Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden
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ABSTRACT |
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Büschges, A.,
M. A. Wikström,
S. Grillner, and
A. El Manira.
Roles of High-Voltage-Activated Calcium Channel Subtypes in a
Vertebrate Spinal Locomotor Network.
J. Neurophysiol. 84: 2758-2766, 2000.
Lamprey spinal cord
neurons possess N-, L-, and P/Q-type high-voltage-activated (HVA)
calcium channels. We have analyzed the role of the different HVA
calcium channels subtypes in the overall functioning of the spinal
locomotor network by monitoring the influence of their specific
agonists and antagonists on synaptic transmission and on
N-methyl-D-aspartate (NMDA)-elicited fictive locomotion. The N-type calcium channel blocker -conotoxin GVIA (
-CgTx) depressed synaptic transmission from excitatory and
inhibitory interneurons. Blocking L-type and P/Q-type calcium channels
with nimodipine and
-agatoxin, respectively, did not affect synaptic transmission. Application of
-CgTx initially decreased the frequency of the locomotor rhythm, increased the burst duration, and subsequently increased the coefficient of variation and disrupted the motor pattern.
These effects were accompanied by a depression of the synaptic drive
between neurons in the locomotor network. Blockade of L-type channels
by nimodipine also decreased the frequency and increased the duration
of the locomotor bursts. Conversely, potentiation of L-type channels
increased the frequency of the locomotor activity and decreased the
duration of the ventral root bursts. In contrast to blockade of N-type
channels, blockade or potentiation of L-type calcium channels had no
effect on the stability of the locomotor pattern. The P/Q-type calcium
channel blocker
-agatoxin IVA had little effect on the locomotor
frequency or burst duration. The results indicate that rhythm
generation in the spinal locomotor network of the lamprey relies on
calcium influx through L-type and N-type calcium channels.
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INTRODUCTION |
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The basic locomotor pattern
generated by spinal networks results from specific synaptic
interactions between different neuronal types and their intrinsic
membrane properties. These are subject to extrinsic and intrinsic
modulatory inputs, thus allowing the locomotor network to operate over
a wide range of frequencies and patterns (Grillner et al.
1998; Rossignol and Dubuc 1994
; Sillar et
al. 1997
). To gain insight into the function and modulation of
networks underlying behavior, it is necessary to characterize individual ion channels in the different neurons and their role in
generating the motor pattern. Voltage-gated calcium channels play a
major role in controlling neuronal and network activity by contributing
to neurotransmitter release and ion channel activation and inactivation
(Hille 1992
). They are also modulated by various transmitters and modulators (Dolphin 1996
; Hille
1994
). Different low-voltage-activated (LVA) and
high-voltage-activated (HVA) calcium channels have been identified in
vertebrate neurons (Fox et al. 1987
; Llinás
et al. 1992
; Mintz et al. 1992
; Nowycky
et al. 1985
; Pearson et al. 1995
; Randall
and Tsien 1995
). HVA calcium channels have been subdivided into
L-, N-, and P/Q-type based on their pharmacology and molecular sequence
(see Birnbaumer et al. 1994
; Snutch and Reiner
1992
; Tsien et al. 1991
). Most studies have concentrated on investigating the role of the different calcium channels in synaptic transmission and the activation of different cellular processes. In the Xenopus embryo spinal cord,
blockade of N-type calcium channels disrupts the locomotor pattern
(Wall and Dale 1994
). In spinal motoneurons of the
turtle, membrane oscillations elicited by
N-methyl-D-aspartate (NMDA) and muscarinic receptor activation have been shown to depend on calcium influx through
L-type calcium channels (Guertin and Hounsgaard 1998
, 1999
).
The in vitro lamprey spinal cord preparation offers an advantageous
experimental model system in which the contributions of specific ion
channels and their modulation to motor pattern generation can be
analyzed. The spinal neuronal circuitry generating swimming has been
characterized (Buchanan 1982; Buchanan and
Grillner 1987
) and can be activated by application of NMDA
(Grillner et al. 1981
). Recently, whole cell patch-clamp
recordings from isolated motoneurons, interneurons, and sensory neurons
from the lamprey spinal cord have shown that their HVA current is
mainly mediated by calcium influx through N-type channels and to a
lesser extent through L- and P/Q-type channels (El Manira and
Bussières 1997
). At the soma, N- and P/Q-, but not
L-type, calcium channels are coupled to activation of calcium-dependent
potassium (KCa) channels underlying the late
afterhyperpolarization (AHP) following the action potential (Wikström and El Manira 1998
), which acts as a
burst terminating factor during locomotor activity (El Manira et
al. 1994
). N- and P/Q-type calcium channels also mediate
synaptic transmission from descending reticulospinal axons
(Krieger et al. 1999
). These studies have thus shown
that the firing properties of neurons and reticulospinal transmission
are primarily controlled by calcium influx through N-type channels. It
is not known, however, whether these calcium channels also play a role
in controlling synaptic transmission from excitatory and inhibitory
network interneurons, or how the changes observed on the cellular and
synaptic mechanisms after blockade of the different calcium subtypes
affect the activity of the spinal locomotor network. In the present
study, we have investigated the role of HVA calcium channel subtypes in
the spinal network generating the swimming motor pattern. We show that
synaptic transmission from excitatory and inhibitory interneurons is
mediated primarily by N-type calcium channels, and that blockade of N- and L-type channels affects the locomotor activity.
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METHODS |
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Preparation, electrophysiology, data storage, and evaluation
Experiments were performed on 20- to 40-cm-long adult sea lampreys (Petromyzon marinus, n = 25) and adult river lampreys (Lampetra fluvialitis, n = 30) in the anadromous stage. We did not detect any difference between the two species with respect to the effect of calcium channel blockers on the locomotor rhythm, and the results of these experiments were therefore pooled. Animals were anesthetized by immersion in a solution containing tricaine methane sulfonate (MS222, 100 mg/l). A 6- to 15-segment-long piece of the spinal cord was dissected from the rostral half of the animal together with the notochord and mounted in a silicone elastomer (Sylgard)-lined experimental chamber continuously perfused with a cooled (8-10°C) physiological solution with the following composition (in mM): 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, 0.5 glutamine, and 2 HEPES. In some experiments the spinal cord was isolated from the notocord. The meninx primativa was removed from the cord using very fine forceps to allow for intracellular recordings.
Fictive locomotion was induced by adding NMDA (100-150 µM) to the
physiological saline, and ventral root activity was recorded with
suction electrodes. After reaching a steady-state frequency, the
locomotor rhythm induced by NMDA remained stable for more than 24 h (see Parker et al. 1998). The effect of calcium
channel agonists and antagonists on the frequency and stability of the locomotor activity was studied by bath applying the different drugs on
the entire spinal cord (Figs. 3-8). In all the experiments of this
study, the different calcium channel blockers were applied at least 30 min after locomotor activity had reached a steady-state frequency.
The effect of calcium channel blockers on the amplitude of locomotor
drive oscillations was analyzed using a split-bath configuration (Dale 1986; Matsushima and Grillner
1992
). In these experiments, the recording chamber was
partitioned into two pools by a petroleum jelly (Vaseline) barrier.
Fictive swimming was induced in the rostral pool by NMDA (100-150
µM), while the caudal pool was perfused with the Ringer. Ventral root
recordings were made in the rostral pool while intracellular recordings
were made from gray matter neurons in the caudal pool. To study
excitatory synaptic transmission, inhibitory glycinergic synaptic
transmission between spinal neurons was blocked by adding 5 µM
strychnine to the caudal pool. The amplitude of the locomotor-driven
synaptic inputs was measured under control conditions and in the
presence of calcium channel blockers. Individual compound excitatory
postsynaptic potentials (EPSPs) were detected and analyzed in highly
amplified sections of the recording in analysis software (DATA-PAC, Run
Technologies). Only EPSPs that appeared as single events were evaluated
over six cycles. The effect of calcium channel blockers on reciprocal glycinergic inhibition was analyzed in the absence of fictive locomotion. Compound inhibitory postsynaptic potentials (IPSPs) were
elicited in contralateral motoneurons and unidentified gray matter
neurons by extracellular stimulating of crossed-caudal projecting
interneurons on the contralateral side of the spinal cord in the
presence of ionotropic glutamate receptor antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and
2-amino-5-phosphonopentanoic acid (AP5); Fig. 2]. A spinal cord lesion
was made on the ipsilateral side caudal to the stimulation electrode,
leaving only the contralaterally projecting axons intact. In this
configuration both small (Ohta et al. 1991
) and
medium-size (Buchanan 1982
) crossed caudally projecting
interneurons were stimulated. The IPSPs occurred at a constant latency
and followed high-frequency (10 Hz) stimulation, suggesting that they
were monosynaptic. Axons were stimulated at a frequency of 0.2 Hz to
obtain IPSPs with stable amplitude. The peak amplitude of IPSPs was
measured under control conditions and in the presence of the different antagonists.
Intracellular recordings from motoneurons (MNs) and interneurons (INs)
were performed using thin-walled glass microelectrodes filled with 3 M
KAc with input resistances of 30-50 M. Intra- and extracellular
signals were amplified and digitized using an A/D interface (Digidata
1200; Axon Instruments) and stored on a PC computer for subsequent
analysis with appropriate software (Axotape, Axoscope; Axon
Instruments). Motoneurons were identified physiologically by their
antidromic activation on stimulation of ventral roots. The membrane
potential of the recorded neurons was not significantly affected by
application of calcium channel blockers (see also Krieger et al.
1999
). During locomotor activity, the duration of a burst was
defined as the interval during which a continuous discharge composed of
several motor units occurred in a ventral root while the contralateral
ventral root was silent. The cycle period of the locomotor rhythm was
measured between the onsets of subsequent ventral root bursts. The
instantaneous frequency was calculated as the inverse of the cycle
period. The coefficient of variation was defined as the standard
deviation of cycle period/mean cycle period. The burst proportion was
defined as the ratio of burst duration and cycle duration.
"N" values in the text represent the number of
experiments or neurons; "n" values represent the sample
size averaged for a given experiment. Mean values of locomotor activity
(cycle period, frequency, burst duration) were calculated from
sequences of 120 s and averaged over 180-350 cycles. The results
are expressed as means ± SD. Means were compared using the paired
Student's t-test from standard statistical software
programs (Plotit, Graphpad). Mean values were considered to be
significantly different with P < 0.05.
Pharmacology
To determine whether individual HVA calcium channel subtypes
play a role in rhythm generation of the spinal locomotor network, we
bath applied the following HVA calcium channel antagonists: 1-3 µM
-conotoxin-GVIA (
-CgTx; Peptide Institute), 0.2 µM
-agatoxin-IVA (
-Aga; Peptide Institute), and 10 µM nimodipine
(RBI). The effect of the L-type channel agonist BayK8644 (2-4 µM,
BayK, RBI) was also tested on the frequency of the locomotor rhythm.
Stock solutions of nimodipine (10 mM) and BayK (10 mM) were prepared in
ethanol. Control experiments revealed that the ethanol amount used to
dilute nimodipine and BayK did not affect cellular, synaptic properties or electrical coupling between neurons (Krieger et al.
1999
; Wikström and El Manira 1998
;
Wikström et al. 1998
). Stock solutions of
-CgTx
(100 µM) and
-Aga (100 µM) were prepared in distilled water and
stored at
20°C. To prevent unspecific binding of the toxins to the
tubes of the perfusion system, 0.5 mg/ml bovine-albumin (Sigma) was
added to the physiological solution.
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RESULTS |
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N-type HVA Ca2+ channels mediate excitatory and inhibitory synaptic transmission in the spinal cord
To test whether calcium influx through N-type channels is involved
in mediating synaptic transmission from propriospinal interneurons, we
performed split-bath experiments (see METHODS). Fictive
locomotion was initiated in the rostral pool by NMDA, while
locomotor-driven excitatory and inhibitory synaptic inputs were
recorded in neurons in the caudal pool in the absence of strychnine
(Fig. 1A). Application of
-CgTx (Fig. 1A; N = 5; 2 MNs, 3 gray
matter neurons) reduced the amplitude of locomotor-driven membrane
potential oscillations, suggesting that N-type calcium channels mediate
synaptic transmission from locomotor network interneurons. The
contribution of N-type channels to excitatory interneurons transmission
was further analyzed after blockade of inhibitory glycinergic
transmission in the caudal pool with strychnine (5 µM), and the
locomotor-driven synaptic inputs are thus arising from excitatory
propriospinal interneurons (Fig. 1, B and C)
(Dale 1986
). Application of
-CgTx (1-2 µM) to the
caudal pool markedly reduced the amplitude of the locomotor-driven excitatory inputs on average by 40.0 ± 9.5% (mean ± SD,
N = 3; 1 MN, 2 gray matter neurons; P < 0.01; 25 cycles evaluated each). The effect of
-CgTx on
individual EPSPs was also analyzed and showed similar properties. For
the sample recording given in Fig. 1, B and C,
the amplitude of the EPSPs evaluated over six cycles ranged between 0.4 and 1.5 mV in control and 0.15 and 0.7 mV in the presence of
-CgTx.
A blockade of N-type channels thus induced a shift of the distribution
histogram toward lower amplitude values, indicating that the amplitude
of EPSPs was reduced.
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The participation of N-type calcium channels to inhibitory synaptic
transmission was also examined. Crossed caudally projecting inhibitory
interneurons (Buchanan 1982; Ohta et al.
1991
) were stimulated extracellularly (see METHODS)
(Alford and Grillner 1991
), while IPSPs were recorded
caudally in the contralateral neurons in the presence of AP5 (100 µM)
and CNQX (10 µM). Within 60 min after the start of the perfusion with
-CgTx (1 µM), the amplitude of the IPSPs decreased by 63.0 ± 7.0% (N = 5; 3 MNs and 2 unidentified;
P < 0.001; Fig. 2,
A and B). These results show that synaptic
transmission from inhibitory propriospinal interneurons is dependent to
a large extent on calcium influx through N-type channels.
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L- and P/Q-type channels do not participate in mediating synaptic transmission from propriospinal neurons
To test whether calcium influx through L- and P/Q-type channels
mediates excitatory synaptic transmission from propriospinal interneurons, locomotor-driven excitatory synaptic inputs were recorded
in neurons in the caudal pool in the presence of strychnine. Blockade
of L- or P/Q-type channels by nimodipine (10 µM; Fig. 3A; N = 3; 1 MN, 2 gray matter neurons) or -Aga (0.2 µM; Fig. 3B;
N = 3; 2 MNs, 1 gray matter neuron) did not affect the
amplitude of locomotor-driven excitation. The effect of blockade of
these channels was also tested on inhibitory synaptic transmission. IPSPs were elicited in neurons by extracellular stimulation of crossed
caudally projecting inhibitory interneurons (Buchanan 1982
; Ohta et al. 1991
) in the presence of AP5
(100 µM) and CNQX (10 µM). Neither nimodipine nor
-Aga affected
the amplitude of the IPSPs, which were blocked by cadmium (200 µM;
N = 5; 3 MNs and 2 gray matter neurons; Fig.
3C). These results indicate that L- and P/Q-type calcium
channels do not mediate synaptic transmission from excitatory and
inhibitory propriospinal interneurons.
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Blockade of N-type channels affects membrane potential oscillations and locomotor rhythm frequency
The influence of -CgTx on membrane potential oscillations
during fictive locomotion was analyzed in motoneurons
(N = 4) and unidentified gray matter neurons
(N = 2). Figure
4A shows a recording from a
motoneuron that displayed membrane oscillations and fired action
potentials in phase with the ipsilateral ventral root burst. A
hyperpolarization of the motoneuron to
85 mV abolished the action
potentials while a phasic synaptic drive was maintained (Fig.
4A). Application of
-CgTx induced a progressive
deterioration of the locomotor activity (Fig. 4, B and
C), although the motoneuron was still able to produce
membrane potential oscillations and fire action potentials. When the
membrane potential of the motoneuron was hyperpolarized, the rhythmic
oscillations were abolished and no synaptic input was observed (Fig. 4,
B and C). These results indicate also that
membrane potential oscillations persist in the absence of synaptic
drive. In some cases, a blockade of N-type calcium channels affected
both the amplitude of the oscillations and the locomotor rhythm. Figure
4D shows recordings from a gray matter neuron, which
exhibited membrane potential oscillations and fired action potentials
during the ipsilateral ventral root burst. Application of
-CgTx
dramatically reduced the amplitude of the oscillations and disrupted
the locomotor rhythm (Fig. 4E). These results establish that
N-type calcium channels play an important role in mediating synaptic
transmission in the spinal cord. The fact that the oscillations in some
neurons disappear on hyperpolarization suggests that they may
correspond to NMDA-induced oscillations (Sigvardt et al.
1985
; Wallén and Grillner 1987
), which are
not abolished by
-conotoxin GVIA (see DISCUSSION).
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In all experiments (N = 16) bath application of the
antagonist -CgTx (1-2 µM) reliably disrupted the pattern of the
locomotor rhythm. The effect was gradual and preceded by a decrease in
the burst frequency (Fig. 5, A
and B). Application of
-CgTx initially decreased the
frequency of the locomotor rhythm and always affected the stability of
the motor pattern which was subsequently disrupted (Figs. 5,
A-D, and 6). The
onset of the disruption of the locomotor rhythm was variable between
preparations and ranged between 15 min and >45 min. In four
experiments displaying a similar time course,
-CgTx significantly
(P < 0.001) decreased the burst frequency (Fig.
5B), while the burst duration (Fig. 5C) and the
coefficient of variation (Fig. 5D) were significantly
(P < 0.001) increased. The change in the locomotor
burst frequency was evaluated in experiments (N = 14)
in which the rhythm was not disrupted after 45 min of perfusion of
-CgTx. The burst frequency significantly decreased to 79.9 ± 11.2% of control (N = 14; P < 0.001;
Fig. 5E).
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The distribution histograms (Fig. 6) show that the cycle duration
increased in the presence of -CgTx (compare control, 20, 30, and 45 min; n = 172 in each histogram) and became more
variable with increasing deterioration of the swimming rhythm (Fig.
6A). After prolonged application of
-CgTx (50-60 min)
the locomotor pattern was disrupted as the left and right ventral roots
did not exhibit any alternation and the bursts could no longer be defined (Fig. 6B). The effect of
-CgTx developed and
became established over time. In some experiments the first changes
were detectable 10 min after the start of the perfusion, whereas in
other experiments changes occurred later, e.g., more than 20 min
following the start of application of
-CgTx (see Fig.
5D). Wash out was tried several times and was successful
only in two cases (2 h wash out), when it was started before the
rhythmic activity was completely disrupted (not shown).
Effects of the L-type HVA-Ca2+ channel antagonist nimodipine and the L-type HVA-Ca2+ channel agonist BayK on NMDA-induced fictive swimming
Bath application of the L-type antagonist nimodipine at a concentration of 10 µM induced a modest but significant decrease of the frequency of the swimming rhythm in 8 of 11 experiments (Fig. 7, A and B). In the other three experiments there was either no change (N = 2) or a small increase (5.4%, N = 1) in the burst frequency. Overall there was a significant decrease in frequency of swimming rhythm to 92.8 ± 8.2% of control (N = 11; P < 0.02, Fig. 7B). The reduction in burst frequency occurred within 30-45 min of application of nimodipine and ranged from 3 to 15% (mean, 13.0 ± 5.0%; N = 8; P < 0.001; Fig. 7). This was obvious from plotting the change in the average normalized burst frequency (Fig. 7B). After 15-30 min of wash out, the frequency of swimming returned to control values (Fig. 7, A and B). The decrease in the swimming frequency was accompanied by a significant increase in the average ventral root burst duration by 4-26% for the individual recordings after 45 min of application of nimodipine. The mean burst duration increased to 111.2 ± 9.5%; N = 10; P < 0.01; Fig. 7C). No significant change was seen in the burst proportion, in the activity phases (left-right alternation) of the segmental roots in the cycle, or in the coefficient of variation (Fig. 7D), which represents a measure of the quality of swimming.
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The effect of the L-type agonist Bay K (2-4 µM) was also tested on the motor pattern during NMDA-induced swimming (Fig. 8). While there was no systematic change on the swimming locomotor pattern in the first 45 min after starting perfusion (Fig. 8, A-C), a significant increase in the frequency was seen after wash out of the agonist in five of six preparations (Fig. 8, A and B). In one experiment, there was no significant change in the frequency. For a given experiment, the increase in the locomotor frequency ranged from 2 to 19% (mean, 109.0 ± 7.2% of control; N = 6; P < 0.05). The increase in burst frequency was accompanied by a significant decrease in the burst duration (Fig. 8C). No significant change was seen on the coefficient of variation (Fig. 8D).
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Effects of blocking P/Q-type HVA Ca2+ channels on NMDA-induced fictive swimming
The role of P/Q-type HVA Ca2+ channels in
generation of the swimming motor pattern was investigated using the
specific antagonist -agatoxin (0.2 µM; N = 7).
Blockade of P/Q-type Ca2+ channels had only minor
effects on the frequency of the locomotor rhythm (Fig.
9, A and B). In
four experiments we observed a small but significant decrease in
frequency, while there was a slight, but significant increase in one
and no change in the other two experiments (Fig. 9B).
Overall there was no significance change in the frequency of swimming
in the presence of
-agatoxin (97.0 ± 6.5% compared with
control, P > 0.2, N = 7; Fig.
9B). No systematic changes were detected for burst duration
(not shown). The regularity of the activity was not affected by
blocking P/Q-type channels as shown by the lack of a significant effect
on the coefficient of variation (N = 5; Fig.
9C).
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DISCUSSION |
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The present investigation set out to elucidate the role that
individual HVA calcium channel subtypes play in the neuronal network
generating locomotor activity in the lamprey by using selective
agonists and antagonists. The main findings are the following:
1) synaptic transmission from excitatory and inhibitory neurons of the spinal locomotor network is mediated by calcium influx
through N-type calcium channels; and 2) the overall
functioning of the lamprey spinal locomotor network relies on calcium
influx through N- and L-type calcium channels. In contrast, excitatory and inhibitory synaptic transmission in the spinal cord are not affected by blockade of L- and P/Q-type calcium channels. In addition, the overall network activity shows a substantial robustness against the
relatively small changes in the firing properties
(Wikström and El Manira 1998;
Wikström et al. 1998
) of spinal neurons induced by
blockade of P/Q-type calcium channels.
N-type calcium channels play an important role in the generation of the locomotor rhythm
Our results show that synaptic transmission from excitatory and
inhibitory propriospinal neurons is mediated primarily by calcium
influx through N-type calcium channels. Similar results have been shown
with regard to synaptic transmission from reticulospinal axons
(Krieger et al. 1999). Blockade of N-type calcium
channels reduced the amplitude of the segmental rhythmic excitatory
synaptic input to motoneurons that has been suggested to correspond to activity of excitatory interneurons (Buchanan et al.
1989
). Crossed inhibitory synaptic input, presumably arising
from small and medium size caudally projecting (CC) interneurons, was
also decreased by blockade of N-type calcium channels. These channels
thus mediate synaptic transmission both from excitatory and inhibitory
propriospinal neurons. The excitatory propriospinal neurons could
correspond to excitatory network interneurons (EINs) (Buchanan
and Grillner 1987
). Dale (1986)
showed that
locomotor drive oscillations are arising from excitatory
propriospinal interneurons with a short rostral projection (1-3
segments) and a long caudal projection (1-9 segments). The
subsequently identified excitatory network interneurons
(Buchanan and Grillner 1987
; Buchanan et al.
1989
) have been shown to project caudally up to five segments
and have suggested to give rise to the locomotor drive. Although our
data suggest that synaptic transmission from network interneurons
relies on calcium entry through N-type channels, a direct demonstration of this requires paired intracellular recordings from identified interneurons and their targets. During fictive locomotion, a blockade of these channels induced an increase in the coefficient of variation and disrupted the motor pattern. The initial decrease of the locomotor frequency observed could therefore be due to a reduced synaptic drive
resulting from the depression of synaptic transmission.
One major factor controlling the burst termination in the lamprey
spinal cord is the late AHP following action potentials that is
mediated through activation of apamin-sensitive
KCa channels (El Manira et al.
1994; Grillner et al. 1998
). The
KCa channels underlying the late AHP are
primarily activated by calcium influx through N-type channels
(Wikström and El Manira 1998
). A blockade of these
channels decreases both the amplitude of the late AHP and spike
frequency adaptation, leading to a prolonged locomotor burst and
thereby a decrease in the frequency of the locomotor rhythm. It is
important to note that specific blockade of KCa by apamin also reduces the locomotor frequency and increases the coefficient of variation of the motor pattern (El Manira et al. 1994
). There is a strong resemblance of the initial effects of inhibition of N-type calcium channels and those of inhibition of
KCa channels on the motor pattern. This suggests
that the decrease of frequency and the increase in coefficient of
variation induced by
-CgTx result, at least partly, from the
decrease of the late AHP. The disruption of the rhythm and especially
the loss of alternation between left and right ventral root bursts is
presumably due to the depression of reciprocal inhibitory synaptic
transmission. Our results thus show that N-type calcium channels are
essential for the function of the spinal network generating locomotion. In the Xenopus embryo spinal cord, inhibition of N-type
channels also disrupts the locomotor pattern by depressing synaptic
transmission and increasing the threshold for firing action potentials
(Wall and Dale 1994
). Although the effect on synaptic
and locomotor activity are similar in the lamprey and
Xenopus, a blockade of N-type channels in the lamprey spinal
cord neurons does not increase the firing threshold but rather
increases the firing frequency of spinal network neurons
(Wikström and El Manira 1998
).
Effects of blocking P/Q- and L-calcium channel subtypes on rhythm generation in the spinal locomotor network
P/Q-type calcium channels contribute to the late AHP by about 20%
(Wikström and El Manira 1998) but do not mediate
excitatory and inhibitory transmission from propriospinal neurons. A
blockade of P/Q-type channels, however, reduces synaptic transmission
from reticulospinal by about 20-30% (Krieger et al.
1999
). P/Q-type channels do not seem to be activated during
NMDA-induced membrane potential oscillations because
-Aga has no
effect on these oscillations (Wikström et al.
1998
). Blocking P/Q-type channels during fictive locomotion did
not result in a consistent significant change in the swimming motor
output. For individual preparations subtle or no changes were observed.
It is possible that the degree to which cellular properties are
affected by P/Q-type calcium channels is below the level that is needed
to affect the network performance. This could be due to the fact that
during each locomotor cycle, neurons fire few spikes and that a small
decrease in the late AHP is not large enough to change the spike
frequency adaptation and thereby the burst termination during fictive
locomotion. Finally, it is conceivable that the effects of blocking
P/Q-type channels only becomes apparent when neurons fire several
action potentials, which can occur during low swimming frequencies.
Blocking L-type channels in NMDA-induced fictive locomotion induced a
significant decrease in burst frequency associated with a concurrent
increase in burst duration in the ventral roots. A potentiation of
L-type calcium channels by BayK resulted in a slight increase in
swimming frequency. Interestingly, after wash out of BayK the burst
frequency increased further. This could be due to an indirect effect of
increasing calcium influx through L-type channels. The effects of
inhibition or potentiation of L-type calcium channels on the locomotor
rhythm does not result from a decrease of synaptic transmission or
spike frequency adaptation because calcium influx through these
channels are involved in neither the activation of
KCa channels (Wikström and El Manira 1998) nor in mediating synaptic transmission from propriospinal neurons and reticulospinal axons in the lamprey spinal cord
(Krieger et al. 1999
). These channels are, on the other
hand, involved in NMDA-induced membrane potential oscillations
(Wikström et al. 1998
). Inhibition of L-type
calcium channels increases the duration of the plateau of NMDA-induced
oscillations, and in some cases a suppression of the oscillations was
observed. Agonists of these channels had the converse effect with a
decrease in the plateau duration. The effects of L-type channel
inhibition and potentiation on the frequency of the locomotor rhythm
could therefore be mediated via modulation of NMDA-induced
oscillations. Other mechanisms may also contribute to the observed
changes in the locomotor rhythm frequency induced by nimodipine. An
involvement of L-type channels in plateau potentials has also been
shown in turtle spinal cord motoneurons (Guertin and Hounsgaard
1998
, 1999
). Membrane oscillations induced by
both muscarinic and NMDA receptors were abolished when calcium influx
through L-type channels was blocked (Guertin and Hounsgaard
1998
, 1999
).
In conclusion, we have addressed the importance of the different
calcium channel subtypes in the generation of locomotor pattern in the
lamprey spinal cord. L-type channels that are mainly involved in
NMDA-induced membrane potential oscillations are involved in controlling the frequency but not the pattern of the locomotor rhythm.
N-type channels, which represent the major component of the total
somatic calcium current (El Manira and Bussières
1997), control both the firing properties of neurons and
excitatory and inhibitory synaptic transmission, and are thus important
in the operation of the locomotor network. These channels may thus be the target of several modulatory systems acting both presynaptically to
affect synaptic transmission and postsynaptically to modify the firing
properties of neurons, and thus modulate the frequency and stability of
the locomotor rhythm.
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ACKNOWLEDGMENTS |
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We thank D. Parker and P. Wallén for comments on the manuscript. We are also grateful to H. Axelgren and M. Bredmyr for excellent technical assistance.
This work was supported by Swedish Medical Research Council Grants 11562 to A. El Manira and 3026 to S. Grillner and a Heisenberg Fellowship of the German Science Foundation to A. Büschges (Bu857/4-1).
Present address of A. Büschges: Zoologisches Institut der Universität Köln, Weyertal 119, 50931 Köln, Germany.
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FOOTNOTES |
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Address for reprint requests: A. El Manira (E-mail: Abdel.ElManira{at}neuro.ki.se).
Received 10 June 2000; accepted in final form 17 August 2000.
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REFERENCES |
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