PROPRIOCEPTIVE REFLEXES CHANGE WHEN AN INSECT ASSUMES AN ACTIVE, LEARNED POSTURE

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1 J. exp. Biol. 107, (1983) 385 Printed in Great Britain 77K Company of Biologists Limited 1983 PROPRIOCEPTIVE REFLEXES CHANGE WHEN AN INSECT ASSUMES AN ACTIVE, LEARNED POSTURE BY SASHA N. ZILL* AND ROBIN R. FORMAN Department of Biology, University of Oregon, Eugene, Oregon 97403, U.SA. {Received 16 February 1983-Accepted 10 June 1983) SUMMARY Imposed changes in activity of a joint angle receptor of the locust leg, the metathoracic femoral chordotonal organ, produce variable, phasic reflex responses in a leg extensor motoneurone in untrained animals. After training the locust to maintain a posture in extension beyond a minimum required joint angle, these reflexes are consistently tonic and excitatory. This plasticity of reflex responsiveness permits the locust to couple motoneurone firing to afferent activity when receptor input is behaviourally relevant. INTRODUCTION The reflex effects of many types of proprioceptive sense organs are not constant but can be enhanced, suppressed or reversed by the central nervous system according to the particular type of behaviour (Wilson, 1965; Ekholm, Eklund & Skoglund, 1960; Bassler, 1976; DiCaprio & Clarac, 1981) or state (Vedel, 1980; Holmqvist & Lundberg, 1961) of an animal. The neuronal mechanisms underlying this reflex plasticity have not been defined because of the large number of proprioceptive interneurones in vertebrates (Jankowska, Johannisson & Lipski, 1981) and the lack of a method of systematically producing reflex changes in the simpler nervous systems of invertebrates. In vertebrates, a number of experiments have shown that reflexes can be consistently altered by instruction or training (Hammond, 1956; Melvill Jones & Watt, 1971; Evarts & Tanji, 1974). While it is known that many invertebrates can learn to assume restricted postures to avoid aversive stimuli (Horridge, 1962; Hoyle, 1976) no studies have employed learning paradigms to examine reflex plasticity. We have used a new learning paradigm (Forman & Hoyle, 1978), utilizing a natural aversive stimulus - heat to the head - to train locusts to maintain a leg posture in extension, and we have studied changes in reflex effects of a leg joint angle receptor, the femoral chordotonal organ (Usherwood, Runion & Campbell, 1968). We find that training is accompanied by systematic changes in the reflex effects of this receptor upon an identified leg extensor motoneurone. This plasticity of reflex responsiveness Present address: Department of Anatomy, University of Colorado Medical School, Denver, Colorado 80262, U.S.A. Key words: Reflexes, learning, plasticity.

2 386 S. N. ZlLL AND R. R. FORMAN Heat lamp 0 T pjex Extend I 180 Lamp on off Femoro-tibial joint angle Fig. 1. (A) Preparation. The locust is mounted so that only the femoro-tibial joint of one hindleg is free to move. A fine wire attached to the tibia is used as the wand of a position transducer. Manipulation of the ligament of the chordotonal organ is effected by a hook attached to a piezoelectric crystal. (B) Records of femoro-tibial joint angle during four consecutive training sessions. The dashed lines indicate the minimum joint angle in extension required to turn off the heat lamp. Black bars indicate when the tibia is held within this range (as sampled twice per second by the computer). The tibia is maintained beyond the required joint angle for progressively longer periods of time in successive training periods (duration of each training session = 4 min).

3 Changes in postural reflexes 387 permits the locust to couple motoneurone firing to afferent activity when receptor Input is behaviourally relevant. METHODS Locusts {Schistocerca americana, Dirsch) were restrained in dental wax so that only the femoro-tibial joint of a single hindleg was free to move (Fig. 1A). The angle of the femoro-tibial joint (normally at rest) was monitored by a modified Sandeman capacitative transducer (Forman & Brumbley, 1980). Animals were required to maintain the tibia in extension within a pre-set range of joint angle (greater than 120 ) to avoid heating of the head or mouthparts by a focussed lamp. Fourminute training sessions were then applied, alternating with 4-min rest periods. The position of the tibia was sampled by an on-line computer that both set the limit of joint angle and controlled the aversive stimulus while storing data for further analysis (Fig. IB). Animals were considered to have learned the required joint angle when they maintained the heat lamp off for at least 1 min in a training session (after 2-14 training sessions). To gain access to the femoral chordotonal organ, a small window was cut into the cuticle of the distal femur and the underlying air sacs were separated to expose the main ligament of the organ (Usherwood et al. 1968). In normal movement, this ligament is stretched as the femoro-tibial joint is flexed and relaxed as the joint is extended (Zill, 1981). We imposed changes in joint angle detected by the chordotonal organ by lifting or dropping the ligament using a fine wire hook attached to a piezoelectric crystal. Rectangular voltage pulses were applied to the crystal producing lifts of the ligament and stretching of the chordotonal organ. A lift of 175 pan resulted in an afferent discharge equivalent to a joint flexion of Displacements of the ligament were maintained for 5-80 s. Releasing the ligament mimicked equivalent joint extensions. Reflex effects of the chordotonal organ were examined in the extensor tibiae muscle by monitoring myographically the activity of the single slow excitatory motoneurone which supplies it. RESULTS Before training, lifts of the chordotonal ligament (N= 19 in seven animals) produced transient, short latency excitatory discharges in the slow extensor motoneurone, resisting the apparent joint movement (Fig. 2A). These discharges were seldom sustained for the duration of the stimulus and often (N 9 in four animals) a decrease of activity followed the initial excitation. Both excitatory and inhibitory responses could be elicited in successive tests in the same animal. After training, lifts of the chordotonal ligament (N = 17 in the same seven animals) consistently produced high frequency, strong resistance discharges that were sustained for the entire duration of the stimulus (Fig. 2B). We compared the slow extensor discharge frequency in the second preceding ligament displacement with that in the fifth second following the onset of maintained displacement in experimental animals both before (Fig. 2C) and after (Fig. 2D) training. The change in discharge

4 388 S. N. ZlLL AND R. R. FORMAN Extensor tibiae Lift ligament s BEFORE TRAINING Extensor tibiae Lift ligament 3s a 1ct of o 100 m N &> c 3 I W * --- \ Extensor frequency (Hz) 1s before lift,' AFTER TRAINING S D? 40 s > 30 N 5. 20! 1 0 I ^ Extensor frequency (Hz) 1 s before lift w Fig. 2. (A) Reflex responses before training. Myograms of the slow extensor tibiae motoneurone show reflex responses to stretches of the chordotonal ligament equivalent to joint flexions of Before training, responses of the extensor motorneurone vary and are not tonically maintained (bars above traces = Is). (B) Reflex responses after training. After training the firing frequency of the extensor always increases in response to stretch of the chordotonal organ. This increase is maintained for the duration of the stimulus. (C, D) Plots of reflex responses. These graphs plot the activity of the slow extensor motoneurone in the second immediately preceding a stretch of the chordotonal organ (abscissa) vs its activity in the fifth second following the onset of stimulation (ordinate) for seven experimental animals. Before training (C) responses are variable. Motoneurone activity may increase, decrease or remain constant. After training (D) motoneurone frequency consistently increases.

5 Changes in postural reflexes 389 Stable 1. Difference in extensor frequency in the second before and the fifth second of sustained lifts of the chordotonal ligament in seven experimental and seven control animals Before training Change in extensor frequency (Hz) (mean ± s.d.) (AT =19) After training Change in extensor frequency (Hz) (mean ± s.d.) (W =17) Experimental -25 ± ±8-2 Control 1-6± ±4-3 Only experimental animals received training contingent upon joint position and showed a consistent increase in reflex response. frequency after training was significantly different for each animal from that occurring before training (P> 0-99 by Student's t test) (Table 1). To establish that these changes in reflex responsiveness were a consequence of training and not merely a change in the excitability of the animal or a general sensitization following intermittent heating of the head and mouthparts, we examined reflex responses in control animals. Controls (seven animals) received local heating and chordotonal stretches identical in duration and timing to those received by experimental animals (obtained by playing back computer records). The aversive and sensory stimuli were, therefore, not contingent upon the joint position of the control animals. Reflex discharges in control animals were not significantly different before and after sham training (Table 1). Responses of controls after sham training were, however, significantly different (P > 0-99 by Wilcoxon matched pair test) from those of trained animals. DISCUSSION We have thus demonstrated that training of the locust to maintain its femoro-tibial joint angle in extension is accompanied by a consistent change in reflexes mediated by the femoral chordotonal organ. Our general finding of plasticity in reflexes of the chordotonal organ is consistent with observations of Bassler (Bassler, 1979; Graham & Bassler, 1981) who surgically altered the insertion of the ligament of the organ to produce erroneous afferent signals of joint position and movement in freely moving locusts. The effects of this operation depended upon the behaviour of the animal, being substantial in walking and jumping but minimal in stridulation. Bassler's conclusion that for the chordotonal organ 'the type of motor program in use determines the response to a particular afference' is supported by the present study. Few studies in invertebrates have examined the alteration of proprioceptive reflexes that may accompany learning. Increases in duration of a defensive reaction, the siphon withdrawal reflex, have been shown to occur in molluscs as a result of sensitization due to the application of prior noxious stimuli (Pinsker, Hening, Carew & Kandel, 1973). In the present experiments, application of the aversive stimulus alone did not produce increased reflex responsiveness as demonstrated in control animals. Our

6 390 S. N. ZlLL AND R. R. FORMAN results are, therefore, not attributable to generalized sensitization but require the specific association of leg position and cessation of the aversive stimulus produced im operant conditioning. The changes of reflex responsiveness found in the present study are, however, strikingly similar to those seen in stretch reflexes of monkeys in training of wrist joint position (Evarts, 1973). In those experiments consistent resistance reflex responses in biceps electromyograms occurred only after the monkey was trained to maintain a joint position. In both the monkey and the locust, therefore, systematic changes in reflex responses to limb proprioceptors accompany learning of joint position. We suggest, in conclusion, that many types of sense organs, in both vertebrates and invertebrates, may show similar plasticity of reflex effects and that learning paradigms may prove useful in studying the underlying neuronal mechanisms. Supported by NSF Grant BNS and NIH Grant 5F32NS REFERENCES BASSLER, U. (1976). Reversal of a reflex to a single motoneuron in the stick insect Carausius momsus. Biol. Cybernetics 24, BASSLEK, U. (1979). Effects of crossing receptor apodeme on the femoral chordotonal organ on walking, jumping and singing in locusts and grasshoppers. J. comp. Phytiol. 134, DICAPRIO, R. A. & CLARAC, F. (1981). Reversal of a walking leg reflex elicited by a muscle receptor. J. exp. Biol. 90, EKHOLM, J., EKLUND, G. & SKOGLUND, S. (1960). On the reflex effects from the knee joint of the cat. Acta physiol. scand. 50, EVARTS, E. V. (1973). Motor cortex reflexes associated with learned movement. Science, N.Y. 182, EVARTS, E. V. &TANJI, J. (1974). Gating of motor cortex reflexes by prior instruction. Brain Res. 71, FORMAN, R. R. & BRUMBLEY, D. J. (1980). An improved capacitative position transducer for biological systems. J. exp. Biol. 88, FORMAN, R. R. & HOYLE, G. (1978). Position learning in behaviorally appropriate situations. Neurosci. Abstr. 4, 193. GRAHAM, D. & BASSLER, U. (1981). Effects of afference sign reversal on motor activity in walking stick insects (Carausius momsus). J. exp. Biol. 91, HAMMOND, P. H. (1956). The influence of prior instruction to the subject on an apparently involuntary neuromuscular response. J. Physiol., Land. 132, 17-19P. HOLMQVIST, B. tc LUNDBERC, A. (1961). Differential supraspinal control of synaptic actions evoked by volleys in the flexion reflex afferents in alpha motoneurons. Acta physiol. scand. 54, 1 51 (suppl. 186). HORRIDGE, G. A. (1962). Learning of leg position by the nerve cord of headless insects. Proc. R. Soc. B157, HOYLE, G. (1976). Learning of leg position by the ghost crab, Ocypode ceratophthalma. Behavl Biol. 18, JANKOWSKA, E., JOHANNISSON, T. tc LIPSKI, J. (1981). Common interneurons in reflex pathways from group la and lb afferents of ankle extensors in the cat. J. Physiol., Land. 310, MELVILL JONES, G. & WATT, D. G. D. (1971). Observations on the control of stepping and hopping in man. J. Physiol., Land. 219, PINSKER, H. M., HENING, W. A., CAREW, T. J. tc KANDEL, E. R. (1973). Long term sensitization of a defense withdrawal reflex in Aplysia. Science, N.Y. 182, USHERWOOD, P. N. R., RUNION, H. I. tc CAMPBELL, J. I. (1968). Structure and physiology of a chordotonal organ in the locust leg.j. exp. Biol. 48, VEDEL, J. P. (1980). The antennal motor system of the rock lobster: competitive occurrence of resistance and assistance reflex patterns originating from the same proprioceptor. J. exp. Biol. 87, WILSON, D. M. (1965). Proprioceptive leg reflexes in cockroaches. J. exp. Biol. 43, ZILL, S. N. (1981). Central modulation of reflex effects of the locust metathoracic femoral chordotonal organ. Nevrosd. Abstr. 7, 252.