Multimodal Proprioceptive Integration in Sensorimotor Networks of an Insect Leg

An animal’s nervous system monitors the actions of the body using its sense of proprioception. This information is used for precise motor control and to enable coordinated interaction with the animal’s surroundings. Proprioception is a multimodal sense that includes feedback about limb movement and loading from various peripheral sense organs. The sensory information from distinct sense organs must be integrated by the network to form a coherent representation of the current proprioceptive state and to elicit appropriate motor behavior. By combining intra- and extracellular electrophysiological recording techniques with precise mechanical sensory stimulation paradigms, I studied multimodal proprioceptive integration in the sensorimotor network of the stick insect leg. The findings demonstrate where, when, and how sensory feedback from load-sensing campaniform sensilla (CS) is integrated with movement information from the femoral chordotonal organ (fCO) in the sensorimotor network controlling movement of the femur-tibia (FTi) joint. Proprioceptive information about distinct sensory modalities (load / movement) and from distinct sense organs of the same sensory modality (trochanterofemoral CS (tr/fCS) / tibial CS (tiCS)) was distributed into one network of local premotor nonspiking interneurons (NSIs). The NSIs’ processing of fCO, tr/fCS, and tiCS was antagonistic with respect to a given NSI’s effect on the motor output of extensor tibiae motor neurons (ExtTi MNs). Spatial summation of load and movement feedback occurred in the network of premotor NSIs, whereas temporal summation was shifted between sensory modalities. Load feedback (tr/fCS / tiCS) was consistently delayed relative to movement signals (fCO) throughout the sensorimotor pathways of sensory afferents, premotor NSIs, and ExtTi MNs. The connectivity between these neuron types was inferred using transmission times and followed distinct patterns for individual sense organs. At the motor output level of the system, the temporal shift of simultaneously elicited load and movement feedback caused load responses to be superimposed onto ongoing movement responses. These results raised the hypothesis that load could alter movement signal processing. Load (tiCS) affected movement (fCO) signal gain by presynaptic afferent inhibition. In postsynaptic premotor NSIs, this led to altered movement parameter dependence and nonlinear summation of load and movement signals. Specifically, the amplitude dependence of NSIs opposing ExtTi MN output was increased, and, consistently, the movement response gain of the slow ExtTi MN was decreased. Movement signal processing in the premotor network was altered depending on the proprioceptive context, i.e. the presence or absence of load feedback. Lateral presynaptic interactions between load (tiCS) and movement (fCO) afferents were reciprocal, i.e. existed from fCO to tiCS afferents and vice versa, and also occurred between sensory afferents of the same sense organ. Additionally, a new type of presynaptic interaction was identified. Load signals increased the gain of directional movement information by releasing unidirectionally velocity- or acceleration-sensitive fCO afferents from tonic presynaptic inhibition. Paired double recordings showed lateral connectivity also at the level of the premotor NSI network. NSIs interacted via reciprocal excitatory connections. Additionally, the activity of individual NSIs was correlated in the absence of external stimuli, and specific types of NSIs showed rhythmic 30 Hz oscillations of the resting membrane potential, indicating an underlying mechanism of network synchronization. Taken together, the results of this dissertation provide an understanding of the integration of multimodal proprioceptive feedback in the sensorimotor network by identifying neuronal pathways and mechanism underlying spatial and temporal signal summation. The local network uses multimodal signal integration for context-dependent sensory processing, thereby providing insights into the mechanism by which a local network can adapt sensory processing to the behavioral context. Initial results clearly highlight the necessity to consider lateral connections along sensorimotor pathways to unravel the complex computations underlying proprioceptive processing and motor control. The findings on the integration of proprioceptive signals, obtained in the resting animal, broaden our understanding of sensorimotor processing and motor control not only in the stationary, but also in the walking animal

Mechanisms for intersegmental leg coordination in walking stick insects

For efficient locomotion, the movements of single legs need to be coordinated during walking, which results in a stepping pattern or gait. This dissertation explores the neural mechanisms underlying the formation of a gait, in particular the neural basis of coupling of ipsilateral leg movements. In a semi-intact walking preparation of the stick insect Carausius morosus, correlations between ipsilateral mesothoracic motoneuron activity and walking movements of a front leg were described. Extracellular recordings showed a dedicated coupling of activity for mesothoracic protractor coxae and extensor tibiae motoneurons. Depressor trochanteris motoneurons showed a more flexible coupling. Mesothoracic retractor coxae and levator trochanteris motoneurons were active in anti-phase with their respective antagonists. Intracellular recordings revealed two different modulations of membrane potentials of mesothoracic motoneurons: a tonic modulation, lasting during the stepping activity of the front leg, and a rhythmic modulation, correlated with individual steps of the front leg. Evidence for tonic excitatory and inhibitory, as well as for rhythmic excitatory and inhibitory inputs were found for different motoneurons. Intracellular recordings of mesothoracic non-spiking interneurons of the pre-motor network revealed that these interneurons receive intersegmental coordinating signals. A tonic as well as a rhythmic modulation of their membrane potential, correlated with the walking activity of the ipsilateral front leg, were found. The non-spiking interneurons were in part morphologically identified and are known to process local sensory information. Hence, they could provide the basis for integration of local sensory and intersegmental signals. Additionally experiments were performed to investigate the origin of intersegmental signals. In experiments with an isolated chain of ganglia which was pharmacologically activated with pilocarpine, interaction between the central rhythm generating networks were studied. Sensory input was excluded in this preparation. No evidence was found for strong coupling of central pattern generators in mesothoracic and metathoracic segments, nor in prothoracic and mesothoracic segments. Two more sets of experiments focused on the role of sensory signals for intersegmental coordination. Signals from the mesothoracic femoral chordotonal organ, measuring position and movement of the femur-tibia joint, showed no clear influence on the activity of metathoracic motoneurons in the 'active' animal. Sensory signals from the metathoracic campaniform sensilla, measuring load on the leg, showed only a weak intersegmental influence on mesothoracic motoneuron activity, but a clear influence on local protractor and retractor motoneuron activity. The latter was found in the resting animal and with reversed effects in the 'active' animal, as well as during rhythmic activity evoked by application of pilocarpine

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

Task-specific modulation of a proprioceptive reflex in a walking insect

The generation of task-dependent and goal-directed walking behaviour requires feedback from leg sense organs for regulating and adapting the ongoing motor activity. Sensory feedback from movement and force sensors influences the magnitude and the timing of neural activity generated in the neural networks driving individual joints of a leg. In many animals, the effects of sensory feedback on the generated motor output change between posture maintenance and locomotion. These changes can occur as reflex reversals in which sensory information, that usually counteract perturbations in posture control, instead reinforce movements in walking. In stick insects, for example, flexion of the femur-tibia joint is measured by the femoral chordotonal organ, which mediates reinforcement of the stance phase motor output of the femur-tibia joint when the locomotor system is active. Flexion signals promote flexor and inhibit extensor motoneuron activity. However, the mechanisms underlying these changes are only partially understood. Therefore, the purpose of the present thesis was to investigate whether the processing of movement and position signals of the FTi joint is task-specifically modified in the generation of adaptive leg movements, which is required when locomotion is adapted to changes in walking direction or in turning movements. To study the role of these task-dependent changes in walking behaviour on the processing of local sensory signals, the generation of reflex reversals mediated by the femoral chordotonal organ in the femur-tibia joint of the stick insect Carausius morosus was measured in a semi-intact walking preparation. In several experimental conditions either in front, in one or both middle or in hind legs, the femoral chordotonal organ was mechanically displaced and the motoneuronal responses in the flexor and extensor tibia were monitored, while the remaining legs performed either forward, backward or curve walking on a slippery surface. I demonstrated that the occurrence of reflex reversals depends on the specific motor behaviour executed. While in forward walking flexion signals from the front leg fCO regularly elicit reflex reversal in the tibial motoneurons, this cannot be observed in backward walking. Similarly, during optomotor-induced curve walking, reflex reversal occurred reliably in the middle leg on the inside of the turn, however not in the contralateral leg on the outside of the turn. Thus, the experiments revealed that the nervous system modulates proprioceptive reflexes in individual legs during task-specific walking adaptation. Furthermore, I showed that nonspiking interneurons, known to be involved in the premotor network of the FTi joint, participate in reflex responses in both the inner and outer middle leg during curve walking. First results show that the reflex response in some interneuron types is altered between the inner and outer leg, while no differences were found in others

Activity of leg motoneurons during single leg walking of the stick insect: From synaptic inputs to motor performance

In the single middle leg preparation of the stick insect, leg motoneurons were recorded intracellularly during stepping movements on a treadmill. This preparation allows investigating the synaptic drive from local sense organs and central pattern generating networks to motoneurons. The synaptic drive comprises rhythmic (�phasic�) excitation and inhibition and a sustained (�tonic�) depolarization. This general scheme was found to be true for all motoneurons innervating the muscles of the three major leg joints. A comparison e.g. with results obtained from deafferented and pharmacologically activated preparations of the stick insect suggests that both tonic depolarization and phasic inhibition originate from central networks, while the phasic excitation is mainly generated by local sense organs. Recruitment of motoneurons was studied on the flexor tibiae muscle as an example of a complexly innervated muscle. It is innervated by ~14 slow, semifast and fast motoneurons that are firing action potentials during the stance phase of the step cycle. During slow steps or steps under small load, less motoneurons are recruited than during fast steps or steps under high load. Fast flexor motoneurons are recruited later during stance phase than slow motoneurons. All motoneurons receive substantial common synaptic drive during walking. They are recruited in an orderly fashion due to the more negative resting membrane potential of the fast motoneurons, which thus require a larger and longer lasting depolarization to reach the threshold for the generation of action potentials. Because walking is not invariable but needs to be adjusted to the behavioral requirements, it was investigated how these adjustments are implemented at the motoneuronal level. The activity of flexor and extensor tibiae motoneurons was analyzed during steps with different velocities. Extensor motoneuron activity during the extension phase of the step cycle (i.e. swing phase) is rather stereotypic and invariant with stance velocity. Flexor motoneurons show two distinct periods of depolarization at the beginning of stance. The initial depolarization is also stereotypic and most likely generated by a release from inhibition that allows the underlying tonic excitation to depolarize the neuron. The subsequent depolarization is larger and faster during fast steps than during slow steps. This indicates that in the single insect leg during walking, mechanisms for altering stepping velocity are becoming effective only during already ongoing stance phase motor output. Since a large portion of the phasic excitation arises from sense organs, it is conceivable that for the generation of different stepping velocities the effectiveness of these pathways are centrally modulated, for example by variations in the degree of presynaptic inhibition

The role of sensory influences in the control of motor activity of a stepping insect leg

For a long time the focus of the discourse in motor control research was on stereotypical movements, such as forward walking. My thesis emphasizes how different levels of neuronal motor control contribute to the processing of task-dependent locomotor behavior. This resulted in three main questions. First, how sensory feedback affects the timing and magnitude of muscle activity in general. Second, how this feedback is processed in changes of task-dependent movement behavior. And third, what mechanisms are used in the neural network to evoke an adaptive capability. Central pattern generators (CPGs) generate a rhythmic, alternating motor activity that is in turn modulated by sensory feedback in timing and in magnitude. In the middle leg of the stick insect Carausius morosus, two major groups of sense organs measure either the load or the movement and positional parameters of the leg. The role of these sense organs was examined in my work. In the first study, the influence of campaniform sensilla (CS) on magnitude and the timing of stance phase muscles was examined. For this purpose, a trapdoor setup with a slippery surface was used. The animal either stepped on a slippery surface with ground contact or they stepped into a hole when the trapdoor was lowered (SIH). Through ground contact, the legs are loaded and thereby activate the leg CS. During SIH this sensory feedback is missing. Through ablation experiments, I was able to show, in addition to CS, an additional sense organ that activated the flexor tibiae (FlxTi) through their sensory information of the touchdown (TD). In all stance phase muscles, except for the depressor trochanteris (DepTr), the strength of muscle activity increased through the TD. In the second study, the control on the timing of activation, especially of the extensor tibiae (ExtTi) muscle by sensory feedback of the femoral chordotonal organ (fCO) was investigated. Here, a focus was set on the processing of this feedback to movement changes, in particular curve walking. The fCO measures various parameters of the knee joint movement (femorotibial [FT] joint) and position. Feedback from the fCO generates resistance reflexes (RR), which are used to maintain the posture. Assistance reflex or reflex reversals (AR), which assist stance phase activity in the active animal, were also observed. During curve walking, the legs on each side of the animal have different kinematics. The leg inside of a curve (inside) is mainly moved around the FT joint, while the leg outside of the curve (outside) is mostly moved in the hip joint (thoracocoxal [ThC] joint). ExtTi motor neurons (MN) were activated during an AR at a certain angular difference. This was independent of the function as inside or outside leg. Further, the fCO stimulation induced ARs more often in the inside leg while RRs occurred more frequently in the outside leg. In addition, more ARs were generated through fCO stimulation at slower velocities and larger starting angles. The protractor coxae (ProCx) showed increased activation through fCO stimulation in the outside leg, while the levator coxae (LevTr) showed no difference in the reaction between the two leg xii functions. Thus, it could be shown in this part of my thesis that feedback from the fCO can cause task-specific motor activity. In the third study, the mechanisms that might be responsible for the decrease in the response to fCO stimulation when the leg is used as the outside leg were investigated. The monosynaptic connection of fCO afferents to ExtTi MNs, as well as to nonspiking interneurons (NSIs), was reduced. Similar observations were made in ExtTi MNs throughout the complete fCO stimulation. Furthermore, ExtTi MNs received greater tonic depolarization and their membrane input resistance was reduced. In my dissertation I could show that motor activity is influenced by various sense organs. These influences can be adapted to changes in movement, whereby I could reveal one mechanism leading to task-dependent motor activity. The influence of NSIs or presynaptic inhibition on changes in task dependent motor output remains unclear

Intersegmental influences contributing to coordination in a walking insect

Locomotion depends on correct interaction of the nervous system, muscles and environment. A key element in this process is the coordinated interplay of multiple body parts to achieve a stable and adapted behavior. Different aspects of intersegmental coordination in the stick insect have been investigated in this thesis: the activation of the walking system, intersegmental information transfer in the connectives and the in uence of load signals. I used a reduced preparation with only single intact front, middle or hind legs. The intact leg(s) performed stepping movements on a passive treadmill, hence providing, both sensory feedback and central input from its active pattern generating networks to the other hemiganglia. The activity of protractor and retractor motoneurons (MNs) was simultaneously recorded extracellularly in the other segments. The preparation allows investigating intersegmental influence of stepping single leg(s) on motoneural activity in the other deafferented hemisegments. The experiments revealed that the stick insect walking system is constructed in a modular fashion. Stepping of a single leg does not imply that the animal is in a locomotor state. In the two leg preparation with two intact legs that stepped on two separate treadmills, stepping of one leg did not imply stepping of the second leg. The legs stepped independent of each other concerning coordination and frequency. In the single leg preparation stepping of a single leg did not activate pattern generating networks in all other hemiganglia. The different hemiganglia were obviously activated independently. Only forward stepping of the front leg and, to a lesser extend, backward stepping of the hind leg, elicited alternating activity in mesothoracic protractor and retractor MNs. Motoneural activity in the other hemisegments increased and was slightly modulated during stepping sequences. Activation of the metathoracic ganglion required both ipsilateral front and middle legs stepping. Furthermore, the stick insect walking system is constructed asymmetrically on the neural level concerning the contribution and importance of the different legs for intersegmental coordination. The influence of middle leg stepping was qualitatively different to the influence of front leg stepping. In the single leg preparation front leg stepping induced alternating activity in ipsilateral mesothoracic protractor and retractor MNs that was most probably shaped by pattern generating networks. Middle leg stepping did not induce alternating activity in MNs of its ipsilateral neighboring segments. In a two leg preparation with front and ipsilateral middle leg stepping the middle leg appears to have no influence on the timing of metathoracic motoneural activity whereas front leg stepping was able to entrain metathoracic MN activity. The processing of intersegmental signals from other stepping legs appears to depend on the state of the receiving ganglion. Signals from the stepping front leg most probably reach the metathoracic ganglion as connective recordings show. If the metathoracic ganglion is active in the sense that the central pattern generating networks are active the signals from a stepping leg are treated differently. If the metathoracic ganglion was not active a general increase in motoneural activity was observed during front leg stepping. In case of an active metathoracic ganglion protractor and retractor MN activity alternated and was influenced by front leg stepping. Sensory signals are particularly important for coordination of the legs in the stick insect. In experiments in which middle leg campaniform sensilla were stimulated during single front leg stepping sequences, mesothoracic levator and depressor motoneuron activity was coupled to the campaniform sensilla stimulation. Stimulation of middle campaniform sensilla pretends increased load on the leg and induced an increase in depressor and a decrease in levator motoneuron activity. In mesothoracic protractor and retractor motoneurons front leg stepping induced alternating activity. Depending on the phase of front leg step cycle middle leg campaniform sensilla stimulation increased retractor and decreased protractor motoneuron activity or the influence was reverse (around 180° of step cycle)

Locomotor system simulations and muscle modeling of the stick insect (Carausius morosus)

It is a matter of fact that even so called "primitive species" (like insects) readily outperform any human locomotive invention with respect to agility, adaptability and reliability - to name the least. The work at hand deals with two aspects that contribute to the pre-eminence of biological, terrestrial locomotor systems, namely motion control and muscle properties. In the first part of this work, a new, biologically well-founded approach for the control of articulated legs is presented. This controller, based on the detailed physiological knowledge of the stick insect's (Carausius morosus) leg control, redundantizes complex forward or backward kinematic calculations by dexterous employment of sensory feedback and muscle properties. This section shows that the collection of segmental coordination rules (which have been studied in the stick insect for several decades) is indeed able to generate periodic, robust middle leg stepping movements in a physical simulation of the animal. Furthermore, the controller is capable of handling stepping in the front and hind leg; although for hind leg stepping minor modifications were necessary. The second part of this work is about muscle modeling and it is divided into three chapters. Lynchpin of any motion is the muscle, and nowadays it is well-accepted that muscle properties are complex and highly variable. Hence, no trivial relationship between motor neuron activity and motion can be expected and typically, computer modeling is required to link the two. This part therefore first describes how a model of the stick insect's extensor tibiae muscle can be developed for individual muscles. The approach presented offers a way to measure and model all properties for the generation of a classical Hill-type model, in a single animal. Therefore it was necessary to reduce the number of measurements, stimulations and the overall time span of the experiment to a degree this muscle could take without severe loss in vitality. After this approach has been described, the next section deals with a possible application of individual muscle modeling. The variation of muscle model parameters is investigated for 10 different individuals. The question of parameter independence is addressed, and in fact it could be shown that there is co-variation between two different pairs of parameters. One correlation was found between two parameters modeling passive static force curve, the other between one parameter of the force-length and one of the force-activation curve. Both correlations suggest that the model can be reduced further. In the final section, isometric and isotonic simulations were performed with different model configurations. It is investigated how far averaging parameters of different animals would influence model performance. This is studied by comparing the error produced by four different model configurations, differing in their share of averaged parameters. Compared to a model entirely composed of averaged parameters, performance of the muscle specific model improves by approximately 40%

Motor flexibility: neuronal control of walking direction and walking speed in an insect

The neuronal basis of locomotion is largely investigated in many different vertebrate and invertebrate species. Especially studying the neuronal control of adaptive locomotor behaviors is important to reveal general insights into nervous system function. In this thesis, the stick insects Carausius morosus and Cuniculina impigra were used to investigate how important parts of the locomotor network generate different walking directions and walking speeds. In order to study which parameters have to be changed to generate the different behaviors, leg muscle, motoneuron, and premotor nonspiking interneuron activity was recorded. In the first part, leg muscle activity during forward, backward, and curve walking was studied in a slippery surface setup, in which the animal is stationary about a slippery substrate and all legs can freely move. Muscle activity and timing was compared during different walking directions. The main change was observed in protractor and retractor muscles which move the leg in anterior and posterior direction. These muscles almost completely reverse their phase of activity with the change of walking direction, and intermediate changes occur in the inside leg during curve walking, depending on the steepness of the curve. In the second and third part, leg motoneuron and interneuron activity was recorded intracellularly in the single-leg preparation, in which only one leg is able to move in the vertical plane on a treadwheel. Fictive forward and backward walking can be reliably elicited in this preparation. It is known that leg motoneurons receive tonic depolarizing synaptic inputs from higher centers throughout walking, and additional phasic excitatory and inhibitory inputs from leg sense organs, as well as phasic inhibitory inputs from the rhythm generating network. It could be shown that similar inputs shape the motoneuron modulation pattern also during backward walking. The phase of the step cycle in which the phasic inputs to protractor and retractor MNs occur reverses during backward walking. It was shown previously that stepping velocity in the single-leg preparation is correlated to flexor MN activity (stance) but not extensor MN (swing) activity. These findings are confirmed in this thesis and also held for backward walking. No such influences could be shown for other stance phase motoneurons. Furthermore, premotor nonspiking interneurons were recorded to investigate their contribution to the generation of different walking directions and walking speeds. These neurons are known to integrate signals from descending, central, and sensory sources and thus contribute to the control of timing and magnitude of the motor output. Previously identified (E3, E4, E5, E7, I1, I2), as well as newly described nonspiking interneurons providing synaptic drive to motoneurons of all leg joints were recorded during forward and backward stepping. Interestingly, neurons could be identified which contribute to the change in protractor and retractor muscle activity. Furthermore it could be shown that all recorded nonspiking interneurons contribute to the motor output during walking in both directions, suggesting that the same premotor network is responsible for the generation of both behaviors. NSI activity also underlies tonic and phasic synaptic inputs. Additionally, the contribution of NSIs to the generation of different stepping velocities was investigated