Tethered stick insect walking: A modified slippery surface setup with optomotor stimulation and electrical monitoring of tarsal contact

Abstract

A modified and improved setup based on Epstein and Graham [Epstein S, Graham D. Behaviour and motor output of stick insects walking on a slippery surface. I. Forward walking. J Exp Biol 1983;105: 215–29] to study straight and curve walking in the stick insect was developed and applications for its use are described. The animal is fixed on a balsa stick and walks freely on a slippery surface created with a thin film of a glycerin/water solution on a black, Ni-coated, polished brass plate. The glycerine/water ratio controls the viscosity of the lubricant and thereby the forces necessary to move the legs of the stick insect. A small amount of NaCl is added to ensure electric conductivity. Walking is induced through an optomotor stimulus given by two stripe-projectors producing rotatory and translatory stimuli to influence walking direction. The walking pattern is monitored in two ways: (1) tarsal contact with the slippery surface is measured electrically using a lock-in-amplifier. The tarsal contact signal allows correlation with the activity in different muscles of the stick insect leg recorded with EMG electrodes; (2) leg kinematics in the horizontal plane is monitored using synchronized high speed video. This setup allows us to determine the coupling of activity in different leg muscles to either swing or stance phase during straight and curve walking in the intact animal or the reduced single-leg preparation with a high time resolution.

Introduction

Walking is the most common form of movement among terrestrial animals. Despite their stereotypical appearance, walking movements show a high degree of variability. In order to gain basic knowledge on the function of different walking systems it is therefore of great importance to understand the neuronal mechanisms that control walking movements and their exact timing which is not apparent from studying either kinematics or muscle activity alone.

The walking stick insect, Carausius morosus has been one of the animal systems that have been very useful for understanding basic principles of joint and leg coordination. It performs a wide range of movements that share major similarities with those performed by vertebrates such as the cat (Büschges, 2005, Pearson, 2004). Additionally, due to the relatively small size and the accessibility of its nervous system, an in depth analysis of the underlying networks that control the single joints is easier than in mammals (Büschges, 2005, Bässler and Büschges, 1998).

The step cycle of the leg of an animal, e.g. stick insect, can be divided into stance and swing phase. During stance the leg is on the ground and, for example during forward walking, the body is displaced anteriorly relative to the position of the tarsus. During swing the leg is lifted off the ground, extended and moved to the front to start a new stance phase (Graham, 1985, Jander, 1982, Wendler, 1964).

A fundamental problem when studying the control mechanisms during walking results from the difficulty in assessing the effects of sensory influences (both, local and intersegmental) and the influences of central neural networks as well as coupling through ground contact in the walking animal (for review see, e.g. Bässler and Büschges, 1998, Grillner, 1981). A solution to this problem for the stick insect walking system was achieved when the single walking leg preparation was introduced by Bässler (e.g. Bässler, 1993, Karg et al., 1991). In this preparation all legs but the one under investigation are amputated at the level of the mid-coxa. The removal of the other legs also removes their sensory organs and, as a consequence, abolishes their sensory driven coordinating intersegmental influences (Cruse et al., 2004) onto the leg in focus. The remaining leg, be it a front, middle, or a hind leg can execute stepping movements on a specially designed treadwheel (Bässler, 1993, Fischer et al., 2001). This preparation allows the investigation of the neural mechanisms underlying the generation of the walking motor output for the single leg and its motoneurons in the absence of coordinating influences from the neighboring legs (e.g. recent review in Büschges, 2005, Bässler, 1993, Bucher et al., 2003, Gabriel et al., 2003, Gabriel and Büschges, in press, Schmidt et al., 2001) and contributed important insights in developing a conceptual model for a neural controller for insect walking (Ekeberg et al., 2004).

One important limitation for this preparation, however, arises from the fact that the stepping movements of the leg investigated are restricted in one plane (for discussion see Fischer et al., 2001, Gabriel et al., 2003). Thus, it does not permit the investigator to study single leg stepping during adaptive walking patterns like curve walking. In order to understand such adaptive behaviors, it is necessary to work in a setup that allows their induction, and on a preparation in which the leg has the degrees of freedom that resemble the in vivo situation in free walking. Therefore, a preparation is needed where it is possible to reliably elicit curve walking, correlate neuro and muscle physiological events of a leg with its behavior, and precisely measure the timing of the single stepping movements.

In order to develop such a preparation we turned to studies by Graham and co-workers (Epstein and Graham, 1983, Graham, 1981, Graham and Cruse, 1981, Graham and Epstein, 1985) who systematically investigated the role of substrate coupling on walking pattern generation in the stick insect. Graham (1981) could show on paired independent treadwheels that the movements of tethered animals were the same as those of freely walking ones. When substrate coupling between the single legs is removed during tethered walking on a mercury surface (Graham and Cruse, 1981), or an oiled glass surface (Epstein and Graham, 1983, Graham and Epstein, 1985), the precise coordination of the legs is maintained. Interestingly, the analysis of phase relationships between the legs when using a slippery surface with silicon oil of a suitable viscosity showed an extremely high concentration of phase values (Epstein and Graham, 1983). Yet, in all the studies cited, the difficulty remains that sensory input to all legs has an impact on the motor activity in all of the other legs even when subtrate coupling is absent. In addition, especially when using the mercury surface, extremely low shearing forces and hazardous conditions can also represent a problem. A combination of the slippery surface preparation with the single leg approach would resolve this problem.

The use of the slippery surface has been extensively used in studying tethered cockroach walking (Camhi and Levy, 1988, Camhi and Nolen, 1981, Tryba and Ritzmann, 2000a, Tryba and Ritzmann, 2000b), and also extended to backward walking for the stick insect (Graham and Epstein, 1985). On the other hand, curve walking in arthropods, including stick insects, has been investigated to a much lesser extent (Cruse and Saavedra, 1996, Dürr and Ebeling, 2005, Franklin et al., 1981, Jander, 1982, Land, 1972, Mu and Ritzmann, 2005, Strauss and Heisenberg, 1990, Zolotov et al., 1975), and so far no systematic studies of curve walking on the slippery surface have been published. It has been known for a long time that curve walking in insects can be induced optically by moving stripe patterns (Burrows and Rowell, 1973, Dvorak et al., 1975, Götz and Wenking, 1973, Jander, 1982, Rowell, 1971a, Rowell, 1971b). Studying curve walking can give insights into the mechanisms that control changes in the coordination of different joints as these are used in a new behavioral context. During free curve walking in the stick insect, the anterior part of the body is pulled into the curve by the inner pro- and mesothoracic legs, while the outer pro- and mesothoracic legs push the body. The metathoracic legs both push slightly against the turning direction. Jander (1982) described the changes in phase relationships of the single joints in detail. The insect achieves the necessary differences of moving speeds between inner and outer legs through variation of the step length. In sharp turns additional differences in step frequency occur (Jander, 1982, Dürr and Ebeling, 2005).

We further advanced a slippery surface design by Epstein and Graham (1983). We have adapted the setup in order to be able to use all the degrees of freedom of the leg, and to produce a high temporal and spacial resolution of the recording in the single-leg preparation as well as in the intact animal. We used an optomotor stimulus to elicit straight and curve walking thereby also improving the tendency to walk. At the same time we monitored the behavior with high speed video, and the tarsal contact electrically. The animal lacks intersegmental feedback through being tethered and through the lack of substrate coupling due to the slipperiness of the walking surface. This method allows us to study muscle activity in single legs of a freely moving, tethered stick insect during different behaviors, and to correlate the recordings with the precise timing of touchdown as well as the position and movement of the leg.