Anke Borgmann

Sensory Feedback Induced by Front-Leg Stepping Entrains the Activity of Central Pattern Generators in Caudal Segments of the Stick Insect Walking System

Legged locomotion results from a combination of central pattern generating network (CPG) activity and intralimb and interlimb sensory feedback. Data on the neural basis of interlimb coordination are very limited. We investigated here the influence of stepping in one leg on the activities of neighboring-leg thorax–coxa (TC) joint CPGs in the stick insect (Carausius morosus). We used a new approach combining single-leg stepping with pharmacological activation of segmental CPGs, sensory stimulation, and additional stepping legs. Stepping of a single front leg could activate the ipsilateral mesothoracic TC CPG. Activation of the metathoracic TC CPG required that both ipsilateral front and middle legs were present and that one of these legs was stepping. Unlike the situation in real walking, ipsilateral mesothoracic and metathoracic TC CPGs activated by front-leg stepping fired in phase with the front-leg stepping. Local (intralimb) sensory feedback from load sensors could override this intersegmental influence of front-leg stepping, shifting retractor motoneuron activity relative to the front-leg step cycle and thereby uncoupling them from front-leg stepping. These data suggest that front-leg stepping in isolation would result in in-phase activity of all ipsilateral legs, and functional stepping gaits (in which the three ipsilateral legs do not step in synchrony) emerge because of local load sensory feedback overriding this in-phase influence.

Plastin 3 ameliorates spinal muscular atrophy via delayed axon pruning and improves neuromuscular junction functionality

F-actin bundling plastin 3 (PLS3) is a fully protective modifier of the neuromuscular disease spinal muscular atrophy (SMA), the most common genetic cause of infant death. The generation of a conditional PLS3-over-expressing mouse and its breeding into an SMA background allowed us to decipher the exact biological mechanism underlying PLS3-mediated SMA protection. We show that PLS3 is a key regulator that restores main processes depending on actin dynamics in SMA motor neurons (MNs). MN soma size significantly increased and a higher number of afferent proprioceptive inputs were counted in SMAPLS3 compared with SMA mice. PLS3 increased presynaptic F-actin amount, rescued synaptic vesicle and active zones content, restored the organization of readily releasable pool of vesicles and increased the quantal content of the neuromuscular junctions (NMJs). Most remarkably, PLS3 over-expression led to a stabilization of axons which, in turn, resulted in a significant delay of axon pruning, counteracting poor axonal connectivity at SMA NMJs. These findings together with the observation of increased endplate and muscle fiber size upon MN-specific PLS3 over-expression suggest that PLS3 significantly improves neuromuscular transmission. Indeed, ubiquitous over-expression moderately improved survival and motor function in SMA mice. As PLS3 seems to act independently of Smn, PLS3 might be a potential therapeutic target not only in SMA but also in other MN diseases.

Control of Stepping Velocity in the Stick Insect Carausius morosus

We performed electrophysiological and behavioral experiments in single-leg preparations and intact animals of the stick insect Carausius morosus to understand mechanisms underlying the control of walking speed. At the level of the single leg, we found no significant correlation between stepping velocity and spike frequency of motor neurons (MNs) other than the previously shown modification in flexor (stance) MN activity. However, pauses between stance and swing motoneuron activity at the transition from stance to swing phase and stepping velocity are correlated. Pauses become shorter with increasing speed and completely disappear during fast stepping sequences. By means of extra- and intracellular recordings in single-leg stick insect preparations we found no systematic relationship between the velocity of a stepping front leg and the motoneuronal activity in the ipsi- or contralateral mesothoracic protractor and retractor, as well as flexor and extensor MNs. The observations on the lack of coordination of stepping velocity between legs in single-leg preparations were confirmed in behavioral experiments with intact stick insects tethered above a slippery surface, thereby effectively removing mechanical coupling through the ground. In this situation, there were again no systematic correlations between the stepping velocities of different legs, despite the finding that an increase in stepping velocity in a single front leg is correlated with a general increase in nerve activity in all connectives between the subesophageal and all thoracic ganglia. However, when the tethered animal increased walking speed due to a short tactile stimulus, provoking an escape-like response, stepping velocities of ipsilateral legs were found to be correlated for several steps. These results indicate that there is no permanent coordination of stepping velocities between legs, but that such coordination can be activated under certain circumstances.

Insect motor control: methodological advances, descending control and inter-leg coordination on the move

Modern approaches, including high performance video, neurophysiology, and neurogenetics, allow to analyze invertebrate behavior on all levels of generation and performance in an unprecedented way. They allow observation and classification of behavior in controlled conditions, dissection of behavioral sequencing, identification of levels of processing and locations of associated sub-networks and, finally, identification of neuronal components and topologies contributing to specific aspects of behaviors. Recently conceptual and methodological progress has contributed to unraveling the neural structures underlying descending control of insect behavior as well as the mechanisms in charge of generating coordinated locomotor movements of the invertebrate extremities during walking. This brief review summarizes some of the most exciting new findings in these areas of research from the past years.

Network modularity: back to the future in motor control.

Optogenetic analysis has revealed the existence of multiple rhythm-generating neural networks that drive leg motoneuron pools in the lumbar spinal cord of rodents. These findings extend the concept of a modular neural network organization for locomotion from invertebrates and lower vertebrates to mammals.

Investigating inter-segmental connections between thoracic ganglia in the stick insect by means of experimental and simulated phase response curves

The neuronal networks that control the motion of the individual legs in insects, in particular in the stick insect, are located in the pro-, meso- and meta-thoracic ganglia. They ensure high flexibility of movement control. Thus, the legs can move in an apparently independent way, e.g., during search movements, but also in tight coordination during locomotion. The latter is evidently a very important behavioural mode. It has, therefore, inspired a large number of studies, all aiming at uncovering the nature of the inter-leg coordination. One of the basic questions has been as to how the individual control networks in the three thoracic ganglia are connected to each other. One way to study this problem is to use phase response curves. They can reveal properties of the coupling between oscillatory systems, such as the central pattern generators in the control networks in the thoracic ganglia. In this paper, we report results that we have achieved by means of a combined experimental and modelling approach. We have calculated phase response curves from data obtained in as yet unpublished experiments as well as from those in previously published ones. By using models of the connected pro- and meso-thoracic control networks of the protractor and retractor neuromuscular systems, we have also produced simulated phase response curves and compared them with the experimental ones. In this way, we could gain important information of the nature of the connections between the aforementioned control networks. Specifically, we have found that connections from both the protractor and the retractor “sides” of the pro-thoracic network to the meso-thoracic one are necessary for producing phase response curves that show close similarity to the experimental ones. Furthermore, the strength of the excitatory connections has been proven to be crucial, while the inhibitory connections have essentially been irrelevant. We, thus, suggest that this type of connection might also be present in the stick insect, and possibly in other insect species.

Dominance of local sensory signals over inter-segmental effects in a motor system: modeling studies

Recent experiments, reported in the accompanying paper, have supplied key data on the impact afferent excitation has on the activity of the levator–depressor motor system of an extremity in the stick insect. The main finding was that, stimulation of the campaniform sensillae of the partially amputated middle leg in an animal where all other but one front leg had been removed, had a dominating effect over that of the stepping ipsilateral front leg. In fact, the latter effect was minute compared to the former. In this article, we propose a local network that involves the neuronal part of the levator–depressor motor system and use it to elucidate the mechanisms that underlie the generation of neuronal activity in the experiments. In particular, we show that by appropriately modulating the activity in the neurons of the central pattern generator of the levator–depressor motor system, we obtain activity patterns of the motoneurons in the model that closely resemble those found in extracellular recordings in the stick insect. In addition, our model predicts specific properties of these records which depend on the stimuli applied to the stick insect leg. We also discuss our results on the segmental mechanisms in the context of inter-segmental coordination.

Intra- and intersegmental influences among central pattern generating networks in the walking system of the stick insect

To efficiently move around, animals need to coordinate their limbs. Proper, context-dependent coupling among the neural networks underlying leg movement is necessary for generating intersegmental coordination. In the slow-walking stick insect, local sensory information is very important for shaping coordination. However, central coupling mechanisms among segmental central pattern generators (CPGs) may also contribute to this. Here, we analyzed the interactions between contralateral networks that drive the depressor trochanteris muscle of the legs in both isolated and interconnected deafferented thoracic ganglia of the stick insect on application of pilocarpine, a muscarinic acetylcholine receptor agonist. Our results show that depressor CPG activity is only weakly coupled between all segments. Intrasegmental phase relationships differ between the three isolated ganglia, and they are modified and stabilized when ganglia are interconnected. However, the coordination patterns that emerge do not resemble those observed during walking. Our findings are in line with recent studies and highlight the influence of sensory input on coordination in slowly walking insects. Finally, as a direct interaction between depressor CPG networks and contralateral motoneurons could not be observed, we hypothesize that coupling is based on interactions at the level of CPG interneurons.NEW & NOTEWORTHY Maintaining functional interleg coordination is vitally important as animals locomote through changing environments. The relative importance of central mechanisms vs. sensory feedback in this process is not well understood. We analyzed coordination among the neural networks generating leg movements in stick insect preparations lacking phasic sensory feedback. Under these conditions, the networks governing different legs were only weakly coupled. In stick insect, central connections alone are thus insufficient to produce the leg coordination observed behaviorally.

Analysis of excitatory and inhibitory interactions at high temporal resolution in core circuits of the respiratory CPG

The pre-Botzinger complex (pre-BotC) is the essential core component of the brainstem respiratory central pattern generator (rCPG) in mammals. Phasic excitatory and inhibitory synaptic inputs during the respiratory cycle are thought to dynamically shape membrane potential trajectories and spiking patterns of pre-BotC neurons. These synaptic inputs reflect the functionally interacting neuron populations involved in rhythm and inspiratory-expiratory pattern generation. The dynamic patterns of these synaptic inputs have not been systematically studied and characterized experimentally. Accurate temporal reconstruction of the synaptic input patterns is important for testing current models of the rCPG that incorporate specific excitatory and inhibitory circuit mechanisms for rhythm and pattern generation. A recent computational model of the rCPG generating a normal “three-phase” respiratory pattern [1] specified patterns of synaptic excitation and inhibition in the preBotC during the respiratory cycle required to produce the behaviorally distinct phases of inspiration (I) and two phases of expiration (E1, or post-inspiration, and E2). This model has not been rigorously tested by experimental data on dynamic changes in phasic postsynaptic excitatory and inhibitory conductances in different populations of pre-BotC inspiratory and expiratory neurons. We developed analytical techniques that allow quantitative reconstruction of the patterns of synaptic conductances at high temporal resolution from intracellular recordings of membrane potential trajectories. Our approach extends previously proposed methods for extracting dynamic patterns of synaptic input conductances [2,3] and provides nearly continuous readouts of inhibitory and excitatory synaptic conductances in electrophysiologically different cell types throughout the respiratory cycle. The membrane potential trajectories of pre-BotC respiratory neurons were obtained by sharp microelectrode current-clamp recording within in situ perfused brainstem-spinal cord preparations of mature rats, which generate a three-phase respiratory pattern and provide mechanically stable conditions for intracellular recordings in functionally intact brainstem circuits. We distinguished different types of inspiratory and expiratory pre-BotC neurons and analyzed the dynamical changes of excitatory (Ge )a nd inhibitory (Gi) conductances. Inspiratory neurons exhibited strong excitatory inputs (large dynamic increases in Ge) during their spiking phase followed by a wave(s) of inhibitory inputs during the expiratory period with large increases in Gi and temporal patterns consistent with two populations of inhibitory neurons providing inputs that coordinate inspiratory to expiratory and expiratory to inspiratory phase transitions. Expiratory neurons exhibited strong inhibition during the inspiratory phase, consistent with the rhythmic alternation of inspiration and expiration, and changes in Ge indicating reciprocal interactions between two major populations of inhibitory neurons coordinating the generation of two (E1 and E2) phases of expiration. The reconstructed patterns of Ge and Gi are generally consistent with previously proposed circuit architecture [1] and also suggest its extension. The techniques that we have developed for high temporal resolution of postsynaptic conductance changes are likely to have broad application for analysis of synaptic interactions in circuit components whenever intracellular recordings of membrane potentials can be