Active Signaling of Leg Loading and Unloading in the Cockroach

Angela L. Ridgel,1 S. Faith Frazier,1 Ralph A. Dicaprio,2 and Sasha N. Zill1

 1Department of Anatomy, Cell and Neurobiology, Marshall University School of Medicine, Huntington, West Virginia 25704; and  2Department of Biological Sciences, Ohio University, Athens, Ohio 45701


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Ridgel, Angela L., S. Faith Frazier, Ralph A. Dicaprio, and Sasha N. Zill. Active signaling of leg loading and unloading in the cockroach. The ability to detect changes in load is important for effective use of a leg in posture and locomotion. While a number of limb receptors have been shown to encode increases in load, few afferents have been demonstrated to signal leg unloading, which occurs cyclically during walking and is indicative of slipping or perturbations. We applied mechanical forces to the cockroach leg at controlled rates and recorded activities of the tibial group of campaniform sensilla, mechanoreceptors that encode forces through the strains they produce in the exoskeleton. Discrete responses were elicited from the group to decreasing as well as increasing levels of leg loading. Discharges of individual afferents depended on the direction of force application, and unit responses were correlated morphologically with the orientation of the receptor's cuticular cap. No units responded bidirectionally. Although discharges to decreasing levels of load were phasic, we found that these bursts could effectively encode the rate of force decreases. These discharges may be important in indicating leg unloading in the step cycle during walking and could rapidly signal force decreases during perturbations or loss of ground support.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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The detection and regulation of forces acting on the body and legs are now considered integral components in the control of posture and locomotion in many animals (Prochazka 1996). In walking, for example, increase in loading after leg contact is signaled by receptors that reflexly adjust the activities of muscles which generate support and propulsion (Bässler et al. 1991; Pearson and Collins 1993). The subsequent decrease in loading at the end of a step is necessary for the initiation of leg lifting in swing (Whelan et al. 1995) and for normal coordination of leg movements (Bässler 1987). The activities of some groups of limb muscles are also strongly correlated with leg unloading during postural perturbations, and decrements in load may be important factors in determining responses to leg slipping (Jacobs and Macpherson 1996; Mcilroy and Maki 1994). However, the specific mechanisms or receptors detecting unloading of a leg have not been identified in many systems (Zill 1993).

We studied the responses of the tibial campaniform sensilla of the cockroach, Periplaneta americana, which detect forces acting on the legs through strains in the exoskeleton (Schnorbus 1971), to determine the specific parameters that the receptors can encode during walking. The responses and locomotor functions of these sense organs have also been incorporated into models and control systems based on insect walking (Schmitz et al. 1995). Campaniform sensilla of insect legs are known to respond directionally to forces that bend the exoskeleton (Delcomyn 1991; Hofmann and Bässler 1986), such as those that occur during leg loading. The directional sensitivity of an individual receptor is correlated with the orientation of its ovoid cuticular cap (Spinola and Chapman 1975). The cap, which is embedded in the exoskeleton, is the site of termination of the sensory dendrite and is thought to be the locus of mechanoelectric transduction (French 1992). The tibial campaniform sensilla are unique in that they form two subgroups (proximal and distal sensilla) with mutually perpendicular cap orientations (Fig. 1B). Each subgroup exhibits discrete responses when forces are applied to the distal tibia with joint movement resisted (Schnorbus 1971). For example, the proximal sensilla respond to bending in the direction of joint extension, whereas the distal sensilla discharge to forced flexions (Zill and Moran 1981). We applied controlled rates and levels of bending to the leg and found that the tibial campaniform sensilla, as a whole, respond not only to increasing levels of force but also exhibit discrete responses to decrements of force. These responses are consistent with the demonstrated directional sensitivities of the receptors and do not represent ON-OFF or bidirectional responses (Dickinson 1992). Instead these new results suggest that, for a given direction of force application, different receptors signal loading or unloading according to the orientation of the cuticular cap.



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Fig. 1. Preparation and identification of unit discharges to applied forces. A: forces were applied to the tibia with joint movement resisted (Pin) via a probe driven by a piezoelectric (PE) crystal. The applied forces were monitored with strain gauges attached to the probe and the activities of the campaniform sensilla were recorded extracellularly (Sens). B: drawing of cuticular caps of tibial campaniform sensilla (after Schnorbus 1971). The receptors are located in 2 subgroups (proximal and distal sensilla), which differ in their directional sensitivity according to their cap orientation. C: bending to forced extension (down on the Force trace) elicited discharges to different units during force increases and decreases (asterisk). D: individual receptors were identified by indenting their cuticular caps (Indent Caps in A) with another probe (arrow), which produced a discharge of equivalent amplitude. E and F: ablating individual receptors (hollow arrow in E) could selectively eliminate recorded responses to force decreases (hollow asterisk in F). G: bending to forced extension (Force) was followed rapidly by indentation of the cuticular cap (arrow) of a distal sensillum. H: coincident cap indentation (arrow) and decline of force application (asterisk) produced a summation in the discharge of the distal sensillum.


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Adult cockroaches, P. americana (n = 21), were anesthetized with carbon dioxide, and the nerves innervating the left metathoracic leg were cut in the thorax. Animals were then restrained with small staples on a resin-coated platform. Pairs of fine (50 µm) wires were implanted in the femoral segment of the denervated leg adjacent to the main leg nerve (nerve 5) or its major branch (nerve 5r8) (Nijenhuis and Dresden 1956). These electrodes recorded the activities of both proximal and distal sensilla, as the axons of receptors of both subgroups travel in the same nerve branches (Schnorbus 1971). Typically, the action potentials of one distal and one or two proximal sensilla were evident in extracellular recordings (n = 17/21 experiments) (Spinola and Chapman 1975). The tarsus (foot) and tibial sensory spines were then severed. The femorotibial joint was immobilized by gluing a pin adjacent to the proximal end of the tibia with the joint at an angle of 90° or in a position of full extension. Forces were applied to the distal tibia as ramp and hold stimuli via a probe that was driven by a piezoelectric crystal. The levels and rates of applied force were monitored through a pair of strain gauges attached to the probe (Fig. 1A). In a typical sequence, we applied 16 different rates, each repeated twice, within a single series, and up to three series were repeated in each experiment. In addition, individual campaniform sensilla were identified by indenting their cuticular caps with a fine-etched tungsten wire attached to another piezoelectric crystal (Fig. 1, A and D) (Dickinson 1992; Spinola and Chapman 1975). All signals were stored on tape for subsequent transcription and data analysis.


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Forces applied to the tibia as ramp and hold stimuli elicited discharges both during the rising and the falling ramp phases (n = 16/17 experiments in which responses of both proximal and distal sensilla were recorded) (Figs. 1, 2, and 3). In all recordings, the amplitudes of the discharges during the two phases were sufficiently distinct to clearly indicate that different units were active during increasing versus decreasing levels of load. We performed a number of controls to confirm that these responses originated from the tibial campaniform sensilla and to identify the individual receptors from which the discharges were derived. After testing responses to bending (Fig. 1C), we mechanically stimulated individual receptors by indenting their cuticular caps with a separate probe (Fig. 1D) and were able to elicit discharges of equivalent amplitude in extracellular recordings to those seen during declining levels of bending force. In most experiments the sensillum was then ablated by increasing the level of indentation until the probe penetrated the cap (Fig. 1E). These ablations could eliminate the entire response to decreasing force levels in a single direction while leaving the discharges to force increases intact (Fig. 1F). Furthermore, in three preparations, we were able to simultaneously apply leg bending and cap indentation, which could show a summation during the responses to declining forces (Fig. 1, G and H). Thus the discharges we recorded to bending were clearly derived from the tibial campaniform sensilla.



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Fig. 2. Responses to ramp stimuli applied in different directions. A and C: bending forces applied in the direction of joint extension (down on the Force trace) elicited discharges of proximal sensilla to force increases and distal sensilla to force decreases. B and D: bending the tibia in the opposite direction (forced flexion, up on the force trace) caused excitation of distal sensilla during force application and proximal sensilla during force declines. E and F: discharges to decreasing forces occurred during the declining ramp and were not due to rebounds in the opposite direction of bending (0 level = no bending force applied) or oscillations in the probe. G: tibial bending applied as repeated increases and decreases without a hold phase approximated the magnitude and time course of ground reaction forces that have been recorded during walking. Bending in the direction of joint extension elicited alternating bursts of activity from the tibial campaniform sensilla, even though bending in direction of forced flexion had not occurred.



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Fig. 3. Encoding of rate of force increase and decrease. A: bending forces were applied as ramps of varying rate but identical amplitude. A series of force extensions (down on Force trace) elicits discharges in proximal and distal sensilla during respective force increases and decreases that varied in frequency with the ramp rate. B: plot of maximum discharge frequencies of a proximal sensilla during the increasing ramp phase of forced extension (, R2 = 0.935, slope = 7.6, y-intercept = 109.3, P < 0.01) and the decreasing phase of forced flexion (open circle , R2 = 0.964, slope = 7.9, y-intercept = 68.6, P < 0.01) during bending tests in a single preparation. Both discharges to increasing and decreasing forces showed rate sensitivities with similar slopes. C: plot as in B for a distal sensillum for tests of rate sensitivities to force increases (, R2 = 0.782, slope = 5.68, y-intercept = 12.8, P < 0.01) and decreases (open circle , R2 = 0.893, slope = 3.85, y-intercept = 18.1, P < 0.01).

The responses of individual afferents to decreasing force levels depended on the direction of force application and the orientation of the receptor's cuticular cap. The distal sensilla fired to decrements in the level of forced extension (Fig. 2, A, C, E, and G), and the proximal sensilla were active during decreases in forced flexion (Fig. 2, B, D, and F). This general pattern occurred stably over time (Fig. 2, A and B) and at a variety of levels of displacement (Fig. 2, C and D) in repetitive bending tests. Discharges to force decrements were initiated during and not after the declining phase of the ramp stimulus (Fig. 2, E and F) and were therefore not the result of resonance in our apparatus or the consequence of inadvertent application of forces in the opposite direction. We also tested responses to bending by using patterns that approximated the durations and magnitudes of ground reaction forces that have been measured during cockroach walking (Full and Tu 1991). Forces applied in the direction of extension as simple, rapid triangle functions, without a substantial hold phase, elicited reciprocal discharges in proximal and distal campaniform sensilla in the same pattern, with consistent bursts during force decrements (Fig. 2G).

Do these discharges simply signal the occurrence of declining force levels, or do they also encode the rate of force decrease? To address this question, we applied ramp and hold stimuli at varying rates of rise and decline (Fig. 3). Firing of proximal sensilla during decreases from forced flexions consisted of intense bursts of activity in the range of 50-200 Hz that showed strong correlation with the velocity of force changes. In contrast, distal sensilla discharged at much lower frequencies with fewer action potentials to ramps declining from forced extensions. At low amplitudes of bending, the distal sensilla fired single spikes and could thus only indicate the occurrence of force decreases. Change in bending levels of higher magnitude, however, could elicit bursts that showed distinct modulation of firing frequency according to the rate of force decline (Fig. 3A). To compare the sensitivities to rate of change in applied force, we plotted the maximum firing frequencies during phasic discharges of single campaniform sensilla. These plots show a dependence on force velocity as a power function in both proximal and distal sensilla during both increasing and decreasing ramps (Fig. 3, B and C). However, similar results were also seen in other experiments (n = 3) in which forces were applied in both directions, and sensitivities to rate of change were observed in all experiments in which sensilla discharged with multiple spikes. Further experiments are necessary to assess these different sensitivities quantitatively and to characterize the effect of force amplitude on afferent firing.


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This study has shown that the tibial group of campaniform sensilla can actively signal both increases and decreases in bending forces. These findings are important in understanding how forces are encoded by these receptors and in evaluating their function as inputs to the cockroach walking control system. Responses to decreasing levels of force have not been previously explicitly reported and studied, but they are evident in some earlier published recordings of campaniform sensilla in the cockroach (Spinola and Chapman 1975; Zill and Moran 1981) and stick insect (Delcomyn 1991). However, the potential information content of these discharges was not systematically examined. Similar responses to declining levels of sinusoidally applied bending were also recently reported for the locust tibial campaniform sensilla (Newland and Emptage 1996), but the individual receptors and the orientation of their cuticular caps were not identified. It was therefore unclear whether those responses implied a bidirectionality in unit discharges. Discharges to decreasing levels of leg loading were demonstrated in groups of cuticular force receptors in other arthropods (Marchand et al. 1995) and may be present in the walking systems of a number of animals.

The mechanisms underlying the generation of discharges to decreasing forces are at present undetermined. Our finding that the orientation of the cuticular cap predicts the directionality of the tibial sensilla to declining forces suggests the parsimonious hypothesis that the cap provides a final common mechanism of transduction for both force increases and decreases. The generation of these diverse responses could then depend on the specific temporal and spatial distribution of strains within the exoskeleton, which can also show viscoelastic properties that could contribute to responses to decreasing forces (Blickhan and Barth 1985). The current findings are in clear contrast to the bidirectional responses obtained from campaniform sensilla of dipteran wings (Dickinson 1992), which differ from leg sensilla in the shape of the cuticular cap and sensory dendrite. Thus the morphological and mechanical properties responsible for the discharges to declining forces remain a subject for further investigation.

What are the potential advantages in actively signaling decreases in load in a walking system? First, the information that forces are declining is prerequisite for the initiation of leg lifting in swing (Bässler 1987; Whelan et al. 1995) and also enhances the placement of other legs in support (Bässler et al. 1991). These functions could be accomplished by monitoring the declining frequency of a receptor that simply encodes the level of load. However, such a signal could be compromised by processes such as adaptation or hysteresis, which are present in campaniform sensilla and common in many sensory systems (French 1992; Zill and Moran 1981). Furthermore, the force that a leg exerts must drop substantially when the foot or point of contact slips on the substrate. The system would therefore be providing an active signal to allow for the initiation of the necessary rapid compensatory reactions (Jacobs and Macpherson 1996). The selective responses of the tibial sensilla to both decreases and increases in load provide a mechanism for anticipating the need for further support and for initiating rapid responses to maintain stable postures. Experiments are planned to test these hypotheses in the cockroach. However, other biological and control systems may utilize signals of force decrements to similar advantage.


    ACKNOWLEDGMENTS

We thank G. Nelson for many helpful discussions and E.-A. Seyfarth and two anonymous reviewers for helpful comments on the manuscript.

This work was supported by Office of Naval Research URISP Grant N00014-96-1-0694.


    FOOTNOTES

Address for reprint requests: A. L. Ridgel, Dept. of Anatomy, Cell and Neurobiology, Marshall University School of Medicine, Huntington, WV 25704.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 August 1998; accepted in final form 20 October 1998.


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