Correlation between ranges of leg walking angles and passive rest angles among leg types in stick insects.

Because of scaling issues, passive muscle and joint forces become increasingly important as limb size decreases.1-3 In some small limbs, passive forces can drive swing in locomotion,4,5 and antagonist passive torques help control limb swing velocity.6 In stance, minimizing antagonist muscle and joint passive forces could save energy. These considerations predict that, for small limbs, evolution would result in the angle range over which passive forces are too small to cause limb movement (called "resting-state range" in prior insect work4 and "area of neutral equilibrium" in physics and engineering) correlating with the limb's typical working range, usually that in locomotion. We measured the most protracted and retracted thorax-femur (ThF) angles of the pro- (front), meso- (middle), and metathoracic (hind) leg during stick insect (Carausius morosus) walks. This ThF working range differed in the three leg types, being more posterior in more posterior legs. In other experiments, we manually protracted or retracted the denervated front, middle, and hind legs. Upon release, passive forces moved the leg in the opposite direction (retraction or protraction) until it reached the most protracted or most retracted edge of the ThF resting-state range. The ThF resting-state angle ranges correlated with the leg-type working range, being more posterior in more posterior legs. The most protracted ThF walking angles were more retracted than the post-protraction ThF angles, and the most retracted ThF walking angles were similar to the post-retraction ThF angles. These correlations of ThF working- and resting-state ranges could simplify motor control and save energy. These data also provide an example of evolution altering behavior by changing passive muscle and joint properties.7.

Neuromodulation Can Be Simple: Myoinhibitory Peptide, Contained in Dedicated Regulatory Pathways, Is the Only Neurally-Mediated Peptide Modulator of Stick Insect Leg Muscle.

In the best studied cases (Aplysia feeding, crustacean stomatogastric system), peptidergic modulation is mediated by large numbers of peptides. Furthermore, in Aplysia, excitatory motor neurons release the peptides, obligatorily coupling target activation and modulator release. Vertebrate nervous systems typically contain about a hundred peptide modulators. These data have created a belief that modulation is, in general, complex. The stick insect leg is a well-studied locomotory model system, and the complete stick insect neuropeptide inventory was recently described. We used multiple techniques to comprehensively examine stick insect leg peptidergic modulation. Single-cell mass spectrometry (MS) and immunohistochemistry showed that myoinhibitory peptide (MIP) is the only neuronal (as opposed to hemolymph-borne) peptide modulator of all leg muscles. Leg muscle excitatory motor neurons contained no neuropeptides. Only the common inhibitor (CI) and dorsal unpaired median (DUM) neuron groups, each neuron of which innervates a group of functionally-related leg muscles, contained MIP. We described MIP transport to, and receptor presence in, one leg muscle, the extensor tibiae (ExtTi). MIP application reduced ExtTi slow fiber force and shortening by about half, increasing the muscle's ability to contract and relax rapidly. These data show neuromodulation does not need to be complex. Excitation and modulation do not need to be obligatorily coupled (Aplysia feeding). Modulation does not need to involve large numbers of peptides, with the attendant possibility of combinatorial explosion (stomatogastric system). Modulation can be simple, mediated by dedicated regulatory neurons, each innervating a single group of functionally-related targets, and all using the same neuropeptide.SIGNIFICANCE STATEMENT Vertebrate and invertebrate nervous systems contain large numbers (around a hundred in human brain) of peptide neurotransmitters. In prior work, neuropeptide modulation has been complex, either obligatorily coupling postsynaptic excitation and modulation, or large numbers of peptides modulating individual neural networks. The complete stick insect neuropeptide inventory was recently described. We comprehensively describe here peptidergic modulation in the stick insect leg. Surprisingly, out of the large number of potential peptide transmitters, only myoinhibitory peptide (MIP) was present in neurons innervating leg muscles. Furthermore, the peptide was present only in dedicated regulatory neurons, not in leg excitatory motor neurons. Peptidergic modulation can thus be simple, neither obligatorily coupling target activation and modulation nor involving so many peptides that combinatorial explosion can occur.

Swing Velocity Profiles of Small Limbs Can Arise from Transient Passive Torques of the Antagonist Muscle Alone.

In large limbs, changing motor neuron activity typically controls within-movement velocity. For example, sequential agonist-antagonist-agonist motor neuron firing typically underlies the slowing often present at the end of human reaches. In physiological movements of large limbs, antagonistic muscle passive torque is generally negligible. In small limbs, alternatively, passive torques can determine limb rest position, generate restoring movements to it, and decrease agonist-generated movement amplitude and velocity maxima. These observations suggest that, in small limbs, passive forces might also control velocity changes within movements. We investigated this issue in stick insect middle leg femur-tibia (FT) joint. During swing, the FT joint extensor muscle actively shortens and the flexor muscle passively lengthens. As in human reaching, after its initial acceleration, FT joint velocity continuously decreases. We measured flexor passive forces during imposed stretches spanning the ranges of FT joint angles, angular velocities, and movement amplitudes present in leg swings. The viscoelastic "transient" passive force that occurs during and soon after stretch depended on all three variables and could be tens of times larger than the "steady-state" passive force commonly measured long after stretch end. We combined these data, the flexor and extensor moment arms, and an existing extensor model to simulate FT joint swing. To measure only passive (flexor) muscle-dependent effects, we used constant extensor activations in these simulations. In simulations using data from ten flexor muscles, flexor passive torque could always produce swings with, after swing initiation, continuously decreasing velocities. Antagonist muscle passive torques alone can thus control within-movement velocity.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties.

We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K2SO4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities ≥2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with (14)C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills.

Using individual-muscle specific instead of across-muscle mean data halves muscle simulation error.

Hill-type parameter values measured in experiments on single muscles show large across-muscle variation. Using individual-muscle specific values instead of the more standard approach of across-muscle means might therefore improve muscle model performance. We show here that using mean values increased simulation normalized RMS error in all tested motor nerve stimulation paradigms in both isotonic and isometric conditions, doubling mean simulation error from 9 to 18 (different at p < 0.0001). These data suggest muscle-specific measurement of Hill-type model parameters is necessary in work requiring highly accurate muscle model construction. Maximum muscle force (F (max)) showed large (fourfold) across-muscle variation. To test the role of F (max) in model performance we compared the errors of models using mean F (max) and muscle-specific values for the other model parameters, and models using muscle-specific F (max) values and mean values for the other model parameters. Using muscle-specific F (max) values did not improve model performance compared to using mean values for all parameters, but using muscle-specific values for all parameters but F (max) did (to an error of 14, different from muscle-specific, mean all parameters, and mean only F (max) errors at p ≤ 0.014). Significantly improving model performance thus required muscle-specific values for at least a subset of parameters other than F (max), and best performance required muscle-specific values for this subset and F (max). Detailed consideration of model performance suggested that remaining model error likely stemmed from activation of both fast and slow motor neurons in our experiments and inadequate specification of model activation dynamics.

Hill-type muscle model parameters determined from experiments on single muscles show large animal-to-animal variation.

Models built using mean data can represent only a very small percentage, or none, of the population being modeled, and produce different activity than any member of it. Overcoming this "averaging" pitfall requires measuring, in single individuals in single experiments, all of the system's defining characteristics. We have developed protocols that allow all the parameters in the curves used in typical Hill-type models (passive and active force-length, series elasticity, force-activation, force-velocity) to be determined from experiments on individual stick insect muscles (Blümel et al. 2012a). A requirement for means to not well represent the population is that the population shows large variation in its defining characteristics. We therefore used these protocols to measure extensor muscle defining parameters in multiple animals. Across-animal variability in these parameters can be very large, ranging from 1.3- to 17-fold. This large variation is consistent with earlier data in which extensor muscle responses to identical motor neuron driving showed large animal-to-animal variability (Hooper et al. 2006), and suggests accurate modeling of extensor muscles requires modeling individual-by-individual. These complete characterizations of individual muscles also allowed us to test for parameter correlations. Two parameter pairs significantly co-varied, suggesting that a simpler model could as well reproduce muscle response.

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.

Slow temporal filtering may largely explain the transformation of stick insect (Carausius morosus) extensor motor neuron activity into muscle movement.

Understanding how nervous systems generate behavior requires understanding how muscles transform neural input into movement. The stick insect extensor tibiae muscle is an excellent system in which to study this issue because extensor motor neuron activity is highly variable during single leg walking and extensor muscles driven with this activity produce highly variable movements. We showed earlier that spike number, not frequency, codes for extensor amplitude during contraction rises, which implies the muscle acts as a slow filter on the time scale of burst interspike intervals (5-10 ms). We examine here muscle response to spiking variation over entire bursts, a time scale of hundreds of milliseconds, and directly measure muscle time constants. Muscle time constants differ during contraction and relaxation, and contraction time constants, although variable, are always extremely slow (200-700 ms). Models using these data show that extremely slow temporal filtering alone can explain much of the observed transform properties. This work also revealed an unexpected (to us) ability of slow filtering to transform steadily declining inputs into constant amplitude outputs. Examination of the effects of time constant variability on model output showed that variation within an SD primarily altered output amplitude, but variation across the entire range also altered contraction shape. These substantial changes suggest that understanding the basis of this variation is central to predicting extensor activity and that the animal could theoretically vary muscle time constant to match extensor response to changing behavioral need.

The extensor tibiae muscle of the stick insect: biomechanical properties of an insect walking leg muscle.

We investigated the properties of the extensor tibiae muscle of the stick insect (Carausius morosus) middle leg. Muscle geometry of the middle leg was compared to that of the front and hind legs and to the flexor tibiae, respectively. The mean length of the extensor tibiae fibres is 1.41+/-0.23 mm and flexor fibres are 2.11+/-0.30 mm long. The change of fibre length with joint angle was measured and closely follows a cosine function. Its amplitude gives effective moment arm lengths of 0.28+/-0.02 mm for the extensor and 0.56+/-0.04 mm for the flexor. Resting extensor tibiae muscle passive tonic force increased from 2 to 5 mN in the maximum femur-tibia (FT)-joint working range when stretched by ramps. Active muscle properties were measured with simultaneous activation (up to 200 pulses s(-1)) of all three motoneurons innervating the extensor tibiae, because this reflects most closely physiological muscle activation during leg swing. The force-length relationship corresponds closely to the typical characteristic according to the sliding filament hypothesis: it has a plateau at medium fibre lengths, declines nearly linearly in force at both longer and shorter fibre lengths, and the muscle's working range lies in the short to medium fibre length range. Maximum contraction velocity showed a similar relationship. The force-velocity relationship was the traditional Hill curve hyperbola, but deviated from the hyperbolic shape in the region of maximum contraction force close to the isometric contraction. Step-like changes in muscle length induced by loaded release experiments characterised the non-linear series elasticity as a quadratic spring.

Different motor neuron spike patterns produce contractions with very similar rises in graded slow muscles.

Graded muscles produce small twitches in response to individual motor neuron spikes. During the early part of their contractions, contraction amplitude in many such muscles depends primarily on the number of spikes the muscle has received, not the frequency or pattern with which they were delivered. Stick insect (Carausius morosus) extensor muscles are graded and thus would likely show spike-number dependency early in their contractions. Tonic stimulations of the extensor motor nerve showed that the response of the muscles differed from the simplest form of spike-number dependency. However, these differences actually increased the spike-number range over which spike-number dependency was present. When the motor nerve was stimulated with patterns mimicking the motor neuron activity present during walking, amplitude during contraction rises also depended much more on spike number than on spike frequency. A consequence of spike-number dependency is that brief changes in spike frequency do not alter contraction slope and we show here that extensor motor neuron bursts with different spike patterns give rise to contractions with very similar contraction rises. We also examined in detail the early portions of a large number of extensor motor neuron bursts recorded during single-leg walking and show that these portions of the bursts do not appear to have any common spike pattern. Although alternative explanations are possible, the simplest interpretation of these data is that extensor motor neuron firing during leg swing is not tightly controlled.

Natural neural output that produces highly variable locomotory movements.

We recorded fast extensor tibiae motor neuron activity during single-legged treadmill walking in the stick insect, Carausius morosus. We used this activity to stimulate the extensor muscle motor nerve, observed the resulting extensor muscle contractions under isotonic conditions, and quantified these contractions with a variety of measures. Extensor contractions induced in this manner were highly variable, with contraction measures having SDs of 12 to 51%, and ranges of 82 to 275%, when expressed as percentages of the means, an unexpectedly wide range for a locomotory pattern. Searches for correlations among the contraction measures showed that, in general, this high variability is not reduced by contraction measure covariation. Comparing responses (to identical input) across animals showed that extensor muscles from different animals generally significantly differed from one another. However, correlation analyses on these data suggested that these differences do not indicate that multiple extensor muscle subtypes exist. Extensor muscles instead appear to belong to a single class, albeit one with high animal to animal variability. These data thus provide another well-quantified example (along with Aplysia feeding) of a repetitive but highly variable motor pattern (in contrast to the high rhythmicity and stereotypy present in most other well-quantified repetitive motor patterns). We suggest this high variability could be an adaptive combination of locomotion, active sensing, and crypsis arising from the relatively low demand for locomotion in Carausius behavior, the highly fragmented environment the animal inhabits, and its need to avoid predatory attention.