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

Intersegmental Coordination of Cockroach Locomotion: Adaptive Control of Centrally Coupled Pattern Generator Circuits

Animals’ ability to demonstrate both stereotyped and adaptive locomotor behavior is largely dependent on the interplay between centrally generated motor patterns and the sensory inputs that shape them. We utilized a combined experimental and theoretical approach to investigate the relative importance of CPG interconnections vs. intersegmental afferents in the cockroach: an animal that is renowned for rapid and stable locomotion. We simultaneously recorded coxal levator and depressor motor neurons (MN) in the thoracic ganglia of Periplaneta americana, while sensory feedback was completely blocked or allowed only from one intact stepping leg. In the absence of sensory feedback, we observed a coordination pattern with consistent phase relationship that shares similarities with a double-tripod gait, suggesting central, feedforward control. This intersegmental coordination pattern was then reinforced in the presence of sensory feedback from a single stepping leg. Specifically, we report on transient stabilization of phase differences between activity recorded in the middle and hind thoracic MN following individual front-leg steps, suggesting a role for afferent phasic information in the coordination of motor circuits at the different hemiganglia. Data were further analyzed using stochastic models of coupled oscillators and maximum likelihood techniques to estimate underlying physiological parameters, such as uncoupled endogenous frequencies of hemisegmental oscillators and coupling strengths and directions. We found that descending ipsilateral coupling is stronger than ascending coupling, while left–right coupling in both the meso- and meta-thoracic ganglia appear to be symmetrical. We discuss these results in comparison with recent findings in stick insects that share similar neural and body architectures, and argue that the two species may exemplify opposite extremes of a fast–slow locomotion continuum, mediated through different intersegment coordination strategies

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

A neuro-mechanical model for the switching of stepping direction and transitions between walking gaits in the stick insect

In this study, a mathematical model for the locomotion of the stick insect is developed. This model takes physiological conditions into account and it is capable of mimicking biological relevant features. The model is predicated on the crucial role, that sensory feedback plays in the coordination of limbs during walking. Central Pattern Generators (CPGs), which produce the rhythm of locomotion, are affected by sensory influences between the segments. The activities of the CPGs are transferred by the motoneurons to the muscles. Starting with existing neuron models and neuronal network models, a neuro-mechanical model is developed that includes the coupling of segments inside of a leg as well as the coupling of multiple legs. Firstly, mechanical models concerning the motion of the three isolated main joints are derived. These mechanical models are fused with the neuronal one. Thus, they represent neuro-mechanical models for the single joints that are coupled via sensory feedback. By means of the introduction of a switching mechanism the model is able to produce forward, backward and sideward stepping of a middle leg. Through the junction of two stepping middle legs to the body of the modeled stick insect, curve walking sequences with different curvatures can be produced. By extending the model to the front and the hind leg, the structure of intersegmental connection between the legs during the tripod and tetrapod gait can be generated. The change of stepping direction can be brought about by changing one single central command. If the middle leg is stepping backwards, the curvature during turning is smaller than in the case of sideward stepping. Weakly inhibitory intersegmental connections show the most accommodating leg coordination during both the tetrapod and the tripod gait

Drosophila modifier screens to identify novel neuropsychiatric drugs including aminergic agents for the possible treatment of Parkinson's disease and depression.

Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae ∼ 1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems

Design of artificial neural oscillatory circuits for the control of lamprey- and salamander-like locomotion using evolutionary algorithms

This dissertation investigates the evolutionary design of oscillatory artificial neural networks for the control of animal-like locomotion. It is inspired by the neural organ¬ isation of locomotor circuitries in vertebrates, and explores in particular the control of undulatory swimming and walking. The difficulty with designing such controllers is to find mechanisms which can transform commands concerning the direction and the speed of motion into the multiple rhythmic signals sent to the multiple actuators typically involved in animal-like locomotion. In vertebrates, such control mechanisms are provided by central pattern generators which are neural circuits capable of pro¬ ducing the patterns of oscillations necessary for locomotion without oscillatory input from higher control centres or from sensory feedback. This thesis explores the space of possible neural configurations for the control of undulatory locomotion, and addresses the problem of how biologically plausible neural controllers can be automatically generated.Evolutionary algorithms are used to design connectionist models of central pattern generators for the motion of simulated lampreys and salamanders. This work is inspired by Ekeberg's neuronal and mechanical simulation of the lamprey [Ekeberg 93]. The first part of the thesis consists of developing alternative neural controllers for a similar mechanical simulation. Using a genetic algorithm and an incremental approach, a variety of controllers other than the biological configuration are successfully developed which can control swimming with at least the same efficiency. The same method is then used to generate synaptic weights for a controller which has the observed biological connectivity in order to illustrate how the genetic algorithm could be used for developing neurobiological models. Biologically plausible controllers are evolved which better fit physiological observations than Ekeberg's hand-crafted model. Finally, in collaboration with Jerome Kodjabachian, swimming controllers are designed using a developmental encoding scheme, in which developmental programs are evolved which determine how neurons divide and get connected to each other on a two-dimensional substrate.The second part of this dissertation examines the control of salamander-like swimming and trotting. Salamanders swim like lampreys but, on the ground, they switch to a trotting gait in which the trunk performs a standing wave with the nodes at the girdles. Little is known about the locomotion circuitry of the salamander, but neurobiologists have hypothesised that it is based on a lamprey-like organisation. A mechanical sim¬ ulation of a salamander-like animat is developed, and neural controllers capable of exhibiting the two types of gaits are evolved. The controllers are made of two neural oscillators projecting to the limb motoneurons and to lamprey-like trunk circuitry. By modulating the tonic input applied to the networks, the type of gait, the speed and the direction of motion can be varied.By developing neural controllers for lamprey- and salamander-like locomotion, this thesis provides insights into the biological control of undulatory swimming and walking, and shows how evolutionary algorithms can be used for developing neurobiological models and for generating neural controllers for locomotion. Such a method could potentially be used for designing controllers for swimming or walking robots, for instance

Modulation of the activity of the locomotor central pattern generator in the rat spinal cord in vitro

The present study has investigated the rhythmic properties of spinal networks in the neonatal rat spinal cord in vitro, by means of intracellular recordings from single motoneurons (MNs) and extracellular recordings from ventral and dorsal roots (VRs;DRs). Distinct subclasses of metabotropic glutamate receptors (mGluRs) on rat spinal neurons mediated complex facilitatory and inhibitory effects. The class I agonist DHPG evoked MN depolarization (via the mGluR1 subtype) mostly at network level and generated sustained, network-dependent oscillations (via the mGluR5 subtype). DHPG also decreased the amplitude of reflex responses induced by DR stimuli, an effect unrelated to depolarization but dependent on glycinergic transmission. Single reflex responses were insensitive to group I mGluRs antagonists, suggesting no phasic activation of group I receptors during this process. Finally, DHPG depressed the glycinergic recurrent IPSP, perhaps by impairing the cholinergic input to Renshaw cells. Thus, the cellular distribution of those mGluRs at strategic circuit connections may determine the functional outcome of the network in terms of excitation or inhibition. Activation of class II or III mGluRs had no direct action on MNs although it strongly blocked evoked synaptic transmission, presumably acting at presynaptic level. To extend our understanding of the network-based properties, which enable a neuronal circuit to produce sustained electrical oscillations, we explored the potential contribution of mGluRs to generate rhythmic discharges. During cumulative depolarization or fictive locomotion, spinal mGluRs were minimally activated by endogenous glutamate, although they could potently modulate these responses once activated by exogenously applied mGluR agonists. Disinhibited bursting was associated with the activation of mGluR1 receptors (facilitating network excitability) and of group II mGluRs (depressing it). We investigated if the K+ channel blocker 4-aminopyridine (4-AP) could facilitate spinal locomotor networks in addition to its well-known effect on motor nerve conduction. 4-AP produced synchronous VR oscillations, which did not develop into fictive locomotion. These oscillations had network origin, required intact glutamatergic transmission and were probably amplified via electrotonic coupling. 4-AP slightly increased input resistance of lumbar MNs, without affecting their action or resting potentials. DR evoked synaptic responses were enhanced by 4-AP without changes in axon conduction. 4-AP accelerated chemically or electrically induced fictive locomotion and facilitated the onset of fictive locomotion in the presence of subthreshold stimuli, that were previously insufficient to activate locomotor patterns. Thus, although 4-AP per se could not directly activate the locomotor network of the spinal cord, it could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli. On DRs, 4-AP induced sustained synchronous oscillations smaller than electrically evoked synaptic potentials, persistent after sectioning off the ventral region and preserved in an isolated dorsal quadrant, indicating their dorsal horn origin. 4-AP oscillations were network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generate 4-AP oscillations, although their intrinsic, disinhibited bursting was accelerated by the substance. Activation of fictive locomotion by either application of neurochmicals or stimulus trains to a single DR reversibly suppressed DR oscillations induced by 4-AP. The present electrophysiological investigation also examined whether the broad spectrum potassium channel blocker tetraethylammonium (TEA) could generate locomotor-like patterns. Low concentrations of TEA induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations evoked alternating discharges between flexor and extensor motor pools, plus a large depolarization of MNs with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABAA and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I mGluRs. Furthermore, TEA also evoked irregular discharges on DRs. The rhythmic alternating patterns elicited by TEA on VRs were relatively stereotypic, had limited synergy with the fictive locomotion induced by DR stimuli, and were not accelerated by 4-AP. Horizontal section of the spinal cord preserved irregular VR discharges and DR discharges, demonstrating that the action of TEA on spinal networks was fundamentally different from that of 4-AP