Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current.

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (IMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Task-dependent modification of leg motor neuron synaptic input underlying changes in walking direction and walking speed.

Animals modify their behavior constantly to perform adequately in their environment. In terrestrial locomotion many forms of adaptation exist. Two tasks are changes of walking direction and walking speed. We investigated these two changes in motor output in the stick insect Cuniculina impigra to see how they are brought about at the level of leg motor neurons. We used a semi-intact preparation in which we can record intracellularly from leg motor neurons during walking. In this single-leg preparation the middle leg of the animal steps in a vertical plane on a treadwheel. Stimulation of either abdomen or head reliably elicits fictive forward or backward motor activity, respectively, in the fixed and otherwise deafferented thorax-coxa joint. With a change of walking direction only thorax-coxa-joint motor neurons protractor and retractor changed their activity. The protractor switched from swing activity during forward to stance activity during backward walking, and the retractor from stance to swing. This phase switch was due to corresponding change of phasic synaptic inputs from inhibitory to excitatory and vice versa at specific phases of the step cycle. In addition to phasic synaptic input a tonic depolarization of the motor neurons was present. Analysis of changes in stepping velocity during stance showed only a significant correlation to flexor motor neuron activity, but not to that of retractor and depressor motor neurons during forward walking. These results show that different tasks in the stick insect walking system are generated by altering synaptic inputs to specific leg joint motor neurons only.

How can motor systems retain performance over a wide temperature range? Lessons from the crustacean stomatogastric nervous system.

Marine invertebrates, such as lobsters and crabs, deal with a widely and wildly fluctuating temperature environment. Here, we describe the effects of changing temperature on the motor patterns generated by the stomatogastric nervous system of the crab, Cancer borealis. Over a broad range of "permissive" temperatures, the pyloric rhythm increases in frequency but maintains its characteristic phase relationships. Nonetheless, at more extreme high temperatures, the normal triphasic pyloric rhythm breaks down, or "crashes". We present both experimental and computational approaches to understanding the stability of both single neurons and networks to temperature perturbations, and discuss data that shows that the "crash" temperatures themselves may be environmentally regulated. These approaches provide insight into how the nervous system can be stable to a global perturbation, such as temperature, in spite of the fact that all biological processes are temperature dependent.

Mapping circuit dynamics during function and dysfunction.

Neural circuits can generate many spike patterns, but only some are functional. The study of how circuits generate and maintain functional dynamics is hindered by a poverty of description of circuit dynamics across functional and dysfunctional states. For example, although the regular oscillation of a central pattern generator is well characterized by its frequency and the phase relationships between its neurons, these metrics are ineffective descriptors of the irregular and aperiodic dynamics that circuits can generate under perturbation or in disease states. By recording the circuit dynamics of the well-studied pyloric circuit in Cancer borealis, we used statistical features of spike times from neurons in the circuit to visualize the spike patterns generated by this circuit under a variety of conditions. This approach captures both the variability of functional rhythms and the diversity of atypical dynamics in a single map. Clusters in the map identify qualitatively different spike patterns hinting at different dynamic states in the circuit. State probability and the statistics of the transitions between states varied with environmental perturbations, removal of descending neuromodulatory inputs, and the addition of exogenous neuromodulators. This analysis reveals strong mechanistically interpretable links between complex changes in the collective behavior of a neural circuit and specific experimental manipulations, and can constrain hypotheses of how circuits generate functional dynamics despite variability in circuit architecture and environmental perturbations.

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.

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.

QUANTITATIVE REEVALUATION OF THE EFFECTS OF SHORT- AND LONG-TERM REMOVAL OF DESCENDING MODULATORY INPUTS ON THE PYLORIC RHYTHM OF THE CRAB, CANCER BOREALIS.

The crustacean stomatogastric ganglion (STG) receives descending neuromodulatory inputs from three anterior ganglia, the paired commissural ganglia (CoGs) and the single esophageal ganglion (OG). In this paper we provide the first detailed and quantitative analyses of the short- and long-term effects of removal of these descending inputs (decentralization) on the pyloric rhythm of the STG. Thirty minutes after decentralization, the mean frequency of the pyloric rhythm dropped from 1.20 Hz in control to 0.52 Hz. Whereas the relative phase of pyloric neuron activity was approximately constant across frequency in the controls, after decentralization this changed markedly. Nine control preparations kept for 5-6 days in vitro maintained pyloric rhythm frequencies close to their control values. Nineteen decentralized preparations kept for 5-6 days dropped slightly in frequency from those seen at 30 minutes after decentralization, but then displayed stable activity over 6 days. Bouts of higher frequency activity were intermittently seen in both control and decentralized preparations, but the bouts began earlier and were more frequent in the decentralized preparations. Although the bouts may indicate that the removal of the modulatory inputs triggered changes in neuronal excitability, these changes did not produce obvious long-lasting changes in the frequency of the decentralized preparations.

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.