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During walking, the leg motor system must continually adjust to changes in mechanical conditions, such as the inclination of the ground. To understand the underlying control, it is important to know how changes in leg muscle activity relate to leg kinematics (movements) and leg dynamics (forces, torques). Here, we studied these parameters in hindlegs of stick insects (Carausius morosus) during level and uphill/downhill (±45deg) walking, using a combination of electromyography, 3D motion capture and ground reaction force measurements. We find that some kinematic parameters including leg joint angles and body height vary across walking conditions. However, kinematics vary little compared with dynamics: horizontal leg forces and torques at the thorax-coxa joint (leg protraction/retraction) and femur-tibia joint (leg flexion/extension) tend to be stronger during uphill walking and are reversed in sign during downhill walking. At the thorax-coxa joint, the different mechanical demands are met by adjustments in the timing and magnitude of antagonistic muscle activity. Adjustments occur primarily in the first half of stance after the touch-down of the leg. When insects transition from level to incline walking, the characteristic adjustments in muscle activity occur with the first step of the leg on the incline, but not in anticipation. Together, these findings indicate that stick insects adjust leg muscle activity on a step-by-step basis so as to maintain a similar kinematic pattern under different mechanical demands. The underlying control might rely primarily on feedback from leg proprioceptors signaling leg position and movement. © 2019. Published by The Company of Biologists Ltd.

[Figure][1] A stick insect ( Carausius morosus ). Photo credit: Department of Biological Cybernetics, Bielefeld University. Anyone who has ever visited the historic city of Cambridge, UK, will know that something is missing: hills. Residents rarely experience the challenge of negotiating

The femoral chordotonal organ (fCO), one of the largest proprioceptive sense organs in the leg of the stick insect, is important for the control of the femur-tibia joint during standing and walking. It consists of a ventral scoloparium with about 80 sensory cells and a dorsal scoloparium with about 420 sensory cells. The present study examines the function of these scoloparia in the femur-tibia control loop. Both scoloparia were stimulated independently and the responses in the extensor tibiae motoneurones were recorded extra-and intracellularly. The ventral scoloparium, which is the smaller of the two, functions as the transducer of the femur-tibia control loop. Its sensory cells can generate the known resistance reflexes. The dorsal scoloparium serves no function in the femur-tibia control loop and its stimulation elicited no or only minor reactions in the extensor motoneurones. A comparison with other insect leg proprioceptors shows that a morphological subdivision of these organs often indicates a functional specialization.

A treadwheel mounted on a torque motor was used to study changes in step coordination as a function of the magnitude and direction of external forces resisting and assisting leg retraction. The apparent contradiction in the literature concerning the shift in the posterior extreme point is shown to depend upon the magnitude of the load. External forces resisting leg retraction generally shift step end-points rostrally and those assisting leg retraction shift them caudally, shifting leg movement so that the active muscles can provide greater force. However, this primary trend reverses for external forces equivalent to or less than the weight of the body. Together, these results indicate that the decision to end stance does not simply depend upon the leg's loading but rather on its efficiency in contributing propulsion. As expected, loads resisting leg retraction decrease retraction speed and increase step period. They also decrease protraction speed, increase swing duration and shift phase relationships of adjacent ipsilateral legs away from alternation towards a metachronal pattern. Moderate to large assisting loads evoke similar changes: they increase swing duration less but shift the phase of middle and rear legs more than the corresponding resisting loads. Thus, by itself, a reduction in the force required during retraction does not transform the gait from metachronal to tripod. The mean phase of contralateral leg pairs is unaffected by load: these legs continue to alternate.

The course of the common apodeme of the tripartite retractor unguis muscle is described for the stick insects Carausius morosus and Acrophylla wulfingii. This apodeme travels through the femoro-tibial joint well outside the axis of rotation of the joint, but movements of the femoro-tibial joint do not affect the position of the tarsal claws, which are moved by the retractor unguis muscle. The independence of tarsal position upon tibial position is not produced by a neural compensation mechanism but by a sophisticated morphological arrangement combined with specialized physiological properties. These mechanisms consist of two parts. (1) Moderate claw flexions are mainly produced by the smaller tibial parts of the muscle and their influence on claw position is not affected by tibial position, because they lie distal to the femoro-tibial joint. (2) The retractor unguis muscle works against strong elastic structures and the claw assumes the position where the elastic force is counterbalanced by the muscle force. The maximum muscle force of the strong femoral part of the muscle is nearly independent of muscle length. Therefore, the force it transfers to the elastic structures is also nearly independent of tibial position.

In the stick insect Carausius morosus (Phasmida) intracellular recordings were made from local nonspiking interneurones involved in the reflex activation of the extensor motoneurones of the femur-tibia joint during ramp-like stimulation of the transducer of this joint, the femoral chordotonal organ (ChO). The nonspiking interneurones in the femur-tibia control loop were characterized by their inputs from the ChO, their output properties onto the extensor motoneurones and their morphology. Eight different morphological and physiological types of nonspiking interneurones are described that are involved in the femur-tibia control loop. The results show that velocity signals from the ChO are the most important movement parameter processed by the nonspiking interneurones. Altering the membrane potential of these interneurones had marked effects on the reflex activation in the extensor motoneurones as the interneurones were able to increase or decrease the response of the participating motoneurones. The processing of information by the nonspiking pathways showed another remarkable aspect: nonspiking interneurones were found to process sensory information from the ChO onto extensor motoneurones in a way that seems not always to support the generation of the visible resistance reflexes in the extensor tibiae motoneurones in response to imposed flexion and extension movements of the joint. The present investigation demonstrated interneuronal pathways in the joint-control loop that show ‘assisting’ characteristics.

The mechanisms by which the legs of a stick insect influence one another during walking were investigated by running the animals on a horizontal plane covered with a thin film of silicone oil to prevent mechanical coupling between the legs. Coupling between ipsilateral legs was investigated by interrupting the retraction (power stroke) of a leg for a short time and observing how the legs return to normal coordination following this disturbance. The results show that three ipsilateral coupling mechanisms exist: (a) a forward-directed influence that inhibits the start of a protraction of the leg as long as the posterior leg is performing a protraction; (b) a forward-directed influence that excites the start of a protraction of the leg when the posterior leg starts a retraction movement; (c) a backward-directed influence that excites the start of a protrac-tion, the influence being stronger the further the anterior leg has moved backwards during its retraction. The latter influence depends on the position but not the phase of the anterior leg.

Behavioral studies of stick insects have identified six mechanisms which coordinate leg stepping. All six are active between ipsilateral leg pairs. As a first step towards locating the neurons mediating these interactions, the present study describes the effects of cutting one of the paired thoracic connectives. After the operation the following changes in step coordination occurred. The ipsilateral leg immediately caudal to the severed connective generally showed weak stepping. In free-walking animals it often remained near its posterior extreme position and dragged along the substratum. During supported walking, rhythmic stepping was common, but the swing phase of this leg was longer and both temporal and spatial coordination were disturbed. When the leg made a pause it usually stopped in the air near the end of its swing movement. During steady walking, the operation interrupted information from the adjacent forward leg normally used to guide the end-point of the swing or to signal errors in leg placement and elicit a corrective treading-on-tarsus reflex. It also interrupted position information affecting the start of the swing. For the leg rostral to the cut, the inhibition during the swing of the posterior leg and the excitation when the latter started its retraction were both interrupted. These results indicate that all six ipsilateral coordination mechanisms are primarily mediated by the ipsilateral connective. In addition, the data show that contralateral coordination within the segmental ganglion is strongest for the front legs, weaker for the rear legs, and not discernible for the middle legs.

Interactions between contralateral legs of stick insects during walking were examined in the absence of mechanical coupling between the legs by studying animals walking on a horizontal plane covered with a thin film of silicone oil. Investigations of undisturbed walks showed that contralateral coupling is weaker than ipsilateral coupling. Two types of influence were found, (i) For each pair of front, middle and rear legs, when one leg started a retraction movement, the probability for the contralateral leg to start a protraction was increased, (ii) For front- and hind-leg pairs, it was found that the probability of starting a protraction in one leg was also increased, the farther the other leg was moved backwards during retraction. Whether such influences exist between middle legs could not be determined. Both ‘excitatory’ mechanisms very much resemble those influences which have been found to exist between ipsilateral legs. However, in contrast to ipsilateral legs, the interaction between two contralateral legs was found to act in both directions.

The apodeme of the femoral chordotonal organ of a middle leg can be moved from its normal position close to the extensor tendon and inserted into a clot cut in the flexor tendon. This inverts the output of the sense organ and produces a ‘wrong’ afference. During walking on a pair of light wheels the operated leg either makes walking steps or is raised and extended in a ‘salute’ posture. The coordination is similar to that for an intact animal if the operated leg walks but changes to the middle leg amputee timing when the operated leg ‘salutes’. The transitions between saluting and the normal walking behaviour of the operated leg can be explained if it is assumed that the animal depends heavily upon the C.O. for determining the tibia position during both walking and the saluting behaviour. Motor output to the levators and depressors of the femur and the protractors and retractors of the coxa shows bursting activity during the salute at the frequency of 3–4 Hz. The depressor bursts are also modulated at a frequency of ∼ 1 Hz and produce strong regular depressions of the femur which are co-ordinated with the movements of the other legs. The maintenance of regular depressor contractions during the salute shows that an important part of the motor output to the saluting leg (depressor activity) arises from an internal oscillation or rhythmic command which maintains its co-ordinated activity when the normal peripheral sensory input to the leg it is attempting to operate is absent. Retractor activity wanes during the salute suggesting that propulsion is much more dependent upon peripheral input than is the support musculature (depressors), The creation of a ‘wrong’ afference can be used to map the importance of the operated organ in different sub-units of behaviour.

In the stick insect Carausius morosus, the role of the chordotonal organ was investigated using a new experimental arrangement which artificially closes the femur-tibia control system. The chordotonal organ was stimulated during voluntary movements by applying trapezoidal ramp stimuli in the closed-loop configuration. The results demonstrate that the feedback loop is used to control the end points of joint movement. In addition, it was found that the control system counteracts experimentally applied velocity changes imposed during the middle part of the movements. Acceleration-sensitive units are shown to contribute to the reaction. The results show that during active voluntary movements reflexes measured in the inactive animal are neither simply incorporated in a servo-system nor suppressed. Instead their characteristics are altered so that the voluntary movements are executed as intended by the animal. Thus reflexes cannot be considered as a fixed behavioural unit; rather their changing role must be analysed in the context of the behaviour studied.

Anterior extreme position (AEP) and posterior extreme position (PEP) of the legs of stick insects were measured during walking on a treadwheel or on a slippery glass plate. In several experiments, either protraction or retraction of a middle or hind leg was interrupted. The AEP of other legs was independent of a protraction interruption but PEP was displaced backward in the leg anterior to the interrupted leg. When a leg was standing on a fixed platform (interruption of retraction) no changes were found in AEP and PEP for the other legs but if the platform was slowly moved, PEP of leg on the platform was moved forward. These results disagree with several published hypotheses. The results suggest the hypothesis of a position-dependent threshold value for protraction which is modulated by co-ordinating influences from other legs.

The coxal leg-stump of a stick insect was rotated about the coxa-thorax joint, by means of a pen-motor, while the animal walked on a self-propelled double treadwheel. Motor activity in the retractor muscles of the legs was recorded for standing and walking animals with the stump of either the middle or hind leg moved forward and backward in a trajectory similar to that used in a walking step. In a standing animal the movement of either leg evokes a weak resistance reflex. If the animal walks with the middle leg-stump held still, then short and weak motor bursts are generated with the periodicity of the walking legs. Front and hind legs alternate in a manner typical of the middle leg amputee and the retractor muscle of the amputated leg is most active during the power stroke of the leg behind. When a middle leg-stump is moved at a different frequency from that of the walking legs, the motor output to the retractor is strongly modulated and depends on the relative timing of the stump and the walking legs. Rearward movement of the hind leg-stump, during walking, is always accompanied by strong motor output in the retractor muscle. In addition, forward movement in this leg-stump produces a resistance reflex similar to that produced in the standing animal.

The denervated thoracic ventral nerve cord produces a motor output which is similar to that observed in the intact animal during irregular leg movements (seeking movements) or rocking, but not walking. When the nerves to some legs are left intact, and the animal walks on a wheel, the motor output in the protractor and retractor motor neurones of the denervated legs is modulated by the stepping frequency of the walking legs. The modulation is similar to that observed in the motor output to a not actually stepping leg of an intact walking animal. When only the crural nerve of one leg is left intact, stimulation of the trochanteral campaniform sensilli induces protractor and retractor motor output to that leg and the leg behind it. In this case the motor output to the ipsilateral leg is in phase. Stimulation of the femoral chordotonal organ influences activity in motor neurones of the extensor tibiae (FETi and SETi) but not those of the protractor and retractor coxae muscles. In a restrained leg of an intact animal stretching of the femoral chordotonal organ excites FETi and SETi as long as the other legs walk (as in a walking leg) and inhibits FETi and SETi (as in a seeking leg) when the other legs are unable to walk.

The femoral chordotonal organ (ChO) of the right middle leg of the inactive stick insect Carausius morosus was stimulated by applying movements having a ramp-like time course, while recordings were made from local and interganglionic interneurones in the anterior ventral median part of the ganglion. Position, velocity and acceleration of the movements were varied independently and the interneurones were categorized on the basis of their responses to the changes in these parameters. Position-sensitivity was always accompanied by responses to velocity and/or acceleration. Velocity-sensitive responses were excitatory or inhibitory and were produced by elongation or relaxation, or by both. In some cases, velocity-sensitive neurones were also affected by position and acceleration. Acceleration responses were always excitatory and were often found in neurones that showed no effects of velocity or position. It is inferred that sensory input from different receptors in the ChO is processed by single interneurones. No interneurone in the recording region was found to be directly involved in the resistance reflex of the extensor tibiae motoneurones, elicited by stimulation of the ChO.

The use of fluorescent dyes for staining neurones has become very common since the introduction of Lucifer Yellow (Stewart, 1978) because it permits high-luminance stainings, for detailed structural analysis, with a minimum of filling time. Lucifer Yellow has been used in a variety of techniques in neurophysiology: in vitro visualization (Stewart, 1978); killing of neurones or parts of neurones (e.g. Miller & Selverston, 1979; Miller & Jacobs, 1984); and in vivo visualization (e.g. Reichert & Krenz, 1986; Kater & Hadley, 1982). We describe a new system for in vivo and in vitro visualization. It can be used with a standard dissecting microscope, features a very simple optical design and costs much less than other systems used previously.

In treadwheel walking, the front and middle legs of the stick insect (Carausius morosus) propel the wheel, while the hind legs exert very little force and can even decelerate the wheel. This result is compared with observations on the function of the legs in different walking situations. Several hypotheses assuming different types of position servo-mechanisms have been proposed in the literature on the control of leg movement during walking in insects. The experimental results support none of these hypotheses. Instead, they indicate that velocity rather than position is the variable controlled during the stance phase.

An experimental arrangement was constructed which is based on the open-loop femurtibia control system of two stick insect species (Carausius morosus and Cuniculina impigra). It could be artificially closed in the following way: the position of the tibia was measured by an optical device and this value was used to drive a penmotor which moved the receptor apodeme of the femoral chordotonal organ in the same way as in intact animals. This arrangement allows direct comparison of the behaviour of the open-loop and the closed-loop system as well as introducing an additional delay. The Carausius system has a phase reserve of only 30° –50° and the factor of feedback control approaches 1 between 1 and 2 Hz. This agrees with the observation that an additional delay of 70–200 ms produces long-lasting oscillations of 1–2 Hz. The Cuniculina system has a larger phase reserve and consequently a delay of 200 ms produced no oscillations. All experiments show that extrapolation from the open-loop system to the closed-loop system is valid, despite the non-linear characteristics of the loop. Consequences for servo-mechanisms during walking and rocking movements are discussed.

In the haemolymph of the stick insect Carausius morosus the concentration of potassium exceeds that of sodium and the concentration of magnesium exceeds that of calcium. The implications of this situation for nerve conduction have been studied. Conduction is maintained in intact and desheathed preparations of the fourth adbominal ganglion under irrigation with a solution resembling haemolymph in ionic composition. Action potentials recorded in response to electrical stimulation of the nerve cord decline in sodium-free solutions, both in intact and in desheathed preparations. Conduction declines slowly under irrigation with magnesium-free solutions both in intact and in desheathed preparations.

The distribution and exchange of inorganic ions between the central and the haemolymph has been studied in the stick insect, Carausius morosus, by flame photometry and radioactive tracers. The exchanges of labelled ions show rapid and slow components which correspond to extracellular and intracellular compartments within the central nervous system. The uptake of sodium from the haemolymph and its concentration in the extracellular fluid is reduced in the presence of metabolic inhibitors. The distribution between haemolymph and extracellular fluid of calcium and magnesium, and also of sodium in poisoned preparations, conforms to a Donnan equilibrium. The distribution of potassium, even in poisoned preparations, does not conform and it is suggested that the activity of this ion may be lower than in free solution. The concentration of magnesium is appreciably greater in the extracellular than in the intracellular compartment. The possible role of magnesium in nervous transmission in this insect is discussed.

5-Hydroxytryptamine (5-HT) and a few of its derivatives will cause an acceleration of secretion by the isolated Malpighian tubules of Rhodnius prolixus and Carausius morosus. Substitution in the terminal amino group of 5-HT causes little loss of effect, but changes in the indole ring and the hydroxy group in general abolish activity. Most derivatives of 5-HT which do not stimulate secretion, and many other related amines, act as inhibitors of secretion in Rhodnius. This inhibition is probably of the competitive type. Substances which stimulate secretion in Rhodnius are easily washed off the Malpighian tubules, although they will stimulate secretion at very low concentrations By contrast, competitive inhibitors wash away relatively slowly and inhibit only at much higher concentrations. It is suggested therefore that stimulation involves only momentary interactions between stimulant and receptor sites, but that competitive inhibitors spend considerably longer attached to the sites. Cyclic 3 ′,5 ′ -adenosine monophosphate (cyclic AMP) will stimulate secretion by tubules of both Rhodnius and Carausius. Tubules of both species are very sensitive to cyclic AMP, measurable effects being produced at concentrations of 4×10−5 M/ l in Rhodnius and 10−4 M/ l in Carausius. Aminophylline (theophylline ethylene diamine) at a concentration of 10−4 M/ l stimulates secretion by Carausius tubules. This compound and other inhibitors of phosphodiesterase have no stimulatory action on Rhodnius tubules, but any such effect may be masked by the adverse reaction of the tubules to the high concentrations necessary. Inhibitors of 5-HT action such as tryptamine and tyramine also inhibit secretion by Rhodnius Malpighian tubules induced by cyclic AMP. This may be due to a dependence of the action of cyclic AMP on the state of the diuretic hormone receptor site. The evidence is consistent with the idea that 5-HT, other stimulant molecules related to 5-HT and the diuretic hormones interact with Malpighian tubule cells of Rhodnius and Carausius at specific sites, probably on the cell membrane facing the haemolymph. As a result of this, secretion is induced, possibly through the action of intracellular cyclic AMP produced as a response.

The effects of variation in the sodium concentration of the bathing media on axonal function has been measured in de-sheathed connectives in the presence of the overlying neural fat-body sheath. The response to solutions of the same sodium concentration as the haemolymph (15 mM/1) was found to be essentially similar to that recorded in de-sheathed connectives in the absence of the fat-body sheath, there being a rapid decline in amplitude of the recorded action potentials in both preparations. On the basis of these observations it is concluded that the neural fat-body sheath is unlikely to be involved in the regulation of the extra-neuronal sodium level.