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.

View larger version (24K):
[in this window]
[in a new window]

View larger version (18K):
[in this window]
[in a new window]
| 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).

View larger version (25K):
[in this window]
[in a new window]
| 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).

View larger version (59K):
[in this window]
[in a new window]
| 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).

View larger version (35K):
[in this window]
[in a new window]

View larger version (37K):
[in this window]
[in a new window]
| 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.
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).

View larger version (49K):
[in this window]
[in a new window]
| 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).

View larger version (34K):
[in this window]
[in a new window]
| 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 |
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.

View larger version (30K):
[in this window]
[in a new window]
| 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. , 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.