PR0PRI0CEPTI0N IN INSECTS

Transcription

1 101 PR0PRI0CEPTI0N IN INSECTS I. A NEW TYPE OF MECHANICAL RECEPTOR FROM THE PALPS OF THE COCKROACH BY J. W. S. PRINGLE, B.A. From the Department of Zoology, Cambridge (Received 14 March 1937) (With Nine Text-figures) INTRODUCTION MANY attempts have been made in the past to determine the nature and extent of the senses of animals. Until recently most of the experiments in this field took the form of observation of the reactions of the animal to some external stimulus that could be controlled and varied, the degree of sensory excitation being deduced from the final muscular response. This method, though yielding much information of a general nature, could rarely, owing to the complexity of the system under study, give any exact indication of the nature of the sensory processes, and it is largely due to the development of a technique for the experimental isolation of sense organs by the electrical detection of impulses in their nerves that we now have a certain amount of knowledge of the workings of this part of the nervous system. The electrical method of investigation extends considerably the range of senses which can be studied, for it is not limited to those whose excitation produces an easily observable response on the part of the animal, nor merely to senses concerned with external stimuli. The work of Matthews (1931 a, 1931 b, 1933) on the physiology of proprioception in the vertebrates provides an example of the exact type of information which can be obtained by this method about a sense which is very difficult to study in other ways, and it opens up a wide range of possibilities for similar work in many parts of the animal kingdom. The present papers describe an investigation by the same method of a new type of mechanical sense in insects. The experimental technique makes possible a study of the physiology of the sense organs concerned quite apart from any consideration of their role in the behaviour of the animal; it is felt that, if the function of a sense is discussed only with a full previous knowledge of its nature and scope, there will be much less risk of misinterpretation. MATERIALS AND METHODS The American cockroach, Periplaneta americana L., has been used throughout the work. This insect has the great advantage of being large and easily obtainable at all times of year. Confirmation of results was obtained with the smaller Blatta

2 102 J. W. S. PRINGLE orientalis, but all descriptions refer to Penplaneta. Evidence will be adduced later to show that the sense is of general occurrence in nearly all insects. The method used is based on that of Matthews (1931 a). Impulses arising from sense organs in the appendages are recorded in the cut sensory nerves at their base by means of fine hook-shaped platinum wire electrodes, a four-stage condenser coupled amplifier and a Matthews (1928) oscillograph. The sense organ is thus experimentally isolated from the rest of the nervous system, and its range of sensitivity can be exactly determined without the complication of any muscular reactions on the part of the animal. An appendage suitable for study was found in the maxillary palp, where the system is sufficiently simple to allow of interpretation of the observations. The cockroach is secured to a wax block by pins through the thorax and abdomen, and with the head held back by a single pin through the labrum, the labium is dissected off to expose the suboesophageal ganglion. The maxillary nerve may then be freed and picked up on the electrodes at any desired point. The preparation can be kept alive with a physiological salt solution made up to the following formula (modified from Lewis & Robertson, 1916): NaCl 9g. KC1 0-2 g. CaCl t 0-2 g. Dextrose g. Water c.c. This solution was found to keep the nerve in an active and healthy condition for many hours, and preparations moistened with it differed in no respect from those where the discharge of body fluid rendered its use unnecessary. RESULTS General morphology of the maxillary palp The maxillary palp of the cockroach (Fig. 1 B) is a slender appendage, consisting of five segments. Of these the basal two are smaller than the rest, and the whole palp measures about 5-5 mm. in length in a typical specimen. The joints are of a primitive type, being formed by a thinning of the chitinous skeleton on one side, towards which bending can then take place. This type of hinge joint, called by Snodgrass (1935) "intrinsic", to distinguish it from-the truly condylic or "extrinsic" type, has a considerable elastic resistance to movement, and the excised palp with the muscles relaxed takes up a fully extended position. The musculature is very simple. The basal joint, which is diarticular, has two antagonistic muscles attached across it, the levator and depressor palpi; each of the other segments is moved by but one muscle, the pull of which is balanced by the elasticity of the intrinsic hinge (Snodgrass (1935) gives an inaccurate figure of the musculature). The direction of movement at the joints varies as shown in Fig. 1 B from the position of the muscle insertions, and in the living insect joint 3-4 is held partially

3 Proprioception in Insects 103 flexed so that contraction of the promotor of 5 moves that segment outwards away from the head. I. pip. I plp~..\ " p. 5 \ N d. pip. Fig. 1. Penplaneta amencana A. Left labial palp. B. Left maxillary palp. A. Muscles / pip. levator palpi; d pip depressor palpi; r. 2, retractor of segment 2; /. 3, p. 3, flexor and promotor of segment 3. B Muscles. / pip levator palpi; d.plp. depressor palpi; r. 2, r. 3, retractors of segments 2 and 3; /. 4, flexor of 4, p 5, promotor of 5. Sensilla in A and B. c lt h la, indicates number of campaniform and hair sensilla and their positions. B Fig 2 Diagram of nerves from suboesophageal ganglion of Penplaneta. hp hypopharyngeal; c <>e c circumoesophageal commissure; mn. mandibular; mx. nerve to basal muscles of maxilla; I., g, P, branches of maxillary nerve to lacinia, galea, and palp; Ib. labial; n. nerve to the neck sclentes; c. connective to first thoracic ganglion The nervous supply to the maxilla is shown diagrammatically in Fig. 2. A small branch (mx.) is given off at the base to the muscles of the cardo and stipes, and the main maxillary nerve forks a little farther out, one part going to the lacinia and galea and the other to the palp. The basal palp muscles are innervated from a branch P lt and the main trunk divides again after entering the palp, one-half

4 104 J* W. S. PRINGLE supplying mainly segments i, 2 and 3; but both branches continue to the tip. A transverse section of the palp nerves shows a few fibres about 10-12/x in diameter and a large number less than 3/z. Sensory physiology With the peripheral end of the maxillary nerve on the electrodes, a big spontaneous discharge is always apparent, especially if all its branches have been included by section close to the ganglion. Most of the activity is from the lacinia and galae, and if these are removed the preparation soon quietens down and the endings in the palp can be studied. The whole of the 5th segment of the palp is covered with hairs which are sensitive to touch. These are so close together that it is impossible to study them singly, and the records are therefore complex owing to the number of active fibres. The amplitude of the impulses is small, and adaptation is rapid and complete. An entirely different sort of discharge is produced by movement of the palp. Each of the joints 1-2, 2-3, 3-4 and 4-5 gives a record as if from a single fibre. The impulses are large, rhythmic, and of constant height, and the frequency declines to a steady level which is undiminished 1 min. after the start of stimulation (Figs. 3, 4 and 5). The discharges are not all similar; the amplitude, and the rate and extent of adaptation vary from one to the other, the largest impulses being from the endings in 1-2 and 3-4. In one experiment in which the whole of the palp branch of the maxillary nerve was on the electrodes, the amplitude of these corresponded to 208 and 86 /xv. respectively, though this is probably only a fraction of the actual spike potentials of the fibres, as the whole of the rest of the nerve is short-circuiting the current. Of the other endings, 2-3 seems always to be small, and 4-5 is usually less than half the amplitude of 3-4; but in one experiment (6 March) 4-5 was unusually large, and 3-4 only just audible under high amplification. Whether this really represented a change in fibre size could not be determined. It is difficult to bend one joint only without affecting the others, but the sensitivity of the endings varies, and 3-4 is by far the most sensitive. With the amplification cut down until 4-5 and 2-3 are inaudible, it is thus possible to get records of 3-4 alone. The impulses from the hairs on 5 do not appear at this amplification, so that there is effectively a single fibre preparation. Most of the observations were made with this arrangement. 1-2, though it has a much bigger amplitude of impulse, is less sensitive and adapts more quickly and completely than 3-4 (see graphs on Fig. 6). It can be studied alone by cutting off the palp at the distal end of 3. Occasionally 2-3 is visible on these records, but can never be mistaken for the large impulses from 1-2. Sensory range The endings can be excited in a variety of ways. They respond, as we have seen, to passive bending of the joint. The effect of active contraction of the muscles is less easy to demonstrate. An attempt was made to stimulate the nerve electrically at

5 Proprioception in Insects 105 one point and record from another, but the amplifier was not suitable for this and the experiment was unsuccessful. Some evidence, however, was got from a few preparations in which the muscles showed spontaneous rhythmic contractions without any stimulation. This occurred twice and on neither occasion was it due to an injury discharge from the cut end of the nerve as found by Barnes (1930). The discharge on cutting was always of short duration whenever it was observed, and the reason for the rhythm remains obscure, but it made possible an investigation of the effect of free flexion of the joint by its own muscle. Fig. 3. Response of ending in joint 3-4 of the maxillary palp of Ptnplaneta to forced flexion of the joint. Tune marker in 1/10 lee.; records read from right to left, and arrow marks the start of bending. B, 5 seconds, and C, 13 seconds after the start of stimulation. One of the preparations which showed this muscular rhythm was the anomalous one of 6 March, when the impulses from the ending of 4-5 were unusually large. The behaviour of this preparation can be summarized as follows: (1) Free movement (by the muscle) of segment 5 gave either no response from the ending, or else very scattered and irregular impulses at the moment of relaxation. (2) Any interference with the movement of segment 5 gave bursts of impulses.

6 Fig. 4. Response of ending in joint i-a of the maxillary palp of Pertplaneta to forced flexion of the joint. Time marker i '10 sec record reads from right to left, and arrow marks start of movement. 3 Fig s Response of enduig in joint 4-5 of the maxillary palp of Penplaneta to pressure on the cuticle of segment 4. Time marker i/iasec.; record reads from right to left, and arrow marks start of stimulation. r

7 Proprioception in Insects 107 (3) With the joint held rigid, bursts of impulses occurred corresponding to the contractions of the muscle. A similar result was later obtained from 1-2 to rhythmic contractions of the retractor muscles of 2 and 3. In this case also the joint was held rigid, but the contractions of the muscles were made visible by the bending of the chitin. In both preparations the discharge ceased completely when the muscle was relaxed. Sideways movement of the joints, i.e. not in their normal plane, produces a more intense sensory discharge than direct flexion. But by far the most effective method -o c u 3 a" Time in seconds from start of stimulation Fig 6 Adaptation curves of the endings in the maxillary palp joints of Pcnplaneta. A Ending in 1 2. Ending in 3-4, two different degrees of bending. Ending in 4 5, stimulated by pressure on the cuticle of 4. The curves for the endings in1 2 and 3-4 were taken from records obtained from the same preparation at a temperature of about 18 0 C That for the ending in 4 5 from the anomalous preparation of 6 March, with temperature about 16 C. of stimulation is not movement of the joints at all, but pressure on the cuticle of the segment next proximal. Particularly in the neighbourhood of the joint the sensitivity to pressure is extremely high, and maximal excitation can be nearly approached by this method. Fig. 5 shows a record from the 4-5 preparation of 6 March to this form of stimulation. At the beginning impulses are being set up at such a rate that they are travelling in the period of incomplete recovery of the nerve, and their amplitude is much reduced. Throughout the record the height of any impulse can be seen to be dependent on the interval between it and the preceding impulse 10

8 io8 J. W. S. PRINGLE (Fig. 7). The temperature was not controlled during this experiment, but was in the neighbourhood of 16 0 C.; but, as remarked above, the preparation was abnormal in the size of the impulses and may have been so in other respects. Certainly the recovery period is not always so prolonged. To sum up the methods of exciting the endings, it may be stated in general that: (1) Free active movement of the segment by its muscle produces only a small excitation (verified only for 1-2 and 4-5). (2) Passive movement with a needle, or resistance to the active movement, are effective stimuli. (3) Sideways movements are more effective. I M V Hi Interval in a since previous impulse Fig. 7. Graph showing the relation between the height of the impulse and the interval between it and the preceding impulse. From the ending in joint 4-5 of the maxillary palp of Penplaneta in the anomalous preparation of 6 March. (4) Pressure on the cuticle round the joint, particularly on the segment next proximal, is the most effective stimulus of all, and is sometimes capable of producing nearly maximal excitation. The identity of the sensory endings In order to find the exact location of the sensory endings, the palp was several times cut back carefully from the tip until the response was abolished. This occurred only when the actual joint had been removed, though cuts just distal to this always produced violent excitation. It thus became necessary to undertake a histological examination of the palp to determine the nature of the sensory endings present. Intra-vitam staining with methylene blue in physiological salt solution was found to be best for this purpose. If the extreme tip of the palp is removed, the solution can be injected at the base with a fine hypodermic needle, and the stain is rapidly picked up by nerves and nerve endings. The chitin has afterwards to be

9 Proprioception in Insects 109 dissected off under a binocular microscope, and the distribution of the larger nerve fibres can then be followed. The sensory endings in the palp joints are of two types, fine hairs on the flexor surface, and campaniform sensilla. These latter need further consideration. Each of the joints 1-2, 2-3, 3-4 and 4-5 has a group of them on the outside (hinge side) of the joint. They vary slightly in number and arrangement, but typically 1-2 has two, one on the distal end of 1, the other on the proximal end of 2; 2-3 has five or more, arranged approximately in line round the distal end of 2; 3-4 has three (Fig. 8) in line near the edge of 3; 4-5 has one on 4. The organs always occur just off the portion of the cuticle which forms the hinge, and their skeletal portions are well seen in potashed preparations, when they appear as oval pits in the chitin. They are extremely small, the largest organ on the palp, the one at the base of 2, measuring about 16 x 6p.. The sense cells of the sensilla stain deeply with methylene blue. Each campaniform sensillum has a single large cell, bipolar, with a short distal process going to Fig. 8. Joint 3 4 of the left maxillary palp of Periplaneta americana. A. Dorsal view, r 1. campaniform sensilla. B Lateral view, h. hair* on flexor surface of joint. the cuticle; the central processes from the cells of each group join up immediately and finally enter the nerve trunk. The largest cell of the 3-4 group measures 20 x 12 ix, and the fibres appear larger than the majority of other fibres in the nerve. It is impossible to be certain from a histological examination of this sort whether the nerves from individual sensilla of a group actually anastomose, but the appearance of these methylene blue preparations is of a single large fibre branching and distributed to all the sensilla of a group (diagram, Fig. 9). It seems evident that these campaniform sensilla are responsible for the observed sensory discharges on bending the joints. Groups of them occur on just those joints from which the response has been obtained, and each group is supplied by a single large fibre such as might give the impulses observed; it remains merely to interpret their sensory range. The various views which have been held as to the function of the campaniform sensilla will be reviewed in a later paper, where also their mode of action will be considered, but from the above observations we can draw only one conclusion, that they must be mechanical receptors sensitive to the strains in the chitinous shell that forms the insect skeleton. The term "stress

10 no J. W. S. PRINGI.E receptors" is proposed, and the following discussion will show how this type of receptor could give the range of sensitivities as set out above. The stresses in the palp skeleton The skeleton of the maxillary palp consists essentially of a hollow tube. At the joints, as we have seen, bending can take place by virtue of the fact that one side of the tube is thin and flexible, but a certain degree of elasticity is also necessary for the other side, for bending also involves some measure of extension on the outside of the bend. If, as can be seen in the palp joints, the hinge portion of the cuticle retains any part of its curvature, then there will be a definite restoring force in the joint, and this will be derived partly at least from a distortion of the whole of the cuticle in the neighbourhood of the hinge. The campaniform sensilla are situated in the cuticle just off the hinge, and if they are sensitive to strains in the skeleton, then the degree of response of the joint to free movement should be determined by the curvature of its hinge. In actual practice we have seen that there is little excitation on free movement of the segment by its muscle, and it is necessary to postulate further that the campaniform sensilla on the palp are sensitive rather to compression forces down the length of the palp than to the extension that is produced on the outside of the bend by free flexion. This assumption will be further justified in the next paper; it is borne out to a certain extent by the observation that the few impulses that do occur during free flexion appear at the moment of relaxation of the muscle, when there would be a sudden removal of tension from the cuticle round the sensilla. On the other hand, any external resistance to the movement of the segment by its muscle produces a definite compression on the hinge side of the joint. Sideways movements involve a lateral shear with a definite compression component, and direct pressure on the chitin of the rather more rigid skeletal tube will produce large surface forces. Thus all these should excite the sensilla. The response obtained to passive bending with a needle may be explained possibly as due to a direct compression on the hinge, but it is more probably owing to the invariable presence of a lateral component in a movement not easy to control very exactly. Labial palps The results described above from the maxillary palp of Periplaneta were confirmed on the labial palps, of which brief mention may be made. These resemble the maxillary palps in general structure (Fig. i A), but have three segments only. Campaniform sensilla are present only in joint 2-3, where there may be five or more in a row on the distal end of 2. Here again the discharge in the nerve is always rhythmic, and the ending may be stimulated by passive bending, by lateral movements, or by pressure on the chitin. Discussion of some points in the physiology of the palp stress receptors It will have been apparent from the above account that although in many cases there are a number of campaniform sensilla at a joint, the discharge in the nerve has always been rhythmic, and the impulses of approximately constant amplitude.

11 Proprioception in Insects 111 This is usually taken to mean that only a single fibre is active. When several fibres in the nerve trunk are discharging impulses, the record is complicated and irregular. A rhythmic discharge like that in Figs. 3, 4 and 5 must come either from a single fibre, or from a number of fibres discharging absolutely synchronously. Synchronous discharges have often been observed from nerve trunks, usually to intermittent stimulation (Wever & Bray, 1930; Pumphrey & Rawdon-Smith, 1936), but also in motor nerves where there is no reason to believe the excitation of the central nervous system to be rhythmic (Adrian & Bronk, 1929). It would therefore be conceivable that in the palp records some factor, possibly the close proximity of the sense organs, might be causing them to discharge synchronously. There are, however, good reasons for rejecting this hypothesis. In joints 2-3 and 3-4 of the maxillary palp, and joint 2-3 of the labial palp, the sensilla are arranged approximately in line across the palp. During sideways bending of the joint, unequal stresses will be set up at the sensilla at opposite extremes. Synchronization of impulses at different stimulating intensities is very difficult to imagine, as the frequency of the discharge depends on the stimulating intensity (see Fig. 7). A discharge in two fibres is easily recognized in the loud-speaker from that in one, as it produces a musical chord, the single fibre discharge being a pure note. The loud-speaker was always in use during experiments, and in no case in some twentyfive preparations was a chord heard from movement of any single joint in the palps. The alternative and more probable explanation is that the fibres from each group of sensilla join up to form a single fibre in the nerve trunk. This is borne out by the histological examination of methylene blue preparations, and would account for the rhythm and constant amplitude of the impulses; but it raises certain difficulties. If one accepts the customary view that the sense cell of the sensillum is the cyton of the sensory nerve, then fibres growing towards the central nervous system must have anastomozed; or else, if the cyton is central in position and the outgrowing nerve fibre has branched to supply the sensilla, the trophic functions must have been taken over by the secondary cells of the sensilla, as no cell bodies have ever been described from anywhere else on the sensory nerve. Developmental studies are obviously necessary to decide between these two possibilities, but in support of the latter, one may recall the type II cells described by Zawarzin (1912) from the joints of the mask (modified labium) of Aeschna larvae. These are large cells, just under the surface, with branched distal processes distributed to the joint membranes. It is conceivable that these represent the primitive type of sensory ending in the joint, and that in the higher insects, where sclerotization is more advanced, special structures have developed at the nerve endings, capable of registering more accurately the stresses in the cuticle. That a system consisting of a single fibre distributed to more than one sense organ would be capable of giving a rhythmic discharge in the nerve is not at once apparent. Take for example the endings in joint 3-4 of the maxillary palp (Fig. 9). The three sensilla have been labelled A, B and C. Suppose now that segment 4 is bent sideways so that A is excited more than B, and B than C. If a, b and c are

12 112 J. W. S. PRINGLE the rates of discharge of impulses from each ending, then a>b>c. Several cases must be considered. (1) Suppose A starts the first impulse. On reaching the point P it will continue in both branches of the fibre similarly at Q and will pass up the nerve to the electrodes. If it reaches B before B has discharged its first impulse, it will reset the rhythm of B (Matthews, 1931 a) so that B's impulse will not occur for another i/b sec. But A's next impulse will reach B after \\a sec. and will again reset the rhythm, since ijckijb. So long then as A continues to discharge faster than B, the recorded rhythm will be that of A alone. (2) Suppose that the impulse from A does not arrive at B until B has itself discharged. Then the two impulses will meet in the stretch PB and cancel each other. A alone will proceed up the nerve. (3) All the endings adapt, and their frequency will decline. The rates of decline may vary. Suppose that at the start A is discharging faster than B, but that A adapts more quickly. The two rates of discharge will gradually approximate, and the point where the impulses meet in PB will move nearer to P. The time will come when it is the impulse from B and not from A that reaches P first and continues up Fig. 9. Diagram of nerves from sense cells of the sensilla in joint 3-4 of the maxillary palp of Periplaneta. Based on a methylene blue preparation. the nerve. At this moment, if PA = PB, their frequencies will be equal, and there will be no abrupt change in the record. The point where the impulses cancel each other will move gradually from BtoP and then down from P to A, until the position is completely reversed. The third ending C will act in the same way with the faster of A or B. The ending with the greatest degree of excitation will always take charge. Function of the palp sensilla Owing to the structure of the primitive insect joint, the campaniform sensilla will act as proprioceptors for the palps. It is interesting to compare them with the only other type of proprioceptor that has been studied in detail, the muscle endings of the vertebrates (Matthews, 1931 a, 1931 b, 1933). Comparison is possible only with the B type of endings of Matthews (1933) which react to the tension in the muscle. There is apparently no parallel mechanism in the cockroach palp to that of the stretch receptors (type A), which are arranged in parallel with the muscle fibres. But with the former type they have much in common, as the compression forces in the cuticle of the joint will be nearly related to the tension in the muscles. The chief difference, perhaps, is that whereas the vertebrate receptors can be influenced only by the state of their own particular

13 Proprioception in Insects 113 muscle, the stress receptors of the palp will react rather to the combined effect of all the palp muscles with a varying degree of emphasis on one or the other. This will result from the great sensitivity to sideways bending; for though such movements cannot be produced by the contraction of the muscle of that particular joint, they will occur, if, as in the palp, the more proximal joints move in a plane at right angles. Thus, even in the absence of any external resistance to the contraction of the flexor of 4 of the maxillary palp, the ending in 3-4 would act as proprioceptor for the depressor muscle at the base, owing to the normally half-flexed position of 3-4. Comparison of the adaptation curves of Fig. 7 with those obtained by Matthews from the frog and cat shows that the general form is the same in both, but that in some of the palp records there is a much greater difference between the initial frequency and the final steady level. Only the ending in 3-4 approximates in this respect to the vertebrate type; the response from 1-2 and 4-5 resembles more closely that of a touch receptor (Pumphrey, 1936). It is possibly significant that only the joint 3-4 is kept tonically flexed in the living cockroach. SUMMARY 1. A group of campaniform sensilla occurs on each of the joints of the maxillary palp of Periplaneta americana. Each group is supplied from a single large sensory nerve fibre. 2. Impulses in the nerves from the sensilla can be recorded at the base of the maxilla. Adaptation is slow and incomplete. 3. The sensilla respond to passive straight or sideways bending of the joint, and also strongly to pressure on the cuticle. They are excited only to a lesser extent when the segment is moved actively by its own muscle. 4. It is shown that the observations are consistent with the view that the campaniform sensilla are "stress receptors" responding to strains in the cuticular skeleton. 5. Their action as proprioceptors is compared with that of the vertebrate tension receptor. I wish to acknowledge my indebtedness to Dr R. J. Pumphrey for much help in the construction of the apparatus used in this work, and also for advice in its early stages. REFERENCES ADRIAN, E. D. & BRONK, D. W. (1929). J. Pkyriol. 67, 119 BARNES, T. C. (1930). J Phynol. 69, 32 P. LEWIS, M. R & ROBERTSON, W. R. B. (1916). Biol. Bull. Wood's Hole, 30, 99. MATTHEWS, B. H. C. (1928). J. Pkysiol. 65, 225. (1931a). J. Phynol. 71, 64. (19316)- J- Pkytiol. 72, 153. (1933) 7- Phystol. 78, 1. PUMPHREY, R. J. (1936). J. Phynol. 87, 6P. PUMPHREY, R. J. & RAWDON-SMITH, A. F. (1936). Proc. roy, Soc. B, 121, 18. SNODGRASS, R. E. (1935). Principle! of Insect Morphology. New York. WEVER, E G & BRAY, C. W. (1930). J. exp. Psychol. 13, 373. ZAWAHZIN, A (1912). Z wiss. Zool 100, 447. JEB-XVI 8