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J Neurophysiol 79: 2964-2976, 1998;
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The Journal of Neurophysiology Vol. 79 No. 6 June 1998, pp. 2964-2976
Copyright ©1998 by the American Physiological Society

Cooperative Mechanisms Between Leg Joints of Carausius morosus I. Nonspiking Interneurons That Contribute to Interjoint Coordination

Dennis E. Brunn

Fakultät für Biologie, Abteilung 4, Universität Bielefeld, 33501 Bielefeld, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Brunn, Dennis E. Cooperative mechanisms between leg joints of Carausius morosus. I. Nonspiking interneurons that contribute to interjoint coordination. J. Neurophysiol. 79: 2964-2976, 1998. Three nonspiking interneurons are described in this paper that influence the activity of the motor neurons of three muscles of the proximal leg joints of the stick insect. Interneurons were recorded and stained intracellularly by glass microelectrodes; motor neurons were recorded extracellularly with oil-hook electrodes. The motor neurons innervate the two subcoxal muscles, the protractor and retractor coxae, and the thoracic part of the depressor trochanteris muscle. The latter spans the subcoxal joint before inserting the trochanter, thus coupling the two proximal joints mechanically. The three interneurons are briefly described here. First, interneuron NS 1 was known to become more excited during the swing phase of the rear and the stance phase of the middle leg. When depolarized it excited several motor neurons of the retractor coxae. This investigation revealed that it inhibits the activity of protractor and thoracic depressor motor neurons when depolarized as well. In a pilocarpine-activated animal, the membrane potential showed oscillations in phase with the activity of protractor motor neurons, suggesting that NS 1 might contribute to the transition from swing to stance movement. Second, interneuron NS 2 inhibits motor neurons of protractor and thoracic depressor when depolarized. In both a quiescent and a pilocarpine-activated animal, hyperpolarizing stimuli excite motor neurons of both muscles via disinhibition. In one active animal the disinhibiting stimuli were sufficient to generate swing-like movements of the leg. In pilocarpine-activated preparations the membrane potential oscillated in correlation with the motor neuronal activity of the protractor coxae and thoracic depressor muscle. Hyperpolarizing stimuli induced or reinforced the protractor and thoracic depressor bursts and inhibited the activity of the motor neurons of the retractor coxae muscle, the antagonistic muscle of the protractor. Therefore interneuron NS 2 can be regarded as an important premotor interneuron for the switching from stance to swing and from swing to stance. Finally, interneuron NS 3 inhibits the spontaneously active motor neurons of both motor neuron pools in the quiescent animal. During pilocarpine-induced protractor bursts, depolarizing stimuli applied to the interneuron excited several protractor motor neurons with large action potentials and one motor neuron of the thoracic depressor. No oscillations of the membrane potentials were observed. Therefore this interneuron might contribute to the generation of rapid leg movements. The results demonstrated that the two proximal joints are coupled not only mechanically but also neurally and that the thoracic part of the depressor appears to function as a part of the swing-generating system.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Arthropod walking requires not only the coordination of all legs but also the cooperative interaction of the different joints of each single leg. The mechanisms coordinating the action of all walking legs have been studied to a great extend (lobster: Chasserat and Clarac 1980; Clarac and Chasserat 1986; crayfish: Müller and Cruse 1991; fly: Straubeta and Heisenberg 1990; stick insect: Bässler 1987; Cruse 1990; Dean 1991; cockroach: Delcomyn 1985; for review see Graham 1985).

Considering the control of movements of an individual leg, most investigations dealt either with the integration of sensory inputs (for review see Bässler 1983; Burrows 1985) or with the control of a single joint (e.g., Bässler 1987, 1988; Burrows 1980; Burrows and Siegler 1978; Büschges 1990). The coordination and cooperation of more than one joint has been investigated only a few times in insects and crustaceans (Burrows 1992; Cruse et al. 1992; El Manira et al. 1991; Vedel and Clarac 1979). These investigations referred only to passive movements set by the experimenters and established the existence of reflexes distributed over several joints of one leg. Investigations with active animals were carried out by Nye and Ritzmann (1992) and Karg et al. (1991). The latter described a coupling between the coxa-trochanter and the femur-tibia joint during searching movements of a front leg with a fixed subcoxal joint.

Cruse and Bartling (1995) studied the cooperation of all three proximal leg joints (the subcoxal, coxa-trochanter, and femur-tibia) of the stick insect not only while walking on a treadwheel and on a slippery surface, but also during free walking on a horizontal platform. They did not find any significant difference between free walking and walking on a treadwheel, which corresponds to earlier results (Cruse 1976), but, more importantly, could show that there are no simple rules governing the combined action of two or three joints.

On the neurophysiological level only a few investigations regarding interjoint coordination exist. Local nonspiking interneurons are well known as crucial elements coordinating the activity of different motor neuron pools (for review see Burrows 1981; Hisada et al. 1984; Siegler 1985; Wilson and Phillips 1983), but again only a few interneurons are described that influence the motor neuron pools of several joints during active behavior for both stick insects and locusts. Burrows (1980) described nonspiking interneurons that excite motor neurons of flexor tibiae, tarsal levator, and coxal adductor muscles leading to a movement of the locust rear leg similar to that in normal behavior. Recently Kittmann et al. (1996) described groups of interneurons influencing motor neuron pools of subcoxal and femur-tibia joints during different motor behaviors. All these interneurons were not identified as single distinctive interneurons. Only very few nonspiking interneurons identified by both morphology and physiology have been described in the context of leg movement control. These investigations described interneurons for which as yet effects are found only on motor neurons of the subcoxal joint [e.g., Schmitz et al. (1991), 4 interneurons] or of the subcoxal and femur-tibia joints during walking and standing [Büschges et al. (1994), interneuron E 4; Wolf and Büschges (1995), 6 interneurons including E 4]. Only interneuron E 4 was identified that influenced motor neurons of the two proximal joints, the subcoxal and the coxa-trochanter joint (Büschges 1995). This interneuron excites protractor coxae, levator trochanteris, and extensor tibiae motor neurons (all involved in swing movement) and was shown to have an inhibitory effect on the motor neurons of the (coxal) depressor trochanteris muscle.

This investigation reports on the influence of three identified local nonspiking interneurons on the motor neuron pools that innervate the following two muscles of the proximal leg joints: 1) the protractor (P in Fig. 1C) of the subcoxal joint and 2) the thoracic part of the depressor trochanteris (D in Fig. 1C). (In 2 cases the activity of the motor neurons of the retractor coxae (R) was recorded instead of the depressor motor neurons.)


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  		FIG. 1. Schematic drawing of a stick insect leg (A) and thoracic muscular anatomy of mesothoracic leg as seen from inside (B, C). C: retractor muscle and some smaller muscles are removed to show the 2 parts of (thoracic) depressor and protractor muscle. D, thoracic depressor trochanteris muscle, innervated by nl4a; P, protractor coxae muscle, innervated by nl2c; R, retractor coxae muscle, innervated by nl5a; Cx, coxa; Tr, trochanter. (B and C adapted with permission from P. Igelmund.)

A companion paper (Brunn and Heuer 1998) will deal with the activity of these two motor neuron pools during walking and will describe the influence of an identified spiking interneuron on these neurons. The results of both studies indicate that the thoracic part of the depressor trochanteris muscle can be regarded as part of those muscles that shape the swing movement.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

All experiments were done with adult female stick insects, Carausius morosus, from our colony at the University of Bielefeld, Bielefeld, Germany. The ventral side of the animals was glued firmly to a wooden beam, which was then attached to the top of a plastic wall in the center of an acrylic vessel. At the beginning of each experiment both rear legs and the right middle leg were fixed. The left middle leg and the front legs remained unfixed.

The thoracic cavity was opened by a longitudinal incision in the tergum extending above the third thoracic ganglion. The gut was transsectioned between the second and the third thoracic ganglion and the parts folded over to the rear and front. A steel platform was inserted under the metathoracic ganglion and the tissue wrapping the ganglion was opened and fixed with insect pins. Thereby the ganglion was sufficiently immobilized in most cases. A ring of petroleum jelly (Vaseline) was set around the ganglion's surface and the ganglion sheath was treated first for 2 min with collagenase (Collagenase/Dispase, Boehringer) and then, without rinsing, for 1 min with protease (Sigma Type XIV). Afterwards the ganglion was thoroughly rinsed with saline (modified after Graham and Wendler 1981) and both the substances and the Vaseline were washed away.

For intracellular recording and staining, thin-wall glass microelectrodes (Clark Electromedical Instruments; 1.2 mm OD × 0.94 mm ID) were used. The tips were filled with 5% Lucifer yellow (Janssen Chimica) dissolved in 0.5 M LiCl2 (Stewart 1978). The electrodes were filled with 1 M LiCl2 and had resistances of 30-50 MOmega . All recordings were obtained from the left half of the metathoracic ganglion of a tethered, nonwalking animal. Because soma recordings in the stick insect C. morosus show only very weak synaptic activity (Godden and Graham 1984) and current injection into the somata normally fails to produce an appropriate output, intracellular recordings were made in the neuropile.

To monitor the activity of the motor neurons, extracellular recordings with oil-hook electrodes (Schmitz et al. 1988) were made from the lateral nerve nl2c (which innervates the protractor muscle and either nl4a, which innervates the thoracic depressor) or nl5a, the nerve that innervates the retractor muscle. The intracellular signals were amplified (L/M-1, List Electronic) and stored on a DAT-recorder (Bio-Logic) together with the extracellular recordings. When a neuron that seemed to influence both motor neuron pools was penetrated, excitatory effects were tested by simply depolarizing the interneuron. Inhibitory effects on others than the spontaneously active neurons could only be monitored after activating the animal by touching it's abdomen or head with a paint brush. If an intracellular recording was considered stable enough, the muscarinic agonist pilocarpine, known to produce rhythmic activity in arthropod motor neuron pools, was used (Büschges et al. 1995; Chrachri and Clarac 1990; Ryckebusch and Laurent 1993). The use of this chemical first allows to test for an inhibitory influence of the interneurons on the motor neurons and also to demonstrate whether a rhythmic modulation of the interneurons exists. Although not shown in the figures, the influences of the three interneurons on the tested motor neuron pools always occurred without spikes. Neither tactile stimulation of the animal, high depolarizing currents, nor fast release from strong hyperpolarization caused spikes in these three interneurons.

Each neuron was stained by applying continuous negative current (-5 nA) for >= 5 min. When the physiological tests were completed, the ganglion was dissected, fixed for 1 h in 5% paraformaldehyde (in phosphate buffer; pH 7.3), and then washed thoroughly with distilled water. After dehydration in an ethanol series (70-90-96% absolute alcohol, 10 min each step) and clearing in methylsalicylate, the ganglion was viewed under a fluorescence microscope and photographed at ×100 magnification in 2.5-µm steps from above and also from the side or front. Line drawings were prepared by tracing the projected negatives.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Anatomic situation

In stick insects the femur and the trochanter are connected rigidly, thus forming the trochantero-femur. The trochantero-femur is moved by two muscles, the levator and the depressor trochanteris. The latter consists of two parts, a coxal and a thoracic (Marquardt 1939). The levator trochanteris and the coxal part of the depressor are located completely within the coxa with the tendon of the levator attaching dorsally and that of both parts of the depressor attaching ventrally. The thoracic part of the depressor does not remain in the coxa, it spans the subcoxal joint and inserts in the thorax at the thoracic wall. The thoracic part is innervated by the lateral nerve nl4a (Graham 1985).

Figure 1A shows a schematic drawing of a stick insect leg. The subcoxal joint between coxa and thorax is used mainly to move the leg in forward-backward direction (angle alpha ) and the up-down movement is performed at the coxa-trochanter joint (angle beta ).

The forward-backward movement of the coxa is carried out by the retractor and protractor coxae, two powerful muscles located in the thorax. The protractor coxae is innervated by the lateral nerve nl2c and the retractor coxae by nl5a. The anatomy of the musculature is shown in Fig. 1, B and C.

The thoracic part of the depressor trochanteris innervated by nl4a is the object of this paper and will be referred to hereafter as the "thoracic depressor." The coxal part of the depressor is innervated by nerve C2, which is not investigated here.

Electrophysiological recordings

The three forms of nonspiking interneurons described here could be distinguished from intracellular recordings within the neuropile of the metathoracic ganglion in 56 of 300 different preparations. They were characterized electrophysiologically and subsequently stained with Lucifer yellow. The first type (denoted NS1) was recorded and stained 18 times, the second (NS2) 21 times, and the third (NS3) was recorded in 17 preparations. Only those preparations where the classification was unambiguous (i.e., where only 1 neuron was stained) were taken into consideration. Soma and neuropile of all three types are restricted to the hemiganglion. All neurons belonging to one type resemble each other in morphology and physiology in such a way that each is considered as one identified interneuron.

Interneuron NS 1

The morphology and some of the features of interneuron NS 1 were described previously (Brunn and Dean 1994).

That publication dealt with quiescent animals only and showed the following traits of the interneuron NS 1: 1) it became more excited as the middle and rear leg approach each other, 2) it was depolarized phasically by tactile stimuli to the rear leg, and 3) it excited several retractor motor neurons when depolarized. It was named DML3 (distance measuring local interneuron in segment 3). Hyperpolarizing stimuli had no effect.

To ease visualization of the following results, the morphology of this neuron is shown in Fig. 2. The soma and the complete neuropile of this neuron are restricted to the anterior half of the ganglion.


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  		FIG. 2. Interneuron NS 1. Morphology in dorsal (A) and frontal (B) plane views. Scale bar, 100 µm.


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  		FIG. 3. Interneuron NS 1. Pilocarpine-activated animal. A: modulation of membrane potential of interneuron NS 1 in synchrony with the bursts of the protractor motor neurons (nl2c). A-C, bottom trace: recordings of retractor motor neurons (nl5a). B: interruption. C: total inhibition of a nl2c (protractor) motor neuron burst. Although in B the compensation was not well, in C this interneuron acted without action potentials. Depol, depolarizing stimulus. Scale bar, 1 s. Note: in this and Figs. 4-9 nl2 is always nl2c, nl4 is nl4a, and nl5 is nl5a. Current is always in the range of 1-6 nA with all stimuli starting from 0 level.

PILOCARPINE-ACTIVATED ANIMALS. When pilocarpine was applied, causing a steady rhythm in the motor neuron pools of nl2c (protractor) and nl5a (retractor), which is usually the case (Büschges et al. 1995), the membrane potential of this interneuron was hyperpolarized in synchrony with the nl2c burst (Fig. 3A). If a depolarizing stimulus is set within a nl2c burst, this burst is disrupted (Fig. 3B) or totally inhibited (Fig. 3C). Moreover, this interneuron inhibited some of the nl4a neurons (not shown).

Interneuron NS 2

The soma of interneuron NS 2 is located in the posterior half of the ganglion (Fig. 4A). Some typical branches that distinguished this interneuron from others that look similar are clearly seen in a frontal view (Fig. 4B, left).


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  		FIG. 4. Interneuron NS 2. Morphology in dorsal (A), frontal (B), and sagittal plane view (C). During the course of the experiments some other neurons were stained that looked very similar, but physiology as well as the neurites (drawn separately in B, left) were distinguishing features. A and B, from same preparation; different than C.

This interneuron showed very weak reactions to tactile stimuli to abdomen and all legs. Its influence on the protractor motor neurons was strongly enhanced when pilocarpine was used.

QUIESCENT ANIMALS. In the inactive animal a depolarization of this interneuron inhibited the activity of the spontaneously active unit in nl2c. A rebound effect occurred, sometimes exciting one nl4a neuron (Fig. 5A).


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  		FIG. 5. Interneuron NS 2. A: hyperpolarizing (Hyperpol) and depolarizing (Depol) stimuli in a quiescent (inactive) animal. The hyperpolarizing stimuli disinhibited the spontaneously active motor neuron in nl2c and 1 motor neuron in nl4a. The depolarizing stimuli inhibited the spontaneously active motor neuron in nl2c. At the end of the stimuli a nl4a unit was excited. B: each of the hyperpolarizing stimuli shown generated a swinglike movement of the leg. For clarity the trace of the interneuron is not shown. Scale bars, 1 s. See note in Fig. 3.

Hyperpolarizing this neuron always excited ("disinhibited") one or two neurons in nl2c and one in nl4a (Fig. 5A, 1st 3 stimuli). The disinhibition of the nl2c motor neurons was enhanced when the animal was activated by touching the abdomen with a pencil. In one preparation with the left rear leg unfixed, the activity of the nl4a motor neurons was unusually high. In this situation each of the hyperpolarizing stimuli shown in Fig. 5B generated a swing-like movement of the leg, i.e., femur and coxa moved from a caudal position rostrad (registered by a camcorder) by ~40°.

PILOCARPINE-ACTIVATED ANIMALS. When pilocarpine was applied the disinhibition also excited several nl2c neurons and sometimes one nl4a neuron. Under these circumstances the hyperpolarizing stimuli frequently prolonged the bursts of the nl2c motor neuron pool for the whole length of stimuli (Fig. 6A). Depolarizing stimuli interrupted ongoing bursts of protractor motor neurons and of the thoracic depressor motor neuron (Fig. 6B). Moreover, in 17 of the 21 preparations the membrane potential of this neuron was modulated with the rhythm of the nl2c and nl4a motor neurons (Fig. 6C). In all cases the beginning of the nl4a burst was delayed by ~300 ms relative to the beginning of the nl2c burst. Hyperpolarizing stimuli reinforced the bursts of both motor neuron pools (Fig. 6C, 1st and 3rd stimuli).


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  		FIG. 6. Interneuron NS 2. Pilocarpine-activated animal. A: Hyperpol stimuli induced and prolonged bursts in nl2c. B: during bursting activity in the nl4a motor neurons a Depol of this interneuron inhibited the bursting activity. C: rhythmic modulation of interneuron NS 2 in synchrony with nl2c and nl4a bursts. Hyperpolarizing stimuli prolonged (1st stimulus) or induced (2nd stimulus) bursts. D: recordings of interneuron (2nd trace) together with the protractor motor neurons (1st trace) and retractor motor neurons (nl5; 3rd trace). Hyperpolarizing stimuli interrupted the ongoing activity of the retractor motor neurons and induced bursts in nl2c. Scale bars, 1 s. See note in Fig. 3.

In one preparation extracellular recordings were made from the nl5a instead of the nl4a. The nl5a innervates the retractor coxae muscle. When pilocarpine was added, burst activity first occurred in nl2c and then to a weaker extent in nl5a (not shown). After some time the nl2c burst activity stopped but activity in nl5a continued. Setting pulses of hyperpolarizing current at this time generated bursts in nl2c with the onset of each stimulus and at the same time stopped the nl5a activity (Fig. 6D). In the same preparation three depolarizing pulses (+8 nA) excited some middle-sized nl5a motor neurons (not shown).

Interneuron NS 3

The soma of interneuron NS 3 is situated in the anterior part of the ganglion, and the neuropile extends through nearly all of the hemiganglion (Fig. 7). Like interneuron NS 2 this interneuron showed very weak reaction to tactile stimuli. Oscillations of the membrane potential were observed neither spontaneously nor under the influence of pilocarpine. No effect on the motor neurons was observed when NS 3 was hyperpolarized.


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  		FIG. 7. Interneuron NS 3. Morphology in dorsal (A) and frontal (B) view. Physiology is shown in Figs. 8 and 9.

QUIESCENT ANIMALS. In the inactive animal a depolarization of this interneuron inhibited the activity of the spontaneously active units in nl2c and those of small size nl4a (Fig. 8A). After a delay of ~280 ms, one large nl4a neuron was activated. Frequently one nl4a unit and up to four nl2c units were activated immediately after the stimulus. Both effects, the inhibition and the activation via a rebound, were dependent on the strength of the stimulus. Sometimes, when a series of stimuli of the same strength was given, the influence changed with the number of the stimuli (Fig. 8B).


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  		FIG. 8. Interneuron NS 3, quiescent animal. A: in an animal that was not activated, depolarizations inhibited the activity of the spontaneously active protractor and depressor motor neurons and excited a large-size motor neuron in nl4a. After the stimulus several motor neurons in nl2c and 1 depressor unit with slightly larger spikes were excited. B: series of stimuli. The 1st 2 stimuli are weaker than the following 4 stimuli. Scale bars, 1 s. See note in Fig. 3.

PILOCARPINE-ACTIVATED ANIMAL. When pilocarpine was added the effect of a depolarizing stimulus given at a time between two nl2c bursts was the same as in a preparation without pilocarpine, namely the spontaneously activated motor neurons with small- and middle-sized spikes were inhibited (Fig. 9A, 1st stimulus). If, however, the depolarizing stimulus was set during an ongoing nl2c burst, several units of large size were excited also (Fig. 9A). To a weaker extent this can already be seen at the end of the first stimulus. (In a preparation without pilocarpine, units of this size were not excitable even with currents of >12 nA.) No such effect was visible in nl4a. The stimulus had either no effect at all or the effect was the same as in a preparation without pilocarpine, i.e., a large unit was excited during the stimulus (Fig. 9B). The strength of the influence on the nl4a motor neurons varied from animal to animal. In a more active animal the influence on the nl4a motor neurons was much stronger (not shown).


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  		FIG. 9. Interneuron NS 3. Effects on the motor neurons in pilocarpine-activated animals. A: 1st depolarization had the same effect as a depolarization in an animal that was not activated (compare with 4th stimulus in Fig. 8B). The following 2 stimuli were set during the bursts and excited the large protractor motor neurons. B: sometimes a large unit in nl4a (3rd trace) was also excited. In this recording from a different preparation from that in A, the difference between nl2c motor bursts with and without depolarization is clearly evident. Scale bars, 1 s. See note in Fig. 3.

In two preparations with a steady rhythm of the nl2c neurons, a depolarizing stimulus was set shortly after the beginning of every third or fourth burst and the number of large-sized units was counted for all bursts. The frequency of the large-size spikes increased from 5.07 ± 4.45 (SD) spikes/s; (15 bursts without stimuli) to 68.8 ± 20.27 (SD) spikes/s; (10 bursts with depolarizing stimuli) in one preparation and from 15.22 ± 8.51 (SD) spikes/s; (9 bursts without stimuli) to 134 ± 17.74 (SD) spikes/s; (5 bursts with depolarizing stimuli) in the second preparation.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

During walking each leg repeatedly performs a step pattern consisting of alternating swing and stance movements. At stance phase the leg is contacting the surface and moves from an anterior position back to push the body forward. During the swing phase the leg is lifted off the ground and returns to a position in front. Depending on whether an insect is walking forward, backward, upside down, on a vertical platform, etc., all the muscles contributing to leg movements are coordinated in slightly different patterns (Duch and Pflüger 1995).

Little is known about the functional role of the thoracic depressor muscle innervated by nl4a. Because of its geometric orientation, Graham (1985) suggested that this muscle might contribute to both the protraction and the retraction. For this reason we investigated interneurons innervating this muscle together with the protractor muscle (or the retractor muscle).

Interneuron NS 1

In a previous paper (Brunn and Dean 1994) this interneuron was classified as a neuron that demonstrates the following properties. 1) It measures both the angle between coxa and thorax of the middle leg and the angle between coxa and thorax of the rear leg. The more these two (ipsilateral) legs approach each other, the more depolarized the membrane potential. 2) The neuron produced strong phasic depolarizations when the rear leg was touched. 3) Depolarizing stimuli excited some of the nl5a (retractor) motor neurons. The conclusion that this neuron plays a role in terminating the swing movement is further supported by the finding that it inhibits the protractor motor neurons (Fig. 2, B and C). No such simple explanation can be given for the inhibition of the thoracic depressor motor neurons, at least not at first glance. This will be discussed below.

The use of pilocarpine made it possible to show the inhibitory influence of interneuron NS 1 on the thoracic depressor and protractor motor neuron pools. Besides the two known depolarizing influences (Brunn and Dean 1994) arising from proprioceptors of the middle and rear legs, pilocarpine experiments revealed a third centrally generated component that influences the membrane potential of this interneuron. If this central influence is also active during walking, it would strongly counteract the depolarizing influence of the sense organs from middle and rear leg at the beginning of the swing. However, toward the end of the swing movement the hyperpolarizing influence would decrease and the depolarizing influence of the sense organs would increase.

Considering the sensory inputs as well as the effects on the two antagonistic coxal muscles, this local interneuron might be able to influence the transition of swing to stance phase by terminating the swing movement and contributing (to a weaker extent) to the start of the stance.

Interneuron NS 2

Like NS 1, this interneuron also affects both antagonistic coxal muscles. But this time it is a disinhibition that excites the protractor and thoracic depressor motor neurons (Fig. 6) and simultaneously inhibits the retractor motor neurons.

Morphology and physiology of this interneuron is very similar to a local interneuron described by Schmitz et al. (1991). During walking the membrane potential of the interneuron was modulated with the motor output of the protractor and retractor in the same way as shown in this study under the influence of pilocarpine. The interneuron showed a strong hyperpolarization during protractor bursts, and depolarizing stimuli---only those were applied---affected the protractor activity in the same way as the interneuron described in this study; they terminated the activity of the protractor. Schmitz at al. (1991) concluded that this interneuron "is an important premotor element that can shape the motor pattern during walking to a great extent." The result of my investigation showed that this interneuron is able to control the motor output of these two muscles (protractor and retractor coxae) and also that of the thoracic depressor muscle, not only while depolarized but also while hyperpolarized, via disinhibition.

Interneurons that excite motor neurons via disinhibition have been described earlier by Burrows and Siegler (1978) and by Simmers and Bush (1980). Both groups reported a nonspiking interneuron inhibiting one single motor neuron when depolarized and enabling the same neuron to spike when hyperpolarized. In both cases single motor neurons were affected. Interneurons affecting more than one motor neuron of antagonistic muscles via inhibition and disinhibition were described by Mendelson (1971) for the ventilatory system of crustaceans. Simultaneous inhibition and disinhibition of several motor neurons is evident in these three and another publication (DiCaprio 1989). Apart from these four, only a few other publications described disinhibition as part of the motor system of arthropods (Burrows and Pflüger 1988; Büschges and Schmitz 1991; Nagayama and Hisada 1987; Ramirez and Pearson 1989).

Recently disinhibition was described for the olfactory system of Manduca sexta (Christensen et al. 1993). Modeling this system demonstrated that disinhibition as a mechanism of neuronal networks is useful as an effective mechanism for neuronal excitation, especially neuronal bursting (Av-Ron and Rospars 1995; Av-Ron and Vibert 1996).

A strong disinhibition like that described here was never before shown for the motor system of the stick insect. Furthermore, no interneuron has hitherto been described that when activated artificially produced movements of a leg. As was seen in one preparation with an animal in a rather active state, a hyperpolarizing stimulation of this interneuron alone was sufficient to generate a swinglike movement of the leg.

Interneuron NS 3

In both the quiescent and the pilocarpine-activated animal, interneuron NS 3 inhibited the spontaneously active small-sized protractor motor neurons. However, excitation of protractor motor neurons by depolarizing this interneuron was never observed in quiescent animals but only in preparations in which pilocarpine induced repetitive bursts (Fig. 9).

Büschges (1995) described a nonspiking interneuron in the mesothoracic ganglion with a similar morphology. In the pilocarpine-activated preparation he showed that depolarizing the interneuron interrupted bursts of the retractor motor neurons and excited protractor motor neurons. However, he did not stimulate during an ongoing protractor burst and the exciting effect he observed was weak, eliciting only a few small-sized spikes in nl2c.

In addition to the inhibiting effect on the spontaneously active motor neurons the results presented here show that the stimulating effect via depolarization to the protractor motor neurons is much more effective when pilocarpine is applied. Obviously pilocarpine enhances the activity level of some motor neurons, which then produce the known bursting rhythm as well as of others that do not spike until another supplementary influence is active. In an actively walking animal, such additional influences may come either from descending influences via interganglionic interneurons and/or from sensory inputs from the same leg and from all other legs (for review see Bässler 1987). A reinforcement of the excitability of motor neurons to the synaptic influence of sensory channels was described by Trimmer and Weeks (1993). They showed that in the tobacco hornworm M. sexta, oxotremorine-M, another agonist of acetylcholine, increased the firing rate of a motor neuron and the threshold for spikes became more negative.

At first glance it might appear contradictory that this interneuron inhibited and excited motor neurons of the same muscle. But by comparing the effects each stimulus caused it is obvious that the inhibiting influence concerns only motor neurons with smaller action potentials as shown in Fig. 9A. The first stimulus shown in Fig. 9A inhibited the tonically active neurons, but at the end of the stimulus a large-sized neuron was excited.

The tonic activity of small-sized slow, excitatory motor neurons contributes little to shortening of muscles. They are mainly involved in the tonic control of posture and frequently innervate the same muscle or muscle fibers as common inhibitory motor neurons do (Bässler and Storrer 1980; Pearson and Iles 1971). A decrease in the activity of the tonically active excitatory motor neurons before the start of muscle contraction increases the rate of muscle relaxation, thus facilitating rapid contraction (relaxation cycles produced by fast fibers), a mechanism similar to that of common inhibitory motor neurons (Iles and Pearson 1971; Wiens and Rathmayer 1985; Wolf 1990).

This interneuron might be involved in the generation of rapid swing movements by removing the tonic tension of muscle fibers and exciting the fast and powerful motor neurons.

The same type of interneuron was recorded and stained in the mesothoracic ganglion. It also inhibited (when depolarized) the tonically active motor neurons in nerve nl2c and nl4a and the slow extensor tibia motor neuron in nerve F2. The same stimuli excited the fast extensor tibia motor neuron, a large motor neuron in nl2c, and the common inhibitor CI 1 (Schneider 1996).

Conclusions

The three local interneurons NS 1, NS 2, and NS 3 all influence the protractor coxae and the thoracic depressor muscles in the same way, i.e., neurons of both motor neuron pools are either excited or inhibited simultaneously (see Fig. 10). This is in contrast to what is known from the coxal part of the depressor trochanteris; for that reason it merits some further consideration.


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  		FIG. 10. Schematic summary of influence of all 3 local interneurons on the motor neurons of protractor and retractor coxae and on those of the thoracic depressor. bullet , exciting influence; |, inhibiting influence.

The coxal part of the depressor trochanteris muscle, innervated by nerve C2, is mainly active during the stance phase (Epstein and Graham 1983). Together with the retractor coxae it provides support and propulsion for the animal's body during walking (Graham 1985). Consequently, when investigating protractor and thoracic depressor motor neuron pools I expected to find interneurons influencing these pools in an opposing rather than in an assisting way. Interneuron E 4, for instance, is involved in generating the swing movement by exciting protractor coxae, levator trochanteris, and extensor tibiae motor neurons and is known to inhibit the motor neurons of the coxal depressor (Büschges 1995).

However, as shown by Cruse et al. (1993) the activity of the nl4a motor neurons during forward walking started with the lifting of the tarsus at the posterior extreme position and increased during the swing movement. In the investigation of Cruse et al. (1993) the protractor activity was not recorded. As will be shown in the companion paper (Brunn and Heuer 1998), the time course of the thoracic depressor activity in all three legs (front, middle, and rear) follows that of the protractor, reaching a peak shortly after that of the protractor activity.

Our provisional conclusion is that the thoracic depressor functionally belongs to the swing generating muscles. Thus a nearly simultaneous inhibition (or excitement) of both motor neuron pools by the same interneuron is sensible.

An investigation we started recently with animals walking on a treadwheel before and after removal of the nl4a supports this assumption. The results of the first experiments, carried out by T. Akay (unpublished observations), a student in our lab, show that removing the nl4a and thus depriving the thoracic depressor from its innervation does not at all lead to an elevated trajectory that would be expected by a trochantero-femur depressor. In most animals the reverse happened; the swing height measured at the proximal end of the tibia was lower after the removal of the nl4a.

    ACKNOWLEDGEMENTS

  This work was supported by Deutsche Forschungsgemeinschaft Grants Cr 58/9-1 and Cr 58/8-3 to Prof. Holk Cruse.

    FOOTNOTES

  Received 20 March 1997; accepted in final form 13 January 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society



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