Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.     

α-Actinin Is Required for the Proper Assembly of Z-Disk/Focal-Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.     

Direct Measurements of Drag Forces in C. elegans Crawling Locomotion

With a simple and versatile microcantilever-based force measurement technique, we have probed the drag forces involved in Caenorhabditis elegans locomotion. As a worm crawls on an agar surface, we found that substrate viscoelasticity introduces nonlinearities in the force-velocity relationships, yielding nonconstant drag coefficients that are not captured by original resistive force theory. A major contributing factor to these nonlinearities is the formation of a shallow groove on the agar surface. We measured both the adhesion forces that cause the worm’s body to settle into the agar and the resulting dynamics of groove formation. Furthermore, we quantified the locomotive forces produced by C. elegans undulatory motions on a wet viscoelastic agar surface. We show that an extension of resistive force theory is able to use the dynamics of a nematode’s body shape along with the measured drag coefficients to predict the forces generated by a crawling nematode.     

Direct Measurements of Drag Forces in C.?elegans Crawling Locomotion

With a simple and versatile microcantilever-based force measurement technique, we have probed the drag forces involved in Caenorhabditis elegans locomotion. As a worm crawls on an agar surface, we found that substrate viscoelasticity introduces nonlinearities in the force-velocity relationships, yielding nonconstant drag coefficients that are not captured by original resistive force theory. A major contributing factor to these nonlinearities is the formation of a shallow groove on the agar surface. We measured both the adhesion forces that cause the worm’s body to settle into the agar and the resulting dynamics of groove formation. Furthermore, we quantified the locomotive forces produced by C. elegans undulatory motions on a wet viscoelastic agar surface. We show that an extension of resistive force theory is able to use the dynamics of a nematode’s body shape along with the measured drag coefficients to predict the forces generated by a crawling nematode.     

A 3D model of the Achilles tendon to determine the mechanisms underlying nonuniform tendon displacements

The Achilles is the thickest tendon in the body and is the primary elastic energy-storing component during running. The form and function of the human Achilles is complex: twisted structure, intratendinous interactions, and differential motor control from the triceps surae muscles make Achilles behavior difficult to intuit. Recent in vivo imaging of the Achilles has revealed nonuniform displacement patterns that are not fully understood and may result from complex architecture and musculotendon interactions. In order to understand which features of the Achilles tendon give rise to the nonuniform deformations observed in vivo, we used computational modeling to predict the mechanical contributions from different features of the tendon. The aims of this study are to: (i) build a novel computational model of the Achilles tendon based on ultrashort echo time MRI, (ii) compare simulated displacements with published in vivo ultrasound measures of displacement, and (iii) use the model to elucidate the effects of tendon twisting, intratendon sliding, retrocalcaneal insertion, and differential muscle forces on tendon deformation. Intratendon sliding and differential muscle forces were found to be the largest factors contributing to displacement nonuniformity between tendon regions. Elimination of intratendon sliding or muscle forces reduced displacement nonuniformity by 96% and 85%, respectively, while elimination of tendon twist and the retrocalcaneal insertion reduced displacement nonuniformity by only 35% and 3%. These results suggest that changes in the complex internal structure of the tendon alter the interaction between muscle forces and tendon behavior and therefore may have important implications on muscle function during movement.     

Mapping motor neuron activity to overt behavior in the leech

As an initial step in constructing a quantitative biomechanical model of the medicinal leech (Hirudo medicinalis), we determined the passive properties of its body wall over the physiological range of dimensions. The major results of this study were:

1. The ellipsoidal cross section of resting leeches is maintained by tonic muscle activation as well as forces inherent in the structure of the body wall (i.e., residual stress).
2. The forces required for longitudinal and circumferential stretch to maximum physiological dimensions were similar in magnitude. Cutting out pieces of body wall did not affect the passive longitudinal or circumferential properties of body wall away from the edges of the cut.
3. The strain (i.e., the percentage change in dimension) of different body segments when subject to the same force was identical, despite differences in muscle crosssections.
4. Serotonin, a known modulator of tension in leech muscles, affected passive forces at all physiological muscle lengths. This suggests that the longitudinal muscle is responsible for at least part of the passive tension of the body wall.
5. We propose a simple viscoelastic model of the body wall. This model captures the dynamics of the passive responses of the leech body wall to imposed step changes in length. Using steady-state passive tensions predicted by the viscoelastic model we estimate the forces required to maintain the leech at any given length over the physiological range.

     

A micromechanical muscle model for determining the impact of motor unit fiber clustering on force transmission in aging skeletal muscle

This study used a micromechanical finite element muscle model to investigate the effects of the redistribution of spatial activation patterns in young and old muscle. The geometry consisted of a bundle of 19 active muscle fibers encased in endomysium sheets, surrounded by passive tissue to model a fascicle. Force was induced by activating combinations of the 19 active muscle fibers. The spacial clustering of muscle fibers modeled in this study showed unbalanced strains suggesting tissue damage at higher strain levels may occur during higher levels of activation and/or during dynamic conditions. These patterns of motor unit remodeling are one of the consequences of motor unit loss and reinnervation associated with aging. The results did not reveal evident quantitative changes in force transmission between old and young adults, but the patterns of stress and strain distribution were affected, suggesting an uneven distribution of the forces may occur within the fascicle that could provide a mechanism for muscle injury in older muscle.

     

A Neuronal Acetylcholine Receptor Regulates the Balance of Muscle Excitation and Inhibition in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three α-subunits, UNC-38, UNC-63 and ACR-12, and two non–α-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf) mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.     

Sexual Selection and Dynamics of Jaw Muscle in Tupinambis Lizards

Sexual dimorphism patterns provide an opportunity to increase our understanding of trait evolution. Because selective forces may vary throughout the reproductive period, measuring dimorphism seasonally may be an interesting approach. An increased male head size may be important in intersexual and intrasexual interactions. In Tupinambis lizards, a big head is attributed in part to a large adductor muscle mass. Competition for mating can differ in species with different sex ratio and different degrees of sexual size dimorphism. We examined sexual differences in mass of the pterygoideus muscle, its temporal variation throughout the reproductive period and the relationship between muscle and reproductive condition in Tupinambis merianae and T. rufescens. We characterized sexual size dimorphism and sex ratio in both species. Mature males had larger jaw muscles than mature females in both species, mainly during the reproductive season. The dimorphism in jaw muscle was due to an increase in muscle mass in sexually active males. Seasonal increases in muscle mass and variation between immature and mature individuals suggest that the jaw muscle might be a secondary sexual character. We propose that the pterygoideus muscle may act as a signal of reproductive condition of males because it is associated with testis size and sperm presence. The patterns of sexual dimorphism in jaw muscle in both species were similar; however, the comparison shows how sexual characters remain dimorphic in different competition contexts and in species with different degrees of body size dimorphism. Our results suggest that jaw muscle as sexual character could be influenced by inter- and intrasexual selective pressures.     

Comparison between in vivo and theoretical bite performance: Using multi-body modelling to predict muscle and bite forces in a reptile skull

In biomechanical investigations, geometrically accurate computer models of anatomical structures can be created readily using computed-tomography scan images. However, representation of soft tissue structures is more challenging, relying on approximations to predict the muscle loading conditions that are essential in detailed functional analyses. Here, using a sophisticated multi-body computer model of a reptile skull (the rhynchocephalian Sphenodon), we assess the accuracy of muscle force predictions by comparing predicted bite forces against in vivo data. The model predicts a bite force almost three times lower than that measured experimentally. Peak muscle force estimates are highly sensitive to fibre length, muscle stress, and pennation where the angle is large, and variation in these parameters can generate substantial differences in predicted bite forces. A review of theoretical bite predictions amongst lizards reveals that bite forces are consistently underestimated, possibly because of high levels of muscle pennation in these animals. To generate realistic bites during theoretical analyses in Sphenodon, lizards, and related groups we suggest that standard muscle force calculations should be multiplied by a factor of up to three. We show that bite forces increase and joint forces decrease as the bite point shifts posteriorly within the jaw, with the most posterior bite location generating a bite force almost double that of the most anterior bite. Unilateral and bilateral bites produced similar total bite forces; however, the pressure exerted by the teeth is double during unilateral biting as the tooth contact area is reduced by half.     

Material Properties of Caenorhabditis elegans Swimming at Low Reynolds Number

Undulatory locomotion, as seen in the nematode Caenorhabditis elegans, is a common swimming gait of organisms in the low Reynolds number regime, where viscous forces are dominant. Although the nematode's motility is expected to be a strong function of its material properties, measurements remain scarce. Here, the swimming behavior of C. elegans is investigated in experiments and in a simple model. Experiments reveal that nematodes swim in a periodic fashion and generate traveling waves that decay from head to tail. The model is able to capture the experiments' main features and is used to estimate the nematode's Young's modulus E and tissue viscosity η. For wild-type C. elegans, we find E ≈ 3.77 kPa and η ≈ −860 Pa·s; values of η for live C. elegans are negative because the tissue is generating rather than dissipating energy. Results show that material properties are sensitive to changes in muscle functional properties, and are useful quantitative tools with which to more accurately describe new and existing muscle mutants.     

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.     

Generating dynamic simulations of movement using computed muscle control

Computation of muscle excitation patterns that produce coordinated movements of muscle-actuated dynamic models is an important and challenging problem. Using dynamic optimization to compute excitation patterns comes at a large computational cost, which has limited the use of muscle-actuated simulations. This paper introduces a new algorithm, which we call computed muscle control, that uses static optimization along with feedforward and feedback controls to drive the kinematic trajectory of a musculoskeletal model toward a set of desired kinematics. We illustrate the algorithm by computing a set of muscle excitations that drive a 30-muscle, 3-degree-of-freedom model of pedaling to track measured pedaling kinematics and forces. Only 10 min of computer time were required to compute muscle excitations that reproduced the measured pedaling dynamics, which is over two orders of magnitude faster than conventional dynamic optimization techniques. Simulated kinematics were within 1 degrees of experimental values, simulated pedal forces were within one standard deviation of measured pedal forces for nearly all of the crank cycle, and computed muscle excitations were similar in timing to measured electromyographic patterns. The speed and accuracy of this new algorithm improves the feasibility of using detailed musculoskeletal models to simulate and analyze movement.     

Dynamic optimization of human walking

A three-dimensional, neuromusculoskeletal model of the body was combined with dynamic optimization theory to simulate normal walking on level ground. The body was modeled as a 23 degree-of-freedom mechanical linkage, actuated by 54 muscles. The dynamic optimization problem was to calculate the muscle excitation histories, muscle forces, and limb motions subject to minimum metabolic energy expenditure per unit distance traveled. Muscle metabolic energy was calculated by slimming five terms: the basal or resting heat, activation heat, maintenance heat, shortening heat, and the mechanical work done by all the muscles in the model. The gait cycle was assumed to be symmetric; that is, the muscle excitations for the right and left legs and the initial and terminal states in the model were assumed to be equal. Importantly, a tracking problem was not solved. Rather only a set of terminal constraints was placed on the states of the model to enforce repeatability of the gait cycle. Quantitative comparisons of the model predictions with patterns of body-segmental displacements, ground-reaction forces, and muscle activations obtained from experiment show that the simulation reproduces the salient features of normal gait. The simulation results suggest that minimum metabolic energy per unit distance traveled is a valid measure of walking performance.     

Load-sharing in the lumbosacral spine in neutral standing & flexed postures – A combined finite element and inverse static study

Understanding load-sharing in the spine during in-vivo conditions is critical for better spinal implant design and testing. Previous studies of load-sharing that considered actual spinal geometry applied compressive follower load, with or without moment, to simulate muscle forces. Other studies used musculoskeletal models, which include muscle forces, but model the discs by simple beams or spherical joints and ignore the articular facet joints.This study investigated load-sharing in neutral standing and flexed postures using a detailed Finite Element (FE) model of the ligamentous lumbosacral spine, where muscle forces, gravity loads and intra-abdominal pressure, as predicted by a musculoskeletal model of the upper body, are input into the FE model. Flexion was simulated by applying vertebral rotations following spine rhythm measured in a previous in-vivo study, to the musculoskeletal model. The FE model predicted intradiscal pressure (IDP), strains in the annular fibers, contact forces in the facet joints, and forces in the ligaments. The disc forces and moments were determined using equilibrium equations, which considered the applied loads, including muscle forces and IDP, as well as forces in the ligaments and facet joints predicted by the FE model. Load-sharing was calculated as the portion of the total spinal load carried along the spine by each individual spinal structure. The results revealed that spinal loads which increased substantially from the upright to the flexed posture were mainly supported by the discs in the upright posture, whereas the ligaments’ contribution in resisting shear, compression, and moment was more significant in the flexed posture.     

Tribendimidine: Mode of Action and nAChR Subtype Selectivity in Ascaris and Oesophagostomum

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.     

A task-specific validation of homogeneous non-linear optimisation approaches

In biomechanics, musculoskeletal models are typically redundant. This situation is referred to as the distribution problem. Often, static, non-linear optimisation methods of the form “min: φ(f) subject to mechanical and muscular constraints” have been used to extract a unique set of muscle forces. Here, we present a method for validating this class of non-linear optimisation approaches where the homogeneous cost function, φ(f), is used to solve the distribution problem. We show that the predicted muscle forces for different loading conditions are scaled versions of each other if the joint loading conditions are just scaled versions. Therefore, we can calculate the theoretical muscle forces for different experimental conditions based on the measured muscle forces and joint loadings taken from one experimental condition and assuming that all input into the optimisation (e.g., moment arms, muscle attachment sites, size, fibre type distribution) and the optimisation approach are perfectly correct. Thus predictions of muscle force for other experimental conditions are accurate if the optimisation approach is appropriate, independent of the musculoskeletal geometry and other input required for the optimisation procedure. By comparing the muscle forces predicted in this way to the actual muscle forces obtained experimentally, we conclude that convex homogeneous non-linear optimisation approaches cannot predict individual muscle forces properly, as force-sharing among synergistic muscles obtained experimentally are not just scaled versions of joint loading, not even in a first approximation.     

Multi-level personalization of neuromusculoskeletal models to estimate physiologically plausible knee joint contact forces in children

Neuromusculoskeletal models are a powerful tool to investigate the internal biomechanics of an individual. However, commonly used neuromusculoskeletal models are generated via linear scaling of generic templates derived from elderly adult anatomies and poorly represent a child, let alone children with a neuromuscular disorder whose musculoskeletal structures and muscle activation patterns are profoundly altered. Model personalization can capture abnormalities and appropriately describe the underlying (altered) biomechanics of an individual. In this work, we explored the effect of six different levels of neuromusculoskeletal model personalization on estimates of muscle forces and knee joint contact forces to tease out the importance of model personalization for normal and abnormal musculoskeletal structures and muscle activation patterns. For six children, with and without cerebral palsy, generic scaled models were developed and progressively personalized by (1) tuning and calibrating musculotendon units’ parameters, (2) implementing an electromyogram-assisted approach to synthesize muscle activations, and (3) replacing generic anatomies with image-based bony geometries, and physiologically and physically plausible muscle kinematics. Biomechanical simulations of gait were performed in the OpenSim and CEINMS software on ten overground walking trials per participant. A mixed-ANOVA test, with Bonferroni corrections, was conducted to compare all models’ estimates. The model with the highest level of personalization produced the most physiologically plausible estimates. Model personalization is crucial to produce physiologically plausible estimates of internal biomechanical quantities. In particular, personalization of musculoskeletal anatomy and muscle activation patterns had the largest effect overall. Increased research efforts are needed to ease the creation of personalized neuromusculoskeletal models.

     

Simultaneous prediction of muscle and contact forces in the knee during gait

Musculoskeletal models are currently the primary means for estimating in vivo muscle and contact forces in the knee during gait. These models typically couple a dynamic skeletal model with individual muscle models but rarely include articular contact models due to their high computational cost. This study evaluates a novel method for predicting muscle and contact forces simultaneously in the knee during gait. The method utilizes a 12 degree-of-freedom knee model (femur, tibia, and patella) combining muscle, articular contact, and dynamic skeletal models. Eight static optimization problems were formulated using two cost functions (one based on muscle activations and one based on contact forces) and four constraints sets (each composed of different combinations of inverse dynamic loads). The estimated muscle and contact forces were evaluated using in vivo tibial contact force data collected from a patient with a force-measuring knee implant. When the eight optimization problems were solved with added constraints to match the in vivo contact force measurements, root-mean-square errors in predicted contact forces were less than 10 N. Furthermore, muscle and patellar contact forces predicted by the two cost functions became more similar as more inverse dynamic loads were used as constraints. When the contact force constraints were removed, estimated medial contact forces were similar and lateral contact forces lower in magnitude compared to measured contact forces, with estimated muscle forces being sensitive and estimated patellar contact forces relatively insensitive to the choice of cost function and constraint set. These results suggest that optimization problem formulation coupled with knee model complexity can significantly affect predicted muscle and contact forces in the knee during gait. Further research using a complete lower limb model is needed to assess the importance of this finding to the muscle and contact force estimation process.     

Feedback control from the jaw joints during biting: An investigation of the reptile Sphenodon using multibody modelling

Sphenodon, a lizard-like reptile, is the only living representative of a group that was once widespread at the time of the dinosaurs. Unique jaw mechanics incorporate crushing and shearing motions to breakdown food, but during this process excessive loading could cause damage to the jaw joints and teeth. In mammals like ourselves, feedback from mechanoreceptors within the periodontal ligament surrounding the teeth is thought to modulate muscle activity and thereby minimise such damage. However, Sphenodon and many other tetrapods lack the periodontal ligament and must rely on alternative control mechanisms during biting. Here we assess whether mechanoreceptors in the jaw joints could provide feedback to control muscle activity levels during biting. We investigate the relationship between joint, bite, and muscle forces using a multibody computer model of the skull and neck of Sphenodon. When feedback from the jaw joints is included in the model, predictions agree well with experimental studies, where the activity of the balancing side muscles reduces to maintain equal and minimal joint forces. When necessary, higher, but asymmetric, joint forces associated with higher bite forces were achievable, but these are likely to occur infrequently during normal food processing. Under maximum bite forces associated with symmetric maximal muscle activation, peak balancing side joint forces were more than double those of the working side. These findings are consistent with the hypothesis that feedback similar to that used in the simulation is present in Sphenodon.