Leg Coordination during Walking in Insects

Locomotion depends on constant adaptation to different requirements of the environment. An appropriate temporal and spatial coordination of multiple body parts is necessary to achieve a stable and adapted behavior. Until now it is unclear how the neuronal structures can achieve these meaningful adaptations. The exact role of the nervous system, muscles and mechanical constrains are not known. By using preparations in which special forms of adaptations are considered under experimental conditions that selectively exclude external influences, like mechanical interactions through the ground or differences in body mass, one can draw conclusions about the organization of the respective underlying neuronal structures. In the present thesis, four different publications are presented, giving evidence of mechanisms of temporal or spatial coordination of leg movements in the stick insect Carausius morosus and the fruit fly Drosophila melanogaster during different experimental paradigms. At first, state dependent local coordinating mechanisms are analyzed. Electromyographic measurements of the three major antagonistic leg muscle pairs of the forward and backward walking stick insect are evaluated. It becomes evident that only the motor activity of the most proximal leg joint is changed when walking direction is changed from forward to backward, which demonstrates that the neuronal networks driving movement in each individual leg seem to be organized in a modular structure. In the second part mechanisms that influence movement speed of the individual leg and coordination of speed between the different legs of the stick insect come into focus. Electrophysiological and behavioral experiments with the intact and reduced stick insect were used to examine relationships between the velocity of a stepping front leg and neuronal activity in the mesothoracic segment as well as correlations between the stepping velocities of different legs during walks with constant velocity or with distinct accelerations. It was shown that stepping velocity of single legs were not reflected in motoneuron activity or stepping velocity of another leg. Only when an increase in walking speed was induced, clear correlation in the stepping velocities of the individual legs was found. Subsequently, the analysis of changes in temporal leg coordination during different walking speeds in the fruit fly reveals that the locomotor system of Drosophila can cover a broad range of walking speeds and seems to follow the same rules as the locomotor system of the stick insect. Walking speed is increased by modifying stance duration, whereas swing duration and step amplitude remain largely unchanged. Changes in inter-leg coordination are gradually and systematically with walking speed and can adapt to major biomechanical changes in its walking apparatus. In the final part it was the aim to understand the role of neuronal mechanisms for the orientation and spatial coordination of foot placement in the stick insect. Placement of middle and hind legs with respect to the position of their respective rostrally neighboring leg were analyzed under two different conditions. Segment and state dependent differences in the aiming accuracy of the middle and hind legs could be shown, which indicate differences in the underlying neuronal structures in the different segments and the importance of movement in the target leg for the processing of the position information. Taken together, common principles in inter-leg coordination where found, like similarities between different organisms and segment specific or state dependent modifications in the walking system. They can be interpreted as evidence for a highly adaptive and modular design of the underlying neuronal structures

Decomposition of 3D joint kinematics of walking in Drosophila melanogaster

Animals exhibit a rich repertoire of locomotive behaviors. In the context of legged locomotion, i.e. walking, animals can change their heading direction, traverse diverse substrates with different speeds, or can even compensate for the loss of a leg. This versatility emerges from the fact that biological limbs have more joints and/or more degrees of freedom (DOF), i.e. independent directions of motions, than required for any single movement task. However, this further entails that multiple, or even infinitely many, joint configuration can result in the same leg stepping pattern during walking. How the nervous system deals with such kinematic redundancy remains still unknown. One proposed hypothesis is that the nervous system does not control individual DOFs, but uses flexible combinations of groups of anatomical or functional DOFs, referred to as motor synergies. Drosophila melanogaster represents an excellent model organism for studying the motor control of walking, not least because of the extensive genetic toolbox available, which, among others, allows the identification and targeted manipulation of individual neurons or muscles. However, their tiny size and ability for relatively rapid leg movements hampered research on the kinematics at the level of leg joints due to technical limitations until recently. Hence, the main objective of this dissertation was to investigate the three-dimensional (3D) leg joint kinematics of Drosophila during straight walking. For this, I first established a motion capture setup for Drosophila which allowed the accurate reconstruction of the leg joint positions in 3D with high temporal resolution (400 Hz). Afterwards, I created a kinematic leg model based on anatomical landmarks, i.e. joint condyles, extracted from micro computed-tomography scan data. This step was essential insofar that the actual DOFs of the leg joints in Drosophila were currently unknown. By using this kinematic model, I have found that a mobile trochanter-femur joint can best explain the leg movements of the front legs, but is not mandatory in the other leg pairs. Additionally, I demonstrate that rotations of the femur-tibia plane in the middle legs arise from interactions between two joints suggesting that the natural orientation of joint rotational axes can extent the leg movement repertoire without increasing the number of elements to be controlled. Furthermore, each leg pair exhibited distinct joint kinematics in terms of the joint DOFs employed and their angle time courses during swing and stance phases. Since it is proposed that the nervous system could use motor synergies to solve the redundancy problem, I finally aimed to identify kinematic synergies based on the obtained joint angles from the kinematic model. By applying principal component analysis on the mean joint angle sets of leg steps, I found that three kinematic synergies are sufficient to reconstruct the movements of the tarsus tip during stepping for all leg pairs. This suggests that the problem of controlling seven to eight joint DOFs can be in principle reduced to three control parameters. In conclusion, this dissertation provides detailed insights into the leg joint kinematics of Drosophila during forward walking which are relevant for deciphering motor control of walking in insects. When combined with the extensive genetic toolbox offered by Drosophila as model organism, the experimental platform presented here, i.e. the 3D motion capture setup and the kinematic leg model, can facilitate investigations of Drosophila walking behavior in the future

Speed-dependent interaction of sensory signals and local, pattern-generating activity during walking in Drosophila

Locomotion in six legged insects requires effective mechanisms for inter-leg coordination. Such mechanisms could be realized by mechanical coupling of the legs via the ground during stance phase, by direct connections between the rhythm generating networks in the ventral nerve cord (VNC) of these animals, or they could rely on an intersegmental exchange of phasic sensory feedback. This thesis investigates the role of local pattern-generating networks and inter-leg sensory influences for the generation of rhythmic motor activity during walking at different speeds in Drosophila. For this purpose, a series of already existing techniques was used in combination for the first time in the model organism Drosophila melanogaster. Single leg amputation was used to reduce sensory feedback of one leg allowing the residual stump to move freely and thereby providing insight into the rhythmic activity of motor pattern-generating networks in the VNC. This approach has already been used to investigate the control mechanisms of walking in several animals, including stick insects (e.g. Wendler, 1964) and cockroach (e.g. Delcomyn, 1988). In the present thesis, oscillation periods, phases, and absolute inter-segmental intervals of movements in the intact legs and single leg stumps were quantified in tethered flies walking on top of an air-cushioned ball. A similar setup has previously been used for several other animals such as cockroaches (Spirito and Mushrush, 1979) and also Drosophila (Seelig and Jayaraman, 2013; Seelig et al., 2010). High-speed video analysis of the walking behavior was performed manually (Strauss and Heisenberg, 1990; Wosnitza et al., 2013) as well as in semi automatic fashion (Branson et al., 2009; Mendes et al., 2013). The nan[36a] mutant (Kim et al., 2003), which has defective chordotonal organs, was used to investigate the influence of sensory feedback from chordotonal organs in the intact legs on movements of the stump. Consistent with findings in cockroaches and stick insects rhythmic oscillatory movements were found for stumps of single front, middle and hind legs during tethered walking in Drosophila. The stumps oscillated with a frequency of approximately 10 Hz that was largely consistent for the whole range of recorded walking speeds. Intact legs showed step periods of 100 ms only during relatively fast walking, thus, during slow walking sequences multiple stump oscillations were found for one step period of the intact legs. Consequently, the phase relation between stumps and intact legs was very variable at low walking speeds. Nevertheless, preferred absolute time intervals were found between intact leg liftoff and subsequent levation or depression onset in the stump, even if the frequency of stump oscillations was much higher than the step frequency of intact legs. With increasing walking speed the stump oscillations became highly coordinated with respect to the intact legs. Interestingly, the transition range to strong coordination occurred at the point where the stepping period in intact legs becomes very similar to the base frequency of the stump oscillations. Single middle leg stumps of nan[36a] mutant flies showed the same high frequency oscillations that were found during experiments with wild type flies. The stumps oscillated almost independently of walking speed with a movement period of about 100 ms. In contrast to wild type flies stump oscillations in the mutant flies failed to entrain to the stepping behavior of the intact legs at high walking speeds and the absolute time intervals between liftoff events in intact legs and subsequent onset of levation or depression in the stump were more variable. These results lead to the following four conclusions: First, a putative descending control of walking speed does not target the rhythm generating networks directly but it probably has an indirect influence by changing the gain factor of sensory signals, for instance. Second, if the relatively high frequency of stump oscillations reflects a high natural frequency of the investigated pattern generating networks this would facilitate the coordination at high walking speeds, where precise coordination is very important. Fourth, coordinating signals from the intact legs influences the stump movements even during slow walking, but it is probably more effective during fast walking where the stump shows a cycle to cycle coupling to the intact legs. This indicates a stronger inter-leg coordination at high walking speeds. Fourth, the chordotonal organs in the intact legs play an important role for this coordination. During the second part of this thesis the speed range and activity pattern of intact Drosophilae and single leg amputees were studied during voluntary untethered walking. For this purpose, a behavioral paradigm was created that allowed for the study of walking behavior in nine individual flies in separate petridish enclosures. Additionally, software was developed to provide a semi- automatic analysis of the recorded videos. It was found that compared to intact animals the occurrence of walking speeds above 5 mm/s was strongly reduced in amputees. During voluntary untethered walking hindleg amputees showed the highest speed range of all amputees. A reduced level of walking activity was found in frontleg and hindleg amputees, whereas middleleg amputees showed the same probability to walk as the intact animals. The flies walked in short bouts of mostly less than two seconds. As previously shown in the literature (Martin, 2004; Valente et al., 2007) the probability for fast walking was higher in the center of the walking arena compared to the area close to the wall of the enclosures, where the flies spend most of the time. In any case walking activity was only found for a maximum 30 % of the recorded time (in R2 amputees)

Intra- and Intersegmental Coordination among Central Pattern Generating Networks in an Insect Walking System

In der thorakalen Ganglienkette der Stabheuschrecke Carausius morosus gibt es verschiedene neuronale Netzwerke, die eine oszillatorische Aktivität in Motoneuronen (MN) auslösen können und die als zentralen Mustergeneratoren (Central Pattern Generators, CPGs) bezeichnet werden. Diese treiben antagonistische Muskeln an den Beingelenken an. Für die Beinkoordination während des Gehens ist eine korrekte Phasenkopplung zwischen Gelenk-CPGs notwendig. Die CPG-Kopplung kann durch intersegmentale Signale und lokale sensorische Rückkopplung vermittelt werden. Es können jedoch auch zentrale Mechanismen existieren und zur CPG-Kopplung beitragen. Hier analysierte ich die Synchronisation, Phasendifferenz und Korrelation zwischen der Aktivität der contralateralen und ipsilateralen Depressor MN-Gruppen der deafferentierten thorakalen Ganglien, als Proxy für die intra- und intersegmentale Kopplung zwischen Coxa-Trochanter (CTr) Gelenk-CPGs, die den Depressor-Muskel von C. morosus antreiben. Ich habe herasusgefunden, dass eine Tendenz zu in- und anti-phasischer Aktivität zwischen kontralateralen Depressor MN Gruppen in den isolierten Meso- bzw. Metathorakalganglien besteht, dass es dagegen keine Hinweise auf eine koordinierte Aktivität zwischen den beiden Hälften des isolierten Prothorakalganglions gibt. In den miteinander verbundenen Ganglien wird die Koordination der Aktivität zwischen contralateralen Depressor MN Gruppen durch intersegmentale Einflüsse modifiziert. Ipsilaterale Depressor-MN-Gruppen der verbundenen meso- und metathorakalen Ganglien sind ebenfalls phasengekoppelt. Diese ipsilaterale Koordination wird darüber hinaus modifiziert, wenn alle drei thorakalen Ganglien miteinander verbunden sind. Die contralaterale Kopplung der Aktivität von Depressor MN Gruppen wird durch die Durchtrennung entweder der hinteren Kommissuren oder eines der Konnektive beeinflusst, jedoch nicht vollständig zerstört. Intrazellulär, zeigt die Depressor MN Aktivität dagegen keine mit dem kontralateralen Depressorzyklus korrelierte Modulation und eine Stimulation des Depressors MN beeinflusst die kontralaterale Aktivität nicht. Zusammenfassend zeigen die Ergebnisse dieser Arbeit in einem Insektenpräparat, dem die phasische sensorische Eingänge fehlen, eine schwache Kopplung zwischen den CTr-Gelenk-CPGs, die die Depressor MN Gruppen antreiben. Die intra- und intersegmentalen Phasenbeziehungen zwischen den MN Gruppen sind jedoch nicht den Mustern ähnlich, die in einer sich verhaltenden Stabheuschrecke beobachtet werden. Daraus kann man schlussfolgern, dass eine zentrale CPG-Kopplung alleine nicht ausreichend ist, um eine Beinkoordination während des Gehens zu erreichen. In einem Nebenprojekt fand ich, dass Oszillationen im Membranpotential von Protractor MN bestehen bleiben, nachdem spannungsaktivierte Na+-Kanäle unter Verwendung des nicht-selektiven Blockers QX 314 blockiert wurden. Dies deutet darauf hin, dass diese Oszillationen nicht auf Aktionspotential-bezogenen ionischen Mechanismen basieren

Serotonergic Modulation of Walking Behavior in Drosophila melanogaster

Walking is an essential behavior across the animal kingdom. To navigate complex environments, animals must have highly robust, yet flexible locomotor behaviors. One crucial aspect of this process is the selection of an appropriate walking speed. Speed shifts entail not only the scaling of behavioral parameters (such as faster steps) but also changes in coordination to produce different gaits, and the details of how this switch occurs are currently unknown. Modulatory substances, particularly small biogenic amine neurotransmitters, can alter the output and even the connectivity of motor circuits. This work addresses the hypothesis that one such neuromodulator – serotonin (5HT) – is a key regulator of walking speed at the level of motor circuitry. To explore this question, I use the model organism Drosophila melanogaster which, like vertebrates, displays complex coordinated locomotion at a wide range of speeds. In Chapter 2, I will describe our efforts to characterize the anatomy of the serotonergic cell populations that provide direct input to motor circuitry. I find that innervation of the neuropil of the ventral nerve cord - a structure roughly analagous to the mammalian spinal cord - is provided primarily by local modulatory interneurons. Using stochastic single cell labeling techniques, I will detail the specific anatomy of individual neuromodulatory cells, and also the distribution of synapses across their processes. In Chapter 3, I will show that optogenetic activation or tonic inhibition of VNC serotonergic neurons produces opposing shifts in walking speed. To analyze behavior, I will use two complementary approaches. On the one hand, I will use an arena assay to holistically assess walking velocity and frequency. On the other, I will use a behavioral assay developed in the lab - the Flywalker - to assess walking kinematics at high resolution. The combination of these technique will give us a broad and specific picture of how the VNC serotonergic system modulates walking. In Chapter 4, I will identify natural behavioral contexts under which serotonin is used to shift walking behavior. I will use a variety of paradigms that induce animals to shift their speed, from changes in orientation and nutrition state, to pulses of light, odor, and a vibration. I will assess the requirement for the VNC serotonergic system under all of these conditions, to build a clearer picture of its role in modulating behavioral adaptation. In Chapter 5, I will describe our efforts, in collaboration with Pavan Ramdya's lab at EPFL, to functionally image VNC serotonergic cells while the animal is walking, to understand how activity is endogenously regulated in this population. Finally, in Chapter 6 I will characterize the circuit elements which might be responsible for serotonin's effect on walking. I will use recently developed mutant lines to identify the particular serotonergic receptors responsible for enacting shifts in walking behavior. Using genetic labeling tools, I will identify potential targets of serotonergic signaling in the VNC, and formulate a model by which action on these targets could adjust locomotor output. Altogether, this work seeks to characterize the anatomy and behavioral role of the VNC serotonergic system in Drosophila. I hope that through this work, I will gain a deeper understanding of not only this particular modulatory system in this particular behavioral context, but also of how static circuits are conferred with essential flexibility in behaving animals

Adaptive Motor Control: Neuronal Mechanisms Underlying (Targeted) Searching Movements

Animals move through a complex environment and therefore constantly need to adapt their behavior to the surroundings. For this purpose, they use sensory information of various kind. As one strategy to gain tactile cues, animals perform leg searching movements when loosing foothold. The kinematics of these searching movements have been well investigated in the stick insect. In this thesis, the modification of stick insect searching movements following a tactile cue are explored as an example of a sensory-motor system that adapts to environmental conditions. Furthermore, the premotor neuronal network underlying the generation of searching behavior is investigated. Searching movements were studied in animals with a single intact leg that was free to move in the vertical plane. After several cycles of searching movements, a stick was introduced into the plane of movements such that animals would touch it with its distal leg. As is known from previous studies, in such a situation stick insects try to grasp the object that they touch. In my experiments, the stick was retracted as soon as a brief contact with the animals' leg had occurred. Therefore, animals could not grasp the stick. I could show that following this short tactile cue, stick insects modify their searching movements to target the former position of the object (PO). Targeting occurs by a change in two parameters of searching movements: animals (i) shift the average leg position of their searching movements towards the PO and (ii) confine searching movements to the PO by a reduction in movement amplitude. These two parameters, position and amplitude, can be changed independently of each other. Searching movements are flexibly adjusted to different locations of the object which demonstrates the targeted response to be a situation-dependent adaptive behavior. The targeted response outlasts the tactile stimulus by several seconds suggesting a simple form of short term memory of the PO as proposed for targeted movements of other insects. Vision is not necessary for a targeted response. Instead, tactile cues from leg sensory organs are important. Two proprioceptive organs, the trochanteral hairplate (trHP) and the femoral chordotonal organ (fCO), are crucial for targeting. Other sensory organs like tactile hairs and campaniform sensilla are dispensable. The brain is not necessary for a targeted response, therefore the adaptation of searching movements is likely to be mediated on the thoracic level. The premotor neuronal network underlying searching movement generation was investigated using the same single-leg preparation as described above. Nonspiking interneurons (NSIs) of the premotor network were recorded intracellularly during searching movements. Additionally, EMG recordings of the four main leg muscles that generate searching movements in the vertical plane were recorded. The membrane potential of previously described, as well as newly identified NSIs providing synaptic drive to leg motoneurons is shown to be phasically modulated during searching. Therefore, NSIs are part of the premotor network for the generation of searching movements. NSIs that were previously described to contribute to the generation of walking behavior are shown to contribute to the generation of searching behavior. When artificially de- or hyperpolarized by current injection, several NSIs are able to induce changes in searching movement parameters like position, amplitude, velocity of movements, or inter-joint coordination. One NSI is able to drive or stop searching movements. Each NSI acts on a specific set of parameters. The same NSIs that were recorded during searching also were recorded during walking behavior. In comparison, NSI membrane potential modulations during searching are smaller in amplitude and more undulated than during walking. In contrast, fast transitions in NSI membrane potential are closely coupled to step phase transitions during walking. The most prominent difference in NSI membrane potential occurs during step phase (when walking) as compared to flexion phase (during searching). This difference might be attributed to load signals from campaniform sensilla. Analogous to results of previous studies in the stick insect, this highlights the importance of sensory feedback in shaping the motor output. Finally, NSIs were recorded intracellularly while animals with their searching leg made contact with the stick that was introduced into the plane of movement. First results indicate that the response of a given NSI to this contact is characteristic and depends on the direction of touch

Encoding of Coordinating Information in a Network of Coupled Oscillators

Animal locomotion is driven by cyclic movements of the body or body appendages. These movements are under the control of neural networks that are driven by central pattern generators (CPG). In order to produce meaningful behavior, CPGs need to be coordinated. The crayfish swimmeret system is a model to investigate the coordination of distributed CPGs. Swimmerets are four pairs of limbs on the animal’s abdomen, which move in cycles of alternating power-strokes and return-strokes. The swimmeret pairs are coordinated in a metachronal wave from posterior to anterior with a phase lag of approximately 25% between segments. Each swimmeret is controlled by its own neural microcircuit, located in the body segment’s hemiganglion. Three neurons per hemiganglion are necessary and sufficient for the 25% phase lag. ASCE DSC encode information about their home ganglion’s activity state and send it to their anterior or posterior target ganglia, respectively. ComInt 1, which is electrically coupled to the CPG, receives the coordinating information. The isolated abdominal ganglia chain reliably produces fictive swimming. Motor burst strength is encoded by the number of spikes per ASCE and DSC burst. If motor burst strength varies spontaneously, the coordinating neurons track these changes linearly. The neurons are hypothesized to adapt their spiking range to the occurring motor burst strengths. One aim of this study was to investigate the putative adaptive encoding of the coordinating neurons in electrophysiological experiments. This revealed that the system’s excitation level influenced both the whole system and the individual coordinating neurons. These mechanisms allowed the coordinating neurons to adapt to the range of burst strengths at any given excitation level by encoding relative burst strengths. The second aim was to identify the transmitters of the coordinating neurons at the synapse to ComInt 1. Immunohistochemical experiments demonstrated that coordinating neurons were not co-localized with serotonin-immunoreactive positive neurons. MALDI-TOF mass spectrometry suggested acetylcholine as presumable transmitter

Investigating Sensorimotor Control in Locomotion using Robots and Mathematical Models

Locomotion is a very diverse phenomenon that results from the interactions of a body and its environment and enables a body to move from one position to another. Underlying control principles rely among others on the generation of intrinsic body movements, adaptation and synchronization of those movements with the environment, and the generation of respective reaction forces that induce locomotion. We use mathematical and physical models, namely robots, to investigate how movement patterns emerge in a specific environment, and to what extent central and peripheral mechanisms contribute to movement generation. We explore insect walking, undulatory swimming and bimodal terrestrial and aquatic locomotion. We present relevant findings that explain the prevalence of tripod gaits for fast climbing based on the outcome of an optimization procedure. We also developed new control paradigms based on local sensory pressure feedback for anguilliform swimming, which include oscillator-free and decoupled control schemes, and a new design methodology to create physical models for locomotion investigation based on a salamander-like robot. The presented work includes additional relevant contributions to robotics, specifically a new fast dynamically stable walking gait for hexapedal robots and a decentralized scheme for highly modular control of lamprey-like undulatory swimming robots

Segment specificity of muscular and neuronal control in insect walking

Locomotion depends on an interplay of neuronal activity, muscle contraction, and sensory feedback. In the limbed animal, the individual legs of the different thoracic segments, as well as the individual leg segments of each leg, and their antagonistic muscles, have to be coordinated with each other. For the stick insect, it is well known that the legs of the pro-, meso- and metathoracic segments have different functions in the straight walking and turning animal (Cruse 1976, Grabowska & Godlewska et al. 2012, Gruhn et al. 2009b). Despite of this, most studies on stick insect walking focused on the mesothoracic legs and their motoneuronal output (Bässler et al. 1996, Borgmann et al. 2007, Guschlbauer et al. 2007, Fischer et al. 2001, Bucher et al. 2003, Gruhn et al. 2016). With my thesis, I wanted to contribute to increase the knowledge for other legs of the stick insect Carausius morosus. Therefore, I investigated thorax- and leg-segment specific differences on the muscular and the neuronal level. In the first part of my thesis, I used mATPase histochemistry to investigate the muscle fiber composition in the six major leg muscles, protractor and retractor coxae, levator and depressor trochanteris, and extensor and flexor tibiae for the pro-, meso-, and metathorax. I demonstrated that most of these muscles contain three different fiber types: a slow, a fast, and an intermediate contracting one. While the proportions of the fiber types differed between the individual muscles, they were mostly consistent for the three analyzed thoracic segments. The only exceptions concerned the depressor trochanteris and retractor coxae, which both had increasing percentages of slow contracting fibers towards the metathoracic segment, suggesting a greater importance of these muscles for posture and tonic force production. In the second part of my thesis, I investigated the motor output of the deafferented meso- and metathoracic leg nerves nl2 (innervates the protractor coxae), nl5 (retractor coxae), C1 (levator trochanteris), C2 (depressor trochanteris), nl3 (extensor tibiae) and of branches of the nervus cruris (flexor tibiae) during front leg turning in a reduced preparation with only two front legs left. I showed, that the neuronal activities of the mesothoracic and metathoracic protractor and retractor coxae are specific for the turning direction, and similar in both thoracic segments. The neuronal activation of the levator and depressor trochanteris, on the other hand, was independent of the turning direction, and showed small thorax-segment specific differences, with a stronger depressor trochanteris activity in the meta- than in the mesothorax. The motoneuron pools of the most distal leg joint were very variable in their motor output. The leg nerves of the extensor and flexor tibiae tended to show direction specific activity in the mesothorax, with a stronger extensor tibiae activity outside, and flexor tibiae activity inside in half of the experiments. Such a tendency was not seen in the metathoracic motoneuron pools. In the third part of my thesis, I analyzed the involvement of local central pattern generators in the thorax-segment, and leg-segment specificity of the turning related motor output. For this purpose, I activated the local CPGs using the muscarinic agonist pilocarpine in a split-bath preparation. I was able to show that the pharmacologically evoked rhythm in the respective antagonistic motoneuron pools was changed towards the activation pattern observed in control turning conditions immediately after the initiation of front leg turning. This suggests that the observed changes in motor output are mediated through influences on the local central pattern generating networks