Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106-7080
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ABSTRACT |
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Tryba, Andrew K. and Roy E. Ritzmann. Multi-Joint Coordination During Walking and Foothold Searching in the Blaberus Cockroach. II. Extensor Motor Neuron Pattern. J. Neurophysiol. 83: 3337-3350, 2000. In a previous study, we combined joint kinematics and electromyograms (EMGs) to examine the change in the phase relationship of two principal leg joints during walking and searching. In this study, we recorded intracellularly from motor neurons in semi-intact behaving animals to examine mechanisms coordinating extension at these leg joints. In particular, we examined the change in the phase of the coxa-trochanter (CTr) and femur-tibia (FT) joint extension during walking and searching. In doing so, we discovered marked similarities in the activity of CTr and FT joint extensor motor neurons at the onset of extension during searching and at the end of stance during walking. The data suggest that the same interneurons may be involved in coordinating the CTr and FT extensor motor neurons during walking and searching. Previous studies in stick insects have suggested that extensor motor neuron activity during the stance phase of walking results from an increase in tonic excitation of the neuron leading to spiking that is periodically interrupted by centrally generated inhibition. However, the CTr and FT extensor motor neuron activity during walking consists of characteristic phasic modulations in motor neuron frequency within each step cycle. The phasic increases and decreases in extensor EMG frequency during stance are associated with kinematic events (i.e., foot set-down and joint cycle transitions) during walking. Sensory feedback associated with these events might be responsible for phasic modulation of the extensor motor neuron frequency. However, our data rule out the possibility that sensory cues resulting from foot set-down are responsible for a decline in CTr extensor activity that is characteristic of the Blaberus step cycle. Our data also suggest that both phasic excitation and inhibition contribute to extensor motor neuron activity during the stance phase of walking.
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INTRODUCTION |
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Many motor tasks require coordination of multiple
motor pools to execute a behavior. For articulated animals, the pattern of activity and phase relationships of motor neurons acting on muscles
at several joints in several legs must be coordinated. Coordination of
motor neuron activity typically results from interactions between
central influences and peripheral sensory feedback (Angel et al.
1996; Bassler 1993
; Brunn 1998
;
Graham and Bassler 1981
; Grillner and Zangger
1979
; Hess and Buschges 1999
; Robertson
et al. 1985
). In insects, interneurons have been described that
receive and integrate central and afferent input and coordinate
activity of motor pools acting at several leg joints (Burrows
1980
, 1981
; Hisada et al. 1984
; Siegler
1985
; Wilson and Phillips 1983
). However, their
contribution to coordination of motor neuron activity has rarely been
described in behaving animals (Kitmann et al. 1995
; Schmitz et al. 1991
). In behaving stick insects, a
parallel and distributed network of interneurons orchestrates
coordination of multiple joints (Kitmann et al. 1995
).
The activity of each of these interneurons either supports or opposes
movements of ongoing active behaviors (walking, searching, rocking) and
postural reflexes (Kitmann et al. 1995
).
While distributed neural networks are adaptive for behavior, they
present several challenges for understanding inter-joint coordination
based on activity of interneurons. First, the output of convergent
interneuron pathways impinges onto a relatively small set of motor
neurons, yet not all of this information is used to shape motor
activity. For example, weak synaptic strengths or presynaptic
inhibition resulting from central or afferent influences may lesson the
contribution of some interneurons to a particular behavior
(Burrows and Matheson 1994; Cattaert et al. 1990
,
1992
; El Manira et al. 1991
; Sauer and
Buschges 1994
; Sillar and Skorupski 1986
;
Wolf and Burrows 1995
). Therefore one could record
activity from interneurons that is consistent with their role in
supporting or opposing an ongoing behavior, yet those interneurons may
in effect not contribute to shaping ongoing motor neuron activity. Second, in addition to input from interneurons, motor neuron activity producing a behavior results from the integration of direct synaptic inputs from afferents and is dependent on the passive and active membrane properties of the motor neuron itself. These influences on
motor neuron activity are separate from those produced by interneurons, are not reflected in recordings made from interneurons, and may have
marked effects on coordination of multiple joints.
These potential problems point to the necessity of analyzing neural
activity associated with a particular behavior while the animal is
performing that behavior. It is technically impractical to use
intracellular techniques to examine the activity from a large group of
interneurons while an insect performs multiple motor tasks.
Nevertheless reasonable progress can be achieved in understanding the
neural basis of multi-joint coordination by recording intracellular
activity from a sample of motor neurons that act at different joints
during ongoing behaviors (Godden and Graham 1984;
Robertson and Stein 1988
; Wolf
1990
). The results of these studies can then be used to
generate testable hypotheses regarding the underlying neural
organization that orchestrates complex behaviors (Robertson and
Stein 1988
). In this study, we recorded from two principal leg
motor neurons while semi-intact tethered cockroaches walked or searched
for a foothold. The activity of these motor neurons results in the
extension of either the coxa-trochanter (CTr) or femur-tibia (FT)
joints. In this paper, we examine synaptic input to these motor neurons
to establish criteria for identifying interneurons that may coordinate
extension of these joints during walking and searching.
Examination of extensor motor neuron activity during both treadmill
(Watson and Ritzmann 1998a) and tethered walking
(Tryba and Ritzmann 2000
) revealed that there are
characteristic intraburst features that include rapid increases and
decreases in frequency at particular points during a step cycle. These
data suggested that both phasic excitation and inhibition coordinate
extensor activity during a rhythmic behavior such as walking. In
contrast, data from fictive locomotion recorded from the deafferented
ganglia of stick insects (Buschges 1998
) and
implications from the flexor burst generator model (Pearson
1976
) suggest that extensor motor neuron activity is primarily
the result of an increase in tonic excitation that is periodically
interrupted by rhythmic inhibition that is centrally generated. The
characteristic phasic modulation of slow depressor coxa neuron (Ds) and
slow extensor tibia neuron (SETi) frequency found in intact walking may
result from both central and sensory influences. Active membrane
properties that are revealed during intact behaviors (see
Ramirez and Pearson 1991
) but not necessarily
observed during fictive locomotion may also contribute to phasic
modulation. To begin to test these hypotheses, we studied whether there
are specific kinematic events such as foot touch down and joint cycle
transitions that may be associated with characteristic changes in motor
neuron firing frequency. We also examined whether we could identify the
principal synaptic inputs that are responsible for the characteristic
phasic modulation of Ds and SETi activity at the end of stance. Because
a similar extensor coordination is observed during searching, we
believe that the same interneurons may coordinate activity of Ds and
SETi at the end of stance and during searching.
When cockroaches lose ground contact, they switch behaviors from
walking to searching (Tryba and Ritzmann 2000). The most prominent differences observed as a result of this switch involved the
joint movements associated with swing and stance phases. During walking, flexion at the CTr and FT joints occurred during swing phase,
whereas when the animal searches, these joints extend during the aerial
phase. In both of these behaviors, leg protraction involves complex
actions of the body-coxa joint that have yet to be examined in detail.
However we did find consistent differences in movements at the more
distal joints of the middle legs when the animal switched from walking
to searching. During walking, the CTr extension precedes extension of
the FT joint, whereas during searching, the onset of CTr extension was
delayed relative to the FT extension. Joint movements that occurred
during searching were coincident with a characteristic
electromyographic (EMG) pattern recorded from CTr extensor (Ds) and FT
extensor (SETi) motor neurons that included high-frequency SETi
activity prior to the onset of Ds activity (Tryba and Ritzmann
2000
). A high-frequency burst in SETi coupled with a decrease
in Ds activity is also seen at the end of stance phase of walking.
However, the kinematics at the FT joint are different in the two
behaviors. We hypothesize that the pattern of high-frequency SETi
activity in conjunction with the cessation of Ds activity either at
extension onset during searching or at the end of stance during walking
involves excitation of SETi and inhibition of Ds. We further propose
that the same interneuron(s) coordinate Ds and SETi activity at those
times. In this study, we recorded intracellularly from Ds and SETi
during tethered walking and searching to test whether we can account for the phase relationship of the CTr and FT joints at the end of the
stance phase and the onset of searching.
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METHODS |
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Animals
Adult male death-head cockroaches (Blaberus discoidalis) were used in all experiments. Cockroaches were raised in our own colony descended from 250 adult animals generously provided by Dr. Larry L. Keeley of Texas A & M University. Cockroaches were housed in 20 l plastic buckets, half filled with aspen shavings, and were held at 27°C in a 12 h light:12 h dark circadian cycle. A commercial dry chicken starter and water were provided ad libitum. Only intact, undamaged cockroaches were used.
Preparation of semi-intact tethered animals for intracellular recording
To gain access to the ganglia of interest, we needed to remove
the dorsal cuticle of the animal, eviscerate the animal, and stabilize
the mesothoracic ganglion. We also needed to stabilize the dorsal
cuticle to keep the remaining cuticle from tearing while the animal
engaged in searching or walking behaviors. Stabilization of the dorsal
cuticle was done by gluing a U-shaped piece of aluminum to the dorsal
cuticle with cyanoacrylate glue. The aluminum was cut into a U-shape
from a soda-pop can pull-tab and glued to the dorsal cuticle while the
animal was under CO2 anesthesia (Fig. 1, A and B). The
top of the U abutted the posterior edge of the animal's pronotum. A
U-shaped insect pin (No. 2) was glued onto the pronotum cuticle and
onto part of the pull-tab. The insect pin was bent so as to form-fit
the shape of the pronotum. The insect pin and pull tab were glued end
to end such that they formed an O shape on the dorsal cuticle to
stabilize it (Fig. 1, A and B). Next, EMG
electrodes were implanted to record from the mesothoracic depressor
trochanteris muscle and extensor tibia muscle as described extensively
by Watson and Ritzmann (1998a) and in the companion paper (Tryba and Ritzmann 2000
). The coxal depressor
muscle is innervated by one slow excitatory motor neuron (Ds), one fast excitatory motor neuron (Df) and three inhibitory motor neurons (Pearson and Iles 1971
). Excitatory innervation
of the extensor tibia muscle includes slow and fast extensor tibia
motor neurons (SETi and FETi).
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The animal was pinned to a cork platform and the dorsal cuticle
circumscribed by the pull tab and pin was removed. Next, the animal was
eviscerated to expose the three thoracic ganglia. The thoracic body
cavity was rinsed and filled with saline solution. Just posterior to
each of the ganglia, above nerve 5 roots, there is some overlying
cuticle that was removed from each of the thoracic ganglia. This
procedure allowed the ganglia to move more independently of the
remaining cuticle. The animal was tethered using two insect pins stuck
through its pronotum on either side of its head as described in the
companion paper (Tryba and Ritzmann 2000) (Fig. 1,
A and B). The tethered animal was then placed on
the glass substrate made slick with microtome oil (Lipshaw
Manufacturing, microtome oil No. 288). The tether was the same as in
our previous study (Tryba and Ritzmann 2000
) with the
exception that the pins were glued to the pronotum where they passed
along the underside of the pronotum and also where they passed through
the pronotum. Gluing the pins to the pronotum provided a more rigid
tethering of the animal so that intracellular recordings could be made. Next the mesothoracic (T2) ganglion was stabilized using a custom built
metal spoon-shaped platform that was form-fitted to the ventral surface
of the T2 ganglion. The ganglion was supported by the platform and
further stabilized by a custom built metal ring that was compressed
onto the dorsal surface of the ganglion to sandwich it between the
ventral platform and dorsal compression ring (Wolf and Pearson
1987
).
Walking was induced by gently tapping on the dorsal abdomen with a
wooden dowel. A walking animal was induced to search for a foothold by
pitching the glass substrate away from the animals' anterior as
described previously (Tryba and Ritzmann 2000). We refer
to this preparation as a semi-intact tethered preparation.
Blaberus saline
The saline solution used for both dissection and intracellular recording consisted of (in mM) 140.0 NaCl, 10.0 KCl, 9.0 CaCl, and 5.0 MgCl in distilled water. The pH was buffered to 7.2 with 3-[N-morpholino]propanesulfonic acid (Sigma) and sodium salt.
Intracellular recordings
Intracellular recordings were made from the Ds, FETi, and SETi
motor neurons. Recordings were made from the neuropile of the second
thoracic ganglia (T2) while the animal engaged in searching or walking
behaviors. We also recorded from interneurons that had processes in the
posterior quadrant of the T2 ganglia, medial to nerve 5 roots.
Intracellular microelectrodes were pulled from single tube capillary
glass (World Precision Instruments). Their tips were filled with a
solution of 4% Lucifer yellow CH (Molecular Probes) in 0.1 M lithium
acetate (Sigma). The remainder of the electrode was back-filled with
1.0 M lithium acetate. Resistances consistently ranged from 60 to 80 M. To facilitate penetration of the sheath surrounding the ganglia
with the recording electrode, we applied a small piece of cotton soaked
with Protease Type XIV (1.0 mg/ml; Sigma) to the ganglia and allowed
the protease access to the sheath for 3 × 5 min. The cotton was
removed and the thoracic cavity was rinsed (4 times) and then filled
with Blaberus saline. The dorsal neurilium was then
penetrated with a recording electrode. The electrode was gradually
advanced into the neuropile, and capacitance ringing of the electrode
tip resulted in motor neuron impalement in the neurite region,
indicated by synaptic activity. Records were stored on a VHS Vetter
recording system (equipped with an A/D converter) and played back for
acquisition and analysis using Axotape recording software (Axon Instruments).
Identification of motor neurons and interneurons
The microelectrodes were filled with Lucifer yellow in the hope of using morphological cues for positive identification of neurons. However, we were unsuccessful at injecting dye into the neurons that we recorded from. Instead motor neuron identification was made by injecting depolarizing and hyperpolarizing current of sufficient magnitude to increase and null the corresponding EMG activity. The action potential activity recorded intracellularly from each motor neuron also matched one for one with recorded muscle potentials (EMGs).
A demonstration of direct connectivity between interneurons and a motor neuron requires simultaneous intracellular recordings from both cells. However, because intracellular recordings in the tethered preparation could only be maintained for short periods, we considered paired intracellular experiments to be unreasonable. We were able to provide indirect evidence that a particular interneuron influences the activity of Ds and/or SETi (directly or indirectly) by injecting hyperpolarizing or depolarizing current into the interneuron and noting changes in motor neuron activity. Of course, the recorded interneurons could also influence the activity of additional motor neurons and/or interneurons that were not monitored. The interneurons were classified as nonspiking interneurons if action potentials were not elicited at any level of depolarization tested despite changes in motor neuron activity.
When recording from motor neurons and interneurons during leg movements, current injections were performed before and after the animals' behavior was examined to ensure the recording electrode remained in the same cell throughout the recording. To control for movement artifacts in the intracellular records taken during behavior, the electrode was removed from the cell, and the animal was again induced to perform walking and searching movements with the electrode just outside the cell. Movement artifact did not appear to contribute to the reported voltage deflections.
Joint kinematics
True joint angles were calculated for the
T2 CTr and FT joints during tethered walking.
Methods used to collect and calculate the joint kinematic data were
described extensively by Watson and Ritzmann (1998a) and
in the companion paper (Tryba and Ritzmann 2000
). To
capture the leg movements, we used a high-speed digital video system
(Redlake HS500) aimed at the lateral projection of the tethered
cockroach. A mirror angled at 45° beneath the glass substrate allowed
us to capture both a ventral and lateral view of the animal (see
Tryba and Ritzmann 2000
). To obtain a very precise
ventral view, we placed the angled mirror as close as possible to the
underside of the glass plate the tethered animal walked on. Placing the
mirror up against the glass plate prevented us from pitching the glass
plate away from the anterior of the animal to induce the animal to
switch to searching (see Tryba and Ritzmann 2000
).
Therefore the combined kinematic and intracellular data reported here
were collected only during walking.
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RESULTS |
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T2 tethered semi-intact walking
RELATIONSHIP OF T2 DS AND SETi ACTIVITY TO JOINT
KINEMATICS.
To establish whether or not the semi-intact tethered preparation yields
biologically relevant walking data, we compared the relationship
between motor neuron activity and joint kinematics for intact tethered,
semi-intact tethered, and freely walking cockroaches. Motor neuron
frequency markedly influences the rate of muscle contraction and in
turn joint angular velocity during horizontal treadmill walking
(Watson and Ritzmann 1998a) and tethered walking
(Tryba and Ritzmann 2000
). Accordingly, during walking there is a linear relationship between mean frequency of slow motor
neuron activity and mean joint angular velocity (Tryba and Ritzmann 2000
; Watson and Ritzmann 1998a
). A
similar relationship should exist between the motor neuron activity and
joint velocity for semi-intact tethered cockroaches during walking if
they walk in a reasonably normal way. To test this, we plotted mean Ds
and SETi frequency and mean CTr and FT joint velocity using EMG data obtained from a single semi-intact preparation during an intracellular recording of Ds (Fig. 2,
A-C). In that case, CTr data shown include 13 CTr joint
extensions, while FT data represent 12 joint extensions (Fig.
2B). We compared the relationship between motor neuron
activity and leg kinematics during: walking on a horizontal treadmill
(Watson and Ritzmann 1998a
), intact tethered walking
(Tryba and Ritzmann 2000
), and semi-intact tethered
walking during an intracellular recording of Ds. During some step
cycles in the semi-intact walking data, fast motor neurons were
recruited (Fig. 2A). Of the 12 SETi bursts examined, six
steps included two FETi potentials, while the remaining six had one
FETi potential per burst. In contrast, only two of the Ds bursts
included fast potentials with each of these bursts having one Df
potential. As with other preparations, the mean CTr or FT joint
extension velocity of the semi-intact preparation was correlated with
the related mean slow (Ds or SETi) motor neuron activity
(r = 0.74 and r = 0.77, respectively; Fig. 2, B and C). A given
mean extensor motor neuron frequency resulted in a lower mean CTr or FT
joint velocity than during treadmill walking. However, it was higher
than the intact tethered walking preparations (Fig. 2, B
and C). We propose possible reasons for these
differences in DISCUSSION.
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INTRACELLULAR RECORDING OF T2 DS DURING TETHERED WALKING.
Having established that the relationship between semi-intact tethered
walking motor neuron activity and joint kinematics is linear, as is the
case for freely walking animals, we then investigated mechanisms
patterning extensor motor neuron activity during walking. We examined
the hypothesis that the pattern of activity for walking found in
extensor motor neurons results primarily from an increase in tonic
depolarization that is periodically interrupted by inhibition (see
Buschges 1998). We marked on the intracellular records
the approximate resting potential of Ds by extending a straight line across the baseline of tonic activity recorded when the animal was not
moving its legs (i.e., prior to walking) to the area of the record that
included walking (Fig. 2A). During bursts of activity associated with walking, the membrane potential of Ds was depolarized above resting conditions. Data collected from Ds recordings represent 58 walking step cycles from four animals.
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INTRACELLULAR RECORDINGS FROM NONSPIKING INTERNEURONS DURING WALKING. Having found that both inhibition and excitation pattern Ds activity, we then searched for evidence of interneurons that could provide this type of synaptic input to Ds during walking. We recorded from two nonspiking interneurons whose activity is consistent with influencing Ds activity during walking. When the animal is not moving its legs, injection of depolarizing current into one of these nonspiking interneurons (NSI-A) results in a decrease in Ds EMG activity, while hyperpolarization increases Ds activity (Fig. 4, A and B).
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RELATIONSHIP OF Ds ACTIVITY TO T2 FOOTFALL AND
CTr JOINT ANGLE.
Phasic modulation of motor-neuron frequency during walking may
result in part from central or sensory influences and in part from
active membrane properties. We examined the relationship among Ds
activity, footfall, and CTr joint angle to determine whether sensory
feedback during specific kinematic events such as foot set-down and
movement through a joint angle is likely to trigger phasic modulation
of motor extensor activity. During walking, Ds motor neuron activity
begins before foot set-down with high-frequency activity within the
first 10% of the burst (Fig. 2C) (see also Watson
and Ritzmann 1998a). Thereafter there is a decline from peak
instantaneous frequency near foot set-down (n = 2 animals, 18 steps) (Watson and Ritzmann 1998a
). Several candidates exist for sensory cues at the time of foot set-down that may
influence this decline in Ds activity. Tactile receptors in the foot or
strain detectors (i.e., campaniform sensilla) in the leg cuticle could
detect foot contact directly. Alternatively, joint angle detectors such
as the chordotonal organs could produce the decline when they detected
a particular joint angle or joint velocity. Still another possibility
is that the decline is centrally patterned.
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Intracellular recording of SETi during tethered walking
As was the case for Ds, the frequency of SETi activity changes in
a characteristic way throughout a burst when the animal walks. Thus we
examined whether or not SETi also receives excitatory synaptic input
that may contribute to modulation of its firing frequency during
walking. At the start of a SETi burst, there is low-frequency activity
that is typically followed by high-frequency activity beginning during
the last 70% of the burst cycle (Fig. 7A) (Tryba and Ritzmann
2000; Watson and Ritzmann 1998a
). As is the case
for Ds when the animal walks, the membrane potential of SETi during a
burst is also depolarized relative to the approximate resting potential
extrapolated from the baseline prior to a bout of walking (Fig.
7A). Quantitative data from the SETi represent intracellular
data recorded for 22 walking cycles from two animals. EPSPs could be
observed to precede some of the SETi action potentials (Fig.
7B).
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Coordination of CTr and FT extensor motor neurons at the end of stance phase of walking
Having examined some of the mechanisms coordinating either Ds or
SETi activity during walking, we then investigated how the activity of
both of these motor neurons might be orchestrated together. Throughout
much of the stance phase of walking, Ds and SETi are simultaneously
active (Figs. 2C and
8A) (Watson and
Ritzmann 1998a). The end of stance phase involves inhibition of
Ds at about the same time high-frequency SETi activity begins (Figs.
2A and 3, B and C). The high-frequency
SETi activity results from rapid depolarization of SETi at the end of
stance (Figs. 2 and 7A).
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There are several neural mechanisms that could account for the nearly simultaneous onset of Ds inhibition and high-frequency SETi activity. One way to achieve this coordinated activity would be to have SETi directly inhibit Ds. This hypothesis can be eliminated, as there did not appear to be a 1 for 1 correlation between SETi spikes and IPSPs in Ds (Fig. 3B).
Alternatively, one or more interneurons could project to both motor neurons where they inhibit Ds and excite SETi. The most direct way to test for this possibility would be to record intracellularly from Ds and SETi simultaneously. However, dual recordings in a tethered preparation during walking would be difficult to achieve. Instead, we evaluated the correlation between the onset of the SETi high-frequency burst and the onset of Ds inhibition. To get a reasonable evaluation of the timing of these events, two individuals visually inspected the SETi records of one animal and independently marked on the records where they perceived there to be a clear increase in SETi frequency. In 2 of 13 bursts examined, there were differences in the perceived onset of high-frequency SETi activity, and these data were not used in the analysis. Next, the time of the last Ds spike was plotted relative to the onset time of SETi high-frequency activity (Fig. 9A). In 3 of the 11 Ds bursts, the last spike was 2 ms before the onset of high-frequency SETi activity. In five cases, the last Ds action potential was within 10 ms after the high-frequency SETi activity began and in the remaining three instances, it was 48-56 ms after the start of the rapid SETi firing (Fig. 9A). Even when the Ds activity continued, the onset of high-frequency SETi was accompanied by a rapid decline in Ds frequency (Fig. 3C). Rapid hyperpolarization of Ds also occurred either concurrently or shortly following the onset of high-frequency SETi activity (Fig. 3, B and C).
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As the high-frequency phase of SETi activity typically extends to the
end of the burst, it is most often seen as a component of a continuous
burst (see Watson and Ritzmann 1998a) (Fig.
9B). However, in two animals, we found three instances where
the high-frequency SETi activity occurred prior to or following the
termination of low-frequency SETi activity (Figs. 7A and 9,
C and D). In one case, the high-frequency
activity occurred as a result of depolarization after the membrane was
repolarized following low-frequency SETi activity (Fig. 9C).
In that case, low-frequency activity in CTr and FT extensor motor
neurons terminated simultaneously (Fig. 9C). When the
high-frequency SETi activity occurred prior to termination of
low-frequency Ds and SETi activity (Fig. 9D), Ds activity
ceased for approximately the same duration as the high-frequency SETi burst, then Ds activity resumed following termination of that event
(Fig. 9D). In another instance where SETi high-frequency activity began following SETi low-frequency activity, as was the case
for Ds, IPSPs contribute to membrane repolarization following low-frequency activity in SETi (Fig. 7B).
It is possible that the cessation of Ds activity and onset of SETi high
frequency is due to sensory feedback from joint angle receptors. If
that is the case, there should be a consistent relationship between the
CTr and/or FT joint angle(s) and onset of the high-frequency SETi
activity. We tested this hypothesis by measuring the CTr and FT joint
angles at the onset of high-frequency SETi activity. The mean CTr and
FT joint angles at that time were 66.3 ± 4.9° and 108.3 ± 3.9° with a range (maximum-minimum values) of 15.8° for CTr and
13.9° for FT (n = 1 animal, 11 cycles). Watson
and Ritzmann (1998a) found that the mean T2 FT and CTr
excursions during treadmill walking were 26.0° and 43.9°,
respectively. Therefore although the standard deviation is relatively
small, the range in FT joint angles measured at onset of high-frequency
SETi activity during semi-intact tethered walking is actually quite
high (60.8% of the total FT excursion during walking). These data
suggest that joint angle detectors do not directly cue the onset of
these events.
Semi-intact tethered T2 leg searching
INTRACELLULAR ANALYSIS OF Ds ONSET DELAY. Having examined how Ds and SETi are coordinated during walking, we then investigated how these motor neurons are coordinated during searching. As with walking, in describing searching, we will focus first on the pattern of Ds activity, followed by the pattern of SETi activity and finally examine how both of these motor neurons may be coordinated during searching.
Combined joint kinematics and EMGs recorded while cockroaches engaged in T2 leg movements established that there was a delay in the onset of Ds activity relative to SETi during searching versus walking (Fig. 8, A and B) (Tryba and Ritzmann 2000
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CONDUCTANCE CHANGE ASSOCIATED WITH INHIBITION OF Ds AT SEARCH
EXTENSION ONSET.
At the onset of each searching extension cycle, Ds is inhibited at the
initiation of the high-frequency SETi burst (Fig. 10B). This
Ds inhibition could result from a decreasing conductance (e.g., to
Na+, or Ca 2+) or an
increase in conductance (e.g., to K+ or
Cl). To test whether Ds inhibition at onset of
searching results from a change in conductance, we injected 0.25 nA
hyperpolarizing square wave current pulses of brief duration (20 ms)
into Ds during searching movements. As a control, we also injected the
same amplitude and duration hyperpolarizing current pulses when the
glass plate was pitched away from the tethered animal but the animal
was not moving its legs. We compared the peak amplitude of the change in membrane potential resulting from current injection during searching
onset and when the animal was not moving its legs.
HIGH-FREQUENCY SETi ACTIVITY AT ONSET OF THE EXTENSION
PHASE OF SEARCHING.
Coupled with Ds inhibition, each extension cycle of searching is also
associated with high-frequency SETi activity often accompanied by FETi
spikes (Tryba and Ritzmann 2000) (Fig.
11A). We recorded intracellularly from SETi while T2 legs engaged in searching. At the
beginning of the extension search cycle, SETi is rapidly depolarized
above rest and exhibits high-frequency activity (13 search cycles,
n = 2 animals) (Fig. 11A).
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DISCUSSION |
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The results of this study indicate that the different phase relationships of CTr and FT joints at the onset of extension during searching and at the end of the stance phase of walking result from direct inhibition of the CTr extensor (Ds) and excitation of FT extensor (SETi) motor neurons. Thus these data provide a clear link between intracellular events in the motor neurons and the execution of behaviors. The observed intracellular events can account for the reversal in delay between CTr and FT extension that occurs when the animal switches between walking and searching (Figs. 2A, 8, A and B, and 10A). They can also account for the relative timing of the termination of CTr and FT extension at the end of the stance phase of walking (Fig. 2A).
The data support the hypothesis that both phasic inhibition and phasic
depolarization contribute to extensor motor neuron activity during
walking. In contrast, Buschges (1998) suggested that in
insects, fictive motor neuron activity is primarily patterned by tonic
excitation coupled with phasic inhibition. We will propose possible
reasons for this discrepancy. Finally, our data showed that a
characteristic feature of Ds activity during the Blaberus step cycle, a decline in frequency from peak firing rate near the time
of foot set-down, is unlikely to result from sensory feedback due to
substrate contact.
Comparison of intact-tethered, semi-intact tethered, and treadmill walking
In the companion paper (Tryba and Ritzmann 2000),
we established the behavioral relevance of the intact tethered walking
preparation by comparing (CTr and FT) joint kinematics and extensor EMG
pattern (Ds and SETi) with those of freely behaving animals. We did not have enough kinematic data collected at the same walking rate from the
semi-intact tethered preparation and treadmill preparation to
quantitatively compare joint kinematics as was done for T2 tethered
versus treadmill data (Tryba and Ritzmann 2000
).
Nonetheless we briefly tested the behavioral relevance of the
semi-intact preparation by examining the occurrence of extensor
motor-neuron potentials (Ds and SETi) within a burst and within a (CTr
or FT) joint cycle (see Tryba and Ritzmann 2000
). The
EMG data shown in Fig. 4, A-C of Tryba and Ritzmann
(2000)
, were data collected from a semi-intact preparation
during an intracellular recording of Ds by methods described in this
paper. These data were consistent with similar data sets collected
during treadmill (Watson and Ritzmann 1998a
) and intact
tethered walking (Tryba and Ritzmann 2000
), suggesting
the semi-intact preparation yields biologically relevant walking data.
The slope of the relationship between mean EMG potentials and joint
velocity was higher for semi-intact than for intact tethered data but
was lower than for treadmill walking (Tryba and Ritzmann 2000). At least two hypotheses can account for the difference in intact tethered and semi-intact tethered walking data. First, in
contrast to intact tethered animals, the semi-intact walking data
included some steps where there were fast muscle potentials (Fig.
2A), and these may have contributed to a higher joint
angular velocity than when only slow motor neurons were active.
However, CTr data shown in Fig. 2B included only two cycles
where Df was active. Even in those cases, it does not appear that there
are data points that include a sudden increase in joint velocity at a
given EMG frequency (Fig. 2B). Therefore it is unlikely that an increase in the slope of the relationship between mean EMG frequency
and joint velocity for semi-intact tethered animals can be completely
explained by the occurrence of fast motor neuron activity during some
of the steps (see also Watson and Ritzmann 1998b
).
There is a second possible reason for the difference between intact and
semi-intact animals. Tethered walking in both cases may involve an
increase in retraction resistance and a reduction in the contribution
of whole body inertia to walking (Tryba and Ritzmann
2000). Compared with intact tethered animals, semi-intact tethered animals were more rigidly tethered and had a lower body mass
due to evisceration. Both of these factors may reduce loading of the
legs and thereby decrease the retraction resistance in the semi-intact
versus intact tethered animals. One would then expect the slope of the
relationship for mean EMG activity and joint velocity for semi-intact
animals to be higher than the intact tethered animals yet lower than
what was observed for treadmill walking, and that is what was found
(Fig. 2, B and C).
Ds and SETi activity during stance phase of walking
During walking, Ds exhibited plateau-like potentials throughout a
burst, whereas SETi did so primarily during high-frequency activity at
the end of the burst (Figs. 2A and 7A). We were
not able to provide critical evidence that these plateau-like
potentials result from active membrane properties rather than summation
of depolarizing synaptic input. However, in a reduced preparation, it
has been demonstrated that Df can produce plateau potentials triggered
as the result of sensory input (Hancox and Pitman 1991, 1993
). Therefore there is reason to suspect that the slow motor neuron plateau-like potentials observed during tethered walking are the
result of active membrane properties of the motor neuron. If this is
the case, then the membrane properties of the extensor motor neurons
may in part be responsible for the intraburst frequency characteristics
observed during walking, but further study is needed to address this issue.
Evidence from fictive locomotion in stick insects suggests that
inhibitory input plays a major role in shaping rhythmic motor activity
(Buschges 1998). Our data support the hypothesis that inhibition contributes to termination of motor neuron bursts as was the
case for fictive locomotion (Buschges 1998
), semi-intact walking (Godden and Graham 1984
), and rhythmic leg
movements (Pearson and Fourtner 1976
). For example, Ds
and SETi were depolarized and remained at a depolarized level
throughout a burst until the Ds burst and low-frequency SETi activity
were terminated at least in part by inhibitory input (Figs.
3B and 7B). We also recorded from a NSI during
walking (NSI-A) whose activity occurred with appropriate timing to
periodically inhibit Ds and contribute to burst termination (Fig.
4C).
In addition to NSI-A, we recorded from NSI-B, which periodically
depolarizes Ds during walking (Fig. 5B). These data suggest at least two alternative hypotheses for excitation of Ds activity during walking. First, NSI-B is tonically depolarized and its depolarization is periodically interrupted by inhibition. In that case,
one might suspect that inhibition is centrally generated (Buschges 1998) via the flexor burst generator
(Pearson 1976
; Pearson and Fourtner
1975
). Second, there may be an interneuron or interneurons that
generate(s) rhythmic excitation of Ds. Our data do not allow us to
distinguish between these two alternatives. For example, we were not
able to determine whether current injection into NSI-B can reset the
phase of extensor motor neuron bursts. However, the data do suggest
that extensor motor-neuron bursts do not simply result from direct
tonic excitation of the motor neuron, that is, periodically terminated
by rhythmic inhibition (see Buschges 1998
;
Pearson 1976
; Pearson and Fourtner 1975
). Further, the finding that NSI-B begins to hyperpolarize prior to the
end of Ds activity during each step also suggests that a reduction in
Ds excitation may contribute to burst termination (Fig. 5B).
It is possible that NSI-B activity is the result of sensory feedback.
That is, during walking, feedback from sensory cues might depolarize
NSI-B, and in turn NSI-B could then depolarize Ds. This is an important
issue as current injection into NSI-B had remarkable (direct or
indirect) effects on extensor burst duration during walking, and at
least one candidate source of sensory information has been shown to
modulate stance duration. In particular, activation of load receptors
in cockroach legs (campaniform sensilla) and muscle force receptors in
vertebrate muscle (Golgi tendon organs) results in excitation of
extensor motor neurons and prolongation of stance phase (Cruse
1976; Cruse and Saxler 1980
; Duysens and
Pearson 1980
; Pearson 1976
; Whelan et al.
1995
). We cannot resolve this issue without
considerably more circuitry analysis than we currently have. However,
it is worth noting that SETi burst duration appeared to also change when NSI-B was depolarized and hyperpolarized, resulting in prolonged or shortened Ds activity (respectively, Fig. 5, C and
D). Presumably, the effect of shortening or lengthening the
burst of the principal leg depressor motor neuron would be to
prematurely unload or prolong leg loading during stance. If that is the
case, the data indirectly support the hypothesis that afferents
signaling leg loading can in part determine extensor burst duration
(Whelan et al. 1995
). SETi activity may also be
influenced by NSI-B activity. Although this effect was not observed
during current injection into NSI-B when the animal was not moving its
legs (Fig. 5A), it might be uncovered during walking (see
also Kitmann et al. 1995
).
Coordinating the phase relationship of Ds and SETi at the end of stance and during searching
As was the case in this study, Krauthamer and Fourtner
(1978) noted that SETi activity is maximally excited at the end
of stance phase at the same time that Ds activity declines. We can only
speculate that the function of this high-frequency SETi activity is to
maintain stance phase support at a time when CTr extensor force would
be expected to decline (Fig. 2A) (see also Krauthamer and Fourtner 1978
). Our data predict that coordination of Ds
and SETi at the end of stance phase involves recruitment of
interneuron(s) that provide inhibitory drive to Ds and also depolarize
SETi at the end of stance (Figs. 3, B and C, and
9, C and D). Although not recorded during
searching or walking, we found one nonspiking interneuron (NSI-D) that
depolarizes SETi and inhibits Ds and could serve this function (Fig.
11D). If this hypothesis is correct, one would expect to
observe high-frequency SETi activity at the end of stance resulting
from phasic depolarization that appears to be independent of that
generating the SETi burst concurrent with Ds excitation throughout much
of stance, and that is what we found (Figs. 9D). The fact
that there is continuation of low-frequency Ds activity following
interruption of the Ds burst at the same time that high-frequency SETi
activity occurs also supports this hypothesis (Fig. 9D).
Along these lines, inhibitory input leading to burst termination
appears to be independent of Ds inhibition that is concurrent with
high-frequency SETi activity and termination of SETi high-frequency
activity (Figs. 9D and both 9C and
7B).
We found that there are components of extensor motor neuron
coordination that appear to occur during both walking and searching. The data are consistent with a modular organization of multi-joint control whereby individual interneurons(s) may coordinate extensor motor neurons during searching and walking. The recruitment of modules
to coordinate multiple joints during an ongoing rhythmic behavior has
also been proposed to occur during rostral scratch in the turtle and is
evident during a variant of rostral scratch termed "extensor
deletion" (Robertson and Stein 1988). Thus use of a
modular neural architecture may represent a general way of coordinating
movements at multiple joints.
In stick insects, a population of interneurons coordinates the activity
of motor neurons acting at several joints (Kitmann et al.
1995). The activity of each interneuron either opposes or
supports an ongoing behavior. While the neural architecture underlying
many behaviors is distributed there is evidence that in stick insects,
"constant functional elements" or "modules" of the distributed
network are recruited to coordinate multiple joints during both
rhythmic and nonrhythmic behaviors (i.e., searching, walking, rocking
and postural reflexes) (Kitmann et al. 1995
). Further,
NSIs that are homologous to those identified in stick insects exist
among other insects (Wolf and Buschges 1995
), so it is
likely that similar interneurons are represented and serve to
coordinate motor activity at multiple joints in cockroaches. In fact,
we recorded from some interneurons that may function to coordinate
extensor motor activity at the CTr and FT joints (Figs. 4, 5, and 11,
B-D). Although there may be exceptions, in stick insects,
current injection into all of the studied interneurons had similar
effects on motor activity when injected prior to leg movements as it
did when the animal engaged in a variety of behaviors (Kitmann
et al. 1995
). Therefore although we did not record from NSI-D
during walking and searching, it is reasonable to suspect that it
functions to inhibit Ds and excite SETi during those behaviors as it
did when the animal was not moving its legs (Fig. 11D).
Our data suggest that interneuron(s) contributing to simultaneous SETi
depolarization and Ds inhibition may coordinate the activity of these
motor neurons at the end of stance and the onset of the searching
extension phase. During walking, "recruitment" of such interneurons
may in fact result from additional activation of a subpopulation of
nonspiking interneurons whose ongoing activity is modulated by central
pattern generators (see also Kittmann et al. 1995).
Along these lines, we found there was a rapid increase in
depolarization of NSI-A at the termination of Ds activity that appeared
to occur in addition to ongoing rhythmic oscillation of its membrane
potential during walking (Fig. 4C) (see also Kitmann et al. 1995
).
As is the case for stick insects (Kittmann et al. 1995),
a distributed population of interneurons is likely to coordinate multiple joints during walking and searching in cockroaches. We recorded from one NSI that contributed to depolarization of SETi during
searching without influencing Ds activity (NSI-C; Fig. 11, B
and C). These data suggest that there is a population of cells that are recruited to coordinate Ds and SETi activity during the
extension phase of searching. This population may be recruited in
parallel with interneurons involved in multi-joint coordination, or
they may represent an overlapping group of cells that are recruited by
interneurons involved in simultaneously coordinating motor neurons
acting at multiple joints (see also Kittmann et al.
1995
).
Relationship of motor neuron activity to joint kinematics and foot contact
We tested several hypotheses regarding the induction of Ds
high-frequency activity before foot set-down and its decline during stance phase of walking. Afferent input from tarsal contact does not
appear to be responsible for the characteristic decline in Ds activity
that occurs near foot set-down during walking (Fig. 6). Similar
conclusions came from experiments on cats in which a decline in ankle
extensor EMG activity still occurred in absence of substrate contact
during walking (Gorassini et al. 1994).
Therefore either different afferent pathways such as joint movement and position information may play a role or this EMG component is centrally
generated as has been suggested for cats (Hiebert et al.
1994
). Our data suggest that it is possible that sensory cues from joint angle receptors initiate the decline in Ds high-frequency activity, although this hypothesis needs to be tested further (see also
Watson and Ritzmann 1998a
). We also examined whether particular CTr and FT joint angles are correlated with the onset of
high-frequency SETi activity. Our data suggested that the onset of
high-frequency SETi activity is not initiated when the CTr and FT
joints extended to a particular joint angle. These data further suggest
that central influences and/or other sensory cues may initiate Ds
inhibition associated with high-frequency SETi activity.
Conclusion
This study was initiated by observing distinct behavioral changes
in leg movements when a cockroach switches from walking to searching
(Tryba and Ritzmann 2000). At the level of motor neurons, we can now account for the principal mechanisms (Ds inhibition concurrent with SETi excitation) that are responsible for the phase
relationship of the CTr and FT joints during the extension phase
searching. Similar mechanisms were found to coordinate these motor
neurons during the end of stance during walking. In both cases, our
data indirectly support the modular concept of multi-joint coordination. We additionally found evidence that searching and walking
extensor motor neuron activity results from the combined input of a
population of interneurons.
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ACKNOWLEDGMENTS |
---|
The authors thank Drs. Sasha N. Zill and Joanne Westin for very helpful comments on the manuscript and Dr. James T. Watson for use of his T2 leg treadmill walking data for comparison and helpful comments. Thanks to A. Pollack for technical support.
This work was supported by Office of Naval Research Grant N0014-99-1-0378 to R. E. Ritzmann.
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FOOTNOTES |
---|
Present address and address for reprint requests: A. K. Tryba,
Dept. of Organismal Biology and Anatomy, The University of Chicago,
Anatomy Bldg., 1027 E. 57th St., Chicago, IL 60637.
E-mail:
Tech10S{at}techsan.org
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 29 November 1999; accepted in final form 13 March 2000.
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REFERENCES |
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