipine
(initial dilution into dimethylsufoxide as 100 mM) and
-conotoxin-GVIA (
-CgTX GVIA). The effect of blocking agents
usually took place in 5-10 min. All chemicals were purchased from
Sigma unless otherwise stated.
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RESULTS |
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The main goals of this study were to isolate currents carried by Ca2+ in rapidly (Type A) and slowly (Type B) adapting mechanosensory neurons in the spider lyriform slit sense organ VS-3 and to determine their physiological functions. We examined the time- and voltage-dependent properties of ICa to relate it to the ICa described in other neurons and to learn if there was any difference in ICa between Type-A and Type-B neurons that could explain their different firing behaviors (Fig. 1). We also used well-known blockers to test for different components of ICa and determine their possible roles in the neurons' dynamic behavior.
Voltage and time dependence of activation and inactivation of ICa
When voltage-activated INa and
IK were blocked (see
METHODS), test potentials of 50-10 mV from a holding
potential of
100 mV produced a transient inward current (Fig.
2A) that was strongly dependent on voltage (Fig. 2B). This
ICa activated at about
45 mV and
reached its maximum amplitude of
1.79 ± 0.65 nA at
27.8 ± 6.7 mV (n = 9) in Type-A neurons and
1.67 ± 0.63 nA at
25.6 ± 7.3 mV (n = 9) in Type-B
neurons. Figure 2B shows the normalized mean (±SD) peak
ICa from nine neurons of each type at
different test voltages. The amplitudes and ranges of voltage where
ICa operated were very similar in
Type-A and Type-B neurons. Statistically significant differences were
not found between the Type-A and Type-B neurons at any recording
voltages.
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Inactivation of ICa was also voltage
dependent. We determined the steady-state inactivation by using 50-ms
conditioning pulses followed by test pulses of fixed amplitude (Fig.
3A). Currents then were
normalized to the maximum current, plotted as a function of test
potential (Fig. 3B) and fitted by the Boltzmann relation
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(1) |
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The time course of activation and inactivation of
ICa at different test potentials was
fitted with the Hodgkin-Huxley equation for an inactivating current
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(2) |
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When the membrane was held at a hyperpolarized potential,
ICa recovered from inactivation with
an exponential time course. In the experiment shown in Fig.
5A,
ICa was activated with a test pulse to
20 mV lasting 100 ms, and a similar test pulse was applied after
varying periods at
100 mV. When peak
ICa elicited by the second test pulse
was normalized to the peak ICa
obtained with the first test pulse and the normalized current was
plotted against time between the two test pulses (Fig. 5B),
the data indicated a fast time constant of recovery for both types of
neurons. The mean (±SD) time constant for Type-A neurons was 23.2 ± 5.2 ms (n = 3) and for Type-B neurons it was
29.0 ± 3.3 ms (n = 3), and there were no
statistically significant differences between the neuron types. In both
cases the recovery was complete in ~100 ms.
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Ba2+ as the current carrier
When extracellular Ca2+ was replaced by an
equimolar (8 mM) concentration of Ba2+, the
inward current inactivated more slowly, revealing a steady component,
but its amplitude did not change significantly (Fig. 6). Peak
IBa was 1.73 ± 0.4 nA for
Type-A neurons and
1.74 ± 0.4 nA for Type-B neurons
(n = 8 in both cases), but there was a shift in the
I-V curve to more negative potentials. In the case of Type-A
neurons, the peak amplitude was reached at
41.3 ± 3.5 mV
(n = 8) and in Type-B neurons, it was at
35.0 ± 7.6 mV (n = 8). When peak and steady-state
IBa were plotted against test potentials (Fig. 6B), they had very similar functional
ranges, suggesting that Ba2+ did not reveal a
different group of Ca2+ channels. When the
inactivation time constants of ICa and
IBa were plotted against membrane
potential (Fig. 6C), the inactivation of
ICa was significantly different at all
recording voltages (P < 0.05). Therefore part of the
inactivation was dependent on the intracellular
Ca2+ concentration. We also tested the function
of 8 mM Sr2+ as a current carrying ion in four
experiments, and it caused similarly long-lasting currents.
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Blocking effect of divalent cations Ni2+ and Cd2+
Divalent cations have been shown previously to function
selectively on different types of Ca2+ channels.
Ni2+ usually blocks LVA channels more readily,
whereas Cd2+ is a more potent blocker of
different types of high-voltage-activated (HVA)
Ca2+ channels (Tsien et al. 1986).
However, both cations do block all types of Ca2+
channels when applied at sufficiently high concentrations. We did not
find clear differences between the effects of
Cd2+ and Ni2+ on the
ICa of VS-3 neurons. In four
experiments with 100 µM Ni2+, ~30% of the
inward current remained, and the peak and steady-state currents were
reduced similarly, as shown in Fig.
7A where the Ni2+ effect on
IBa is illustrated. With long (>20
min) incubation times, the same concentration of
Ni2+ blocked all of the inward current. The
Ni2+ effect was always stronger at more negative
potentials (Fig. 7B), and the reduction of inward current
was smaller at more depolarizing test voltages. The effect of 50 µM
Cd2+ on ICa is
demonstrated in Fig. 8, A and
B. In this cell, ICa did
not have a clear steady-state component, but when
Ba2+ was used as the current carrying ion in
other experiments, there was a steady-state current and we did not see
any difference between the effects of Cd2+ on
peak and steady-state currents. The average amplitude of remaining inward current after 10 min in Cd2+ was 40% of
control, and as with Ni2+, most of the current
was blocked by application times of 20 min.
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Because LVA-ICa has been suggested to
be an important factor in the regulation of action potential thresholds
and the regulation of action potential firing frequencies
(Llinas and Yarom 1981), we tested the effects of
Ni2+ and Cd2+ on the
voltage responses at several different holding potentials in both
Type-A and Type-B neurons. Figure 7C illustrates the small effect that Ni2+ had on one Type-B
neuron at a holding potential of
80 mV. In this typical experiment,
the number of action potentials was not affected by application of 100 µM Ni2+ and the threshold current was not
significantly changed either, although the potential at which action
potentials were fired was slightly more positive in the
Ni2+-treated cell than in the control situation.
Figure 8C illustrates the effect of 50 µM
Cd2+ on a Type-A neuron. In this recording, the
neuron fired only one action potential after Cd2+
application, compared with two in the control situation. This was the
strongest effect we ever saw after Cd2+
application. These results indicate that
ICa does not affect the threshold of
VS-3 neurons nor does it change the firing behavior in these neurons,
because if this was the case, the threshold for action potentials would
increase significantly after ICa is blocked. Although we did see small changes in the threshold in some
experiments, they were in both directions. Similar changes often occur
during long recordings, when the membrane seals better around the
recording electrode or when the electrode clogs slightly.
Ni2+ and Cd2+ also would block IK(Ca) under the same conditions as were used in the current-clamp experiments, which would be expected to accelerate firing. We did not observe any such effect. This results supports our observations with specific blockers of IK(Ca), iberiotoxin, charybdotoxin, and apamin (results not shown), which have no effect on the dynamic behavior of these neurons. In addition, these neurons do not show the afterhyperpolarization at the end of a spike train that is usually produced by IK(Ca).
Ca2+ spikes
VS-3 neurons fire Na+ spikes when they are
bathed in normal spider saline (Seyfarth and French
1994). When 1 µM TTX is added to the saline, these neurons
completely cease firing, but a small rapidly decaying depolarization is
seen with high depolarizing voltages (e.g., Juusola and French
1998
). When we blocked voltage-activated K+ currents with TEA and 4-AP, action potential
amplitudes increased significantly and even Type-A neurons started
firing several action potentials as shown in Fig.
9. This is a typical effect of blockade of repolarizing outward currents that is observed in many neurons. In
all eight experiments here, the threshold for firing action potentials
decreased when 4-AP and TEA were added to the saline. When we also
added 1 µM TTX in addition to the K+-channel
blockers, Type-A and Type-B neurons still fired one large and wide
action potential, but neither of them generated repetitive responses.
The threshold for this action potential was always higher than for the
Na+ spikes, but still lower than in control
recordings. Although Ca2+ spikes in the presence
of K+-channel blockers are not a new finding
(reviewed by Hagiwara and Byerly 1981
), their uniformity
in VS-3 neurons suggests that the difference in normal spiking behavior
is not caused by ICa.
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Effect of toxins on ICa
To further explore the identity of
ICa in the VS-3 neurons, we used two
specific blockers of Ca2+ channels, nifedipine
and -CgTX GVIA, in several concentrations. These blockers have been
shown to act specifically on HVA-Ca2+ channels,
and
-CgTX GVIA even more specifically on the N-type channels
commonly found in neurons (Swandulla et al. 1991
). There are no specific blockers of LVA-ICa,
but their identification is commonly based on exclusion.
-CgTX GVIA
at 3 and 5 µM concentrations did not have any effect on either
current or voltage responses of VS-3 neurons confirming that no part of
the ICa belonged to the N-type HVA group.
Nifedipine did not have any effect on ICa in VS-3 neurons when applied at low (10-50 µM) concentrations, but when 100 µM concentration was used it did block approximately one-half of the ICa as shown in Fig. 10, and in most experiments, this effect was at least partially reversible. In several experiments, 100 µM nifedipine also caused a loss of voltage control in the voltage-clamp experiments; this may have occurred because of increased activity. Nifedipine had a clear effect on voltage response, decreasing the threshold for action potentials and increasing firing frequency as shown in Fig. 10C. This effect was more pronounced when neurons were held at hyperpolarizing voltages. This effect cannot be explained by a blockade of Ca2+ channels but could have been caused by agonistic effects on them.
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DISCUSSION |
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LVA-ICa has been suggested to
modify neuronal firing patterns by lowering the threshold for action
potentials and inducing bursting behavior. The presence of
LVA-ICa in the paired spider mechanoreceptor neurons led us to hypothesize that this current was
responsible for the difference between the rapidly and slowly adapting
members of each pair. However, our results do not support this
hypothesis. On the contrary, we found that
LVA-ICa in VS-3 neurons does not play
any part in shaping the voltage response. What other functions could
this prominent current have in these neurons?
LVA-Ca2+ channels often are localized at major
sites of synaptic input; dendrites and somata (e.g., Huguenard
1996; Kaneko et al. 1989
) where excitatory
synaptic input can activate ICa. VS-3
neurons are peripheral sensory neurons but they have extensive synaptic connections from the CNS (Fabian-Fine et al. 1999a
,b
).
Some of these synaptic connections are probably excitatory, but
LVA-Ca2+ channels in VS-3 neurons are more likely
to open in response to depolarizing receptor potentials and especially
during action potentials, increasing intracellular
Ca2+ concentration during firing for other
cellular purposes.
Kinetic properties of ICa in VS-3 neurons
ICa in VS-3 neurons had rapid
kinetics of activation and inactivation, and it also recovered from
inactivation faster than in most other neurons.
ICa activated at potentials positive
to about 45 mV and reached maximum amplitudes at
27.8 and
25.6 mV
in Type-A and Type-B neurons, respectively (Fig. 2). These values are
in the range previously outlined for
LVA-ICa (Gu and Spitzer
1993
; Huguenard 1996
; Kaneko et al.
1989
; Liman and Corey 1996
; Moolenar and
Spector 1979
; Rennie and Ashmore 1991
;
Yoshii et al. 1998
). The rate of activation of
ICa in both types of VS-3 neurons was voltage
dependent and varied from 0.3 to 2 ms (Fig. 4A), somewhat
faster than values of 2-50 ms previously reported for
LVA-ICa (Huguenard
1996
; Tsien et al. 1986
). Inactivation time
constants (Fig. 4B) in Type-A (6-38 ms) and Type-B (71-114 ms) neurons were similar to values obtained from other sensory and
central neurons, where they vary between 10 and 400 ms (Bossu and Feltz 1986
; Huguenard 1996
; Liman and
Corey 1996
). When Ca2+ was replaced with
Ba2+, the inactivation became significantly
slower (Fig. 6), indicating its dependence on intracellular
Ca2+ concentration. Although
Ca2+-dependent inactivation is a property of
HVA-Ca2+ channels, it is not usually associated
with LVA channels (Fox et al. 1987
). However, it has
been observed in some invertebrate neurons, e.g., transient
ICa in cockroach dorsal unpaired
median (DUM) neurons has Ca2+-dependent
inactivation (Wicher and Penzlin 1997
).
Recovery from inactivation is the most variable feature of
LVA-ICa, with its time constant
varying from ~100 ms to several seconds (Bossu and Feltz
1986; Carbone and Swandulla 1989
;
Huguenard 1996
; Kaneko et al. 1989
). In
VS-3 neurons, the recovery was very rapid with a full recovery in
~100 ms and time constants of 20-30 ms, and there were no
statistically significant differences between the two neuron types.
Steady-state inactivation of LVA-ICa
in VS-3 neurons had slope factors close to 4 mV (Fig. 3), in the low
range of values reported for central and sensory neurons (Gu and
Spitzer 1993; Huguenard 1996
; Liman and
Corey 1996
; Moolenar and Spector 1979
; Tsien et al. 1986
; Wicher and Penzlin
1997
). However, V50 values of
steady-state inactivation in Type-A and Type-B VS-3 neurons were
35.1
and
30.3 mV, respectively, significantly more positive than the
V50 values of
50 to
95 mV reported
for other neurons (Huguenard 1996
; Kaneko et al.
1989
; Liman and Corey 1996
; Moolenar and
Spector 1979
; Yoshii et al. 1998
), with the
lower values being more common in sensory neurons.
The rapid kinetics of ICa allows a fast neural response, a property that may be needed when VS-3 neurons respond to natural stimuli, such as substrate vibrations. Differences in the rate of inactivation and recovery from inactivation could be explained if Type-A neurons could respond more readily to high-frequency stimuli, whereas Type-B neurons would provide more time for Ca2+ to enter at the cost of reduced high-frequency sensitivity.
Ca2+-channel blockers
We found no evidence for Ca2+-channel types
other than LVA on the somata of VS-3 neurons. Peak and steady-state
currents were equally sensitive to blockers, and their activation
ranges were similar. -cgTX GVIA had no effect on VS-3 neuron
ICa. At high concentrations,
Ni2+ and Cd2+ block all
types of Ca2+ channels as well as other
voltage-gated channels. At lower concentrations, Cd2+ is less effective on LVA than HVA channels
(Mogul and Fox 1991
; Ozawa et al. 1989
)
and Ni2+ acts more strongly on LVA channels
(Hagiwara et al. 1988
). In VS-3 neurons, the effect of
Ni2+ was slightly stronger on
LVA-ICa than that of
Cd2+, but both blocked the current completely
when application times were long. Current-clamp experiments with
Cd2+ and Ni2+ indicated
that ICa does not decrease the
threshold of VS-3 neurons (Figs. 7C and 8C).
Ni2+ and Cd2+ also block
KCa channels, so it is impossible to draw firm
conclusions about their physiological effects when they are tested on
neurons that have IK(Ca) (e.g.,
Kawai et al. 1996
). Because the somata of VS-3 neurons
do not have IK(Ca), we could reliably
show that although Ni2+ and
Cd2+ both block
ICa, the current does not control the
spiking of these neurons.
Nifedipine like other dihydropyridines (DHP) is a blocker of
HVA-ICa (Tsien et al.
1986). However, at high concentrations DHPs can have
nonspecific effects on Ca2+ and also on
Na+ and K+ channels
(reviewed by Carbone and Swandulla 1989
). DHP block is
more efficient at depolarizing potentials, with hyperpolarizing potentials tending to remove the block. This might explain the excitatory effect of nifedipine on the voltage response of VS-3 neurons
at hyperpolarizing potentials. DHP-sensitive
LVA-Ca2+ channels are found in e.g., rat
hypothalamic neurons (Akaike et al. 1989
).
Ca2+spikes
Ca2+ spikes are common in invertebrate
muscle cells (Fatt and Katz 1953) and in developing
neurons (Hagiwara and Byerly 1981
). When quaternary
ammonium ions are present, many vertebrate neurons also are able to
fire Ca2+ spikes (Fain et al.
1977
; Horn 1978
; Koketsu et al.
1959
). Although both VS-3 neurons could fire one
Ca2+ spike when TEA and 4-AP were present (Fig.
9), this spike had higher threshold than Na+
spikes under similar conditions and repetitive firing did not occur. In
most other neurons, Ca2+ spikes also have higher
thresholds than Na+ spikes (e.g., Llinas
and Yarom 1981
).
Functional significance of LVA-ICa in VS-3 neurons
ICa activated and peaked at
slightly more negative potentials in Type-A than Type-B neurons, but
this difference was not statistically significant. In Type-A neurons,
the depolarization level at which the cells fired action potentials was
about 31 mV and in Type-B neurons
39 mV. A similar, statistically
significant, difference was found before when threshold depolarizations
of large numbers of VS-3 neurons were studied under similar conditions
(Sekizawa et al. 1999
). These findings argue against the
hypothesis of ICa lowering the
threshold for action potentials because in that case, ICa in Type-B neurons would activate
at lower voltages.
The consequence of lowering the threshold for Na+
spikes in vertebrate central neurons is assumed to be promotion of
burst firing (Huguenard 1996). However,
LVA-ICa is not only found in bursting
neurons. In fact, its existence is not well correlated with firing
behavior. For example, LVA-ICa is
present in mouse vomeronasal chemosensory neurons that fire tonically
(Liman and Corey 1996
) and in mouse retinal bipolar
neurons (Kaneko et al. 1989
) that do not fire action
potentials. However, it is not found in cultured rat olfactory neurons
(Trombley and Westbrook 1991
) that only fire one action
potential in response to a steady stimulus or amphibian olfactory
neurons that fire phasically (Liman and Corey 1996
).
Here, we found LVA-ICa in two phasic
sensory neurons.
In vertebrate saccular hair cells, ICa
and IK(Ca) interact to enhance the
frequency tuning to mechanical stimuli. However, in contrast to VS-3
neurons, ICa in hair cells flows
through HVA channels, and these neurons do not fire action potentials
(Hudspeth 1986). The rapid activation and inactivation
kinetics of ICa in VS-3 neurons seem
likely to allow fast cellular responses, which may be significant when
there are high stimulus frequencies. Using action potential waveforms
as stimuli, McCobb and Beam (1991)
demonstrated that
LVA-ICa activated early during the
rising phase of the action potential and peaked after the spike peaks.
This behavior makes it possible to rapidly increase intracellular
Ca2+ concentration during the brief spike.
In contrast to earlier suggestions that LVA-ICa is an important regulator of neuronal firing properties, we found no evidence that LVA-ICa is involved in controlling the firing patterns of Type-A and Type-B mechanosensory neurons in the spider VS-3 organ. This leaves two important questions that we will address in the future: what is the function of this prominent current in these sensory neuron pairs and what is the origin of their different firing patterns? These questions and their answers seem certain to have wider implications for other neurons.
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ACKNOWLEDGMENTS |
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We thank J. Nason for maintaining the animals.
This work was supported by Medical Research Council of Canada.
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FOOTNOTES |
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Address reprint requests to P. H. Torkkeli.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 August 1999; accepted in final form 14 October 1999.
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REFERENCES |
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