Zoologisches Institut, Universität zu Köln, 50923 Cologne, Germany
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ABSTRACT |
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Schmidt, Joachim, Hanno Fischer, and Ansgar Büschges. Pattern Generation for Walking and Searching Movements of a Stick Insect Leg. II. Control of Motoneuronal Activity. J. Neurophysiol. 85: 354-361, 2001. In the stick insect, Cuniculina impigra, intracellular recordings from mesothoracic motoneurons that control flexion and extension of the tibia and depression and levation of the trochantero-femur were made while the leg performed walking-like movements on a treadband or stereotyped rhythmic searching movements. We were interested in how synaptic input and intrinsic properties contribute to form the activity pattern of motoneurons during rhythmic leg movements without sensory feedback from other legs. During searching and walking, motoneurons expressed a rhythmic bursting pattern that was formed by a depolarizing input followed by a hyperpolarizing input in the inter-burst interval. This basic pattern was similar in all fast, semi-fast, and slow motoneurons that were recorded. Hyperpolarizations were in synchrony with activity in the antagonistic motoneurons. De- and hyperpolarizations were associated with a decrease in input resistance. All motoneurons showed spike frequency adaptation when depolarized by current injection to a membrane potential similar to that observed during walking. In the hyperpolarizing phase of fast flexor motoneurons, the initial maximum hyperpolarization was followed by a sag in potential toward more depolarized values. Consistent with this observation, only fast flexor motoneurons developed a depolarizing sag potential when hyperpolarized by injection of constant negative current.
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INTRODUCTION |
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In a walking animal, the
coordinated activity of muscles that control movements of different leg
joints depends on the precise timing of the activity of motoneurons
that innervate these muscles (recent reviews: Orlovsky et al.
1999; Pearson 1993
). The timing and magnitude of
motor activity during walking is known to result from interaction of
sensory signals and the output of central rhythm generating networks
(recent reviews: Büschges and El Manira 1998
;
Clarac 1991
; Duysens et al. 2000
;
Pearson 1995
). The complete structure of central walking
rhythm generating networks is not known in any animal (Orlovsky
et al. 1999
), although in insects some interneurons have been
identified that are part of premotor networks involved in postural
control and locomotion (see Bässler and Büschges
1998
; Burrows 1996
). However, not only is the
information about central premotor elements fragmentary, but there is
little information about the membrane potential changes in motoneurons during walking, which would show the pattern of synaptic inputs (cockroach: Pearson and Fourtner 1975
; Tryba and
Ritzmann 2000
; stick insect: Godden and Graham
1984
: locust: Wolf 1990
, 1992
). In addition,
there is only sparse data on the intrinsic properties of insect
motoneurons that contribute to forming their activity pattern
(David and Pitman 1996
; Hancox and Pitman 1991
,
1993
; Mills and Pitman 1999
; Ramirez and
Pearson 1991
).
The membrane potential of leg motoneurons has been studied in
preparations of the cat, the rat, and the locust in which walking-like motor activity has been induced by electrical stimulation of higher locomotor regions (Shefchyk and Jordan 1985) or by
application of drugs (Cazalets et al. 1996
;
Ryckebusch and Laurent 1993
). Under these conditions,
rhythmic activity of motoneurons results from alternating excitatory
and inhibitory synaptic input. These data are consistent with the
observation in the cockroach that phasic inhibition and phasic
depolarization contribute to activity of a coxal motoneuron and an
extensor tibiae motoneuron during walking (Tryba and Ritzman
2000
) Application of the cholinergic agonist pilocarpine onto
stick insect ganglia activates central neuronal networks that generate
alternating rhythmic activity in antagonistic leg motoneurons
(Büschges et al. 1995
). However, under these
conditions, rhythmic activity in mesothoracic flexor and extensor
motoneurons appears to be based on tonic excitation and cyclic
hyperpolarizing synaptic input (Büschges 1998
). It is the objective here to study the intracellular activity pattern in,
and the role of synaptic inputs to, stick insect motoneurons in a
semi-intact walking preparation that is suitable for long-term intracellular recordings (see companion paper, Fischer et al. 2001
).
Along with synaptic inputs, the activity pattern of motoneurons is also
sculpted by their intrinsic membrane properties. Spike frequency
adaptation (SFA) and depolarizing sag potentials and plateau potentials
affect the activity pattern of motoneurons in different systems (SFA:
lamprey: El Manira et al. 1994; phrenic motoneurons of
cat: Jodkowski et al. 1988
; hypoglossal motoneurons of
rat: Sawczuk et al. 1995
; depolarizing sag potentials:
stomatogatric nervous system: Kiehn and Harris-Warrick
1992
; hypoglossal and facial motoneurons of rat: Bayliss
et al. 1994
; Magariños-Ascone et al. 1999
;
plateau potentials: for review, see Hultborn 1999
; Kiehn 1991
). Plateau potentials in insect motoneurons
have been shown in cockroach leg motoneurons (Hancox and Pitman
1991
, 1993
) and locust flight motoneurons (Ramirez and
Pearson 1991
), but it is not known whether insect leg
motoneurons exhibit SFA and depolarizing sag potentials. We have looked
for both properties in stick insect motoneurons because they might
affect the motoneuronal activity pattern during walking. SFA would
decrease spike frequency during depolarizations and depolarizing sag
potentials could counteract membrane hyperpolarizations.
In the companion paper (Fischer et al. 2001), we have
introduced a preparation in which after amputation of all legs but one middle leg, the remaining leg performs walking movements on a treadband
or stereotyped rhythmic searching movements when ground contact is
absent. We made intracellular recordings from mesothoracic motoneurons
of the coxa-trochanteral joint and the femur-tibial joint and
investigated the overall synaptic drive underlying patterning of
motoneuronal activity in the locomotor cycle. We found that in all
motoneurons recorded rhythmic activity during walking and searching
movements was due to alternating inhibitory and excitatory synaptic
inputs. When studying cellular properties we found that slow,
semi-fast, and fast motoneurons exhibited marked spike frequency adaptation. In addition, fast flexor tibiae motoneurons were found to
develop a depolarizing sag potential when hyperpolarized by injection
of constant current.
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METHODS |
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All experiments were carried out on adult female stick insects, Cuniculina impigra Redthenbacher (syn. Baculum impigrum Brunner) that were raised at the Zoological Institut, University of Cologne. The experiments were performed at room temperature of 20-22°C under dimmed light conditions.
The experimental setup and the methods of preparation are described in
detail in the accompanying paper (Fischer et al. 2001). In short, the animals were mounted on a platform using dental wax. All
legs but one middle leg were amputated between trochanter and coxa. The
stumps were glued to the platform to prevent movements and the coxa of
the remaining leg was glued to the body wall to prevent pro- and
retraction movements. After opening the thoracic cavity, the
mesothoracic ganglion was placed on a platform, and all nerve roots on
the side of the leg stump and the nerves nl2 and nl5 were crushed.
The activity of motoneurons was recorded intracellularly from their
neuropilar arborizations in the ipsilateral hemi-ganglion with
microelectrodes (1 mm OD, 0.78 mm, Science Products, Hofheim, Germany)
that had resistances of 15-25 M and were filled with a solution of
3 MKAc/0.05 M KCl. An NPI-10 L amplifier (npi electronic GmbH, Tamm,
Germany) was used in bridge or discontinuous current-clamp mode.
Monopolar hook electrodes were used for extracellular recording from
nerve roots. Electromyographic recordings were obtained by inserting
two 50-µm copper wires into the respective muscles of the leg
segments. Motoneurons were identified either by a one-to-one relationship of their action potentials with muscle potentials in EMG
recordings and/or by induction of leg movements on injection of
depolarizing current. Each spike in a fast motoneuron evoked a distinct
fast movement. Individual spikes in semi-fast motoneurons evoked
movements that were much smaller and hardly detectable by eye. However,
a spike train in semi-fast motoneurons evoked a smooth movement that
was clearly faster than movements evoked by spike trains in slow
motoneurons. A single spike in a slow motoneuron was never able to
evoke a movement of a joint.
For data processing, see Fischer et al. (2001). For
creating x, y data plots, we used PlotIT for Windows
(Scientific Programming Enterprises, Haslett, MI). PlotIT was used for
smoothing the y values in x, y data using a
smoothing factor of 0.6. Measurements are given as means ± SE. We
used a modified t-test (Dixon and Massey
1969
) to compare data sets and samples were regarded
significantly different for P < 0.05.
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RESULTS |
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Activity pattern of motoneurons during walking and searching movements
Electromyographic recordings (EMG) from leg muscles have shown
that the motoneurons of the coxa-trochanteral joint and the femur-tibial joint generate bursts of action potentials when the middle
leg performs walking-like movements on a treadband or performs searching-like movements. For a discussion of some differences between
these walking- and searching-like movements of the single middle leg
and walking and searching leg movements in intact animals that result
from the constrains of the single leg preparation, see companion paper
by Fischer et al. (2001). For the sake of readability,
we will refer to walking- and searching-like movements further on as
walking and searching.
The intracellular recording of the fast extensor tibiae motoneuron
(FETi) gives an example of the basic features of the typical rhythmic
motoneuronal pattern. During walking, FETi (n = 5)
generated bursts of action potentials (Fig.
1A). Between bursts, FETi
repolarized and the membrane potential settled at 67 mV, 1 mV more
negative than its potential at rest. Each burst in FETi indicated the
swing phase of a walking cycle (Fischer et al. 2001
). A
similar rhythmic activity pattern was generated in FETi during
searching movements of the leg (Fig. 1B). Spike frequency
was generally lower and cycle period was faster during searching than
during walking. During searching, FETi did not hyperpolarize below its
potential at rest between bursts. However, when FETi was held at a more depolarized membrane potential (
40 mV) by current injection during searching, the membrane hyperpolarized below the new resting potential (Fig. 1C), indicating that FETi not only repolarized due to
decreasing depolarizing input but also received inhibitory synaptic
input in the inter-burst interval.
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During walking, extensor motoneurons also appear to receive inhibitory
input. Bursting of SETi activity during walking was formed by
depolarizations and hyperpolarizations around the potential at rest
(n = 5; Fig. 2). It was a
general pattern for all motoneurons that the hyperpolarization was in
synchrony with burst activity in the antagonistic motoneurons. Starting
from a potential at rest of 50 mV, SETi hyperpolarized by about 4 mV
during flexor activity (stance phase) and generated bursts of action
potentials during swing phase. The walking sequence shown started with
a hyperpolarization of SETi because the walking cycle started with a
stance phase.
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Flexor motoneurons expressed the same basic activity pattern during
walking and searching as extensor motoneurons as is shown in Fig.
3. The membrane of a slow flexor
motoneuron (sFlex, Fig. 3A) hyperpolarized some 4 mV below
the resting membrane potential of 50 mV in the interburst interval in
synchrony with activity in extensor motoneurons. The walking sequence
shown started with a hyperpolarization of sFlex because the sequence
started with a swing phase. In this preparation, the rhythm paused for
about a second after each swing-stance cycle. The pause was
characterized by a repolarization of the membrane to resting potential
and an increase in input resistance. A sudden decrease in input
resistance and further hyperpolarization indicated the onset of
inhibitory synaptic input (see
).
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During walking and searching (Fig. 3B), the hyperpolarizing
phase in all motoneurons tested was associated with a decrease in input
resistance. The mean input resistance of 6.5 ± 0.91 M [n = 7, 5 slow and 2 fast motoneurons (MNs)] at rest
was reduced to 3.1 ± 0.61 M
in the inhibitory phase of a
walking or searching cycle. The depolarizing phase during walking and
searching is also associated with a decrease in input resistance (Fig.
3B). During searching, a semi-fast flexor motoneuron was
rhythmically depolarized by 2-3 mV associated with a decrease in input
resistance of about 40%.
Depolarizations and hyperpolarizations around the resting potential
also formed the activity pattern of depressor and levator trochanteris
motoneurons during searching (Fig. 4). A
fast depressor trochanteris motoneuron (fDprTr) generated bursts of
action potentials during the down stroke of the leg and repolarized
back to its resting potential between bursts. When depolarized by
current injection, fDprTr hyperpolarized below the new resting
potential between bursts (Fig. 4A). These hyperpolarizations
were not due to K currents activated by a preceding depolarization
because the sequence started with a hyperpolarization of fDprTr. When the membrane potential was held more negative than the resting potential of 65 mV, the membrane did not reach the new resting potential between bursts indicating a reversal potential of
65 mV for
the inter-burst hyperpolarization (Fig. 4A). The fLevTr motoneuron in Fig. 4B usually did not reach spike threshold
when depolarized and did not hyperpolarize below its resting potential of
66 mV during leg depression. Again, when depolarized by current injection the membrane hyperpolarized below the new resting potential between depolarizations, indicating hyperpolarizing synaptic input during depression of the leg.
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Only 8% of the fast motoneurons (n = 14) that were
recorded while the animal performed searching movements hyperpolarized below the resting potential between bursts. When depolarized by current
injection, however, all neurons hyperpolarized below the new resting
potential. A far greater percentage of slow motoneurons, 42%,
hyperpolarized below rest during searching movements (n = 26). Of those slow motoneurons that were held at a more positive membrane potential by current injection (n = 7), all
but one hyperpolarized below the new resting potential. A similar
picture emerged for semi-fast motoneurons. The mean resting potential
of the neurons that hyperpolarized below rest was 49 ± 2.0 mV
(n = 17), whereas the resting potential of neurons that
did not hyperpolarize below the resting potential during searching was
58 ± 1.2 mV (n = 45) and thus significantly
more negative. These data suggest that a hyperpolarization below rest
was more often observed in slow and semi-fast motoneurons because the
mean resting potential of those was significantly more positive,
51 ± 1.0 mV (n = 47) and
52 ± 1.6 mV
(n = 27), respectively, than that of fast motoneurons, which was
62 ± 1.2 mV (n = 35; see also Table
1).
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Our data suggest that in all fast, semi-fast, and slow extensor,
flexor, depressor trochanteris, and levator trochanteris motoneurons
that were recorded, the activity pattern during searching and walking
appeared to be formed by depolarizing and hyperpolarizing synaptic
input. Rhythmic activity in motoneurons during walking and searching
appears to be based on the same mechanisms; the only difference was
that spike activity during searching was usually less strong and cycle
period was faster than during walking (see also Fischer et al.
2001).
Spike frequency adaptation
The activity pattern of neurons is not only determined by their
synaptic input but also by intrinsic cellular mechanisms, one of which
is spike frequency adaptation (SFA). All motoneurons that were tested
exhibited SFA (Table 1). Figure
5A shows the adaptation of
the instantaneous spike frequency in nine different motoneurons that
assumed an initial frequency of 100 ± 15 Hz on onset of a
depolarizing current pulse (2-5 nA). The spike frequency reached a
steady state about 500 ms after stimulus onset. Differences in SFA
between different types of motoneurons were not apparent, and the
steady-state spiking rate within one type was quite large. For example,
in two different slow flexor tibiae motoneurons that were depolarized
by current injection of 3 nA to a holding potential of 44 and
41
mV, respectively, the initial instantaneous spike frequency was 102 Hz
in both cells and adapted to a steady-state frequency of 47 ± 1.4 and 26 ± 1.2 Hz, respectively (Fig. 5A).
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The magnitude of adaptation depended on the membrane potential, as
shown for a slow flexor motoneuron in Fig. 5, B and
C. A current pulse of 2 nA for 2 s depolarized the
membrane from 56 to
49 mV and initially evoked an instantaneous
spike frequency of 43 Hz that was reduced by 42% when reaching a
steady state of 25 Hz after about 500 ms. When the membrane was
depolarized to a potential of
44 mV, the instantaneous frequency was
reduced by 51% when reaching a steady state, and a reduction by 65%
was observed when the membrane was depolarized to a membrane potential of
39 mV.
During the stance phase of a step cycle (Fig. 5D), the same
neuron depolarized to a membrane potential of 44 mV and generated spikes with a slightly higher maximum instantaneous frequency, which
was 125 Hz. Similar examples could be shown for other motoneurons, indicating that SFA is likely to contribute to determining spike frequency in the excitatory phase during walking activity.
Restorative depolarizing sag
Figure 6 shows rhythmic activity in
a fast flexor motoneuron (fFlex) during walking. fFlex received
hyperpolarizing input during extensor activity (Fig. 6, A
and B). The membrane was initially hyperpolarized by 4-5 mV
at the onset of extensor activity and slowly repolarized by 3-4 mV
until extensor activity stopped (Fig. 6B). A similar decline
of hyperpolarization during swing phase was observed in two other fFlex
motoneurons but not in any other motoneuron. In a variety of systems,
there are neurons that when hyperpolarized by constant current
injection reveal a restorative sag in membrane potential that
depolarizes the cells (Pape 1996). We tested the leg
motoneurons for such a sag potential by injecting negative current
pulses (
1 to
4 nA) of 1-2 s. Only the fast flexor motoneurons
(n = 6) of all the motoneurons that were recorded exhibited a depolarizing sag potential when hyperpolarized (Fig. 7, Table 1). The depolarizing sag in the
inhibitory phase of fast flexor motoneurons during walking is
consistent with this observation.
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DISCUSSION |
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Walking movements of a single middle leg on the treadband
consisted of stance and swing phases in the transversal plane with successive steps sometimes separated by short pauses. The coordination pattern of leg movements in the step cycle was somewhat different from
those observed during straight walking of intact animals (for detailed
discussion, see companion paper, Fischer et al. 2001)
but similar to those used for forward walking movements of front legs
in the intact stick insect (Bässler 1986
), and it
resembled the coordination pattern in the middle leg during curve
walking (Jander 1982
; Rixe and Dean
1995
).
Alternating excitatory and inhibitory synaptic drive sculpts rhythmic locomotor activity of leg motoneurons
During walking and searching movements of a leg, rhythmic activity
patterns in motoneurons innervating muscles of the coxa-trochanteral joint and the femoro-tibial joint control the movements of these joints. We found that the rhythmic bursting pattern in these
motoneurons is based on alternating depolarizing and hyperpolarizing
synaptic inputs to the motoneurons associated with an increase in input resistance. This finding was true for all fast, semi-fast, and slow
motoneurons that were recorded (Table 1). During walking, leg
motoneurons always received hyperpolarizing synaptic input in synchrony
with activity in the antagonistic motoneuron pool. Our findings
substantiate previous data from Godden and Graham (1984), who observed termination of bursts by strong
hyperpolarization in coxal motoneurons recorded in a semi-intact
six-legged preparation of the stick insect that walked on a tread
wheel. The depolarizing input that we observed during walking was not
described probably because Godden and Graham (1984)
could not obtain a stable threshold for spike initiation in their
recordings. Similar to the stick insect, extensor and depressor
activity in the cockroach during walking appears to be patterned by
inhibitory and excitatory synaptic input (Tryba and Ritzmann
2000
). Therefore in both, stick insect and cockroach, the
active and the inactive phase of leg motoneurons during walking is
under control of premotor neurons.
We do not know how the alternation of inhibitory and excitatory input
to motoneurons and switching between antagonists is controlled during
walking. It is quite conceivable that signals from proprioceptors of
the leg play an important role, such as the femoral chordotonal organ
(fCO), which measures parameters of movement of the femoro-tibial joint
or force sensors in the cuticle, like campaniform sensilla in the
trochanter and femur. For example, when the stick insect locomotor
system is active, flexion signals from the fCO inhibit extensor
motoneurons and activate flexor motoneurons (Bässler
1988). At a rather flexed position of the femoro-tibial joint,
the influence of fCO signals on tibial motoneuron activity is reversed
as extensor motoneurons are excited and flexor motoneurons are
inhibited (Bässler 1988
). The switch from
inhibition to excitation in extensor motoneurons and from excitation to
inhibition in flexor motoneurons is very similar to the activity switch
in these motoneurons at the transition from stance to swing. Thus
signals from the fCO may contribute to the control of synaptic input to
flexor and extensor motoneurons during walking.
In a variety of isolated nerve systems of different species, it
is possible to pharmacologically evoke rhythmic activity patterns in
motoneurons that resemble activity patterns during locomotion. Some of
these preparations show motor activity that is also based on
alternating excitatory and inhibitory synaptic input to motoneurons, for example, swimming in lamprey (Russel and Wallén
1983) and tadpole (Soffe and Roberts 1982
) and
fictive walking in rat (Cazalets et al. 1996
) and locust
(Ryckebusch and Laurent 1993
). In the stick insect,
application of the muscarinic agonist pilocarpine evokes alternating
activity of antagonistic motoneurons (Büschges et al.
1995
). In such a preparation, rhythmic activity in extensor and
flexor motoneurons appeared to be based on tonic depolarization and
cyclic hyperpolarizing synaptic input to the motoneurons
(Büschges 1998
). The apparent lack of rhythmic
depolarizing input in this preparation is in contrast to the
motoneuronal activity patterns observed during real walking or
searching movements of the leg.
Membrane properties of motoneurons
The resting membrane potential of fast motoneurons was on average
62 mV and thus about 10 mV more negative than that of semi-fast and
slow motoneurons. In the light of numerous functional and physiological
differences between fast and slow motoneurons (Burrows 1996
), the difference in resting membrane potential is likely to contribute to differences in excitability, for example during sensory input. Elongation of the femoral chordotonal organ excites slow
and fast extensor tibiae motoneurons in nonactive stick insects (Bässler 1983
). Suprathreshold activation of the
fast extensor tibiae motoneuron occurs at high stimulus velocities,
whereas the slow extensor tibiae motoneuron responds over a wider range of stimulus velocities. Similar observations have been made in the
locust (Field and Burrows 1982
). Burrows
(1996)
suggested that sensory neurons coding different
velocities make different connections with members of a motor pool,
e.g., with the fast motor neurons receiving greater inputs from neurons
coding higher velocities. In addition, the more negative resting
membrane potential of fast motoneurons might contribute to the
different responses of slow and fast motoneurons to input from the
femoral chordotonal organ. Depolarizing input from the same source will
most probably evoke more spikes in slow motoneurons than in fast
motoneurons due to their lower spike threshold.
All motoneurons tested exhibited SFA when depolarized by current
injection. Differences among slow, fast, or semi-fast motoneurons were
not apparent. SFA was found to be effective at membrane potentials and
spike frequencies that occur during walking and is therefore likely to
affect the activity pattern of motoneurons during their phase of
activity in the locomotor cycle. SFA has also been found in a variety
of vertebrate motoneurons (Del Negro et al. 1999; El Manira et al. 1994
; Magariños-Ascone et
al. 1999
; Sawczuk et al. 1995
), although the
functional implication of SFA in stick insect motoneurons is not yet
apparent. As a general function of SFA, the prevention of excessive
discharge is discussed by Sawczuk et al. (1995)
. In the
lamprey locomotor network, a calcium-dependent potassium current that
causes SFA plays a critical role in burst termination (El Manira
et al. 1994
). In any case, SFA is likely to shape the final
output of the motoneurons during burst activity within the locomotor cycle.
During walking, fast flexor motoneurons were hyperpolarized in the swing phase of the leg movement. After the maximum initial hyperpolarization, the membrane potential declined steadily until burst activity started. A similar behavior was never observed in any of the other neurons. Consistent with this observation is that fast flexor motoneurons expressed a depolarizing sag potential when hyperpolarized by intracellular current injection. However, we cannot exclude decreasing hyperpolarizing input to fFlex that contributes to the depolarizing sag. The depolarizing sag potential that develops during hyperpolarization in swing phase would support a fast transition to depolarization in the subsequent stance phase.
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ACKNOWLEDGMENTS |
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The authors thank Drs. U. Bässler and R. A. DiCaprio for helpful comments on the manuscript.
This project was supported by the Deutsche Forschungsgemeinschaft (Bu857/2 and 857/6).
Present address of H. Fischer: School of Biology, Div. of Biomedical Sciences, Bute Medical Bldg., University of St. Andrews, Fife KY16 9TS, Scotland, UK.
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FOOTNOTES |
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Address for reprint requests: J. Schmidt, Zoologisches Institut, Universität zu Köln, Weyertal 119, D-50923 Cologne, Germany (E-mail: Joachim.Schmidt{at}uni-koeln.de).
Received 10 July 2000; accepted in final form 28 September 2000.
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REFERENCES |
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