Department of Medical Physiology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark
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ABSTRACT |
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Perrier, Jean-François and Jørn Hounsgaard. Ca2+-Activated Nonselective Cationic Current (ICAN) in Turtle Motoneurons. J. Neurophysiol. 82: 730-735, 1999. The presence of a calcium-activated nonspecific cationic (CAN) current in turtle motoneurons and its involvement in plateau potentials, bistability, and windup was investigated by intracellular recordings in a spinal cord slice preparation. In the presence of tetraethylammonium (TEA) and tetrodotoxin (TTX), calcium action potentials evoked by depolarizing current pulses were always followed by an afterdepolarization associated with a decrease in input resistance. The presence of the afterdepolarization depended on the calcium spike and not on membrane potential. Replacement of extracellular sodium by choline or N-methyl-D-glucamine (NMDG) reduced the afterdepolarization, confirming that it was mediated by a CAN current. Plateau potentials and windup were evoked in response to intracellular current pulses in the presence of agonist for different metabotropic receptors. Replacement of extracellular sodium by choline or NMDG did not abolish the generation of plateau potentials, bistability, or windup, showing that Na+ was not the principal charge carrier. It is concluded that plateau potentials, bistability and windup in turtle motoneurons do not depend on a CAN current even though its presence can be detected.
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INTRODUCTION |
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In the spinal cord, plateau potentials have
been described in motoneurons (Bennett et al. 1998a,b
;
Hounsgaard et al. 1988
; Hounsgaard and Kiehn
1985
; Hounsgaard and Mintz 1988
; Lee and Heckman 1998a
,b
) and several subtypes of interneurons
(Hounsgaard and Kjærulff 1992
; Morisset and Nagy
1996
; Russo and Hounsgaard 1996
). Currents
responsible for plateau potentials are modulated by metabotropic
receptors for glutamate, serotonin (5-HT), and acetylcholine
(Delgado-Lezama et al. 1997
; Svirskis and
Hounsgaard 1998
). Windup (i.e., the gradual increase of the
response to repeated depolarizations) has also been described in
several types of spinal cord neurons (Morisset and Nagy
1996
; Russo and Hounsgaard 1994
; Svirskis
and Hounsgaard 1997
). Both phenomena depend on
Ca2+ influx through L-type
Ca2+ channels, which triggers the plateau
potential (Hounsgaard and Mintz 1988
) and windup of the
plateau potential during repeated depolarizations (Russo and
Hounsgaard 1994
; Svirskis and Hounsgaard 1997
).
It is not known, however, whether the Ca2+ influx
is the charge carrier for the plateau potential or merely the trigger
for a noninactivating Ca2+-activated conductance
generating the current underlying the plateau potential. The
nonselective calcium-activated cationic current (ICAN) is a possible candidate because
CAN channels, usually permeant for Na+,
K+, and also Ca2+, do not
undergo voltage- or Ca2+-dependent inactivation
and thus provide a potential mechanism for maintaining depolarization
and Ca2+ entry in the cell (Partridge et
al. 1994
). Recent studies have shown an involvement of such a
ICAN in plateau potentials in several types of neurons. In the dorsal gastric motor neuron of the
stomatogastric ganglion in the crab for example,
ICAN current plays an important role
in generation and maintenance of plateau potentials (Zhang et
al. 1995
). In rostral ambiguus neurons in the brain stem of newborn mice, a ICAN current is the
main charge carrier for plateau potentials (Rekling and Feldman
1997
). In rat deep dorsal horn neurons of the spinal cord,
ICAN represents a fraction of the plateau current, the rest being mediated by noninactivating calcium channels (Morisset and Nagy 1999
). Moreover,
ICANs are modulated by intracellular
pathways compatible with metabotropic modulation of plateau potentials.
For example, ICAN currents in
Helix burster neurons are modulated by cyclic AMP-dependent
membrane phosphorylation (Partridge et al. 1990
); in CA1
hippocampal neurons, ICANs are activated by metabotropic receptors for glutamate (Crepel et al. 1994
). Finally, cumulative increase in the concentration of
intracellular Ca2+ is a plausible mechanism for
windup of a ICAN in response to repeated depolarizations.
Several questions remain to be answered. 1) Is such a ICAN expressed in turtle motoneurons? 2) Does it contribute significantly to plateau potentials? 3) Does metabotropic modulation, known to regulate generation of plateau potentials, act on ICAN currents? 4) Is ICAN current involved in windup?
In the present report we show that ICANs are expressed in turtle motoneurons. We also show that the contribution of ICAN to plateau potentials, bistability, and windup is insignificant, and that modulatory pathways known to regulate the generation of plateau potentials do not affect ICANs.
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METHODS |
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Transverse slices (1.5-2 mm thick) were obtained from the
lumbar enlargement of adult turtles (Chrysemys picta)
anesthetized by intraperitoneal injection of 100 mg sodium
pentobarbitone and killed by decapitation. The surgical procedures
comply with the Danish legislation and are approved by the controlling
body under The Ministry of Justice. Experiments were performed at room
temperature (20-22°C) in a solution containing (in mM) 120 NaCl, 5 KCl, 15 NaHCO3, 2 MgCl2,
3CaCl2, and 20 glucose saturated with 98%
O2-2% CO2 to obtain pH
7.6. Intracellular recordings in current-clamp mode were performed with
an Axoclamp 2B amplifier (Axon Instruments). Pipettes were filled with
a solution containing 1 M K-acetate. Motoneurons were selected for
study if they had a stable membrane potential of more than 60 mV. The
data were sampled at 16.6 kHz with a 12-bit A/D converter (DIGIDATA
2000 from Axon) and displayed by means of Axoscope software and stored
on a hard disk for later analysis.
Low-sodium medium was prepared by substituting choline chloride or N-methyl-D-glucamine chloride (NMDG; Sigma) for sodium chloride. The pH of medium prepared with NMDG was carefully adjusted to 7.6 by addition of the necessary amount of HCl. This was necessary because medium added NMDG-HCl stock solution often had a pH 8-8.4 when saturated with carbogen. In four of four experiments, high pH reduced or abolished plateau potentials. In normal medium an increase in pH above 8.0, produced by adding HEPES sodium salt, greatly increased the threshold for plateau potentials (n = 2). In low sodium ringer a high pH value had the same effect (n = 2).
Plateau potentials were facilitated directly with an agonist for L-type
Ca2+ channels (BayK8644; 2 µM; Sigma) or
indirectly by activation of group I metabotropic glutamate receptors
with cis-(±)-1-aminocyclopentane-1,3-dicarboxylic acid
(cis-ACPD; 40 µm; Tocris), muscarine receptors (Muscarine; 20 µM; Sigma), or serotonin receptors (5-HT; 10 µM; Sigma), all known to promote the generation of plateau potentials
(Delgado-Lezama et al. 1997; Hounsgaard and Mintz
1988
; Svirskis and Hounsgaard 1998
). Ca spikes
and plateau potentials were facilitated by addition of
tetraethylammonium (TEA; 10 mM) to the medium (Hounsgaard and Mintz 1988
).
Other drugs that were used (all from Sigma) are as follows: tetrodotoxin (TTX, 2 µM); Nifedipine (10 µm); cobalt; HEPES sodium salt (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]).
Data are presented as means ± SE.
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RESULTS |
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ICAN current in turtle motoneurons
One way to test whether a CAN current
(ICAN) is present in motoneurons is to
record the effect of a transient increase in the level of intracellular
calcium. We have taken advantage of the ability of motoneurons to
generate calcium action potentials in the presence of TEA (10 mM) and
TTX (2 µM) (Hounsgaard and Kiehn 1993;
Hounsgaard and Mintz 1988
) to induce a transient calcium increase. Under these conditions, a Ca2+ spike
was evoked by a depolarizing current pulse of sufficient amplitude
(Fig. 1A). The involvement of
calcium channels in this regenerative response was confirmed by its
disappearance when 2 mM Co2+ was added to the
medium (n = 2). In all the cells recorded from, calcium
spikes were followed by a slow depolarizing potential, lasting >500 ms
(arrow in Fig. 1B; n = 26). The existence of
this afterdepolarization was strictly correlated to the presence of calcium spikes and was not dependent on voltage, because it was possible to induce it even when the cell was hyperpolarized by a strong
negative bias current. As shown in Fig. 1C calcium spikes were always followed by a prolonged afterdepolarization independent of
the membrane potential. This is consistent with mediation by a
calcium-activated inward current. This afterdepolarization was not
mediated by L-type Ca channels because it persisted in the presence of
nifedipine (10 µM; n = 4; see Fig.
2).
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Application of trains of low-amplitude, negative current pulses following calcium spikes showed that the afterdepolarization was associated with a decrease in input resistance by up to 60% and lasting up to 600 ms (n = 6), i.e., the same duration as the afterdepolarization. (Fig. 1D).
The amplitude of the afterdepolarization following calcium spikes
increased with increasing levels of hyperpolarizing holding current
(Fig. 2A1). The relation between the amplitude of the afterdepolarization and the holding potential was linear (Fig. 2A2), showing that the current mediating the
afterdepolarization was not voltage sensitive. The afterdepolarization
was reversible in response to depolarizing holding current (Fig.
2A). The reversal potential was 55 ± 2.2 mV
(means ± SE; n = 11), suggesting that different
ion species were involved. As shown in Fig. 2B the
afterdepolarization was partly mediated by Na+
ions because its reversal potential was shifted to more hyperpolarized levels when extracellular NaCl was replaced by choline chloride or NMDG
(n = 7).
Taken together these data showed that the afterdepolarization following calcium spikes in turtle motoneurons was due to activation of a CAN with an amplitude linearly related to the membrane potential.
ICAN current not necessary for plateau potentials
If the ICAN makes a significant contribution to plateau potentials, then their amplitude should be sensitive to the concentration of extracellular Na+. We tested this by subjecting motoneurons, facilitated to generate plateau potentials in normal medium, to low sodium medium. In all motoneurons, independent of the way in which plateau potentials were promoted, the generation of plateau potentials was uninhibited by low sodium medium (n = 16 of 16; 7 with choline medium and 9 with NMDG medium). In particular the generation of plateau potentials was not shifted to more hyperpolarized membrane potentials. These findings are illustrated in Fig. 3. In normal medium the train of spikes evoked by a depolarizing current pulse did not outlast the duration of the stimulus (Fig. 3A1). In the presence of 40 µM ACPD, the same stimulus evoked a sustained discharge only terminated by a hyperpolarizing current (Fig. 3A2). This bistable response pattern was maintained when sodium chloride was replaced with NMDG (Fig. 3A3). Similar results were obtained when plateau potentials were promoted by serotonin (Fig. 3B; n = 3), by muscarine (n = 3), or BAY K (n = 2; not illustrated). These experiments show that ICAN current is not a major contributor to plateau potentials in turtle motoneurons.
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ICAN current not necessary for windup
We finally tested whether windup of plateau potentials was
preserved in low-sodium medium. With the same protocol as used previously (Russo and Hounsgaard 1994; Svirskis
and Hounsgaard 1997
), we found that the plateau potential,
generated by a depolarizing current of near threshold intensity,
increased in amplitude and decreased in latency with each of the first
few pulses in a stimulus train (n = 8). Facilitation
was present in all motoneurons in low-sodium medium whether plateau
properties were induced by cis-ACPD (n = 2;
Fig. 4A), 5-HT
(n = 3; Fig. 4B), or muscarine
(n = 3; not illustrated). Therefore we conclude that
ICAN is not necessary for windup.
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DISCUSSION |
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Our experiments show that ICAN is
present is turtle motoneurons. Two experimental observations suggest
that this current is activated by calcium. First, under our
experimental conditions, the presence of this current was strictly
correlated with the presence of calcium spikes. Second, when calcium
influx was removed by addition of cobalt, the current was absent. The
ions carrying ICAN include
Na+, because its removal shifted the reversal
potential substantially. K+ ions probably also
contribute because the reversal potential for the CAN-mediated
afterdepolarization is near 55 mV in normal medium and because the
reversal shifts toward the equilibrium potential for potassium when
NaCl is replaced by NMDG chloride. In agreement, at the resting
membrane potential the CAN-induced afterdepolarization was converted to
an afterhyperpolarization in NMDG medium.
Our experiments also show that removal of a depolarizing component of
ICAN (i.e., the
Na+ inward current) does not affect the ability
of motoneurons to generate plateau potentials and windup. These results
demonstrate that Na+ is not the main charge
carrier for plateau potentials or windup in turtle motoneurons. The
increased conductance during plateau potentials is incompatible with
decreased Cl or K+
currents. Therefore our results support the hypothesis that plateau potentials in turtle motoneurons are not only triggered but also generated by a calcium current.
Modulation of plateau properties induced by activation of metabotropic
receptors for glutamate, serotonin, or acetylcholine was not
significantly affected by low-sodium Ringer, showing that pathways
ending on CAN channels are not essential in this process. Moreover,
experiments in the presence of TEA and TTX showed that the reversal
potential for CAN current is around 55 mV, which is below the
threshold for activation of plateau potentials in turtle motoneurons
(above
50 mV) (Svirskis and Hounsgaard 1997
). Above
55 mV,
ICAN is outward and therefore
hyperpolarizes the cell and thus counteracts plateau potentials.
Finally the absence of voltage sensitivity of
ICAN does not make it a good candidate for mediating plateau potentials and windup. Both phenomena have been
shown to be extremely sensitive to voltage. Several studies show that
brief hyperpolarizing current pulses is sufficient to turn off plateau
potentials (Hounsgaard and Mintz 1988
) (see also Fig.
3). The linear relationship between the holding potential and
ICAN amplitude (Partridge et
al. 1994
) (see Fig. 2) also allows us to exclude its
involvement in the nonlinear termination of plateau potentials.
However, our data do not exclude the possibility that a sustained
ICAN may assist in holding the cell
near the threshold for action potentials.
Our results are at odds with recent findings suggesting that CAN
currents mediate plateau potentials in rostral ambiguus neurons in the
brain stem of newborn mice (Rekling and Feldman 1997) or in rat deep dorsal horn neurons of the spinal cord (Morisset and Nagy
1999
) and do not support involvement of a TTX-sensitive sodium channel
as in trigeminal motoneurons (Hsiao et al. 1998
). These differences undoubtedly reflect differences in the functional sets of
ion channels in dendrites and cell bodies in different types of
neurons. One obvious difference between Ca2+- and
Na+-mediated plateau potentials is that
Ca2+ accumulation may serve as second messenger
and also be potentially harmful.
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ACKNOWLEDGMENTS |
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This work was funded by The Danish Medical Research Council, The Lundbeck Foundation, and The NOVO-Nordisk Foundation. J.-F. Perrier is recipient of a Marie Curie Research Training Grant ERB4001GT970998.
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FOOTNOTES |
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Address for reprint requests: J. Hounsgaard, Dept. of Medical Physiology, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
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 9 February 1999; accepted in final form 9 April 1999.
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REFERENCES |
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