1Zoologisches Institut, Universität zu Köln, 50923 Cologne; 2Fachbereich Biologie, Universität Kaiserslautern, 67653 Kaiserslautern; and 3Chamissostrasse 16, 70193 Stuttgart, Germany
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ABSTRACT |
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Akay, Turgay,
Ulrich Bässler,
Petra Gerharz, and
Ansgar Büschges.
The Role of Sensory Signals From the Insect Coxa-Trochanteral
Joint in Controlling Motor Activity of the Femur-Tibia Joint.
J. Neurophysiol. 85: 594-604, 2001.
Interjoint coordination in multi-jointed
limbs is essential for the generation of functional locomotor patterns.
Here we have focused on the role that sensory signals from the
coxa-trochanteral (CT) joint play in patterning motoneuronal activity
of the femur-tibia (FT) joint in the stick insect middle leg. This
question is of interest because when the locomotor system is active,
movement signals from the FT joint are known to contribute to
patterning of activity of the central rhythm-generating networks
governing the CT joint. We investigated the influence of femoral
levation and depression on the activity of tibial motoneurons. When the locomotor system was active, levation of the femur often induced a
decrease or inactivation of tibial extensor activity while flexor motoneurons were activated. Depression of the femur had no systematic influence on tibial motoneurons. Ablation experiments revealed that
this interjoint influence was not mediated by signals from movement
and/or position sensitive receptors at the CT joint, i.e., trochanteral
hairplate, rhombal hairplate, or internal levator receptor organ.
Instead the influence was initiated by sensory signals from a field of
campaniform sensillae, situated on the proximal femur (fCS). Selective
stimulation of these fCS produced barrages of inhibitory postsynaptic
potentials (IPSPs) in tibial extensor motoneurons and activated tibial
flexor motoneurons. During pharmacologically activated rhythmic
activity of the otherwise isolated mesothoracic ganglion (pilocarpine,
5 × 104 M),
deafferented except for the CT joint, levation of the femur as well had
an inhibitory influence on tibial extensor motoneurons. However, the
influence of femoral levation on the rhythm generated was rather labile
and only sometimes a reset of the rhythm was induced. In none of the
preparations could entrainment of rhythmicity by femoral movement be
achieved, suggesting that sensory signals from the CT joint only weakly
affect central rhythm-generating networks of the FT joint. Finally, we
analyzed the role of sensory signals from the fCS during walking by
recording motoneuronal activity in the single middle leg preparation
with fCS intact and after their removal. These experiments showed that
fCS activity plays an important role in generating tibial motoneuron
activity during the stance phase of walking.
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INTRODUCTION |
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Locomotion in legged organisms results from the
interaction of central rhythm-generating networks in the nervous system
with sensory organs that provide information about actual movements and
forces generated by the appendages and changes in body posture and
equilibrium (e.g., Bässler 1983;
Bässler and Büschges 1998
; Cruse
1990
; Graham 1985
; Grillner 1981
;
MacPherson et al. 1997
; Orlovsky et al.
1999
; Pearson 1995
; Wendler
1964
). In multi-jointed appendages, the emerging locomotor
pattern results from the coordinated action of several joints. The
control of the motor output in walking systems encompasses three
different levels: intrajoint control, interjoint coordination, and
intersegmental coordination (for summaries, see Clarac
1991
; Cruse et al. 1995
; Grillner
1981
; Orlovsky et al. 1999
; Pearson
1995
; Stein and Smith 1997
).
In insects and crustaceans, considerable detailed knowledge is
available on intrajoint information processing in posture and movement
control (crayfish: ElManira et al. 1991a; locust:
summary in Burrows 1996
; stick insect:
Bässler and Büschges 1998
). However, there
is less known about the neuronal mechanisms responsible for interjoint
information processing, specifically for the generation of coordinated
activities during locomotion (e.g., Bässler 1993b
; Büschges et al. 1995
; ElManira et al.
1991b
; Hess and Büschges 1997
). In walking
systems that have been reported to have a highly centralized structure,
i.e., one common central locomotor pattern generator for the entire
appendage, e.g., the locust (Ryckebusch and Laurent
1993
) and the crayfish (Chrachri and Clarac
1990
), evidence suggests that this central pattern generator
organizes the motor output for all leg joints automatically. The
situation, however, is more complicated in the case of walking systems
that are less centrally organized and that contain neuronal networks governing the individual leg joints that are only loosely coupled, like
the stick insect walking system (summary in Bässler and Büschges 1998
). Here the question arises as to how the
activities of adjacent leg joints are coupled together during
locomotion and what role sensory and central signals might play in coordination.
A previous investigation (Hess and Büschges 1997,
1999
) has shown that proprioceptive signals from one leg joint
affect central rhythm generation of the adjacent leg joint when the
stick insect locomotor system is active and the joint control networks
are in the movement control mode ("active" behavioral state) (for definition, see Bässler 1983
; Bässler
and Büschges 1998
). Movement signals from the
femur-tibia (FT) joint e.g., flexion signals, induce specific
transitions in activity of rhythm generating networks of the
coxa-trochanteral (CT) joint, e.g., by eliciting levator and
terminating depressor activity. Extension movements induced the
opposite response. During locomotion this could facilitate the onset of
leg levation due to flexion signals from the FT joint or the onset of
leg depression during extension signals from the FT joint. From these
studies, the question emerges as to whether such specific influences
represent a common mechanism for interjoint coordination in the
multi-jointed limb. Therefore we chose to investigate the influence of
signals from sense organs at the CT joint on the activity of tibial
motoneurons and muscles in the active stick insect. We applied levation
and depression movements to the femur, while the locomotor system of
the stick insect was active, i.e., in the locomotor state, and
monitored changes in the activity of extensor tibiae and flexor tibiae
motor neurons. Using ablation experiments, we analyzed which sense
organs at the CT joint affect motoneuronal activity in tibial
motoneurons and muscles. We also investigated whether the observed
interjoint influences were mediated purely by "reflex-like"
interactions or whether sensory signals from the CT joint have access
to the central rhythm-generating premotor networks of the FT-joint. In a final series of experiments we investigated the role of this interjoint influence in controlling motor activity in the FT-joint during walking.
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METHODS |
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The experiments were performed on adult female stick insects (Carausius morosus and Cuniculina impigra) from our breeding colonies at the University of Cologne and Kaiserslautern and were carried out under daylight conditions and at room temperature (20-22°C). Most of the experiments were carried out on both species. We did not detect any difference between C. morosus and C. impigra. In the text we specify for each given experimental protocol and figure the species used.
Preparation
EXPERIMENTS INVESTIGATING THE INFLUENCE OF FEMORAL LEVATION AND
DEPRESSION.
The experimental animal was fixed dorsal side up with dental cement
(Protemp II, ESPE) along the edge of a foam platform with only the left
middle leg left intact and fixed perpendicular to the thorax. The
thoraco-coxal joint (TC joint) was immobilized with the coxa-trochanter
joint (CT joint) being free to move. A small window was cut in the
meso- and metathoracic tergum. The gut, fat, and connective tissue were
removed to expose the ventral nerve cord. The innervation of the middle
leg was either left intact or the leg was completely denervated, except
for the CT joint, whose sensory innervation was left intact (Fig.
1). To exclude indirect sensory
influences, the following lateral and leg nerves of the mesothorax were
crushed or cut: nl2, nl3, nl4, nl5, C1, C2. In experiments in which the
activity of the flexor tibiae muscle was recorded by EMGs, the
innervation of the flexor-tibiae muscles via the nervus cruris was left
intact. The activity of the tibial extensor motoneurons was recorded
extracellularly with a hook electrode from nerve nl3 proximal to the
site where it was crushed. The femur of the middle leg was moved by a
pen motor between ±20 deg around the horizontal resting position of
the CT joint (Fig. 2A). Sense
organs at the CT joint were ablated by the following procedures: the
trochanteral and the femoral campaniform sensilla (fCS, trCS) were
destroyed with a fine insect pin (Schmitz 1993), which
was heated and pushed into the cuticle at their location; the hair
fields, i.e., the trochanteral hairplate (trHP) and the rhombal
hairplate (rHP) were ablated with a microblade under visual control
(Dean and Schmitz 1992
; Schmitz 1986a
);
the signals from internal levator stretch receptors (levSR), were eliminated by crushing the nl3 nerve distal to the recording hook electrodes (Bässler 1983
; Tartar
1976
).
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EXPERIMENTS WITH RESTRAINED MIDDLE LEGS.
In experiments in which the influence of sensory stimulation on tibial
muscle activity was investigated, the nervus cruris was left intact to
allow EMG and nerve recordings in the femur. The lateral nerves, i.e.,
nl2, nl4, and nl5 were cut. In experiments with recordings from
extensor motoneurons from nerve F2 in the femur, the nervus lateralis 3 (nl3) was left intact. The trochanteral campaniform sensilla (trCS)
were destroyed with a fine insect pin (Schmitz 1993),
that was heated and pushed into the cuticle at their location. The FT
joint and CT joint were both immobilized with dental cement. In these
experiments, femoral campaniform sensilla were selectively stimulated
mechanically (see experiment shown in Fig. 8A) by means of a
low-voltage piezo-electrical element (PI Physic). The femoral
chordotonal organ (fCO) was stimulated as well (C. morosus),
according to established procedures (Büschges 1989
). In short, a small window was cut into the dorsal side of the femur. The apodeme of the fCO was exposed and then fixed in the
clamp of a stimulation device, described in detail in Hofmann et
al. (1985)
. Elongation and relaxation stimuli were applied to
the fCO with an amplitude of 300 µm (equivalent to 60° movement of
the FT joint) (Weiland et al. 1986
).
EXPERIMENTS ON CENTRALLY GENERATED RHYTHMIC MOTOR
ACTIVITY.
For pharmacologically activated rhythmic preparations, the anterior and
posterior connectives were also cut and pilocarpine (5 × 104 M)
(Büschges et al. 1995
) was applied in the Ringer
solution (Weidler and Diecke 1969
).
Electrophysiology
The activity of the tibial extensor motoneurons (MN) in
nerves nl3 or F2 was recorded with hook electrodes (Schmitz et
al. 1991). The activity of the flexor tibiae muscle was
monitored by an electromyographic (EMG) recording with copper wires of
65 µm diameter, insulated except for the tip. To perform
intracellular recordings from the neuropilar processes of a motoneuron,
the mesothoracic ganglion was fixed on a wax-coated platform with fine cactus spines according to the established procedures
(Büschges 1989
). The ganglion sheath was
treated with Pronase (Merck KGaA, 64271 Darmstadt) for 60 s.
Recordings were made in bridge mode with thin-walled microelectrodes
with a resistance of 20-25 M
when filled with 3 M KAc or 0.05 M
KCl/2 M KAc. Extensor motoneurons were identified by a one-to-one
correlation of their action potentials in both the intracellular
recording and extracellular recording from the extensor nerve.
Behavioral analysis
For behavioral analysis, the single middle leg preparation was
used (Fischer et al. 2001; Karg et al.
1991
). In this preparation, all legs except a middle leg of the
animal were removed. The middle leg was fixed perpendicular to the
thorax of the animal extending over the rim of a foam platform. The TC
joint was immobilized, and the more distal leg joints were free to
move. The leg performed well coordinated walking movements on a
treadband or searching movements in the absence of ground contact.
Sequences of walking movements were elicited by touching the abdomen
with a paintbrush. We recorded the activity of tibial extensor
motoneurons extracellularly from nerve nl3 and tibial flexor activity
by means of EMGs from the muscle. The EMG signals were recorded also as
rectified and integrated (time constant, 40 ms) records. The animals
were tested in three situations, 1) control situation: the
nerves innervating the muscles as well as the sense organs of the leg
joints were left intact. In all of the behavioral experiments, the
motor pattern was recorded in control situation first (number of
experiments, N = 12). 2) After removal of
the fCS: the fCS of the leg was removed by destroying the field on the
cuticle with an insect pin where the fCS is located (N = 9). And 3) sham-operated animals: in this group, instead
of destroying the fCS, we only made a small hole on the anterior side
of the femur (N = 3). Therefore any changes in motor
activity in extensor and flexor MN pools due to the surgery at the
femur could be monitored.
Scanning electron microscopy
The left middle legs of adult stick insect C. morosus were removed carefully to prevent damage of the TC joint. We than separated the tibia and tarsus. The preparations first were dehydrated with alcohol series (10, 30, 40, 50, 60, 70, 80, 90, and 98%), in which the preparations remained for 8 min each. The solution with 30% alcohol was applied twice as in this application the cuticle was cleaned in ultrasonic bath. After dehydration the preparations were dried and coated (SEM Coating Unit PS3, Agar Aids for Electron Microscopy) with gold (150 A°). The preparations of the leg were then inspected and analyzed with a scanning electron microscope (Hitachi, S520).
Reconstruction of serial sections of the femur
Serial sections, 10 µm thick and stained with
hematoxilin-eosin, were used. Reconstructions were made from two middle
legs and two hind legs of C. morosus (for details, see
Bässler 1977).
Data analysis
Extracellular recordings, EMGs, and intracellular recordings were stored on a DAT-Recorder (SONY, PC 116) or on FM-tape recorder (RACAL Store 7DS). Analog-to-digital conversion was performed off-line on a CED 1401plus interface (Cambridge Electronic). The recordings were analyzed with the Spike2 software (Version 3.13). Statistical evaluation of data and plotting of graphs was done with PlotIt and Excel 97. In the text N gives the number of experiments and n gives the sample size. Differences in means of samples were tested by using the Student's t-test (Excel 97). Means were regarded as significantly different with P < 0.05.
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RESULTS |
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Influence of movements of the trochanterofemur on the activity of muscles and motoneurons of the FT joint
In a first set of experiments, we investigated the influence of
movements of the CT joint on activity of muscles and motoneurons of the
FT joint in the active animal (Figs. 2 and
3). The influence of movements of the CT
joint were investigated with the thoraco-coxal (TC) and joint
deafferented to exclude any indirect influences on tibial motoneuron
and muscle activity. After activating the animal, extensor and flexor
tibiae muscles exhibited alternating activity (Fig. 2A). The
femur of the middle leg was moved up and down at an angle of ±20°
around the horizontal center position (Fig. 2). In both species used
(C. impigra and C. morosus) upward movement of
the femur applied during extensor activity led to an inactivation or to
a decrease of motor activity. A quantitative evaluation of nine
experiments and 127 stimulus presentations revealed this influence in
69% of the stimulus presentations. This is exemplified by the
extracellularly recorded extensor activity in nerve nl3, in which the
biggest units belong to the fast extensor tibiae motoneuron (FETi, Fig.
2A, ). The probability of occurrence for termination of
extensor activity could vary considerably from animal to animal with
the extreme values ranging from 39 to 100% for individual
preparations. Inactivation of extensor activity was in most cases
accompanied by an activation of flexor motoneurons (Fig. 2A,
1st and 2nd stimulus). The inhibitory influence of femur levation on
extensor activity is also shown in a peri-stimulus time (PST) histogram
of extensor activity during upward movement of the femur (Fig.
2B). Differential PST histograms for SETi and FETi showed
that both excitatory extensor motoneurons, i.e., SETi and FETi, were
affected, with the influence being stronger for FETi (Fig.
2C). In some cases, inactivation of extensor motoneurons did
not outlast the end of femur levation (Fig. 2A, 3rd levation stimulus). Intracellular recordings from the neuropilar processes of
FETi (N = 4) revealed that inactivation induced by
femur levation resulted from a hyperpolarization of this motoneuron
(Fig. 3, A and B). Levation of the femur during
ongoing flexor activity did not result in any change of activity (not
shown). Depression of the femur did not elicit systematic changes in
motoneuronal activity. Only occasionally, when the femur was depressed
during extensor activity, a slight increase in extensor activity was observed (Fig. 2A, 1st depression).
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This influence of movements of the femur on the activity of extensor and flexor motoneurons in the active animal differed clearly from the situation in the inactive, resting animal. In the inactive animal, both levation and depression of the femur regularly had only a weak influence on the activity of extensor and flexor motoneurons. Figure 3 shows this for the response of FETi and SETi to levation and depression of the femur. Both movements elicited one or two action potentials in SETi as visible from the extracellular recording of nl3 (Fig. 3A). FETi received in general small transient hyperpolarizing synaptic inputs of 1-2 mV during levation of the femur (N = 9; Figs. 3, A and B, and 4). In four of these recordings, we observed as well small depolarizing synaptic inputs elicited by both levation and depression of the femur (Fig. 3, A and B). In none of our recordings did we detect any change in flexor muscle activity in response to levation or depression of the femur (not shown) in the resting animal.
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Influence of sense organs at the CT joint on muscles and motoneurons supplying the FT joint
To determine which sense organs are responsible for the observed interjoint influence, we selectively ablated sense organs at the CT joint. The ablation of sense organs measuring movements and forces at the CT joint was performed in different combinations and sequences. We then investigated the influence of levation and depression of the femur on the activity of tibial motoneurons in the active animal. Similar results were collected for both C. morosus and C. impigra.
Movement and force at the CT joint of the stick insect are measured by
four different sense organs located around the CT joint: the
trochanteral hairplate (trHP, Fig.
5A) located on the dorsal surface of the trochanter responds to levation movement of the trochanterofemur in relation to the coxa (Schmitz
1986a-c
; Tartar 1976
); the rhombal hairplate
(rHP, Fig. 5B), is located on the ventral surface of the
trochanter (Tartar 1976
), and its hairs are bent by the
joint membrane on depression of the trochanter; changes in cuticular
stress at the trochanter are signaled by three different fields of
trochanteral campaniform sensilla (trCS, Fig. 5, A and
B) (Delcomyn 1991
; Hofmann and
Bässler 1982
; Tartar 1976
); cuticular
stress at the proximal femur is detected by a field of femoral
campaniform sensilla (fCS) (Hofmann and Bässler 1982
; Tartar 1976
) located on the posterior side
of the femur close to the borderline of the trochanter (Fig.
5B). Finally, a preliminary study reports the existence of
an internal levator stretch receptor organ (levSR) (Schmitz and
Schöwerling 1992
) that is situated inside the coxa
parallel to the levator trochanteris muscle. This sense organ detects
length changes of the levator trochanteris muscle, similar to the one
in the locust (Bräunig and Hustert 1985
).
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As there was no previous information available on the innervation of the fCS, we traced their innervation in semi-thin transections (10 µm) of two middle legs and two hind legs in C. morosus (Fig. 6). From these reconstructions, it became clear that the fCSs are innervated by a side branch of nerve F2 arising in three cases proximal to the merging point of nerve nl3 (the motor root of F2) and nerve F2 (sensory root of F2) close to the autotomization point at the fusion region of trochanter and femur. In one middle leg, this side branch originated just distal to the merging point of nerves nl3 and F2.
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In various sets of experiments, we subsequently removed the sense organs around the CT joint either by ablation (trHP, rHP, trCS, fCS) or by cutting the innervating nerves close to the sense organ (levSR, trHP, trCS). We then activated the experimental animal and recorded the influence of femoral levation and depression on the activity of the extensor tibiae (Fig. 7, A and B). For example, in the experiment shown in Fig. 7A termination of extensor activity with femur levation was still present after removal of all sense organs (i.e., trCS, trHP, and rHP) except the fCS. After ablation of the fCS, termination of extensor activity is no longer induced by leg levation. The results of the different sets of experiments are summarized in Table 1. These experiments revealed that only removal of the fCS abolished the interjoint influence of trochanteral movements on tibial motoneuron activity (Fig. 7, A and B). The relevance of fCS signals for the interjoint influence was exemplified by removal of those prior to the experiment (Fig. 7C).
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Role of fCS signals in controlling activity of extensor tibiae motoneurons
In further experiments, we focused on the detailed influence of signals from the fCS on extensor motoneurons. We intended to selectively stimulated the fCS by applying pressure on the femoral cuticle at their location to investigate their influence on tibial motoneuron activity. At first we recorded the activity generated by the fCS in response to application of pressure on the femoral cuticle (Fig. 8A; N = 3; C. morosus). To monitor their activity, an extracellular recording was made from the nervus cruris in the coxa while it was cut proximal to the electrode and distal to the fCS in the femur. Application of pressure to the femoral cuticle at the location of the fCS induced barrages of spikes in sensory neurons of the fCS field (top trace). This response was abolished after destroying the fCS (bottom trace), verifying that only the fCS afferents were firing in response to cuticular pressure (Fig. 8A). These experiments revealed that application of pressure to the femoral cuticle could be used as a tool to induce activity in fCS neurons and to investigate their influence on motoneurons selectively.
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Applying pressure to the fCS in the resting, inactive animal always decreased spontaneous activity in SETi, while no influence was detectable on the flexor EMG recording (Fig. 8B). In these experiments, the femur was deafferented, except for the fCS and the fCO, and de-efferented, except the flexor muscle. In the active behavioral state, pressure applied to the fCS terminated activity in extensor motoneurons (Fig. 8B) and elicited activity in flexor motoneurons, as obvious from the flexor EMG recording (arrows Fig. 8B). Intracellular recordings from FETi also revealed the inhibitory synaptic drive caused by fCS stimulation in the inactive and active behavioral state (Fig. 8C). The inhibitory influence of fCS signals was also manifested as a change in the strength of intrajoint reflex activation of extensor motoneurons by stimulation of the femoral chordotonal organ (Fig. 8, D and E), indicating that extensor motoneuron activity is determined by signals from both intra- and interjoint sensory signals.
The preceding results show that signals from the fCS affect
pattering of activity of extensor tibiae motoneurons. In the light of
previous findings on the role of sensory signals in interjoint information flow (Hess and Büschges 1999), the
question arose as to whether signals from the fCS have access to the
central rhythm generating networks of the FT joint. Therefore we tested the influence of signals from the fCS elicited by levation and depression of the femur on the activity of centrally generated rhythmic
activity in tibial motoneurons. Relatively regular rhythmic activity
can be elicited by topical application of the muscarinic agonist
pilocarpine (Büschges et al. 1995
) at a final bath
concentration of 5 × 10
4 M. We stimulated the
CT joint in the otherwise isolated mesothoracic ganglion (C. morosus; C. impigra) and monitored the influence of joint
movements on the rhythmic activity of tibial extensor motoneurons (Fig.
9). In 57% (n = 77) of
the trials (N = 5, n = 135), levation
of the femur resulted in a change of rhythmic activity in extensor
motoneurons. Levation of the femur in these cases led to a shortening
(Fig. 9A, left) of tibial extensor activity. In
about 20% of the trials, shortening of extensor activity was followed
by a long interburst interval in extensor activity (Fig. 9A,
right). In these cases (57%), extensor burst duration (BD, Fig. 9B, left) was correlated with the latency
between extensor burst onset and the onset of the stimulus.
Interestingly, not only extensor burst duration but also interburst
interval (IBI) between extensor bursts was altered by the stimulus as
it became more variable and increased compared with control (Fig.
9B, middle). The duration of the affected cycle
depended on the time of stimulus, however, with a high degree of
variability. Nevertheless cycle period was significantly correlated
with the time between burst onset and stimulus onset. From generating
phase response plots for these experiments (Fig. 9C), it
became clear that femoral levation had a significant phase dependent
influence on rhythmicity, however, with a considerable amount of
variability. As judged from the regression line, femoral levation
delivered early in the cycle had a general tendency of shortening the
cycle, while this influence reversed to a general tendency of a slight
lengthening toward higher phase values. In the remaining cases, i.e.,
in 43% of the trials (N = 5, n = 135),
no change in motor activity could be detected. These results indicate
that sensory influences mediated by signals from the fCS during leg
levation do affect patterning of extensor motoneuron activity, however,
in a rather variable and labile manner. Furthermore depression of the
femur was never observed to induce any detectable changes in tibial
extensor activity (not shown).
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Role of fCS in walking pattern generation in the middle leg
We have shown that stimulation of the fCS inhibited
extensor motoneurons and elicited activity in flexor motoneurons (e.g., Fig. 8B). In a final set of experiments, we examined the
role of the fCS in controlling tibial motoneuron and muscle activity in
walking. We choose to investigate this question in the single middle
leg preparation (Fischer et al. 2001) (C. impigra) by ablating this sense organ and analyzing any changes in
the walking motor pattern. This preparation is appropriate for such
investigations because segmental mechanisms of walking pattern
generation can be investigated without the influence of intersegmental
coordinating influences from the other legs.
Figure 10A shows recordings from the middle leg preparation during walking with the fCS intact (Fig. 10A, left) and after their removal (Fig. 10A, right). The most obvious difference between these situations is the reduced flexor activity during stance after removal of the fCS compared with control. This is seen both in the flexor EMG as well as from the rectified and integrated EMG activity. We found such a decrease in flexor activity during stance in all animals tested (N = 9). Removal of the fCS also caused a slight but significant change in the mean flexor burst duration during walking (Fig. 10B), increasing from 1.31 ± 0.58 s (n = 486) to 1.51 ± 0.94 s (n = 475).
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The decrease in flexor activity during stance was also obvious from plotting the average amplitude of the rectified and integrated EMG within a normalized burst comparing intact animals and following removal of the fCS (Fig. 10C). After removal of the fCS, the amplitude of flexor activity was significantly reduced in all bins except for the first six bins at the begin of stance (see Fig. 10, C and D; 1st 6 bins in C and 1st bin in D). The same was true for the average normalized amplitude of flexor activity of all investigated animals for the normalized flexor burst duration (N = 9; Fig. 10D) as well as over time (N = 4; Fig. 11A). For doing so bin values were normalized to the maximal bin value of the intact situation ("fCS intact"). No significant decrease in flexor activity, however, could be observed in sham-operated animals (N = 3, Fig. 10E). In contrast, no changes were detectable in extensor activity during leg swing, as measured by the mean spike activity of the extensor motoneurons, FETi and SETi (Fig. 11A).
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We further investigated whether sensory information from the fCS plays a role in generating the step phase transitions from stance to swing and vice versa during walking on the treadband. We compared the average latencies between the activities of the two antagonistic tibial motoneuron pools at both transitions with intact fCS and after fCS removal (Fig. 11B). For the transition from swing to stance, we measured the time between termination of tibial extensor motoneuron activity in an extracellular recording from the extensor nerve nl3 and the start of tibial flexor activity on the flexor EMG. We found no significant change in latency (Fig. 11B, left) in any of the tested animals (N = 4). The same was true for the transition from stance to swing (Fig. 11B, right), although in one animal the overlap between the end of flexor and the begin of extensor activity was significantly smaller after the removal of the fCS.
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DISCUSSION |
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We have shown that sensory signals from the CT joint affect
activity of tibial motoneurons. Levation of the femur was able to
inactivate tibial extensor motoneurons and to initiate activity in
tibial flexor motoneurons (Fig. 2A). Ablation experiments
revealed that this influence was not mediated by sensory signals from
any of the known movement and/or position sensitive receptors located at the CT joint (Table 1). Instead femoral movements were signaled by
cuticular stress-sensing sense organs, i.e., by a group of campaniform
sensilla located at the proximal femur close to the CT joint, the fCS
(Fig. 7). Selective stimulation of the fCS revealed that they have an
inhibitory influence on tibial extensor motoneurons and an excitatory
effect on tibial flexor motoneurons (Fig. 8B). The
experiments on pharmacologically activated rhythmic preparations show
that fCS signals can affect centrally generated activity in motoneurons
supplying the FT joint (Fig. 9), however in a rather labile and
variable manner, compared with the influence from fCO signals from the
FT joint on motor activity driving the CT joint (Hess and
Büschges 1999). Our results suggest that influences from
movements at the CT- onto FT joint motoneuron activity are mediated to
a large extent by reflex-like influences and to a lesser extent by
influences on the central rhythm-generating networks of the FT joint.
Finally, we were able to show that signals provided by fCS contribute
to the generation of flexor activity during stance in walking (Fig.
10).
Interjoint information flow between the CT joint and the FT joint
Our results show that moving the femur influences activity of
motoneurons supplying the FT joint due to inputs from the fCS and not
due to movement and/or position sensitive receptors at the CT joint.
The trochanteral hairplate (Bässler 1983;
Tartar 1976
), the rhombal hairplate
(Bässler 1983
; Tartar 1976
), and the levator stretch receptor organ (Schöwerling
1993
) did not affect patterning of activity in tibial
motoneurons. However, from the design of our experiments, we cannot
exclude more subtle influences of these sense organs on the magnitude
of activity in motoneurons of the FT joint (e.g., Bässler
1993a
). Even though our results clearly differ from previous
findings on the influence of proprioceptive signals from the FT joint
on motor activity in the CT joint (Hess and Büschges
1999
), our present results together with these previous
findings suggest the following scheme for information flow between the
FT joint and the CT joint: proprioceptive information is unidirectional
in interjoint coordination, i.e., from the FT joint onto the CT joint.
Thus our results indicate an asymmetry for the role of proprioceptive
signals in interjoint coordination.
Signals from cuticular stress detecting sense organs, the fCS, affected
the activity of motoneurons of the FT joint. In contrast to
proprioceptive signals, which report position or movement of the joint
they arise from, signals from CS are produced by strain of the cuticle
(recent review in Duysens et al. 2000). As such, activity of fCS afferents, for example, could arise from forces in the
CT joint elicited by femoral movements or from activation of muscles in
the leg. They could as well be induced by forces generated in the FT
joint or by forces at other locations on the thorax that induce torsion
of the proximal femoral cuticle (see also Ridgel et al.
2000
).
The presented findings differ from results reported by Bässler on
the stick insect foreleg. Bässler (1993a) showed
that proprioceptive signals from the trochanteral hairplate at the CT
joint influenced interjoint coordination between the CT and FT joint,
an influence that was not detectable in our studies on the middle leg.
There are four possible factors that may account for this difference.
First, the differing results may be due to differences in mechanisms of
interjoint coordination between the middle leg and the foreleg and/or a
differing architecture of the trochanter and femur basis in these legs.
Second, influences of the trochanteral hairplate on tibial motoneuron
activity may only be effective during the production of leg movements
and may not be effective in reduced preparations. Third, in the
experiments of Bässler (1993a)
, the levSR was left
intact. Finally, the efficacy of proprioceptive signals from the CT
joint on activity of the FT joint may depend on the actual phase of
motoneuronal activity in the TC and CT joint, a possibility that was
not controlled in the present investigation. At present, we have no
indications that this is the factor, but this aspect will be in the
focus of subsequent investigations.
Influence of femoral campaniform sensilla on FT-joint motoneurons
We have shown that when the locomotor system of the stick insect
is active, signals from the fCS inhibit extensor motoneurons and
activate flexor tibiae motoneurons. At present the neuronal pathways
mediating this influence are not known. It is quite conceivable that
direct connections from the afferents and pathways via intercalated interneurons are mediating this influence similar to the situation for
the influence of CS signals on coxal motoneurons of stick insect
(Schmitz and Stein 2000) and locust (Newland and
Emptage 1996
). Recent experiments indicate that in fact
individual identified pathways via nonspiking interneurons of the
FT-control network (for review on this network see
Büschges et al. 2000
) are involved in mediating
the influence of fCS on the activity of tibial motoneurons (Akay and
Büschges, unpublished data).
There are four fields of CS at the CT joint, three on the trochanter
and one on the very proximal femur that have been treated in the past
mostly as "one" sensory system (e.g., Hofmann and Bässler 1982; Schmitz 1993
; Schmitz
and Stein 2000
). Here we report a specific influence of one of
these four fields, i.e., the fCS on the generation of motor activity.
No such influence was detectable for the three fields of trCS. In this
context another observation appears interesting (Gerharz
1999
; Akay, Gerharz, and Büschges, unpublished
observation): levation and depression of the CT joint also affected
activity of motoneurons supplying the thoraco-coxal (TC) joint.
However, in this case this influence from CT joint movements arose from
the trCS, only. In conjunction with the present results, there appears
to be a segregation of function in the CS fields at the CT joint with
the fCS affecting motoneuron activity of the FT joint and the trCS
affecting motoneuron activity of the TC joint.
Role of sensory signals from the campaniform sensilla in controlling motor activity during locomotion
By ablation experiments, we could show that the sensory signals
from the fCS affecting tibial motoneurons influence the magnitude of
activity in the flexor muscle during stance in the single walking leg
preparation. These results are consistent with previous findings on the
role of CS in controlling the stance phase motor output of the leg that
indicated that sensory information from CS about load on the leg
reinforces stance phase motor output (e.g., Cruse et al.
1983; Pearson 1972) and specifies the role of
one field of CS in this functional task. Interestingly, we did not find an influence of fCS ablation on the timing of step phase transitions in
the walking cycle, neither for the transition from stance to swing
(e.g., Bässler 1977
; Newland and Emptage
1996
) nor for the transition from swing to stance (e.g.,
Cruse and Bartling 1995
).
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ACKNOWLEDGMENTS |
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We are grateful to Drs. H. Cruse, R. DiCaprio, H. Scharstein, J. Schmitz, and G. Wendler for valuable discussions throughout the course of the work as well as to Dr. R. DiCaprio for suggestions on an earlier draft of the manuscript. We also thank Ch. Graef and H. P. Bollhagen for excellent technical support.
This project was supported by the Deutsche Forschungsgemeinschaft (Bu857/6-1 and 2).
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FOOTNOTES |
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Address for reprint requests: A. Büschges, Zoologisches Institut, Universität zu Köln, Weyertal 119, 50923 Cologne, Germany (E-mail: Ansgar.Bueschges{at}uni-koeln.de).
Received 27 July 2000; accepted in final form 31 October 2000.
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REFERENCES |
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