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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 work(4) 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 postretraction 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)

Producing context-appropriate motor acts requires integrating multiple sensory modalities. Presynaptic inhibition of proprioceptive afferent neurons(1-4) and afferents of different modalities targeting the same motor neurons (MNs)(5-7) underlies some of this integration. However, in most systems, an interneuronal network is interposed between sensory afferents and MNs. How these networks contribute to this integration, particularly at single-neuron resolution, is little understood. Context-specific integration of load and movement sensory inputs occurs in the stick insect locomotory system,(6,8-12) and both inputs feed into a network of premotor nonspiking interneurons (NSIs).(8) We analyzed how load altered movement signal processing in the stick insect femur-tibia (FTi) joint control system by tracing the interaction of FTi movement(13-15) (femoral chordotonal organ [fCO]) and load(13,15,16) (tibial campaniform sensilla [CS]) signals through the NSI network to the slow extensor tibiae (SETi) MN, the extensor MN primarily active in non-walking animals.(17-19) On the afferent level, load reduced movement signal gain by presynaptic inhibition. In the NSI network, graded responses to movement and load inputs summed nonlinearly, increasing the gain of NSIs opposing movement-induced reflexes and thus decreasing the SETi and extensor tibiae muscle movement reflex responses. Gain modulation was movement-parameter specific and required presynaptic inhibition. These data suggest that gain changes in distributed premotor networks, specifically the relative weighting of antagonistic pathways, could be a general mechanism by which multiple sensory modalities are integrated to generate context-appropriate motor activity.

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.

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.

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 C-14-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm- matched electrode fills.

In many animals, individual legs can either function independently, as in behaviors such as scratching or searching, or be used in coordinated patterns with other legs, as in walking or climbing. While the control of walking has been extensively investigated, the mechanisms mediating the behavioral choice to activate individual legs independently are poorly understood. We examined this issue in stick insects, in which each leg can independently produce a rhythmic searching motor pattern if it doesn't find a foothold [1-4]. We show here that one non-spiking interneuron, 14, controls searching behavior in individual legs. One 14 is present in each hemi-segment of the three thoracic ganglia [5, 6]. Search-inducing sensory input depolarizes 14. 14 activity was necessary and sufficient to initiate and maintain searching movements. When substrate contact was provided, 14 depolarization no longer induced searching. 14 therefore both integrates search-inducing sensory input and is gated out by other sensory input (substrate contact). Searching thus occurs only when it is behaviorally appropriate. 14 depolarization never elicited stepping. These data show that individual, locally activated neurons can mediate the behavioral choice to use individual legs independently. This mechanism may be particularly important in insects' front legs, which can function independently like vertebrate arms and hands [7]. Similar local command mechanisms that selectively activate the pattern generators controlling repeated functional units such as legs or body segments may be present in other systems.