Multipolar stretch receptors and the insect leg reflex

J, Insect Physiol., 1967,Vol. 13, pp. 1637 to 164-4. Pcrgamon Press Ltd.

MULTIPOLAR

Printed in Great Britain

STRETCH RECEPTORS AND THE INSECT LEG REFLEX D. M. GUTHRIE

Department of Zoology, Marischal College, University of Aberdeen (Received 17 June 1967) Abstract-The cockroach walking leg contains multipolar stretch receptor: near the joints, hitherto ignored by students of insect leg reflexes. The large cell near the condyle of the femoro-tibia1 articulation responds with low frequency, slow adapting trains of impulses to tibia1 levation, and this appears to be associated with slow contractions in the tibia1 depressor muscle. Fast adapting receptors are also believed to lie in this region. These sense cells are physiologically similar to multipolar receptors described in Limulus.

INTRODUCTION RECENT WORK on the correlative aspects of leg reflexes (HORRIDGE,1962; WILSON, 1965 ; RIJNION and USHERWOOD, 1966) has emphasized the need for more detailed

information as to the units involved. Most attention has been focussed on the contribution of campaniform sense organs (PRINGLE, 194Q), chordotonal organs (BECHT,1958; RUNION and USHERWOOD, 1966), and spines and hairplates (PRINGLE, 1938; WILSON, 1965). Relatively little is known about multipolar neurones in the insect leg. ZAWARZIN (1912) figures such a cell in the trochanter of Aeschna, and similar types of a cell have been noted in termite limbs by RICHARD(1950) and DENIS (1958) and in Trichoptera by BARBIER(1961). These cells differ a good deal in detail, but they are generally similar to the receptors described both physiologically and morphologically by FINLAYSONand L~WENSTEIN( 195 8) in the abdominal segments of various insects. Surprisingly, the closest point of comparison was provided by the study of multipolar neurone function in the walking leg of Limuhs by PRINGLE (1956). This brief report describes observations on the multipolar cells of the cockroach (Periplaneta americana L.) made during a study on the reflex control of the tibia1 depressor muscle. METHODS Receptors were stained using methylene blue for whole mounts and Hansen’s Trioxyhaematin for sectioned material. Recordings were made with conventional amplifying apparatus, applied tensions being monitored by means of a strain gauge transducer. 1637

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Most attention was focused on the large stretch receptor near the tibia1 condyle (Fig. l,c), but isolation of the response proved difficult. Recordings were made with the leg pinned out and the roots of nerves 3 and 5 crushed or sectioned.

FIG. 1. Diagram of the femur and trochanter, and parts of the coxa and tibia, to indicate the position of the multipolar sense cells. a-trochauteral cell, b-the two dorsal femoral cells, c-the ventral femoral cell. The main femoral chordotonal organ (fch), and various groups of campamform organs (ca), are also shown. Other abbreviations cx-coxa, co-condyle, f-femur, t-tibia.

The tibia had to be cut down to a small stump in order to remove the campaniform organs in the base (Fig. 1,ca). In many cases the apodemes of the major femoral muscles were also cut to prevent chordotonal organ stimulation. A glass hook inserted in the tibia1 stump was used to rotate it. THE

MORPHOLOGY

OF THE

RECEPTORS

A large cell with a centripetal axon and at least twenty branching processes lies near the anterior ventral condyle of the femoro-tibia1 articulation (Figs. 1,c and Za). The processes appear to penetrate the soft transparent cuticle lying about the condyle. The axon has a thick sheath round it extending over the cell body, like that of other stretch receptors (FINLAYSON and LOWENSTEIN, 1958; GUTHRIE, 1962). A similar cell of rather smaller dimensions lies against the femoro-trochanteral articulation (Fig. 1,a and 2b). This has only three or four branches arising from the cell body, although these have numerous fine rami ending in small clubbed terminals. These terminals lie in the anterior part of the articulation. More recent work demonstrates the presence of a second dorsal trochanteral receptor similar to, and lying very close to the one illustrated in Fig. 1,a. In addition to these two elements, there exist a pair of cells of smaller size whose assignment to the multipolar sense cell type is less certain. They lie above the postero-dorsal condyle of the femoro-tibia1 joint (Fig. 1,b). The unbranched processes of these cells appear much shorter and thicker than in the other cells, and only two or three processes are visible.

FIo. 2a. The ventral femoral receptor (cell c).

FIc. 2b. The tro,chanteral receptor (cell a).

FIG. 3. Electrical responses of the main femoral chordotonal organ (upper trace) to forced tibial levation (lower trace). Increased tension upwards. M a x i m u m deflection on u p p e r trace 250/zV, on lower trace 2"5 g.

MULTIPOLARSTRETCH

RECEPTORS AND THE INSECT LEGREFLEX

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Considerable difficulty was encountered in recording from cells c and b due to the circuitous path their axons take in order to join nerve 5. RESPONSES

TO

MECHANICAL

STIMULATION

The most easily reproducible of all the sensory responses connected with the movement of the knee joint (i.e. excluding spine and hair organ responses) are those deriving from the large chordotonal organs of the middle of the femur (Fig. 1,fch). These send axons into ramus 8 of nerve 5 (NIJENHUIS and DRESDEN, 1952) and can be easily stimulated by forced levation of the tibia1 segment (Fig. 3). The characteristics of this type of response have been described by BECHT (1958) in the trochanteral organs and are usually high frequency, non-adapting, large amplitude, high threshold responses.

0.5%X FIG. 4. Electrical responses of the ventral femoral receptor in four trials. In each record the sense cell response is shown in the upper trace while forced levation of the tibia is indicated on the lower trace. Levation and increased tension downwards.

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There appear to be other responses from the more distal regions of the femur which may come from chordotonal organs contributing to ramus 8 but these seldom approached this type of response. As the chordotonal organs are typically dependent on the tension of muscles applied to the cuticle their response is essentially a delayed one as far as tibia1 flexion is concerned. Two kinds of response were observed in fibres of the posterior nerve, into which axons from the large receptor (c) and the small receptors (b) appear to run. A very brief, rapidly adapting response, and a slow adapting response operating over a very low frequency range. The latter appears to be unitary response, and was believed to originate in cell c. The records illustrated in Fig. 4 show the response of this receptor to forced levation of the tibia (increased pressure, downwards). The monitor illustrates changes in applied pressure to a maximum of approximately 2 g. The small initial change of 100 to 200 mg required to overcome the inertia of the hinge evokes no response, but the beginning of the levation movement produces an increase in frequency more or less proportional to the applied pressure. This dies away hardly at all at a maintained pressure (Fig. 4 b). Rate of pressure change may also be involved. Increasing the rate of change by four times about doubles the discharge frequency in a number of records. In most preparations frequencies seldom rise above 20 per set or interspike intervals of about 50 msec. Smaller intervals (down to 10 msec) were encountered when pressure was suddenly relaxed or applied, and in some instances a brief adapting train of impulses with an interval sequence dropping from 10 to 50 msec occurred, but this high frequency response lasts less than 100 msec.

FIG. 5. A graph showing the relation between impulse interval and applied tension in two discharges of the ventral femoral receptor.

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In the graph (Fig. 5) frequency in terms of impulse intervals is plotted against the tension applied to the stump of the tibia in order to levate it on the femur. Tension rather than angular movement was taken as a stimulus parameter due to ease of registration, although it was realized that tibia1 angle is likely to be the significant aspect of the information. The two impulse trains used in the graph were obtained under slightly different conditions of lateral stress on the condyle, and the difference in the exponential curves obtained was believed to relate to this. The overstretch phenomenon noted by FINLAYSONand LOWENSTEINin their material was not observed, and is clearly unlikely to occur in this receptor due to its situation.

b

O-25 set

I

FIG. 6. Responses in the posterior femoral nerve to tibia1 pressure believed to originate in the two smal1 “b” celk of the femur. Maximum deflection on the upper trace 20 ,uV, on the lower trace 1.0 g.

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Very brief fast adapting responses were occasionally detected in branches of the posterior nerve following tibia1 levation, and these often have the appearance of the response of two elements (Fig. 6 a and b). It is tempting to see these as the activity of the two “b” cells found near the tibia1 levator apodeme, but they were not systematically investigated. REFLEX

FUNCTION

Due to the low level of impulse production by the stretch receptors of the knee joint their responses are lost in the greater activity of the campaniform and chordotonal organs when recordings are taken from proximal regions of n.5. To some extent this is also true of stretch receptor dependent changes in intramuscular recordings taken from the tibia1 levator and depressor, although small trains of low value muscle potentials can be seen to accompany forced levation. These have the same amplitude as potentials that can be assigned to the slow fibre system in other preparations. If the tibia1 depressor apodeme is cut and attached to a transducer thus preventing stimulation of the main femoral chordotonal organ the effect of tibia1 levation

FIG. 7. Mechanical response of the tibia1 levator muscle (lower trace) to forced tibia1 levation (shown as solid shading on the upper trace). Upper trace: small and large strokes al1 one second intervals.

is to produce a contraction rising in 0.5 set about 1.5 g, and thereafter declining slowly despite continued levation. This is shown in Fig. 7, where the effect of fast and then slow levation can be seen. CONCLUSION

The morphology of the cockroach sense cells described above differs in detail, although they all seem to belong to the multipolar stretch receptor type illustrated by FINLAYSON and LOWENSTEIN (1958) in muscular and connective tissue strands in the abdomen of various insect species. The processes of the leg receptors are less consistently orientated in one axis than those of the abdominal receptors due to the nature of their insertions in cuticular folds. The trochanteral receptor as figured by ZAWARZIN (1912) in Aeschna, and as it occurs in Periplaneta, has a very rich development of fine dentritic processes, and is perhaps more elaborate in this respect than the abdominal cells.. On the other hand the b cells with their few short simple processes appear less complex.

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Physiologically the same degree of difference between the abdominal receptors and the large c cell can be observed. A low frequency, small delay slow adapting response occurs if the receptor cell is placed under tension in both Instances, but in the abdominal cells the frequency range is rather more elevated, in that frequencies of 40 to 50 impulses/set may be maintained for several seconds. The discharge from cell c only rises above 20 impulses/set during rapid movement or at the high tensions required to produce extreme tibial deflection. It must be noted, however, that the abdominal receptors were silent until placed under a slight preliminary tension. The very brief responses believed to be associated with stimulation of the b cells in the leg ar: quite different to abdominal cell discharges. A remarkable similarity appears to exist between the knee joint receptors in Periplaneta and multipolar sense cells in the same region of the walking leg of Limzdus. PRINGLE (1956) demonstrated tonic elements responding to joint flexion with changes in the frequency of discharge between 5 and 20 impulses/set, and phasic cells responding with very brief bursts. The activity of the tonic cells appears to drive slow neuromuscular elements. The tonic or slow adapting receptors were believed to lie against the cuticle of the condyle, the phasic cells or fast adapting receptors were situated more internally. The c cell in Periplaneta appears to differ hardly at all from the tonic sense cell of LimuZus, and although a very limited amount of information was obtained about the b cells they appear to resemble Limuhs phasic cells in the paucity of the impulses in a response train. A much more sophisticated organ of tension registration has been described recently in the metathoracic femur of the locust by RUNION and USHERWOOD(1966). This highly compact chordotonal organ lying internally, contains both fast and slow adapting elements connected to muscular and cuticular structures within the limb, and may represent the other end of the scale of specialization. The function of the large chordotonal organ of the cockroach femur appears to be to inform the central nervous system of excessive tensions in the origins of the tibia1 depressor muscle by means of a “door bell” type of response. The tibia1 levator may also be involved in the stimulus, but this was not clear. The cuticle overlying the origin of the tibia1 depressor muscle can be seen to buckle inwards as the electrical response starts. An interplay of muscle activity and tibia1 angle are clearly both involved in the stimulus. The resulting motor output is complex but contains a major “fast” component. The multipolar stretch receptors provide an independent source of information concerning gradual movements at the femorotibia1 condyle and their motor connexion involves slow contractions. Finally, it may be pointed out that the attribution of slow muscular activity following forced movements of the limbs to campaniform organ function (PRINGLE, 1940) was made without the contribution of multipolar sense cells being taken into account. Acknowledgements-It is a pleasure to thank Mr. L. PANKOfor his technical assistance and Professor L. H. FINLAYSON for drawing my attention to a number of references. This work was aided by a grant from the Science Research Council.

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D. M. GUTHRIE REFERENCES

BARBIERR. (1261) Contribution B 1’Ctude de l’anatomie sensori-nerveuse des insectes trichopteres. Ann. Sci. nut. (Zool.) 12, 173-183. BECHTG. (1958) The influence of DDT and Lindane on chordotonal organs in the cockroach. Nature, Land. 181, 777-779. DENIS C. (1958) Contribution B 1’6tude de l’ontogenese sensori-nerveuse du termite Culotermes flavicolla’s Fab. Insectes sot. 5, 171-188. FINLAYSONL. H. and LOWENSTEINL. (19.58) The structure and function of abdominal stretch receptors in insects. Proc. R. Sot. (B) 148, 433449. GUTHRIED. M. (1962) Control of the ventral diaphragm in an insect. Nature, Lond. 196, 1010-1012. HORRIDGEG. A. (1962) Learning in headless insects. Proc. R. Sot. (B) 157, 35-51. NIJENHUISE. D. and DRESDEND. (1952) A micromorphological study on the sensory supply of the mesothoracic leg of the American cockroach Peripkmeta americana. Koninkl. med. Akad. Wet. (C) 55, 300-310. PRINGLEJ. W. S. (1938) Proprioception in insects III. The function of; the hair sensillae at the joints. J. exp. Biol. 15, 467-478. PRINGLEJ. W. S. (1940) The reflex mechanism of the insect leg. J. exp. Biol. 17, 8-17. PRINGLEJ. W. S. (1956) Proprioception in Limulus. J. exp. Biol. 33, 658-667. RICHARD G. (1950) L’innervation et les organes sensoriels de la patte du termite a cou jaune. Ann. Sci. nut. (Zool.) 12, 65-83. RUNION H. I. and USHERWOODP. N. R. (1966) Responsiveness of a mechanoreceptor (chordotonal organ) in the locust leg to static displacement and velocity of stretch. y. Physiol., Lond. 187, 40-41 P. WILSON D. M. (1965) Proprioceptive leg reflexes in cockroaches. y. exp. Biol. 43, 397-410 ZAWARZINA. (1912) Histologische studien iiber Insekten II. Das sensible Nervensystem. der Aeschnalarven. 2. wiss. 2001. 12, 65-83.