School of Biomedical Sciences, Gatty Marine Laboratory, University of St. Andrews, Fife KY16 8LB, United Kingdom
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ABSTRACT |
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Mills, Janette D. and Robert M. Pitman. Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude.
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INTRODUCTION |
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It has become clear in recent years that the electrical properties
of some neurons can be changed radically by chemical modulators (Harris-Warrick and Marder 1991; Kiehn
1991
); for example, some neurons, when isolated from synaptic
influences, may be electrically silent, spike regularly, generate
spikes in regular bursts, or produce plateau potentials, depending on
the presence or absence of specific neuromodulatory compounds. Such
transitions in properties can have a profound effect not only on the
output of individual neurons but also of the networks in which they
participate. It is important, therefore, to establish the ionic
mechanisms by which such changes may take place. The soma of the fast
coxal depressor motoneuron (Df) of the cockroach,
Periplaneta americana, third thoracic ganglion can display
two distinct types of electrical activity. In response to long-duration
depolarizing pulses or synaptic stimulation, it can generate plateau
potentials, each of which may last >1 s and reach a mean membrane
potential of
37 mV (Hancox and Pitman 1991
, 1993
).
Neuron, Df, is also capable of generating calcium-dependent
action potentials, but these normally are seen only between 1 and
4 h after dissection of the CNS (Hancox and Pitman
1992
). Earlier expression of spiking can be brought about
pharmacologically; for example, Df can generate spikes
after intracellular injection of calcium chelating agents or after
extracellular application of the K channel blocker tetraethylammonium
(TEA+) (Pitman 1979
). One possible mechanism
underlying the transition in membrane response from a plateau potential
to an action potential therefore could be a fall in the magnitude of K
currents. In this preparation, however, TEA+ appears to
suppress both voltage-dependent (IK,V) and
Ca-dependent (IK,Ca) K conductances
(David and Pitman 1995a
), so it gives no indication of
the roles of each component of the K current in determining the
membrane properties of the neuron. Reduction in [Ca2+]i by intracellular injection of calcium
chelators, does suggest, however, that Ca-dependent K channels may be
involved in the appearance of spiking activity; this procedure probably
acts in two ways: first, reduction in [Ca2+]i
would be expected to increase the magnitude of calcium currents both by
increasing the inward gradient of this ion and also by reducing any
Ca-mediated Ca inactivation (Mills and Pitman 1997
). Second, the fall in [Ca2+]i also reduces
IK,Ca, which constitutes a major component of the outward current in this neuron, (David and Pitman
1995a
; Thomas 1984
). It has been proposed that
IK,Ca may be large enough to shunt inward
calcium currents and thereby prevent the cell spiking (Hancox
and Pitman 1992
; Pitman 1979
; Thomas
1984
).
There is a diversity of potassium channels (Jan and Jan
1990) each of which may influence the electrical properties of
neurons in different ways. For example, calcium-dependent potassium
conductances can contribute toward the resting membrane potential,
spike repolarization, afterhyperpolarizations, modulation of repetitive
firing, spike frequency adaptation, and termination of bursts
(Blatz and Magleby 1987
; Hermann and Erxleben
1987
; Latorre et al. 1989
; Rudy
1988
; Sah 1996
). Calcium-dependent potassium
conductances can be classified according to characteristics such as
kinetics, calcium sensitivity, and single channel conductance.
Different types of IK,Ca also have been
distinguished in a number of species by their sensitivity to the
neurotoxins apamin and charybdotoxin (ChTX). Apamin, a bee venom
neurotoxin, is known to block a low-conductance calcium-dependent potassium channel (Hugues et al. 1982
; Lazdunski
1983
; Romey et al. 1984
), whereas ChTX, a
peptide neurotoxin isolated from the venom of the scorpion,
Leiurus quinquestriatus, (Gimenez-Gallego et al.
1988
; Smith et al. 1986
) has been shown to block
a variety of calcium-dependent potassium channels of differing
conductances (35-250 pS) (Anderson et al. 1988
;
Garcia et al. 1991
; Hermann and Erxleben
1987
; Miller et al. 1985
; Moczydlowski et
al. 1988
; Schäfer et al. 1994
). This toxin
has been shown to block a Ca-dependent potassium current in dorsal
unpaired median cells of the cockroach (Grolleau and
Lapied 1995
; Wicher et al. 1994
).
The voltage-dependent potassium channels are another group of potassium
channels that are activated by changes in the membrane potential and
not by calcium. This group includes the delayed rectifiers that were
described first by Hodgkin and Huxley (1952) in the
squid giant axon and are responsible for action potential repolarization. The "A" potassium current was first described in
molluscan neurons (Connor and Stevens 1971a
) and has a
lower threshold, faster kinetics and displays steady-state inactivation at more negative membrane potentials than other potassium channels. Currents with similar properties to the A current have been described in other preparations, but there appears to be some variation in the
properties of these channels, particularly in the voltage-dependence of
inactivation and inactivation rates (review by Rudy
1988
). These currents can regulate the latency to first spike
during depolarization, spike frequency and action potential
repolarization. 4-Aminopyridine (4-AP) is a potassium channel blocking
agent that has its most potent effect on these channels.
In this paper, we have used different techniques to investigate the effects of potassium channel blockers and shown that ChTX blocked a large proportion of outward current in this cell while apamin was without effect. The ChTX-sensitive K current is important in determining the electrical properties of Df somata in the cockroach because it can determine whether the cell responds to depolarization by plateauing or spiking. A smaller ChTX-insensitive IKCa and a transient 4-AP-sensitive current also may be involved in the fine tuning of electrical activity.
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METHODS |
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All experiments were performed on the metathoracic "fast"
coxal depressor motoneuron, Df (Pearson and Iles
1970), of adult male cockroaches (P. americana).
Animals were decapitated, the mesothoracic and metathoracic ganglia and
the first three abdominal ganglia were dissected out, and the
metathoracic ganglion desheathed for electrophysiological recording
(see Pitman 1975
). Experiments were performed in
circulating oxygenated saline containing (in mM) 214.0 NaCl, 3.1 KCl,
9.0 CaCl2, and 10 TES buffer (pH 7.2). Stock solutions (10 µM) of ChTX and apamin (Latoxan, Rosans, France) were made up in
saline and stored at
4°C. 4-AP (Sigma-Aldrich, Poole, U. K.) was made up daily as a 10 mM stock solution. Aliquots (20 or 200 µl) of these agents were added to a side compartment of the
chamber (total volume 2 ml), where the oxygenation system mixed and
diluted them before they reached the preparation. Concentrations are
expressed as final values after mixing in the experimental chamber.
Experiments were carried out at room temperature (20-23°C).
Df somata were penetrated with two thin-walled,
fiber-filled borosilicate glass microelectrodes (Clark Electromedical,
Pangbourne, U. K.). Microlectrodes contained 2 M
potassium acetate as electrolyte and, for current-clamp recording, had
resistances of 12-20 M.
Voltage clamp
For voltage-clamp recordings, the voltage electrode (for
monitoring membrane potential) had a resistance between 8 and 15 M,
whereas the current electrode (used to apply current) had a resistance
between 5 and 10 M
. Current was monitored using a laboratory-built
"virtual earth" amplifier circuit connected to the reference
electrode in the experimental chamber. Data from current-clamp
experiments were recorded on tape using a DTR 1204 digital tape
recorder (Biological Science Instruments) and displayed on a Gould 1604 oscilloscope. A CED 1401 Plus computer interface (Cambridge Electronic
Design) and associated software were used for generating voltage
command pulses, recording digitized data, and off-line analysis.
Hardcopy data were downloaded from tape or computer using a Gould
Colorwriter 6120 plotter or a Hewlett-Packard Laserjet 4P printer.
Trains of negative iontophoretic pulses [300 ms, sufficient to
hyperpolarize the membrane by 40 mV (2-10 nA) delivered at 0.1 Hz for
20 min] were used to inject the calcium-chelator, 1,2 bis(2-aminophenoxy)ethane-N',N, N',N'-tetraacetic acid
(BAPTA, Sigma-Aldrich) into the cells. In such experiments, electrodes were filled with solution containing 100 mM BAPTA and 100 mM KCl. All
statistical data are presented as means ± SE.
Action potential clamp
The procedure in these experiments was to voltage clamp the
Df neuron (as described in the preceding section) but using
a command signal identical to the electrical activity exhibited by the
cell (Doerr et al. 1990). Action potentials or plateau potentials, evoked under current-clamp conditions by applying depolarizing pulses, were collected digitally. The same cell then was
voltage clamped at its resting membrane potential, and a typical sample
of activity was used as the command signal (using software kindly
supplied by John Dempster, University of Strathclyde, Glasgow). A small
sustained current was recorded in control conditions which corresponded
to the stimulus current that was injected under current-clamp conditions to reach threshold for activity. Because the cell then was
clamped by its own action or plateau potential, no other current was
recorded. When, under these conditions, a current was blocked pharmacologically, the clamp amplifier had to apply a compensation current corresponding to the contribution of the blocked current to the
electrical activity.
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RESULTS |
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Time-dependent changes in electrical properties in Df
Because the mechanically isolated soma of the fast coxal depressor
motoneuron (Df) can show a time-dependent change in its electrical response to depolarization (Hancox and Pitman
1992), experiments were designed to determine which currents
are responsible for the observed transition in membrane properties.
Figure 1A shows the typical
time-dependent change in the response to depolarization which is
observed in the soma of Df. The resting membrane potential was
74.6 ± 0.7 mV (n = 107 neurons) and did not
change significantly during the course of the experiment. Initially
after dissection, Df generated plateau potentials on
depolarization (Fig. 1Ai). These events had a threshold
between
60 and
40 mV (mean:
50.9 ± 0.74, n = 68 neurons) and reached a mean membrane potential of
37.2 ± 1.1 mV (n = 45 neurons). The duration of plateau
potentials varied between preparations and depended on the strength of
the applied current pulse. In some preparations, such plateau
potentials were sustained throughout a 800-ms depolarization, whereas
in other preparations they could be <200 ms in duration.
Small-amplitude current injections sometimes produced plateau
potentials that inactivated during the pulse, whereas higher amplitude
current injections in the same cell produced longer-duration plateau
potentials. Over time, an intermediate transitional stage was observed
frequently in which oscillations of declining amplitude were
superimposed on the plateau (Fig. 1Aii). One to 4 h
after dissection, the Df soma generated action potentials
rather than plateau potentials when depolarized (Fig.
1Aiii). The action potentials had a threshold of
50.5 ± 1 mV (n = 14 neurons) and normally did not overshoot zero (mean membrane potential reached:
21 ± 2.5 mV,
n = 12 neurons; Fig. 1Aiii). Action
potentials recorded from Df somata mechanically isolated
from their processes similarly fail to overshoot zero (Hancox
and Pitman 1992
). Plateau potentials and time-dependent spikes
are calcium dependent because both can be blocked by 1 mM
Cd2+ but not by tetrodotoxin (
1 µm) (Hancox and
Pitman 1991
, 1992
). Two components of the calcium current have
been identified: one that activates at membrane potentials positive to
60 mV and is blocked by nifedipine and a second component that
activates at membrane potentials positive to
40 mV and is blocked by
micromolar cadmium ions (Mills and Pitman 1997
). The
nifedipine-sensitive component appears to underlie the plateau
potential, and both the nifedipine- and Cd2+-sensitive
components contribute to spikes.
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Although action potentials normally cannot be evoked from the neuron
shortly after dissection, external application of the potassium channel
blocker, TEA+ (10 mM, n = 9) or
intracellular injection of calcium-chelating agents, such as BAPTA
(n = 19), could enable Df to spike within minutes (Fig. 1, B and C) (cf. Pitman
1979). To eliminate the possibility that the effects observed
resulted from the hyperpolarizing pulses used to inject the BAPTA
rather than from the reagent itself, control trains of hyperpolarizing
pulses were applied to microelectrodes filled with 2 M K acetate. These
had no effect on membrane properties. The spikes produced in the
presence of bath-applied TEA+ or after injection of BAPTA
reached a mean membrane potential of
4.8 ± 2.2 (n = 9 neurons) and
15.7 ± 2.3 (n = 18 neurons), respectively. The effects of these
agents on electrical activity indicate that K currents may have an
important role in dictating the type of electrical activity generated
by this neuron; BAPTA may operate both by enhancing
ICa and by depressing
IK,Ca, whereas TEA+ causes a
nonselective suppression of most, if not all, components of K current
(personal observations). It is likely, therefore, that one or more
components of K current may suppress spiking by shunting the
depolarizing effects of ICa; a progressive
reduction in K current thus could contribute to the time-dependent
transition of the somatic response of Df to depolarizing
stimuli. To establish the role of different components of K current in
determining the type of electrical activity generated by Df
at any time, we have investigated the effects of different K channel
blockers under current-clamp, voltage-clamp, and action-potential-clamp conditions.
Effect of potassium channel blockers on electrical activity recorded under current-clamp conditions
Because IK,Ca forms a large proportion of
depolarization-induced outward current in this neuron (David and
Pitman 1995a; Thomas 1984
) and experimental
reduction of [Ca2+]i can enable the soma of
Df to generate action potentials, it is very likely that
this current is normally at least partially responsible for suppression
of spiking. To establish whether suppression of
IK,Ca alone could mimic the time-dependent
transition in the electrical characteristics normally observed in
Df, we have used the bee venom neurotoxin, apamin, and the
scorpion neurotoxin, charybdotoxin (ChTX), each of which blocks a
different class of calcium-dependent potassium channel. Depolarization
of the cell shown in Fig. 2A
produced plateau potentials of similar amplitude, duration and
threshold in control saline and 10 min after application of 1 µM
apamin. Similar results were obtained in a total of six preparations.
ChTX, on the other hand, rapidly caused neurons to change their
response to depolarization from plateau potentials to spikes (Fig.
2B, n = 8). The neuron shown in Fig.
2B initially generated plateau potentials in control saline.
Within 30 s of applying ChTX (100 nM), the soma responded to
depolarization by generating shorter-duration, higher-amplitude plateau
potentials on which were superimposed oscillations (n = 4, c.f. Fig. 1Aii). After 1 min in ChTX, nonovershooting
spikes reaching an absolute membrane potential of
20 mV were
generated. After 2 min, the spikes began to overshoot 0 mV. While the
action potentials illustrated in Fig. 2B attained a positive
potential, this was not true of all neurons; the mean membrane
potential reached by spikes after 5 min in ChTX was
7.9 ± 5 mV
(n = 8). Although the absolute level of the action
potential peak potentially also could be influenced by changes in the
resting membrane potential, this did not have to be considered here
because 100 nM ChTX produced no detectable change in the resting
membrane potential (n = 8 neurons). In effect, ChTX,
caused cells to rapidly undergo a transformation in excitable properties similar to that seen over a period of hours in control neurons (Hancox and Pitman 1992
). This suggests that the
time-dependent change in activity seen in cells in normal saline at
least in part could be due to a progressive decrease in the
ChTX-sensitive component of the calcium-dependent potassium
conductance.
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Because motoneuron Df is known to have complex dendritic arborizations, experiments were performed on cell bodies that had been separated mechanically from their processes to eliminate the possibility that the effects of ChTX resulted from differential effects of this agent on the soma and dendrites of the neuron or from poor space-clamping. Under these conditions, ChTX still could transform the response of the soma to depolarization from a plateau potential to a series of action potentials (n = 4).
In preparations that had been allowed a sufficient interval from the
time of dissection to enable them to generate spikes when depolarized
(2 h), application of ChTX caused an increase in spike amplitude
(n = 2 neurons). This is shown in Fig. 2C, in which spikes generated in both control and ChTX saline are superimposed. Depolarization induced spikes in control saline, which
reached an absolute membrane potential of 15 mV. After application of
100 nM ChTX for 1 min, the spike amplitude increased to overshoot 0 mV
(Fig. 2C). The spike duration of 7 ms (measured at half
spike amplitude) and the absolute membrane potential reached by the
afterhyperpolarization (
55 mV) were not changed in the presence of
ChTX. Although these observations may suggest that this conductance
does not play a significant role in spike repolarization and
afterhyperpolarization, the increase in spike amplitude could cause
greater activation of other voltage-dependent outward conductances, which may mask any change in the afterhyperpolarization produced by
suppression of IK,Ca.
Many different types of neuron possess a low-threshold, transient
current (Rogawski 1985; Rudy 1988
) that
was described first in molluscan neurons (Connor and Stevens
1971a
) and termed the "A current." This current
influences the excitable properties of a number of different types of
neuron. For example, it regulates the frequency with which neurons
produce repetitive spiking (Connor and Stevens 1971b
),
delays the onset of spiking during depolarization (Byrne
1980
; Getting 1983
), produces spike frequency
adaptation (Partridge and Stevens 1976
), and contributes
to spike repolarization (Belluzzi et al. 1985
;
Storm 1987
). Because this current can have such a
significant effect on the excitable properties of neurons, we applied
the selective A current blocking agent 4-AP to motoneuron Df to determine whether it influences its excitable
properties. Under current-clamp conditions, application of 1 mM 4-AP
leads to an increase in synaptic activity (n = 7) and
slight depolarization of the resting membrane potential (2.8 ± 0.5 mV, n = 6). In neurons that displayed a plateau
potential on depolarization, 4-AP increased the amplitude of the
superimposed oscillations (n = 3, Fig.
3A). In 4-AP, the oscillations
reached a more positive absolute membrane potential (by
5 mV) and
were followed by a more pronounced phase of repolarization. Application
of 4-AP to neurons that already spiked when depolarized, increased
action potential amplitude 1-3 mV (n = 5) and latency
to first spike was decreased (Fig. 3B, i and ii).
In some preparations, spike afterhyperpolarizations developed more
slowly in the presence of 4-AP (Fig. 3B, i and ii,
n = 3). In other preparations, this was not so
noticeable (n = 2). In some preparations, 4-AP
increased the frequency of spikes evoked by depolarizing pulses of a
given amplitude (n = 3 of 5).
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In some experiments in which ChTX and 4-AP had been coapplied to block outward currents, (Fig. 3Ci) subsequent administration of TEA+ (50 mM) caused a marked increase in both the amplitude and duration of action potentials (Fig. 3Cii). The magnitude of the spike afterhyperpolarization also was increased significantly.
Time dependence of outward currents measured under voltage-clamp conditions
In physiological saline, depolarizing command pulses elicited an outward current, mainly attributable to flux of potassium ions, which was sufficiently rapid and large to obscure a smaller inward calcium current. Altering external chloride concentration had no observable effect on the current-voltage relationship, suggesting that there was no significant chloride conductance in these cells.
To determine whether there is a fall in outward current that
corresponds with the transition in the electrical events recorded from
the neuron, the amplitude of this current was monitored over time.
Within half an hour of dissection, the neuron in Fig.
4 was displaying plateau potentials on
depolarization. Electrical activity and the amplitude of outward
current then were measured every 15 min. With increasing time from
dissection, a decrease in outward current was observed
(n = 3; Fig. 4). During this time, activity induced in
this neuron by depolarization under current-clamp conditions changed
from plateauing into spiking (not shown). The peak amplitude of inward
calcium currents in this neuron is normally <200 nA (Mills and
Pitman 1997) in recordings made up to several hours after
dissection. Inward sodium currents are too small to be detected. Any
increase in these currents therefore would be too small to produce
sufficient direct contribution to membrane currents to account for the
comparatively large (>1-µA reduction when measured at a command
potential of
10 mV) time-dependent decrease in net outward current
observed. The explanation for this reduction in net outward current
therefore is almost certainly a fall in the potassium current. It is
possible, however, that this fall in outward current could result
indirectly from a small change in inward current amplitude; a small
change in Ca influx could have a profound effect on
IK,Ca because this neuron appears to be very
sensitive to changes in [Ca2+]i
(Thomas 1984
).
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Effects of apamin and ChTX on outward currents
Because apamin and ChTX differed in their ability to alter the
type of activity produced by Df under current-clamp
conditions, the effect of these toxins on outward currents was
determined. Figure 5 shows currents and
current-voltage (I-V) relationships obtained from
Df before and after application of apamin. The holding potential was 70 mV, which is close to the normal resting potential of the neuron; 50-ms-duration command pulses, applied every 15 s,
stepped the membrane potential to values between
60 and +100 mV in
10-mV increments. The control I-V relationship of the
Df soma has a characteristic form; outward currents
increase steeply between about
40 and +80 mV. Beyond approximately
+80 mV, the net outward current shows a sharp decline before undergoing
a further increase at still more positive potentials (not shown). This
N-shaped I-V relationship is due to the presence of a large IK,Ca in this neuron (cf. David and
Pitman 1995a
; Thomas 1984
). The I-V
relationship illustrated in Fig. 5 covers this range to demonstrate the
form that is diagnostic of IK,Ca, even though membrane potentials between
70 and 0 mV are most physiologically relevant in this neuron.
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Apamin (1 µM) did not depress outward currents measured under these conditions nor did it alter their time course (Fig. 5). This is consistent with lack of effect of this toxin observed under current-clamp conditions. Application of the Ca-channel blocker Cd2+ in the presence of apamin reduced a large proportion of the outward current and abolished the decline in outward current seen as the command potential steps are increased from +80 and +100 mV (responsible for conferring the N shape to the I-V relationship of the neuron). This confirms the presence of a large Ca-dependent outward current component in this neuron; the lack of effect of apamin, therefore, cannot be attributed to absence of IK,Ca.
The effect of ChTX on currents evoked at different membrane potentials
is shown in Fig. 6. At potentials between
40 and +70 mV, ChTX (100 nM) reduced the net outward current by
66.1 ± 1.2% (7 neurons) (measured at the peak outward current).
At membrane potentials more positive than approximately +80-100 mV,
however, there was a fall in the proportion of the total outward
current blocked by ChTX (Fig. 6, A and C). This
is presumably because the contribution of IK,Ca
to the total outward current declines as the membrane potential
approaches the calcium equilibrium potential. In the presence of a dose
of ChTX (100 nM) sufficient to produce its maximal effect on
IK,Ca, addition of 0.5 mM Cd2+ still
could cause a further reduction in the outward current measured at some
membrane potentials. Combined application of ChTX and Cd2+,
for example, blocked 82 ± 7% (n = 8) of the peak
outward current at +70 mV; the current that has been blocked represents
IK,Ca. The additional block produced by
Cd2+ suggests that there may be a ChTX-insensitive
component of IK,Ca in Df. When
Cd2+ was applied before ChTX (n = 3, not
shown), no further depression of outward current was produced by ChTX
showing that ChTX was not affecting any cadmium-insensitive outward
conductances.
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To investigate different components of outward current, those recorded
in the presence of a drug were subtracted from those obtained in
control conditions. The ChTX-sensitive current obtained in this way is
shown in Fig. 6Bi. The IK,Ca that was
insensitive to ChTX but sensitive to cadmium ions (ChTX-insensitive
IK,Ca) was obtained by subtracting the currents
in the presence of both ChTX and Cd2+ from those in the
presence of ChTX alone (Fig. 6Bii; note that these currents
do not give an indication of the whole current that is sensitive to
Cd2+, which also would include the ChTX-sensitive
component). Difference currents (Fig. 6Bi) show that ChTX
blocked the sustained outward current as well as the peak outward
current. This also is reflected in the I-V relationships
measured at 45 ms (Fig. 6Ci) and at 8 ms (Fig.
6Cii) after the beginning of a 50-ms pulse. The kink in the
I-V curve seen as command pulses are increased from +80 to
+120 mV (which normally gives the characteristic N-shaped
I-V relationship of the neuron) is reduced by ChTX and
abolished by Cd2+ (currents sampled at 8 and 45 ms; Fig.
6C). The ChTX- and Cd2+-resistant outward
current, which activated at potentials more positive than 40 mV, also
showed transient and sustained components; the transient component
became more prominent at very positive membrane potentials (Fig.
6A, *) where it peaked at 3 ms after the beginning of the
depolarizing pulse. Because ChTX and Cd2+ have little
effect on currents recorded 3 ms after the onset of command pulses
(Fig. 6, A and Ciii), it appears that the initial rapid rise outward current in Df is primarily calcium insensitive.
Effect of 4-AP on outward currents
The fact that electrical activity recorded under current clamp is
influenced by 4-AP provides indirect evidence for a transient potassium
current in Df. In fact, the current that remained in the
presence of Cd2+ was blocked by 1 mM 4-AP (Fig.
7A). It was essential to use
relatively low concentrations of 4-AP to observe this effect; at
concentrations >5 mM, 4-AP caused an unexpected increase in an outward
conductance that obscured the block and resulted in complex effects on
the I-V relationship (not shown). In some molluscan neurons,
4-AP has been shown to display a similar concentration-dependent
effect; at low concentrations, it suppresses outward current, but at
high concentrations, it increases IK,Ca
(Hermann and Gorman 1981). 4-AP (1 mM) decreased the
slope of the I-V relationship (recorded in the presence of
Cd2+) measured at 3 and 8 ms but had little effect on
current measured at 45 ms after the beginning of the depolarizing pulse
(Fig. 7B), reflecting the rapid activation and inactivation
kinetics of the 4-AP-sensitive currents. The time to the peak of the
current was between 3 (at command potentials >0 mV) and 4 ms (at more
negative command potentials), and the current decay could normally be
fitted using a single exponential that was faster at more positive
command potentials (Table 1). The fast
kinetics and sensitivity to 4-AP suggest that this current may be
similar to the fast transient A current originally described in
molluscan neurons by Connor and Stevens (1971a)
and
subsequently observed in a number of different preparations (review by
Rudy 1988
).
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Steady-state inactivation of the outward current
Typical A currents display almost complete steady-state
inactivation at 40 mV, whereas their threshold for activation is normally lower than those for other K currents (Rudy
1988
). The inactivation properties of outward currents in
Df therefore were investigated. Current subtractions were
used to study Cd2+- and 4-AP-sensitive currents. The
Cd2+-sensitive current consisted of rapidly and slowly
decaying transient components and a sustained component (Fig.
8Ai). The 4-AP-sensitive current was comparatively small and appeared to consist of a single component that almost completely decayed during 50-ms command pulses
(Fig. 8Aii).
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Steady-state inactivation was determined by applying 800-ms
conditioning prepulses to step the membrane potential to values between
100 and 0 mV (in 10-mV increments) from a holding potential of
70
mV. This duration was sufficient to allow inactivation to reach a
steady level. Each prepulse was followed by a 50-ms test pulse to 0 mV
(Fig. 8B). As the conditioning pulse was made more positive,
the amplitude of the total outward current evoked by a test pulse
decreased. To represent this graphically, the peak current recorded
after a given prepulse (I) was normalized to the peak
current observed after a prepulse to
100 mV
(Imax), then plotted against prepulse potential
(Fig. 8C). Steady-state inactivation curves were determined
for total outward current, Cd2+-sensitive current,
4-AP-sensitive current, and the current that was insensitive to both
4-AP and Cd2+ ions, by subtracting the currents remaining
in the presence of channel blockers from currents recorded in normal
saline solution (Fig. 8C). The mean steady-state
inactivation curves were fitted by a Boltzmann distribution. The total
outward current displayed steady-state inactivation that reached a
maximum of between 50 and 60% at prepulse potentials more positive
than
15 mV and had an E0.5 (which is the potential at
which inactivation is half completed) of
33 mV. The Cd-sensitive
current also displayed partial steady-state inactivation, which reached
a maximum of ~70% at potentials more positive than
10 mV and had
an E0.5 of
26 mV. The 4-AP-sensitive current displayed
slight inactivation at the resting membrane potential of
70 mV in
some preparations (in 4 of 8 preparations), but a conditioning pulse to
55 mV normally was required before inactivation occurred in the
remaining preparations; maximum steady-state inactivation was observed
at potentials more positive than
15 mV, where it approached 100%;
the E0.5 was
36 mV.
Action potential clamp
This method is a powerful tool for observing the contribution made
by specific ion conductances toward different components of electrical
events recorded from excitable cells (Doerr et al. 1988,
1989
). Using this technique, representative plateau potentials or action potentials were recorded from Df under current
clamp, then used as the command signal applied to the same cell under voltage-clamp conditions. When the neuron is clamped by its own activity, no net current will be measured once activity has
synchronized. However, if a specific current is blocked
pharmacologically, the clamp amplifier has to compensate for the
contribution that this current would normally make to the electrical
activity. The compensation current therefore should be identical to the
original contribution of the blocked ion conductance (Doerr et
al. 1989
). An example of the effects of ChTX on a
Df neuron that was clamped by one of its typical plateau
potentials is shown in Fig. 9. On
depolarization under current-clamp conditions, this neuron produced
plateau potentials similar to that shown in Fig. 9iii. This
typical plateau potential was used as the command signal under
voltage-clamp conditions. Because motoneuron Df is not
normally spontaneously active, it must be depolarized by injection of a
small amount of current. The onset of this current pulse is visible on
the records (see arrows). Under control conditions, application of the
prerecorded plateau potential produced only a very small compensation
current (at arrow) that represents the current injected under
current-clamp conditions to depolarize the cell to threshold (Fig.
9i). After application of 100 nM ChTX to the preparation
[which under current-clamp conditions converted the response of the
neuron to depolarization from a plateau potential to an action
potential (not shown; c.f. Fig. 2)], a downward compensation current
was observed because the clamping amplifier had to inject negative
current to compensate for the absence of the current blocked by ChTX
(Fig. 9, ii-iv). The ChTX-compensation current activated
during the depolarizing phase of the plateau potential, but peaked
during the repolarizing phase of the oscillations (Fig.
9iv). Under current-clamp conditions, the ChTX-sensitive
current would shunt the inward current underlying these events and
thereby limit their amplitude. Activation of this current also could
account for the inability of the Df soma to spike at short
intervals after dissection (see preceding text). The compensation
current declined in amplitude to almost zero by the end of the plateau
potential, suggesting that this ChTX-sensitive conductance is unlikely
to be responsible for plateau potential termination.
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The effects of ChTX on cells that were already spiking indicated that
this conductance cannot be primarily responsible for spike
repolarization and afterhyperpolarization because spikes evoked in the
presence of ChTX still repolarized rapidly and had distinct
afterhyperpolarizations (see Fig. 2). The action-potential-clamp method
was used to test whether the ChTX-insensitive, calcium-dependent potassium conductance, on the other hand, does contribute toward these
events. The current-clamp recording shown in Fig.
10A was made from
Df in the presence of ChTX, which caused this neuron to
spike on depolarization. After application of micromolar
Cd2+, the amplitude of the spike and afterhyperpolarization
were decreased and the rate of depolarization and repolarization were
slowed (Fig. 10A, spikes in Cd 2+ marked by *).
These effects may not necessarily be due to a decrease in a
calcium-dependent potassium conductance but rather from a reduction in
voltage-dependent potassium conductances resulting from the decrease in
spike amplitude. This problem is circumvented by using the
action-potential-clamp technique; the membrane potential excursion
produced by a control action potential as the command signal will be
the same before and after administration of Cd2+.
Interpretation of resultant current therefore will not be complicated by variations in the extent to which voltage-dependent conductances are
activated. A series of typical action potentials was used as the
command signal when the neuron was held under voltage-clamp conditions
(Fig. 10Biii). The control response consisted of a small sustained upward compensation current (corresponding to the injected current that was required under current-clamp conditions to reach the
spike threshold). After application of 20 µM Cd2+,
biphasic transient compensation currents were observed. The relatively
small upward current, which coincided with spike depolarization and the
peak of the action potentials resulted from block of the Cd2+-sensitive calcium current (Mills and Pitman
1997). The larger transient downward compensation currents,
coinciding with the spike repolarizations, were due to block of a
calcium-dependent potassium current. Because the cell already was
bathed in ChTX, the potassium current responsible for these
compensation currents must be the ChTX-insensitive,
Cd2+-sensitive IK,Ca.
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We already have indicated that 4-AP increases the amplitude of oscillations superimposed on plateau potentials but does not cause a transition to spiking (see Fig. 3). To establish the contribution of the 4-AP-sensitive current to plateau potentials, we used the action-potential-clamp method. Figure 11 shows the results of applying this technique to the neuron used in Fig. 3. A typical control plateau potential was used as the command signal under action-potential-clamp conditions (Fig. 11Aiii). An upward compensation current was recorded, which reflects the current applied to evoke the template plateau potential; the plateau potential signal itself evoked no compensation current (Fig. 11Ai). In the presence of 1 mM 4-AP, transient downward compensation currents were observed that activated during the membrane potential oscillations and peaked during the repolarizing phase of the oscillation (Fig. 11A, ii-iv). The effect of the 4-AP-sensitive current, like that of the ChTX-sensitive current, therefore would be to shunt the inward currents underlying the oscillations superimposed on plateau potentials. The amplitude of the 4-AP-compensation current is considerably smaller than the ChTX-compensation current. This probably accounts for the inability of 4-AP to cause Df to undergo the transition from plateau potential activity to spikes. During the sustained phase of the plateau potential, no significant compensation current was observed, suggesting that this conductance does not contribute to plateau termination.
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Application of 1 mM 4-AP to a spiking neuron under current-clamp conditions can decrease the latency to first spike, increase spike amplitude, and slow the development of the spike afterhyperpolarization (see Fig. 3B). This same preparation was clamped by a series of typical action potentials (Fig. 11Biii). In control responses, each template action potential was associated with an extremely small compensation current (Fig. 11Bi). The compensation currents observed in the presence of 4-AP are shown in Fig. 11Bii; these developed during the depolarizing phase of each action potential and peaked during spike repolarization (Fig. 11Bii). The 4-AP-sensitive current therefore would tend to reduce spike amplitude and contribute toward the afterhyperpolarization.
These experiments show that outward conductances in the soma of Df can influence the electrical activity displayed by this neuron. A ChTX-sensitive IK,Ca is the major outward conductance in this cell and is important in determining whether the cell spikes or displays plateau potentials on depolarization. Other smaller conductances such as the ChTX-insensitive IK,Ca and the 4-AP transient currents contribute toward spike repolarization and appear to play a role in the fine-tuning of electrical activity.
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DISCUSSION |
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In this paper, current-clamp, voltage-clamp. and
action-potential-clamp techniques have been used to explore the roles
of potassium conductances in determining the type of electrical
activity displayed by the cockroach Df motoneuron soma.
Depolarizing pulses evoke plateau potentials at short intervals after
dissection; after ~1 h, however, depolarization elicits action
potentials (Hancox and Pitman 1992). Similar changes are
seen if the soma is divided mechanically from its processes
(Hancox and Pitman 1992
). This indicates that the
observed transition in electrical properties occurs in the soma itself
and is not an indirect result of a change in the length constant or
other characteristics of the neuron that enable the dendrites to exert
a greater effect on somatic recordings. It was suggested that, shortly
after dissection, [Ca2+]i is relatively high
and that this results in elevation of calcium-dependent potassium
current (IK,Ca), which, in turn, shunts inward
currents, so preventing the soma from spiking (Hancox and Pitman
1992
). In line with this proposal, we have found that there is
a decrease in net outward current with increasing time from dissection,
which correlates well with the change in electrical properties of the soma. We propose that plateau potentials can occur shortly after dissection, despite enhancement of IK,Ca,
because this current is not strongly activated at the membrane
potentials attained during plateau potentials. The inward current
underlying the plateau potential is probably the low-threshold,
nifedipine-sensitive calcium current we have described recently
(Mills and Pitman, 1997
). We have shown that the
Df soma possesses IK,Ca, a transient 4-AP-sensitive and a delayed rectifier current, each of which may have
a different role in regulating the electrical properties of the neuron.
Calcium-dependent potassium current
IK,Ca is the largest current recorded from
the soma of this neuron. Because apamin had no effect on membrane
currents in Df, it appears that, like Aplysia
neurons (Hermann and Hartung 1983) and larval muscle
fibers of Drosophila (Elkins et al. 1986
;
Gho and Mallart 1986
), this neuron lacks an
IK,Ca similar to the apamin-sensitive low-conductance subtype found in a range of vertebrate and invertebrate preparations (Lazdunski 1983
). ChTX, on the other hand,
almost completely blocked IK,Ca recorded from
the soma of Df. Experiments with isolated somata indicates
that the ChTX current is present on the soma itself. Application of
different concentrations of ChTX indicates that there is a small
ChTX-insensitive component of IK,Ca that can be
blocked by Cd2+. A slight possibility remains, however,
that ChTX produces incomplete block of a single class of current. When
ChTX was applied after Cd2+, there was no additional
inhibition of IK,Ca, indicating that ChTX does
not affect any calcium-independent (Cd2+-insensitive) conductances.
Although the identity of the small ChTX-resistant component of
IK,Ca has not been established, it may be
similar to one of the different subtypes of
IK,Ca that have been identified in insect neurons. For example, in Drosophila muscle and "giant"
neurons, two subtypes of IK,Ca have been
identified; a fast transient and a slow non-inactivating component
(Elkins et al. 1986; Gho and Mallart
1986
; Saito and Wu 1991
; Salkoff
1983
; Singh and Wu 1989
). A
Drosophila mutant, slowpoke (slo), lacks a component of
IK,Ca in muscle and giant neurons (Elkins
et al. 1986
; Saito and Wu 1991
; Singh and
Wu 1989
); these cells are more excitable and display broader
action potentials than the wild-type, indicating that, in these
preparations, IK,Ca is important in spike
repolarization (Elkins et al. 1986
; Singh and Wu
1990
). Irregular regenerative responses, including oscillations
of variable amplitude superimposed on slow waves of depolarization have
been recorded from some subsets of neurons in slo mutants (Saito
and Wu 1991
). In Kenyon cells from the honeybee
(Schäfer et al. 1994
), and in cockroach DUM neurons (Grolleau and Lapied 1995
), a ChTX-sensitive
IK,Ca with fast transient and sustained
components has been identified. Although the ChTX-sensitive
IK,Ca identified in Df displays some
inactivation during a sustained depolarization, a fast ChTX-sensitive
transient component was not observed. It is possible, however, that
such a component may have been present but extremely small compared with the sustained component.
Application of ChTX to Df rapidly causes the neuron to generate spikes instead of plateau potentials when depolarized. This transition in response is similar to the time-dependent change that normally develops in this preparation during a period of hours, indicating that this conductance may be important in determining electrical properties of the neuron. The gradual fall in K currents with time from the onset of recording (perhaps due to falling levels of intracellular calcium), which we report here, may be responsible for the appearance of spikes, and their subsequent gradual increase in amplitude as a function of time from dissection.
The activation characteristics of IK,Ca,
determined in voltage-clamp experiments, could explain why
Df is initially capable of generating plateau potentials
but not spikes. The potential reached by a plateau potential rarely
exceeds 30 mV, whereas the membrane potential reached during a spike
can be close to 0 mV. At
30 mV, IK,Ca may not
be large or fast enough to shunt the inward current underlying the
plateau potential. At more positive membrane potentials, however,
IK,Ca activation is faster and larger and is
more likely to be capable of shunting the currents responsible for
spiking. When IK,Ca is reduced, either by direct
pharmacological block or by lowering intracellular calcium levels, the
inward currents underlying spiking would be shunted to a smaller
extent, thereby allowing the cell to spike. The action-potential-clamp technique, makes it possible to establish the contribution of individual conductances to electrical events with a precision not
possible with conventional voltage-clamp method. For example, this
method has enabled us to show that the ChTX-sensitive conductance activates sufficiently fast at the onset of a plateau potential to
shunt inward currents, so, at the same time, limiting plateau potential
amplitude and preventing action potential generation. During plateau
potentials, this current decays to such an extent that it could not be
responsible for termination of these events.
In Aplysia, ChTX produces different effects on action
potentials depending on the cell type; in bursting cells, ChTX prolongs the action potential duration but it has no effect on beating or silent
cells after direct stimulation (Hermann and Erxleben 1987). Hermann and Erxleben (1987)
found that
ChTX also could depolarize the resting membrane by blocking a resting
potassium conductance, thus causing an increase in firing frequency. In Df, however, ChTX had no effect on the resting membrane
potential. ChTX did not appear to affect spike duration or reduce the
amplitude of the spike afterhyperpolarization in Df
even though the activation characteristics of the ChTX-sensitive
current are such that this conductance should contribute toward
speeding spike repolarization. In fact, application of ChTX to neurons
that were already able to generate action potentials increased the
afterhyperpolarization amplitude, probably because ChTX produced an
increase in action potential amplitude, so causing stronger activation
of the voltage-dependent K currents (cf. effects of 4-AP). A
similar phenomenon is probably responsible for the initial shortening
of plateau potential duration by ChTX, since it causes an increase in
the amplitude of plateau potentials amplitude.
4-AP-sensitive current
The small amplitude of the 4-AP compensation current compared with that for ChTX may explain why 4-AP did not convert activity in Df from plateauing to spiking but merely increased the amplitude of the oscillations that normally are superimposed on plateau potentials. The small depolarization observed after application of 4-AP most probably resulted from synaptic input impinging on Df because this drug increased the level spontaneous synaptic activity. This is supported by the observation that 4-AP did not appear to influence voltage-dependent currents activated at the resting membrane potential of Df.
Although the 4-AP-sensitive current did not induce spiking in the neuron soma, it did contribute toward spike repolarization and afterhyperpolarization as demonstrated in current-clamp and action-potential-clamp experiments. Under current clamp, the effects of 4-AP on spike afterhyperpolarization varied from preparation to preparation; in some, it was prolonged, whereas in others it was unaffected. The explanation for this is probably the same as that for the effect of ChTX on spike repolarization and afterhyperpolarization; 4-AP produces a small increase in spike amplitude in some preparations by reducing shunting of inward conductances. In those preparations where this occurred, activation of other voltage-dependent outward conductances, and hence afterhyperpolarization, may be increased. Under action-potential-clamp conditions, the 4-AP-sensitive compensation current activated during the depolarizing phase of the action potential and peaked during spike afterhyperpolarization, confirming that this conductance does contribute toward the afterhyperpolarization. In addition to this current, a small, ChTX-insensitive component of IK,Ca also contributes to repolarization.
The threshold of activation and inactivation of the 4-AP-sensitive
current in Df occur at more positive membrane potentials than those of a typical A-type current and are more similar to the
"high-threshold A-type" currents that have been observed in some
preparations (review by Rudy 1988) such as the current
observed in Drosophila muscle [A1 transient current that is
removed by the Shaker mutation: (Salkoff and Wyman 1981
;
Solc et al. 1987
; Zagotta 1988
)]. The
transient conductance in Df appears to influence the
latency to first spike, spike repolarization, and firing frequency as
does the A current in many preparations. These observations contrast
with those made on another cockroach neuron, a basalar/coxal depressor
motoneuron known as cell 3, which is a partial synergist of
Df (Nightingale and Pitman 1989
). Although
cell 3 and Df have many similarities, when studied either
under current or voltage clamp, little or no transient outward current
was observed in cell 3 when command steps were applied from a holding
potential less negative than
70 mV. When the membrane potential was
held at more negative values, however, command steps to
45 mV
generated transient outward currents, the amplitude of which increased
as the holding potential was made more negative. Inactivation of the
transient outward current in cell 3, therefore appears to occur at more
negative potentials than similar currents in other neurons. Such
currents in Df, on the other hand, inactivate at less
negative potentials than is usual.
Delayed rectifier current
The current remaining after application of Cd2+ and 4-AP displayed activation and inactivation properties of a typical delayed rectifier current. We did not further explore the influence of this current on electrical activity in Df mainly because we found no means to manipulate this current pharmacologically. As a result, this current could only be investigated by first eliminating all other outward currents; it was impossible, therefore, to directly determine the contribution made by the delayed rectifier current to the electrical properties of the neuron. For this reason, it was not studied in depth.
To summarize, the ChTX-sensitive current is the largest component and
is important in determining whether the neuron responds to
depolarization by spiking or by producing plateau potentials; when this
conductance is high, the cell cannot spike. The other smaller
conductances appear to be important in the fine tuning of electrical
activity. Obviously any change in inward conductances would be
important in determining electrical activity not only directly but also
via their indirect effect on outward conductances. For example, the
calcium conductance in this cell shows calcium-dependent inactivation
(Mills and Pitman 1997). Therefore high intracellular calcium levels would not only decrease the amplitude of
ICa but also would enhance
IK,Ca and thus may disable spiking in the soma of Df shortly after dissection via these two synergistic mechanisms.
Although it is likely that the transition from plateauing to spiking
may result from a gradual fall in [Ca2+]i, it
is not clear whether the neuronal properties observed immediately after
dissection represent a normal state or whether they are consequences of
trauma associated with dissection (e.g., temporary hypoxia or excessive
neuronal activity produced by stress associated with handling and
dissection which could cause release of abnormally large amounts of
neurotransmitters and neuromodulators). If this were the case, the
postoperational change in neuronal properties could reflect gradual
recovery. Any neuromodulator that induces a change in intracellular
calcium levels may be expected to exert an important influence on
output from this motoneuron. For example, muscarinic agonists have been
shown to elevate intracellular calcium levels (David and Pitman
1996a) and decrease ICa (David
and Pitman 1995b
). These agents convert activity in
Df from spiking to plateauing (unpublished data). Increases
in intracellular cyclic AMP inhibit IK,Ca in
Df (David and Pitman 1996b
) so that
neuromodulators that increase cyclic AMP also would be expected to
alter electrical activity. The possibility cannot be excluded, however,
that, under physiological conditions, the soma of Df does
not normally spike and that development of somatic action potentials
reflects an early response to axotomy. If this were the case, the
transition in properties could result either from of redistribution of
ion channel proteins or from de novo synthesis of new
channel proteins (cf. Pitman et al. 1972
). Whatever the
case, a similar change in excitability has not be found in motoneuron
Ds, a synergist of Df or in inhibitory
motoneurons, indicating that the transition we have observed in
Df must be selective for certain neurons.
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ACKNOWLEDGMENTS |
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We thank Dr. John Dempster, University of Strathclyde, Glasgow, for providing software for action potential clamp experiments.
This work was supported by the Biotechnology and Biological Sciences Research Council.
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FOOTNOTES |
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Address reprint requests to R. M. Pitman.
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 23 December 1997; accepted in final form 26 January 1999.
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REFERENCES |
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