Zill Lab NSF Research

RESEARCH PROJECT: DISCRETE SENSORY SIGNALS OF LOAD DECREASES IN THE LEGS OF FREELY MOVING COCKROACHES S.N. Zill*, E.R. Duke, B.R. Keller, Dept. Anat. and Pathol., J.C. Edwards Sch. Med., Marshall Univ., Huntington, WV, USA

ABSTRACT - Decreases in load are important cues in the control of posture and walking, but the specific receptors that signal leg unloading have been identified in only a few systems. We recorded activities of the tibial campaniform sensilla, receptors that monitor forces as strains in the exoskeleton, in freely moving cockroaches. The effective body load was changed via magnets attached to the thorax and currents applied to a coil below the substrate. Body position was monitored by high speed video recording. The tibial sensilla are organized into proximal and distal subgroups. Recent studies have demonstrated that the anatomical arrangement and number of receptors is similar in all legs. The proximal and distal sensilla showed differential responses to changes in load in freely standing animals. Tests in which loading was suddenly increased produced short latency firing of proximal sensilla of the middle leg, while imposed forces that decreased body load elicited bursts from the distal sensilla. In both increases and decreases in load, sensory discharges more closely approximated the time of peak velocity of body movement than the maximum change in body position. Tests in which forces were applied for longer durations showed that the proximal sensilla discharged tonically and reflected the magnitude of load increases, while the distal sensilla typically responded only phasically to sustained unloading. Distal sensilla also had a higher threshold than proximal receptors, and the threshold depended upon both the rate and amplitude of unloading. Thus, these studies strongly suggest that 1) small variations in load are signaled by modulation of firing of proximal sensilla, which can reflexly excite extensor motoneurons to provide support of body load; 2) large or rapid load decreases specifically excite distal sensilla in freely standing animals, which can inhibit extensor firing. These decreases in load should occur prior to leg lifting in swing or when a leg slips and loses contact with the substrate. We are presently recording the activities of leg muscles that support body load to examine how sensory cues of decreased load are integrated into motor outputs. Although specific sense organs that detect unloading have not been identified in vertebrates, similar functions could be fulfilled by receptors of the feet in signaling load decreases in posture and locomotion. Support Contributed By: NSF Grant IBN-0235997

LOAD DECREASES IN POSTURE AND WALKING - Our project is studying how forces are detected and controlled in posture and locomotion. Forces must be generated to support and stabilize the body weight when standing and to provide propulsion when walking. Decreases in force, particularly those occurring during changes in load, can be important indicators of the effectiveness of leg use and provide cues for the coordination of limb movements. Activities of some limb muscles are strongly correlated with leg unloading in postural perturbations, and decrements in load may be determining factors in eliciting responses to leg slipping. In addition, the decrease in loading at the end of the stance phase of walking is necessary for the initiation of leg lifting in swing. However, sensory receptors that detect load decreases have rarely been identified.

DECREASES IN LOAD PRODUCE MODULATION OF MUSCLE ACTIVITIES -Several recent studies have shown that load decreases can modulate activities of leg muscles in walking in addition to their effects in eliciting rapid reactions or phase transitions. Tonically decreasing body weight during treadmill walking (by a pulley and winch) substantially reduced the amplitude of bursts of a leg extensor muscle. Mazzaro et al. mimicked decreases in load by producing small changes in the ankle joint angle with a robotic actuator. These experiments applied stimuli at relatively slow rates and small amplitudes to approximate ongoing load changes that can occur in the absence of large perturbations. Decreasing dorsiflexion, as would occur with unloading, produced a rapid decrease in firing in the soleus muscle. Several control experiments suggested that these effects were mediated by Golgi tendon organs, which monitor forces in the leg. These experiments support the idea that receptors that detect loads can contribute to setting motor activities during normal, unperturbed walking.

SUPPORT OF BODY LOAD IN COCKROACHES - Our previous studies have shown the tibial campaniform sensilla, which detect forces in the legs, are strongly activated by sudden increases in body load. Some receptors (distal sensilla) discharge as loading returns to baseline, suggesting that they can actively encode force decreases. Our working hypothesis is that the tibial sensilla can contribute to compensatory reactions by detecting changes in forces before substantial changes in body position have occurred. We have also found that the trochanteral extensor muscle is used in support of body load in all legs. Activity of this muscle is strongly affected by reflex connections of the tibial sensilla. In the present experiments, we examined how sensory and motor activities are affected by load decreases, including perturbations of relatively small amplitudes.

ANATOMY AND RESPONSES OF TIBIAL CAMPANIFORM SENSILLA - A campaniform sensillum consists of a sensory neuron whose dendrite inserts upon a cuticular cap embedded in the exoskeleton. The tibial sensilla are organized in proximal and distal subgroups whose caps are mutually perpendicular in orientation. We have recently examined the cuticular caps of the tibial sensilla from each of the segmental legs of individual animals (N=5). The mean number and size of the sensilla are similar in the legs of all segments (the only statistically significant difference was found that the hind legs had more proximal sensilla than the front legs). Histological sections and physiological recordings suggest that the distal sensilla contain the largest sensory neurons. The tibial sensilla show differential responses to forces imposed upon the legs and discrete reflex effects in restrained preparations. Distal sensilla are excited when forces that bend the tibia toward extension are decreased. Distal receptors also show a higher threshold than proximal receptors and elicit reflexes that inhibit the trochanteral extensor muscle.

DECREASES IN LOAD ARE PRODUCED WITH A MAGNET AND COIL - An electrical coil is positioned below the arena and activated by currents that are generated by computer. A magnet is glued to the dorsum of the animal. Currents to the coil produce magnetic fields that repel the magnet and decrease the load upon the legs. Animals are videotaped at high speed to monitor body position. Extracellular recordings are taken of sensory or motor activities and are synchronized with kinematic data. The forces generated by the coil in the Z axis are calibrated using magnets attached to a force transducer.

LOAD DECREASES PRODUCE MODULATION OF EXTENSOR ACTIVITIES - Our previous studies have shown that the trochanteral extensor muscle produces force that supports the body weight in upright posture. In the sequence above, currents were applied using rapid half sine waveforms that decreased or increased load and then returned to baseline. Load decreases produced short latency inhibition of firing of the extensor. Synchronization of kinematic data with muscle recordings showed that the inhibition was initiated at approximately the same time as the peak of velocity of body movement.

DISTAL SENSILLA FIRE WHEN LOAD RAPIDLY DECREASES - In the sequence below, currents to the coil were applied repeatedly using half sine wave forms that rapidly decreased the body weight. The distal sensilla fired intense bursts at short latency during the initial rising phase when load was decreasing, while the proximal sensilla discharged as the load began to increase from the peak of unloading. The recordings are synchronized with measurements of the body position and velocity. Voltage to the coil that decreased the body weight produced an increase in the height of the body above the substrate. Identification of units as tibial sensilla was confirmed by ablating the cuticular caps. Cap ablation eliminated sensory activities recorded in response to bending of the tibia and in subsequent tests of load decreases.

SENSILLA DISCHARGES OCCUR CLOSE TO PEAK VELOCITY OF BODY MOVEMENT IN BOTH LOADING AND UNLOADING - These recordings show the responses to tests of unloading and loading in the same animal (half sine duration = 150 ms). The graphs below plot the mean responses of proximal and distal sensilla and changes in body position to application of transient decreases and increases in load (n = 127 tests unloading, 123 increased loading, N = 5 animals; position = 29 unload, 34 load). The graphs were normalized from the stimulus onset and also include a time period (50% of stimulus duration) prior to and after the perturbation. In unloading, the distal sensilla fired a burst when body load was decreasing, and the proximal sensilla discharged when load was returned to baseline. The reverse pattern of activation of subgroups was seen when load was rapidly increased. The proximal receptors fired to load increases, while the distal receptors discharged in the declining phase. In both graphs, the sensory discharge substantially precedes the maximum or minimum body movement and more closely approximates the peak of body velocity. The histograms above plot the times of peak change in position and velocity of movement after the onset of the stimulus for individual trials within the same data. They also show both the latency to the maximum firing frequency and the time to 30% of that value (indicated by the arrows in the upper figures) in individual tests. Both latency values are similar for distal sensilla discharges to body unloading, indicating that firing accelerated rapidly within a burst. The onset of bursting of proximal sensilla in tests of increased load was even more rapid but was slower to reach a maximum. These data are consistent with previous findings that proximal receptors have lower thresholds than distal sensilla. However, in all perturbations that change body load, the discharges of sensory receptors monitoring force occurred more closely in time with peak velocity than with maximum change in body position.

VARIABILITY IN DISTAL FIRING FREQUENCY AND THRESHOLD - The firing frequency of the distal sensilla in half sine perturbations depended upon the rate of unloading and was consistent when single postures were held. In the sequences shown above, the stimulus duration was 250 ms and resulted in lower rates of firing of the distal sensillum. However, the same stimulus applied in another posture produced a lower rate of firing, and in other postures only single or pairs of spikes were elicited. Tests were also performed using half sine waves at different peak currents. Small amplitude perturbations produced only inhibition of proximal firing and the distal sensillum threshold was reached at higher levels of unloading.

DISCHARGES OF DISTAL SENSILLA ARE PREDOMINANTLY PHASIC WHEN UNLOADING IS SUSTAINED BUT CAN ENCODE THE RATE OF LOAD DECREASE - The recordings below show the sensory discharges that occurred when ramp and hold waveforms were applied to the coil to change body load. When loading was increased, proximal sensilla fired intensely during the rising phase and then adapted to a tonic discharge frequency that was sustained at a level higher than baseline throughout the load increase. The distal sensilla fired upon return to the initial level. When loading was decreased, the firing of proximal sensilla was inhibited. The distal sensilla typically only fired phasically during the ramp phase and the discharge was not sustained. In some cases, single or pairs of distal spikes occurred during the hold phase. The proximal sensilla fired as the load returned to baseline during the return phase of the ramp. The lower recordings and graphs show the results of unloading at different rates but the same amplitude. The firing frequency of the distal sensilla increased at higher rates of unloading. These findings support the idea that the distal sensillum discharges indicate that unloading is occurring and the rate of unloading. Distal receptors do not signal the new, lower level of load.

UNLOADING AT DIFFERENT AMPLITUDES: SMALL PERTURBATIONS PRODUCE MODULATION OF PROXIMAL SENSILLUM FIRING; LARGE PERTURBATIONS ELICIT DISTAL SENSILLUM DISCHARGES - Mazzaro et al. (2005) proposed that responses to small perturbations reflect adjustments to variations during normal behaviors, while larger perturbations might activate different neural circuits. We tested the responses of animals to unloading applied as ramp functions at different amplitudes but at the same rate of rise and decline. The recordings below show tests in which ramp load decreases were applied at progressively increasing amplitudes in a single trial while the graphs at bottom plot the mean discharge rates of the proximal and distal sensilla. In the smallest perturbations, the firing frequency of the proximal sensillum declined during the load increase and then showed a rebound during the ramp return when load was reapplied. The distal sensillum began firing when the amplitude of unloading was higher. These findings are consistent with the idea that small changes in load produce modulation of proximal sensillum firing, while large load decreases recruit distal sensilla. The reflex effects of the sensilla should therefore 1) decrease excitatory drive to extensors during small perturbations and 2) produce active inhibition of extensors during larger or more rapid perturbations.

CONCLUSIONS:

1) The tibial campaniform sensilla are organized into proximal and distal subgroups in all legs. Anatomical studies have examined the cuticular caps and shown that the numbers of receptors and cap sizes are similar in all serially homologous legs.

2) Studies that have recorded activities of the receptors during perturbations that changed body load in freely-standing animals have directly demonstrated that the distal tibial campaniform sensilla respond to stimuli that decrease body load. This finding confirms hypotheses from studies in restrained preparations and shows that insect sense organs actively signal leg unloading.

3) Tests that used very rapid half sine perturbations showed that the distal sensillum discharge precedes peak body displacement and more closely approximates the maximum body velocity. A similar correlation was found for discharges of proximal receptors to increased load. These findings support the idea that perturbations can be countered multimodally using information from force and velocity receptors.

4) Perturbations using ramp and hold functions have demonstrated that the proximal sensilla discharge tonically and reflect the magnitude of load increases, while the distal sensilla typically respond only phasically to sustained unloading. The threshold of distal sensilla is higher than that of proximal receptors, and depends upon both the rate and amplitude of unloading.

5) Tests were applied in which only the amplitude of the hold phase was varied. In the smallest perturbations, the firing of proximal sensilla declined during the load decrease and then showed a rebound during the ramp return when load was reapplied. The distal sensilla began firing at higher amplitudes of unloading. These findings are consistent with the idea that small changes in load produce modulation of proximal sensillum firing, while distal sensilla are recruited in large load decreases. The reflex effects of the sensilla should therefore decrease excitatory drive during small perturbations and produce active inhibition during larger perturbations.

6) Future tests will examine how these sensory signals are incorporated into motor outputs by recording activities of leg muscles during load decreases in posture and walking. Perturbations applied to standing animals produce inhibition of the trochanteral extensor muscle of the middle leg consistent with the proposed functions of the receptors. While specific sense organs that detect load decreases have not been identified in vertebrates, similar functions could be fulfilled by receptors of the feet in signaling the effects load decrements in posture and locomotion.

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