Zoological Institute, University of Cologne
Gruhn, M., Rosenbaum, P., Bollhagen, H., Bueschges, A. Studying the Neural Basis of Adaptive Locomotor Behavior in Insects. J. Vis. Exp. (50), e2629, doi:10.3791/2629 (2011).
Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch & Pflüger, 1995; Böhm et al., 1997; Gruhn & Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.
1. The Walking Surface
The black walking surface is made up of two nickel coated brass plates permanently joined side by side and electrically insulated from one another by a 2mm wide strip of 2-component epoxy glue (UHU plus, UHU GmbH, Germany) directly underneath the longitudinal axis of the animal. They produce a total surface area of 13.5x15.5cm (Figure 1). The separation of the half-planes for left and right legs allows independent tarsal contact monitoring for a single leg on each side.
2. Optical Stimulation Setup
3. Preparing the Experimental Animal
4. Placement of EMG Electrodes
Muscle activity of different leg muscles such as, in this example, the protractor and retractor coxae muscles, which are located in the thorax and cause protraction (forward movement) and retraction (backward movement) of the leg, is recorded by means of EMG electrodes implanted into the thorax. All EMG recordings are differentially amplified. The signal was amplified 100x in a preamplifier (electronics workshop, Zoological Institute, Cologne), band-pass filtered, (100Hz-2000Hz), further amplified (10x), and imported through an AD converter into spike2 (Vers.5.05, CED, Cambridge, UK).
5. Recording of the Tarsal Contact
Tarsal contact is always recorded together with EMG traces. Since the slippery surface is split into two halves, a maximum of two contralateral tarsal contacts can be registered at the same time. For this purpose, the leg is used as a "switch" to close a circuit between the plate and the amplifier. Current can be applied to the plates separately through two sockets at the base of each plate. We can generate two square wave signals with 2-4mV amplitudes with a pulse generator (Model MS501, electronics workshop, Zoological Institute, Cologne) that are phase-delayed by 90°. The signals are attenuated by 60dB (/1000) and applied to the two halves of the slippery surface. In this experiment we only use one of the two signals. It is simultaneously fed into a lock-in-amplifier as a reference signal. The 2-phase lock-in-amplifier (electronics workshop, Zoological Institute, Cologne) selectively amplifies only signals of the same frequency and phase as the provided reference signal. A second channel only detects the 90° phase delayed signal. Noise signals at frequencies other than the reference frequency are rejected and do not affect the measurement. The amplifier output signal is fed into an AD converter (Micro 1401k II, CED, Cambridge, UK) and digitalized using Spike2. Between touch-down and lift-off of the tarsus onto and from the slippery surface, current flows from the plate through tarsus and tibia into the copper wire (see below: 5.3-5.8). The chosen amplifier has a high input resistance (1MΩ) and the signal voltage is very small in order to avoid affecting the walking behavior of the animal. The current passing through tarsus and tibia is between 2 and 4nA. The signal lead time during touch-down, between the entry into the lubricant and the surface contact is less than 1ms. The contact signal transition during lift-off is less steep and more delayed. This is due to the delayed tearing of the lubricant from the tarsus by a capillary rise effect and due to movement of the leg without complete lift-off in swing phase.
6. Optical Recording of Leg Movements for Digital Analysis
The stepping frequency of stick insects on slippery surfaces can reach 3-4Hz (Graham and Cruse, 1981).
7. Recording of Walking Sequences
8. Representative Results:
Figure 1. Experimental setup; A: photograph of a stick insect tethered above the slippery surface, marked with fluorescent pigments for leg tracking and wired for EMG recording and tarsal contact measurement. B: wiring diagram for dual leg tarsal contact recording during walking on the slippery surface.
Figure 2. Positions for the placement of EMG electrodes in the protractor and retractor muscles in the thorax of the stick insect middle leg. ML: middle leg; FL: front leg
Figure 3. Stick insect with EMG wires in the pro- and retractor muscles of the middle leg in place; front and hind legs have been removed to study the effect of the presence of neighboring legs on the kinematics and muscle activity of the middle legs in the reduced preparation.
Figure 4. Example trace of retractor EMG activity with reference signal of tarsal contact in an inside middle leg during turning. At the beginning of the trace (first blue box) the retractor is active during stance, while the leg has ground contact ("closed circuit"), indicating that the leg makes forward steps; after a short switch (second blue box), the retractor is active during swing, while the leg is in the air ("open circuit", third blue box), showing the occurrence of backwards directed steps.
Figure 5. Screen shot of a recording from the protractor (top trace) and retractor (middle trace) muscles and the simultaneously recorded tarsal contact trace (bottom trace). Note the alternating activity in the EMG traces which corresponds to the activity of the two muscles in the step cycle, that is protractor activity just before and during swing, when the tarsal contact trace signals loss of ground contact, and retractor activity after and during touch-down of the tarsus, when leg is in stance. The insert shows the matching video file corresponding to this recording that is used to verify the behavior of the animal.
We have described a setup that allows the optically induced generation of turning behavior and permits to a large degree the uncoupling of neuronally generated walking activity from the passive effects caused by the mechanical displacement of the other walking limbs on the ground. Potential information flow between the legs through the nervous system about ground reaction forces or tarsal contact, on the other hand, is still possible and allows the experimenter to study the influence of such information in the reduced preparation. Major advantages of the slippery surface setup include that the animals show a very high tendency to walk, and contrary to walking or stepping on a treadwheel, the animal can perform swing and stance phase movements in all the directions of natural walking. In addition, the degrees of freedom for all the legs allow the animal to perform curve walking whether it is an intact or semi-intact preparation. Because the legs cannot be passively moved simply by the forward movement of the animal or the movement of the substrate underneath, every movement reflects the motor output of that leg (Cruse, 1976; Graham and Wendler, 1981). The setup is highly suitable to investigate the neuronal basis of adaptive behaviors such as turning or forward vs. backward walking, because one can combine electrophysiological recordings of motor activity with the analysis of limb movement kinematics.
We used the stick insect's optomotor response to elicit walking. The responses of the animals to the rotating stripe pattern show their readiness to perform curve walking while tethered over the slippery surface. Most surprisingly for us, single legs in single-leg preparations qualitatively show the same moving pattern as in the intact animal. We thus have reason to believe that the control of curve walking can function largely without coordinating sensory input from neighboring legs. It will be important to test in further experiments whether the activity of the motor neurons of the removed legs is also influenced by the optomotor pattern. The setup can easily be modified to allow the study of other tasks such as straight forward and backward walking by placing a single stripe in front of the animal or gently pulling the antennae.
The precise measurement of ground contact allows us to correlate muscle activity and leg position. The high time resolution of this electric contact signal is better than 1ms and leads us to a new insight into the timing of the switch from swing to stance phase. The resolution is worse for the stance to swing transition due to the delay in shearing of the conducting lubricant and a lack of need for a complete lift-off during protraction on the slippery surface. Nevertheless, the knowledge on the precise swing to stance transition is a particularly useful first step if we want to understand the mechanisms that control muscle timing and the coordinated activities of leg muscles in different behavioral contexts (see also: Büschges et al., 2008; Büschges & Gruhn 2008).
As an example, we used the retractor and protractor coxae muscle of the middle leg and precisely correlated its activity with the switching from swing to stance phase while we simultaneously monitored the behavioral context in which the leg was used. For this purpose, we induced walking and recorded the muscle activity continuously. A given leg can be an inside or an outside leg, depending on the turning direction. In the stepping middle leg, acting as an inside leg in the functional sense, it can be observed that retractor and protractor muscles can both work as functional stance muscles because the leg intermittently produces backward steps in addition to forward directed steps (see Fig.4).
The electromyograms (EMGs) from both muscles were rectified and normalized to the time of touch-down and the latency of the first muscle spikes was calculated. Interestingly, the latencies of both muscles with respect to lift-off and touch-down depend on the function of the muscle as respective swing or stance muscle (see Fig.4) and not on the muscle itself, and show only minor alterations in the timing of activity onset. Most explanations for the change of state from swing to stance assume that sensory signals of tarsal contact trigger the start of stance. The interesting question of how the short latencies between touch-down and muscle activation in the stick insect are brought about and on what sensory information they depend can now be addressed with the modified setup.
In summary, we show a slippery surface setup that allows us to reliably elicit straight and curve walking in stationary stick insects. Kinematics, muscle activity and the timing of tarsal touch-down and lift-off can be monitored and correlated in the two different behavioral contexts at the same time. This gives us an excellent tool to study the detailed connection between muscle activity and behavioral context for a single leg as well as in the intact animal and the underlying mechanisms.
No conflicts of interest declared.
We thank Michael Dübbert, Oliver Hoffmann, Hans Scharstein, Jan Sydow and Anne Wosnitza for excellent technical assistance. This study was supported by DFG grant Bu 857/8,10 to A.B.
|2-component epoxy glue||UHU plus, UHU GmbH, Germany|
|glass screens (diameter 130mm)||Linos Photonics|
|dental cement||ProTemp II, 3M ESPE, Seefeld, Germany||3M Id : 70-2011-0358-0 Catalog Number : 46430||Available through 3M (http://www.3m.com/)or dental suppliers|
|fluorescent pigments||Dr. Kremer Farbmühle, Aichstetten, Germany||Cat.#s: i.e 56200 Fluorescent Pigment Golden Yellow 56350 Fluorescent Pigment Flame Red||http://kremer-pigmente.de/en or http://www.kremerpigments.com/|
|histoacrylic glue||3M||supplier: WPI|
|coated copper wire||Elektrisola Eckernhagen||http://www.elektrisola.com/|
|electrode cream||Marquette Hellige, Freiburg, Germany||Product is now discontinued, we suggest for example: www.grasstechnologies.com|
|pulse generator||Model MS501, electronics workshop, Zoological Institute, Cologne, Germany|
|AD converter||Cambridge Electronic Design|
|high speed video camera||Allied Vision Technologies|
|UV LED arrays||Zoological Institute||30-50V DC, electronics workshop
|Digitalizing software Spike2||Vers.5.05, CED, Cambridge, UK|
|motion tracking software||Mikromak service, Berlin, Germany||WINanalyze, Vers.1.9|