Body side-specific changes in sensorimotor processing of movement feedback in a walking insect.

Feedback from load and movement sensors can modify timing and magnitude of the motor output in the stepping stick insect. One source of feedback is stretch reception by the femoral chordotonal organ (fCO), which encodes such parameters as the femorotibial (FTi) joint angle, the angular velocity, and its acceleration. Stimulation of the fCO causes a postural resistance reflex, during quiescence, and can elicit the opposite, so-called active reaction (AR), which assists ongoing flexion during active movements. In the present study, we investigated the role of fCO feedback for the difference in likelihood of generating ARs on the inside vs. the outside during curve stepping. We analyzed the effects of fCO stimulation on the motor output to the FTi and the neighboring coxa-trochanter and thorax-coxa joints of the middle leg. In inside and outside turns, the probability for ARs increases with increasing starting angle and decreasing stimulus velocity; furthermore, it is independent of the total angular excursion. However, the transition between stance and swing motor activity always occurs after a specific angular excursion, independent of the turning direction. Feedback from the fCO also has an excitatory influence on levator trochanteris motoneurons (MNs) during inside and outside turns, whereas the same feedback affects protractor coxae MNs only during outside steps. Our results suggest joint- and body side-dependent processing of fCO feedback. A shift in gain may be responsible for different AR probabilities between inside and outside turning, whereas the general control mechanism for ARs is unchanged.NEW & NOTEWORTHY We show that parameters of movement feedback from the tibia in an insect during curve walking are processed in a body side-specific manner, and how. From our results it is highly conceivable that the difference in motor response to the feedback supports the body side-specific leg kinematics during turning. Future studies will need to determine the source for the inputs that determine the local changes in sensory-motor processing.

Identification of the origin of force-feedback signals influencing motor neurons of the thoraco-coxal joint in an insect.

Force feedback from Campaniform sensilla (CS) on insect limbs helps to adapt motor outputs to environmental conditions, but we are only beginning to reveal the neural control mechanisms that mediate these influences. We studied CS groups that affect control of the thoraco-coxal joint in the stick insect Carausius morosus by applying horizontal and vertical forces to the leg stump. Motor effects of ablation of CS groups were evaluated by recording extracellularly from protractor (ProCx) and retractor (RetCx) nerves. Extracellular recordings showed that the effects of stimulating the sensilla were consistent with their broad ranges of directional sensitivity: for example, RetCx firing in response to posterior bending could be reduced by ablating several groups of trochanteral CS, whereas ablation of tibial and femoral sensilla had little effect. In contrast, ProCx motor neuron activity upon anteriorly directed stimuli was affected mainly by ablating a single CS group (G2). Dye fills of trochanteral, femoral and tibial CS groups with fluorescent dyes revealed a common projection pattern with little group specificity. These findings support the idea that the influences of CS feedback are determined by the activities of pre-motor interneurons, facilitating fast and task-dependent adaptation to changing environmental conditions.

Fiber-type distribution in insect leg muscles parallels similarities and differences in the functional role of insect walking legs.

Previous studies have demonstrated that myofibrillar ATPase (mATPase) enzyme activity in muscle fibers determines their contraction properties. We analyzed mATPase activities in muscles of the front, middle and hind legs of the orthopteran stick insect (Carausius morosus) to test the hypothesis that differences in muscle fiber types and distributions reflected differences in their behavioral functions. Our data show that all muscles are composed of at least three fiber types, fast, intermediate and slow, and demonstrate that: (1) in the femoral muscles (extensor and flexor tibiae) of all legs, the number of fast fibers decreases from proximal to distal, with a concomitant increase in the number of slow fibers. (2) The swing phase muscles protractor coxae and levator trochanteris, have smaller percentages of slow fibers compared to the antagonist stance muscles retractor coxae and depressor trochanteris. (3) The percentage of slow fibers in the retractor coxae and depressor trochanteris increases significantly from front to hind legs. These results suggest that fiber-type distribution in leg muscles of insects is not identical across leg muscles but tuned towards the specific function of a given muscle in the locomotor system.

The role of leg touchdown for the control of locomotor activity in the walking stick insect.

Much is known on how select sensory feedback contributes to the activation of different motoneuron pools in the locomotor control system of stick insects. However, even though activation of the stance phase muscles depressor trochanteris, retractor unguis, flexor tibiae and retractor coxae is correlated with the touchdown of the leg, the potential sensory basis of this correlation or its connection to burst intensity remains unknown. In our experiments, we are using a trap door setup to investigate how ground contact contributes to stance phase muscle activation and burst intensity in different stick insect species, and which afferent input is involved in the respective changes. While the magnitude of activation is changed in all of the above stance phase muscles, only the timing of the flexor tibiae muscle is changed if the animal unexpectedly steps into a hole. Individual and combined ablation of different force sensors on the leg demonstrated influence from femoral campaniform sensilla on flexor muscle timing, causing a significant increase in the latencies during control and air steps. Our results show that specific load feedback signals determine the timing of flexor tibiae activation at the swing-to-stance transition in stepping stick insects, but that additional feedback may also be involved in flexor muscle activation during stick insect locomotion. With respect to timing, all other investigated stance phase muscles appear to be under sensory control other than that elicited through touchdown.

Segment-specific and state-dependent targeting accuracy of the stick insect.

In its natural habitat, Carausius morosus climbs on the branches of bushes and trees. Previous work suggested that stick insects perform targeting movements with their hindlegs to find support more easily. It has been assumed that the animals use position information from the anterior legs to control the touchdown position of the ipsilateral posterior legs. Here we addressed the question of whether not only the hindleg but also the middle leg performs targeting, and whether targeting is still present in a walking animal when influences of mechanical coupling through the ground are removed. If this were the case, it would emphasize the role of underlying neuronal mechanisms. We studied whether targeting occurred in both legs, when the rostral neighboring leg, i.e. either the middle or the front leg, was placed at defined positions relative to the body, and analyzed targeting precision for dependency on the targeted position. Under these conditions, the touchdown positions of the hindlegs show correlation to the position of the middle leg parallel and perpendicular to the body axis, while only weak correlation exists between the middle and front legs, and only in parallel to the body axis. In continuously walking tethered animals, targeting accuracy of the hindlegs and middle legs parallel to the body axis barely differed. However, targeting became significantly more accurate perpendicular to the body axis. Our results suggest that a neural mechanism exists for controlling the touchdown position of the posterior leg but that the strength of this mechanism is segment specific and dependent on the behavioral context in which it is used.

Thorax-Segment- and Leg-Segment-Specific Motor Control for Adaptive Behavior.

We have just started to understand the mechanisms underlying flexibility of motor programs among segmental neural networks that control each individual leg during walking in vertebrates and invertebrates. Here, we investigated the mechanisms underlying curve walking in the stick insect Carausius morosus during optomotor-induced turning. We wanted to know, whether the previously reported body-side specific changes in a two-front leg turning animal are also observed in the other thoracic leg segments. The motor activity of the three major leg joints showed three types of responses: 1) a context-dependent increase or decrease in motor neuron (MN) activity of the antagonistic MN pools of the thorax-coxa (ThC)-joint during inside and outside turns; 2) an activation of 1 MN pool with simultaneous cessation of the other, independent of the turning direction in the coxa-trochanteral (CTr)-joint; 3) a modification in the activity of both FTi-joint MN pools which depended on the turning direction in one, but not in the other thorax segment. By pharmacological activation of the meso- or metathoracic central pattern generating networks (CPG), we show that turning-related modifications in motor output involve changes to local CPG activity. The rhythmic activity in the MN pools of the ThC and CTr-joints was modified similarly to what was observed under control conditions in saline. Our results indicate that changes in meso- and metathoracic motor activity during curve walking are leg-joint- and thorax-segment-specific, can depend on the turning direction, and are mediated through changes in local CPG activity.

Terminal H-reflex Measurements in Mice.

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS). In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

Dominance of local sensory signals over inter-segmental effects in a motor system: experiments.

Legged locomotion requires that information local to one leg, and inter-segmental signals coming from the other legs are processed appropriately to establish a coordinated walking pattern.However, very little is known about the relative importance of local and inter-segmental signals when they converge upon the central pattern generators (CPGs) of different leg joints.We investigated this question on the CPG of the middle leg coxa­trochanter (CTr)-joint of the stick insect which is responsible for lifting and lowering the leg.We used a semi-intact preparation with an intact front leg stepping on a treadmill, and simultaneously stimulated load sensors of the middle leg.We found that middle leg load signals induce bursts in the middle leg depressor motoneurons(MNs). The same local load signals could also elicit rhythmic activity in the CPG of the middle leg CTr-joint when the stimulation of middle leg load sensors coincided with front leg stepping. However, the influence of front leg stepping was generally weak such that front leg stepping alone was only rarely accompanied by switching between middle leg levator and depressor MN activity. We therefore conclude that the impact of the local sensory signals on the levator­depressor motor system is stronger than the inter-segmental influence through front leg stepping.

Optical inactivation of a proprioceptor in an insect by non-genetic tools.

BACKGROUND: The specific role of sensory organs in locomotor pattern generation is traditionally investigated by means of mechanical ablation in arthropods that currently do not allow genetic manipulation. Mechanical ablation is irreversible, and may lead to injury discharges and changes in the structural integrity of the cuticle. NEW METHOD: Here, we present a new method to temporarily or permanently deprive parts of an insect nervous system of sensory feedback from leg proprioceptors by means of blue light application. We illuminated campaniform sensilla (CS) with a blue LED (420-480 nm) or a 473 nm laser at different light intensities to optically eliminate sensory and motor neuron responses to mechanical stimulation. RESULTS: We were able to eliminate all stimulus-evoked responses of CS. Individual CS groups were precisely and selectively inactivated without affecting nearby proprioceptors, using an optical fiber (Ø 200 µm) to guide the light. Our results demonstrated that lower light intensities significantly increase the required exposure time, but also the chance for recovery, thus making the effect reversible. COMPARISON WITH EXISTING METHODS: In contrast to mechanical ablation, optical inactivation of individual sensory organs is non-invasive and does not affect the behavioral state of the animal, nor does it induce escape behavior. This is especially relevant in non-model system experimental animals where optogenetic manipulation cannot be used, due to a lack of established methods of access. CONCLUSION: Our results show that the proposed method is a reliable alternative to mechanical ablation and can be successfully applied to the CS, as it fulfills all requirements regarding selectivity, efficiency, and reproducibility.

Introduction to spasticity and related mouse models.

Although spasticity is one of the most common causes of motor disability worldwide, its precise definition and pathophysiology remain elusive, which to date renders its experimental targeting tricky. At least in part, this difficulty is caused by heterogeneous phenotypes of spasticity-causing neurological disorders, all causing spasticity by involving upper motor neurons. The most common clinical symptoms are a series of rapid muscle contractions (clonus), an increased muscle tone (hypertonia), and augmented tendon reflex activity (hyperreflexia). This muscle overactivity is due to disturbed inhibition of spinal reflexes following upper motor neuron dysfunction. Despite a range of physical and pharmacological therapies ameliorating the symptoms, their targeted application remains difficult. Therefore, to date, spasticity impacts rehabilitative therapy, and no therapy exists that reverses the pathology completely. In contrast to the incidence and importance of spasticity, only very little pre-clinical work in animal models exists, and this research is focused on the cat or the rat spastic tail model to decipher altered reflexes and excitability of the motor neurons in the spinal cord. Meanwhile, the characterization of spasticity in clinically more relevant mouse models of neurological disorders, such as stroke, remains understudied. Here, we provide a brief introduction into the clinical knowledge and therapy of spasticity and an in-depth review of pre-clinical studies of spasticity in mice including the current experimental challenges for clinical translation.

Neural control of unloaded leg posture and of leg swing in stick insect, cockroach, and mouse differs from that in larger animals.

Stick insect (Carausius morosus) leg muscles contract and relax slowly. Control of stick insect leg posture and movement could therefore differ from that in animals with faster muscles. Consistent with this possibility, stick insect legs maintained constant posture without leg motor nerve activity when the animals were rotated in air. That unloaded leg posture was an intrinsic property of the legs was confirmed by showing that isolated legs had constant, gravity-independent postures. Muscle ablation experiments, experiments showing that leg muscle passive forces were large compared with gravitational forces, and experiments showing that, at the rest postures, agonist and antagonist muscles generated equal forces indicated that these postures depended in part on leg muscles. Leg muscle recordings showed that stick insect swing motor neurons fired throughout the entirety of swing. To test whether these results were specific to stick insect, we repeated some of these experiments in cockroach (Periplaneta americana) and mouse. Isolated cockroach legs also had gravity-independent rest positions and mouse swing motor neurons also fired throughout the entirety of swing. These data differ from those in human and horse but not cat. These size-dependent variations in whether legs have constant, gravity-independent postures, in whether swing motor neurons fire throughout the entirety of swing, and calculations of how quickly passive muscle force would slow limb movement as limb size varies suggest that these differences may be caused by scaling. Limb size may thus be as great a determinant as phylogenetic position of unloaded limb motor control strategy.

Activity patterns and timing of muscle activity in the forward walking and backward walking stick insect Carausius morosus.

Understanding how animals control locomotion in different behaviors requires understanding both the kinematics of leg movements and the neural activity underlying these movements. Stick insect leg kinematics differ in forward and backward walking. Describing leg muscle activity in these behaviors is a first step toward understanding the neuronal basis for these differences. We report here the phasing of EMG activities and latencies of first spikes relative to precise electrical measurements of middle leg tarsus touchdown and liftoff of three pairs (protractor/retractor coxae, levator/depressor trochanteris, extensor/flexor tibiae) of stick insect middle leg antagonistic muscles that play central roles in generating leg movements during forward and backward straight walking. Forward walking stance phase muscle (depressor, flexor, and retractor) activities were tightly coupled to touchdown, beginning on average 93 ms prior to and 9 and 35 ms after touchdown, respectively. Forward walking swing phase muscle (levator, extensor, and protractor) activities were less tightly coupled to liftoff, beginning on average 100, 67, and 37 ms before liftoff, respectively. In backward walking the protractor/retractor muscles reversed their phasing compared with forward walking, with the retractor being active during swing and the protractor during stance. Comparison of intact animal and reduced two- and one-middle-leg preparations during forward straight walking showed only small alterations in overall EMG activity but changes in first spike latencies in most muscles. Changing body height, most likely due to changes in leg joint loading, altered the intensity, but not the timing, of depressor muscle activity.

Loss of miR-210 leads to progressive retinal degeneration in Drosophila melanogaster.

miRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. We used small RNA sequencing to identify tissue-specific miRNAs in the adult brain, thorax, gut, and fat body of Drosophila melanogaster One of the most brain-specific miRNAs that we identified was miR-210, an evolutionarily highly conserved miRNA implicated in the regulation of hypoxia in mammals. In Drosophila, we show that miR-210 is specifically expressed in sensory organs, including photoreceptors. miR-210 knockout mutants are not sensitive toward hypoxia but show progressive degradation of photoreceptor cells, accompanied by decreased photoreceptor potential, demonstrating an important function of miR-210 in photoreceptor maintenance and survival.

Tethered stick insect walking: a modified slippery surface setup with optomotor stimulation and electrical monitoring of tarsal contact.

A modified and improved setup based on Epstein and Graham [Epstein S, Graham D. Behaviour and motor output of stick insects walking on a slippery surface. I. Forward walking. J Exp Biol 1983;105: 215-29] to study straight and curve walking in the stick insect was developed and applications for its use are described. The animal is fixed on a balsa stick and walks freely on a slippery surface created with a thin film of a glycerin/water solution on a black, Ni-coated, polished brass plate. The glycerine/water ratio controls the viscosity of the lubricant and thereby the forces necessary to move the legs of the stick insect. A small amount of NaCl is added to ensure electric conductivity. Walking is induced through an optomotor stimulus given by two stripe-projectors producing rotatory and translatory stimuli to influence walking direction. The walking pattern is monitored in two ways: (1) tarsal contact with the slippery surface is measured electrically using a lock-in-amplifier. The tarsal contact signal allows correlation with the activity in different muscles of the stick insect leg recorded with EMG electrodes; (2) leg kinematics in the horizontal plane is monitored using synchronized high speed video. This setup allows us to determine the coupling of activity in different leg muscles to either swing or stance phase during straight and curve walking in the intact animal or the reduced single-leg preparation with a high time resolution.

Body side-specific control of motor activity during turning in a walking animal.

Animals and humans need to move deftly and flexibly to adapt to environmental demands. Despite a large body of work on the neural control of walking in invertebrates and vertebrates alike, the mechanisms underlying the motor flexibility that is needed to adjust the motor behavior remain largely unknown. Here, we investigated optomotor-induced turning and the neuronal mechanisms underlying the differences between the leg movements of the two body sides in the stick insect Carausius morosus. We present data to show that the generation of turning kinematics in an insect are the combined result of descending unilateral commands that change the leg motor output via task-specific modifications in the processing of local sensory feedback as well as modification of the activity of local central pattern generating networks in a body-side-specific way. To our knowledge, this is the first study to demonstrate the specificity of such modifications in a defined motor task.

An implantable electrode design for both chronic in vivo nerve recording and axon stimulation in freely behaving crayfish.

A chronically implantable electrode design permitting alternate extracellular nerve recording and axon stimulation in freely behaving crayfish was developed. The electrode consists of a double hook made from 20 microm thin platinum wire that can be fitted to various nerve diameters, and is easily implantable. A fast curing, flexible two-component silicone was used for insulation. The double hook was connected to plugs and fixed on the carapace of a crayfish allowing the animals to roam freely. The setup also allows for repeated dis- and re-connection of the crayfish for alternating recording and stimulation. Two channel recordings were used to determine directionality and to discriminate between afferent activity of the two stretch receptor neurons and efferent activity of several motor neurons. In addition, they were also used to determine the conduction velocity of the recorded efferent activity. Stable two-channel recordings could be obtained for up to 5 months and 15 days without apparent effects on the animal. In vivo stimulation could be performed for at least 3 1/2 weeks. The implantable double hook is suitable for widespread use in invertebrate neurobiology.

A laser-supported lowerable surface setup to study the role of ground contact during stepping.

We introduce a laser-supported setup to study the influence of afferent input on muscle activation during walking, using a movable ground platform. This approach allows investigating if and how the activity of stance phase muscles of an insect (e.g. stick insect) responds to a missing ground contact signal. The walking surface consists of a fixed and a lowerable part, which can be lowered to defined levels below the previous ground level at any time point during a walking sequence. As a consequence, the leg under investigation finds either a lower ground level or no ground support at all. The lowerable walking surface consists of a 49 mm × 34 mm stainless steel surface, made slippery and equipped for tarsal contact monitoring, similar to the system that was described by Gruhn and colleagues (Gruhn et al., 2006). The setup controller allows pneumatic lowering of the surface and subsequent detection of tarsal entry into the previous ground level with the help of a thin sheet of laser light and a corresponding detector. Here, we describe basic properties of the new setup and show the results of first experiments to demonstrate its use for the study of sensory and central influences in stepping of a small animal. In the experiments, we compare the effect of ground-support ("control") with either steps into the hole (SiH), ground support at a lower surface level, or the amputation of the tarsus on the onset of EMG activity in the flexor tibiae muscle of the stick insect.

Studying the neural basis of adaptive locomotor behavior in insects.

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch & Pflüger, 1995; Böhm et al., 1997; Gruhn & Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006). We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

Straight walking and turning on a slippery surface.

In stick insects, walking is the result of the co-action of different pattern generators for the single legs and coordinating inter-leg influences. We have used a slippery surface setup to understand the role the local neuronal processing in the thoracic ganglia plays in the ability of the animal to show turning movements. To achieve this, we removed the influence of mechanical coupling through the ground by using the slippery surface and removed sensory input by the successive amputation of neighboring legs. We analyzed the walking pattern of the front, middle and hind legs of tethered animals mounted above the surface and compared the kinematics of the straight walking legs with those of the curve walking inside and outside legs. The walking pattern was monitored both electrically through tarsal contact measurement and optically by using synchronized high-speed video. The vectors of leg movement are presented for the intact and a reduced preparation. Animals showed the ability to walk in a coordinated fashion on the slippery surface. Upon change from straight to curve walking, the stride length for the inside legs shortens and the vector of movement of the inner legs changes to pull the animal into the curve, while the outer legs act to pull and push it into the turn. In the reduced two-leg and in the single-leg preparation the behavior of the legs remained largely unchanged in the behavioral contexts of straight walking or turning with only small changes in the extreme positions. This suggests that the single stepping legs perform given motor programs on the slippery surface in a fashion that is highly independent not only of mechanical coupling between but also of the presence of the other legs.

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.