plasticity and proprioception in insects - the journal of

J. exp. Biol. 116, 435-461 (1985) 4 3 5Printed in Great Britain © The Company of Biologists Limited 1985

PLASTICITY AND PROPRIOCEPTION IN INSECTSI. RESPONSES AND CELLULAR PROPERTIES OF INDIVIDUAL RECEPTORS

OF THE LOCUST METATHORACIC FEMORAL CHORDOTONAL ORGAN

BY SASHA N. ZILL*

Department of Biology, University of Oregon, Eugene, Oregon 97403, U.SA..

Accepted 12 October 1984

SUMMARY

1. The metathoracic femoral chordotonal organ is a joint angle receptor ofthe locust hindleg. It consists of 45—55 bipolar sensory neurones locateddistally in the femur and mechanically coupled to the tibia.

2. Responses of receptors of the organ were examined by extracellular andintracellular recording. The organ as a whole encodes the angle of the femoro-tibial joint but shows substantial hysteresis. Tonic activity is greatest at theextremes of joint position.

3. The organ possesses no direct linkage to tibial muscle fibres and shows noresponse to resisted muscle contractions in most ranges of joint angle.However, responses to extensor muscle contractions are obtained when thetibia is held in full flexion due to specializations of the femoro-tibial joint.These responses could be of importance in signalling preparedness for ajump.

4. Intracellular soma recordings of activity in individual receptors indicatethat the organ contains two types of receptors: phasic units that respond tojoint movement and tonic units that encode joint position and also show someresponse to movement. All units are directionally sensitive and respond onlyin limited ranges of joint angle.

5. Some phasic units increase firing frequency with increasing rate ofmovement and thus encode joint velocity. Other phasic units fire only singleaction potentials and can encode only the occurrence and direction of jointmovement. All tonic units increase activity in the extremes of joint positionand show substantial hysteresis upon return to more median positions.

6. Direct soma depolarization produces different responses in differenttypes of units: phasic receptors show only transient discharges to currentinjection; tonic receptors exhibit sustained increases in activity that arefollowed by periods of inhibition of background firing upon cessation ofcurrent injection.

7. Receptors of the chordotonal organ are separable into two major groups,based upon their response characteristics, soma location and dendriticorientation: a dorsal group of receptors contains tonic units that respond inranges of joint flexion (joint angle 0-80°) and phasic units that respond to

•Present address: Department of Anatomy, University of Colorado Medical School, Denver, Colorado80262, U.S.A.

Key words: Joint angle receptors, proprioception, cellular physiology.

436 S. N. ZILL

flexion movements; a ventral group of sensilla contains tonic units active inranges of joint extension (joint angle 80-170°) and phasic receptors thatrespond to extension movements.

8. The response properties of these receptors are discussed with referenceto the potential functions of the chordotonal organ in the locust's behaviouralrepertoire.

INTRODUCTION

Many sense organs, in both vertebrates and invertebrates, precisely monitor theangles of joints of appendages (Granit, 1955; Mill, 1976). While the responses of jointangle receptors of vertebrates have been studied in detail (Boyd & Roberts, 1953;Ferrell, 1977), little is known about how information provided by these receptors isincorporated into behaviour (Grigg, Harrigan & Fogarty, 1978; Baxendale & Ferrell,1981). In contrast, recent work in invertebrates has shown that during certainbehaviour, such as walking (Graham & Bassler, 1981) and jumping (Steeves &Pearson, 1982), input from joint angle receptors can extensively modify postureand movement. However, other more stereotyped behaviour, such as stridulation(Bassler, 1979) and flight (Wilson, 1961), can be performed in the absence of inputsfrom joint angle receptors or with this input experimentally disrupted. Despite theadvantageous accessibility of invertebrate nervous systems for study at a cellularlevel (Hoyle & Burrows, 1973), the neuronal mechanisms underlying this impliedplasticity of behavioural effects of joint angle receptors have not yet been determined.

The present series of investigations, therefore, were undertaken to study a single,identified group of joint angle receptors of the locust hindleg, the metathoracicfemoral chordotonal organ. The goals of these investigations are to define theproperties of sensory and motor elements of the locust nervous system that determinethe functions of this group of receptors and permit flexibility of coupling in behaviour.

The locust metathoracic femoral chordotonal organ was first described byUsherwood, Runion & Campbell (1968), who examined the morphology andresponses of the organ and identified it as a joint angle receptor. However, a number ofbasic questions remained as to the responses of the organ. As noted by Burrows &Horridge (1974, p.59): 'No doubt many motorneuron responses to joint motion aredue to the chordotonal organ, but inferences about its central action are limitedbecause we lack the following details: (a) whether some or all units are directional intheir response, (b) whether the two directions of motion excite the same or differentunits in different parts of the range, (c) whether vibration at different parts of therange excites different units which could thus signal position although not tonically.'Further, a number of experiments by Bassler (1968), utilizing lesion or disruption ofafferent input, have shown that the organ can substantially affect walking andjumping. However, the interpretation of some of these experiments has been confusedby a lack of knowledge about the effects of these operations on chordotonal organoutput. For example, Bassler (1968) showed that after cutting one of the ligaments ofthe organ, jumping could not be elicited. Heitler & Burrows (1977) attributed this

Insect proprioception 437

effect, not to a discrete function of the chordotonal organ in the jump, but rather to thefact that the locust perceived its joint as fully extended. In contrast, Pearson, Heitler& Steeves (1980) postulated that the chordotonal organ provided inputs thatspecifically triggered jumping, although the mechanism by which a joint anglereceptor could trigger a movement in a joint held immobile by the co-contraction ofantagonist muscles was not determined.

The first paper in this series, therefore, re-examines the basic morphology andresponses of the chordotonal organ, and also studies the responses of individualreceptors by intracellular recording. Subsequent papers utilize the informationprovided by this study to investigate the specific effects of the chordotonal organ uponmotoneurones and interneurones.

Few previous studies have examined the responses of chordotonal sensilla byintracellular recording (Mendelson, 1963, 1966), owing in part to technical problems.The locust femoral chordotonal organ has proved amenable to such studies. Thecellular properties of these receptors may provide insight into the functions of jointangle receptors in behaviour.

METHODS

Adult male locusts {Schistocerca gregaria), provided by the University of BritishColumbia and maintained in laboratory cages at 25°C, were used in all experiments.

Anatomy

Animals (N = 7) were induced to autotomize one metathoracic leg that was pinnedout in a Sylgard resin-coated dish, with the femoro-tibial joint angle at 80°. Afterremoving the tarsus, the leg was gently perfused with Karnovsky's fixative for 30 min.The parts of the distal femur and proximal tibia containing the femoral chordotonalorgan and its attachments were then cut out, dehydrated and embedded in Spurr'sresin. Serial sections (2 fim) were taken either perpendicular or parallel to the long axisof the leg, stained with toluidine blue, and examined by light microscopy. Individualsections were traced through a drawing tube onto acetate sheets and compositeoverlays were constructed.

Physiology

Extracellular recordings

Intact animals were restrained in wax so that the outer surface of the femur was heldin a horizontal plane facing upwards. A small window was cut into the distal femur toexpose the main ligament and flexor attachment of the organ and to cut nerves distal tothe receptors. Another window was cut 8-10 mm proximal to the organ and multi-unit recordings were taken from the whole nerve 5bl (Campbell, 1961) using hookelectrodes. The femoro-tibial joint angle was monitored either by a potentiometerattached to the tibia (Young, 1970) or with a capacitative transducer (Sandeman,

438 S. N. ZILL

1968). Movements of the tibia were generated manually or with a piezoelectric crystal(as described below). All data were stored on tape for subsequent analysis.

Intracellular recordings

Animals were mounted dorsal side up and the metathoracic legs held in wax so thatthe inner surface of one femur was in a horizontal plane (Fig. 1). An insect pin, whoseends had been bent into small loops, was glued to the proximal tibia. One of theseloops served as a swivel joint when linked to another pin that was attached to the end ofa piezoelectric crystal. The crystal generated smooth movements of the tibia whendriven by voltages derived from a wave-form generator. Movements were monitoredby a photoelectric cell placed close to a flag that was attached to the tibia.

To expose the femoral chordotonal organ, a small window was cut in the cuticle ofthe distal femur. The tibia was then fully extended and a section of the tendon of theextensor tibiae muscle was removed. The joint was then moved to an angle of 45 ° and a

PE crystal

Intracellular electrode

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Fig. 1. Diagram of preparation for intracellular recording. A locust is mounted in wax so that onemetathoracic leg lies in a horizontal plane with its inner (medial) surface upwards. Changes in tibialposition are generated by a piezoelectric crystal (PE crystal) linked to the tibia by a small pin.Displacements are monitored by a photocell placed close to a flag attached to the distal tibia. A smallwindow is cut in the distal femur to expose the femoral chordotonal organ (CO, enlarged for clarity).The organ is stabilized by a small platform and penetrated with microelectrodes.


portion of the flexor tibiae tendon proximal to the attachment of the chordotonal organwas excised. This dissection exposed the main body of the chordotonal organ andeffectively left the tracheal supply of the organ intact as it derives from the outersurface of the organ. The presence of an intact tracheal supply and its aeration by theanimal proved to be essential for prolonged recordings from the chordotonal organ. Inearly experiments, performed on isolated legs or those involving ventral dissection ofthe leg and disruption of the tracheal supply, preparations rapidly deteriorated asjudged from decline in resting potential and poor mechanical response. In contrast,recordings from dorsally dissected organs showed stable resting potentials and con-sistent responses could be elicited from single receptors for up to 1 h. A small amountof saline was placed over the preparation to prevent drying and to link it to a groundwire placed close to the opening. The saline used was kept to a minimum and was seento mix rapidly with the animal's own circulating haemolymph.

A small platform was manoeuvred under the proximal portions of the organ formechanical support. Single bipolar neurones of the organ were then penetrated withglass microelectrodes filled with either ZmolP1 potassium acetate (50— 70 MQ) or a5 % solution of Lucifer Yellow (kindly provided by Dr Walter Stewart). For dye-filling, negative current pulses of 1-3/iA were applied for 500 ms at 1 Hz.Chordotonal organs were then fixed, cleared and examined conventionally.

In other experiments, intracellular recordings were taken from axons of chordo-tonal sensilla in nerve 5 close to the metathoracic ganglion. The ganglion was exposed,supported and perfused with saline according to the method of Hoyle & Burrows(1973). Individual receptors were identified by their short latency responses to liftingof the main or flexor ligaments of the organ (exposed as above) by means of a smallhook attached to the piezoelectric crystal.

RESULTS

Anatomy

The femoral chordotonal organ is composed of 45-55 (mean 48-8, N =1) bipolarneurones located in the distal femur 6-7 mm proximal to the femoro-tibial joint.Axons of these neurones join nerve 5bl (Campbell, 1961) by a short branch to projectto the central nervous system. The organ is firmly attached to the outer (anterior) wallof the femur by a short attachment ligament (Fig. 2). In light micrographs, twostructures were consistently found that couple the receptors to the next distal legsegment, the tibia: a main ligament that terminates adjacent to the insertion of thetendon of the extensor tibiae muscle, and a flexor ligament that attaches directly to thetendon of the flexor tibiae muscle, approximately 5 mm proximal to the femoro-tibialjoint. While Usherwood et al. (1968) state that several other small ligaments link theorgan directly to fibres of the extensor and flexor muscles (based upon examination ofwhole mount preparations) these ligaments could not be identified in seven prep-arations serially sectioned and examined by light microscopy in the present study. Asmall nerve was regularly found to continue distally from the organ and branch in the

440 S. N. ZILL

; *' 0®fr *

Fig. 2. Structures of the chordotonal organ and individual receptors. (A) Composite lightmicrograph showing neurones and ligaments of the chordotonal organ. The organ is composed of twogroups of bipolar neurones (dorsal and ventral) whose axons join nerve Sbl (n5bl). An attachmentligament (a) anchors the organ to the cuticle (c) of the femur. Two ligaments link the organ to thetibia: a main ligament (m) and a flexor ligament (/"). A small nerve proceeds distally from the dorsalsurface of the organ to innervate hairs of the distal femur. (B) Individual chordotonal sensilla (filledwith Lucifer Yellow) differ in their dendritic orientation. Dendrites of receptors of the dorsal group(left) are orientated parallel to the long axis of the femur (arrow); receptor dendrites of the ventralgroup (right) are angled with respect to the same axis. Calibration bar: A, 100^m; B, 75 ftm.

region of the extensor muscle. This nerve, which contains only axons from small hairslocated on the distal femur (as indicated by extracellular recordings) could have beenmistaken for a ligamentous attachment. Thus the only mechanical coupling of theorgan appears to be by two ligaments that link it to the tibia. Movement of the tibiaproduces selective stretching of the organ by each of these ligaments that is readily


discernible in dissected preparations (Fig. 3A): decreasing joint angle in flexionprogressively stretches the organ by the main ligament; increasing joint angle inextension stretches the organ by the flexor ligament.

Receptor cell somata and scolopale terminations of receptor dendrites in individualsections were drawn on acetate sheets (by means of a drawing tube) and compositeoverlays constructed from serial sections. Several features were noted in each of theseven composites constructed. First, the cell bodies of the receptors often formeddiscrete dorsal and ventral groups. Within these groups the largest somata were

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• Tibia

Axis of femurAttachment

Main ligamentI

Fig. 3. (A) Diagram of insertions of chordotonal ligaments. The femur and tibia are shown inoutline. The joint is hinged and can move only in flexion or extension (in the plane of the diagram).Two antagonist muscles produce these movements, the extensor and flexor tibiae (their tendons aredrawn as dark lines). The chordotonal organ (enlarged) has two ligaments that link it to the tibia: themain ligament is stretched during joint flexion, the flexor ligament is stretched during joint extension.(B) Orientations of dendritic insertions in the organ. A composite diagram, constructed from acetateoverlays of drawing of serial sections of one chordotonal organ, indicates the orientations of thedendritic terminations (scolopales): scolopales of dorsal neurones are orientated parallel to the longaxis of the femur; scolopales of ventral sensilla are angled with respect to the femoral axis.

442 S. N. ZILL

located proximally, smaller cells generally were found more distally. Often the axonsof the dorsal and ventral groups formed separate bundles that joined nerve 5b 1individually. Second, the scolopales of the receptors showed two distinct orientations(Fig. 3B): scolopales located dorsally were orientated parallel to the long axis of theleg, while ventral scolopales were angled (30-35° with the leg fixed at 90°) withrespect to the same axis. These observations suggest that, anatomically, the femoralchordotonal organ is composed of two discrete groups of receptors.

Physiology

Extracellular recordings

Tonic discharge and hysteresis. Tonic activity of the chordotonal organ wasexamined by extracellular recording from nerve 5b 1, with all nerve branches distal tothe organ cut while the femoro-tibial joint was set at different angles. The upper plotin Fig. 4A shows the tonic activity of the organ when the tibia was moved away fromthe mid-position (80°) progressively into extension (160°) and flexion (0°). The organas a whole shows a corresponding increase of activity in each range away from the restposition. However, this discharge showed considerable hysteresis and spiking activitydepended upon the direction from which a particular joint angle was approached.When the tibia was moved back toward the mid-range of joint angle a sudden decreasein firing rate occurred that remained depressed for periods up to 1 min (Fig. 4A, lowertrace). This hysteresis depended both upon the magnitude of joint angle traversed inreturn and the specific range of angle, being greater in joint flexion than extension.Similar hysteresis has been noted in other chordotonal organs (Burns, 1974) and otherjoint receptors in insects (Coillot & Boistel, 1969).

Contributions of different ligaments to tonic response. To evaluate the effects of thetwo insertions of the chordotonal organ, each ligament was individually cut in thedistal femur and discharge at different joint angles was recorded extracellularly (Fig.4B). Severing the main ligament eliminated the increase in firing rate in joint flexionbut did not affect the discharge to joint extension. Cutting the flexor ligamentproduced a discharge upon joint flexion that was unchanged and almost completelyeliminated the increment of response in joint extension. A small increment occurredin the most extreme range of joint extension. It should be noted that after theseablations the chordotonal organ discharge in the affected range did not fall to zero butremained approximately equivalent to that seen at 80°.

Phasic discharges. To examine the responses of the chordotonal organ to jointmovement recordings were taken from n5bl while the tibia was moved at differentrates through angles of 10-15° using sinusoidal or ramp waveform inputs to thepiezoelectric crystal. Fig. 5 shows the results of one experiment using sinusoidalfrequencies of 0-2, 0-5 and 5 Hz over the range of 90-105° of joint extension. Severalcharacteristics of the response to joint movement can be noted. First, discretedischarges occur during rapid joint movement. Inspection of the second and thirdtraces in Fig. 5 clearly shows that units of different size respond to each direction ofmovement. Similar discharges were observed in all ranges of joint angle. Thus, the


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Fig. 4. (A) Tonic responses of the chordotonal organ. Activity of the organ was recordedextracellularly while the tibia was moved from an angle of 80° in 20° steps progressively into flexionand extension. All action potentials above baseline were counted. The graph plots the dischargefrequency (+S.D.) for 20 consecutive seconds, 30 s after tibial displacement. Discharges increase asthe tibia is moved away from the median range. Return movements show substantial hysteresis andmuch lower firing rates are attained. (B) Contribution of different ligaments to tonic discharge. One ofthe ligaments of the organ was cut in each of two animals and discharge of the organ was tested asabove. Severing the main ligament (dashed line) eliminates the increase in firing rate in ranges of jointflexion but leaves intact the discharge to joint extension. Cutting the flexor ligament (solid line)produces normal responses to joint flexion but almost completely eliminates the increase of activity injoint extension.

chordotonal organ appears to contain phasic units that respond to any movement atany starting position. Secondly, units in the size range of those that were tonicallyactive at rest (top trace) showed a modulation of their frequency during jointmovement, increasing in movements away from 80° (joint extension) and decreasingupon return (joint flexion). At higher frequencies, complete inhibition of tonicactivity occurred during return (flexion) movements. Similar inhibition was notedfollowing step displacements, corresponding to the hysteresis previously described.

444 S. N. ZlLL

Responses to vibration. To examine the potential responses of the femoralchordotonal organ to transmitted vibration, extracellular recordings were taken from

CO

Extend

0-2 Hz

Stim.

0-5 Hz

5 Hz

Fig. 5. Discharges during joint movement. With the tibia at rest (joint angle 110°) irregular firing oftonic units is seen. During sinusoidal movements (15° at 0p2, 0-5, 5-0Hz) phasic units are active.Units with different sized spikes fire in response to joint flexion or joint extension. Activity of tonicunits is inhibited by joint flexion (up on position trace) and increases upon joint extension. In veryrapid movements tonic activity is completely inhibited during flexion movements. Calibration: firsttrace, 0-3 s; second trace, 1-25 s; third, fourth trace, 0-1 s. CO, chordotonal organ.


the organ with the femoro-tibial joint completely immobilized with epoxy resin whilesinusoidal mechanical stimuli were applied at the distal tibia with a piezoelectriccrystal. No responses were obtained in these preparations in the absence of jointmovement. In other experiments, where the joint was free to move, vibratory stimuliwere applied by tapping on the experimental mounting or by placing a variable speedmotor adjacent to the preparation. Again, no consistent responses occurred withoutvisible joint movement. Although some frequencies of vibration may be transmittedby the organ in freely moving animals, sensitivity is probably severely limited byviscosity of the tibial muscles. A lack of responsiveness to vibration has also beennoted for other joint chordotonal organs (Hustert, 1982). In general, chordotonalorgans that respond to vibration do not span leg joints (Schnorbus, 1971).

Response to muscle contractions. A number of chordotonal organs have beenshown to be directly mechanically linked to leg muscles (Burns, 1974; Clarac, 1968).Usherwood et al. (1968) also speculated that the metathoracic femoral chordotonalorgan responded to resisted muscle contractions. In the present series of experimentsactivity was recorded from the organ during spontaneous movements by the animaland during stimulation of the nerves to the extensor and flexor tibiae muscles.Chordotonal organ activity during spontaneous movements (Fig. 6Ai,ii) was similarto that seen during imposed displacements and there was no indication of a resetting ofafferent activity by muscle contractions, as occurs in vertebrate muscle spindles(Matthews, 1972). Also, with the joint completely immobilized, no change of activitywas recorded during stimulation of either the extensor or flexor nerves (with oneexception, described below). Thus, through most of the range of joint angle there isno direct efferent control of chordotonal organ activity.

A change in chordotonal activity was produced by extensor muscle contractionswhen the joint was placed in full flexion and movement of the tibia blocked by a smallpin or held by spontaneous flexor activity. Repeated twitch contractions of theextensor muscle produced an increase in chordotonal organ activity (Fig. 6B). Thisfiring was not due to a direct effect of fast extensor activity upon the organ but rather tothe effect of extensor contractions upon the femoro-tibial joint. Resisted contractionsof the extensor muscle at full joint flexion have been shown to produce bending of thedistal end of the femur, storing energy for the jump (Bennet-Clark, 1975; Heitler,1977). These contractions also result in a bending of the proximal end of the tibia anddisplacement of the insertion of the main ligament of the organ (50-60/xm asmeasured by an optical micrometer). The resultant discharge is of importance asthe chordotonal organ can thus monitor the extent of tibial bending and signalpreparedness for the jump.

Intracellular recordings

Individual chordotonal sensilla regularly showed resting potentials of 50—65 mVin intracellular recordings. The most stable penetrations were obtained from theproximal portions of the organ, that is, the region containing the receptor cell bodies.In this area, overshooting action potentials (70-90 mV in amplitude) could regularly

446 S. N. ZILL

Ai

Joint angle Extend

co y

CO

TM

Fig. 6. Chordotonal organ activity (CO, lower traces) recorded extracellularly during spontaneousmovements of the tibia. The femoro-tibial angle (upper traces) was monitored by a Sandemantransducer. Movements generated by the animal produced activity similar to that seen during imposedchanges in joint angle. (Ai) A large joint flexion (80°-0°) produces phasic activity during themovement and sustained firing when the tibia is held flexed, (ii) A joint flexion is followed by a largejoint extension (80°-140°). Return to the initial joint angle is accompanied by hysteresis and slowrecovery. (B) Activity during spontaneous repetitive extensor contractions. With the tibia held in fullflexion and joint movement prevented, a burst of activity in the fast tibial extensor motoneurone (largespikes in tibial myogram, TM, lower trace) produces intense afferent activity that subsides aftermotoneurone activity ceases. Calibration: A, 500 ms; B, 100ms.

be recorded. Cells in different parts of this proximal region showed responses indifferent ranges of joint angle (see below). The entire angular range of response of asingle receptor could not be examined in soma recordings since large changes in jointangle produced a stretch of the organ and a shift in soma position sufficient to pull the

Insect proprioception 447recording electrode out of the cell. Consequently, responses could be routinelyexamined in soma recordings only through arcs of 10-15°. Some receptors wereexamined over their full response ranges through axon recordings (see below).However, single preparations were studied in several different ranges of joint angleand cell populations that did not show mechanical responses at some joint angles wereactive in others. The results presented below represent recordings from 72 cells in 18preparations with as many as nine cells recorded from a single organ.

Phasic units. Many cells (N = 49) did not exhibit activity at rest but showedvigorous phasic responses to joint movements (Fig. 7). Phasic units were alwaysdirectionally sensitive, responding to either extension or flexion movements but not

AStim.

CO

-ill J L

Ei Eii

Fi Fii

Fig. 7. Phasic unit responses. Individual phasic receptors are directionally sensitive in their responsesto joint movement firing to extension (A) or flexion (B) but not both directions. Some receptorsproduce multiple spikes to joint movements (C) while others fire only single action potentials at allfrequencies of oscillation (D). Many phasic units show increases in firing frequency and decreases ofburst duration during increasing frequencies of sinusoidal (E) and ramp (F) movements. Calibration:vertical displacement A,B, 25°; C,D, 20°; E,F, 10°; voltage: 90mV; horizontal A,B,E,0-5s;C,D, 0-1 s ;F , 0-2s.

448 S. N. ZILL

both. However, in all ranges of joint angle different individual receptors could belocated that responded to either movement, so the organ as a whole detects jointdisplacements in any direction in agreement with observations based on extracellularrecordings.

The responses of individual phasic units depended upon their position withinthe organ. Cells responding to flexion movements were found in the dorsal part ofthe organ, while cells sensitive to extension movements were located ventrally, close tothe origin of the flexor ligament. Examination of phasic units filled with LuciferYellow showed that receptors with different directional sensitivities were alsomorphologically distinct in their dendritic orientation (Fig. 2B). Cells of the dorsalgroup had dendrites that inserted nearly parallel to the long axis of the leg. Receptorsof the ventral group had dendrites that were angled (25-35°) with respect to the legaxis and inserted close to the origin of the flexor ligament. Thus the responses ofindividual phasic receptors were correlated with both their position and theirdendritic orientation. Somata of phasic receptors were often quite large (maximumdiameter llO^m) and their dendrites quite long (maximum 170/im).

Some phasic units (N— 14) showed only single spikes to all frequencies of jointmovement (Fig. 7A,B,D) and thus function solely as movement detectors. Theseunits were often, however, extremely sensitive and spiking could be elicited by jointmovements as small as 0-3/im (approximately l-2minutes of an arc). Other units(N = 14) (Fig. 7C,E,F) fired repetitively to single movements and the frequency offiring depended upon the velocity of joint movement. Fig. 8 is a plot of the responsesof two different phasic units, one extension and one flexion sensitive, to alternatingmovements of 12° at different frequencies. At low velocities of joint movement, theseunits respond to increasing rate of joint movement with a linear increase in firingfrequency. At frequencies greater than 50—75 Hz these units show responsesaturation. Similar response saturation has been found in phasic receptors of otherchordotonal organs (Bush, 1965).

Tonic units. Many units in the chordotonal organ (TV = 23) showed tonic activity.This firing was always irregular over time and patterned activity or regular groupingof spikes did not occur (Fig. 9Ai). All tonic units increased their activity when thetibia was moved away from 80°, i.e. to increasing flexion or extension (Fig. 9Aii).Individual units, however, were directionally sensitive, increasing activity to eitherextension or flexion but not both. All tonic units also showed some phasic properties:small-to-moderate step displacements produced an initial high firing rate thatadapted, with some irregularity over time (Fig. 9Aiii,iv). Step returns to the initialtibial position were often accompanied by an inhibition of background activity. Thiswas particularly apparent in large displacements where firing could be completelyinhibited for several seconds and full recovery could take up to 1 min (Fig. 9Av).This inhibition paralleled the hysteresis shown to step displacements by the organas a whole, although unit responses to large displacements producing prolongedinhibition could not be studied in soma recordings. Tonic units also showed direc-tionally sensitive modulation of their firing frequency during imposed movements ofthe tibia. Slow ramp movements resulted in irregular increases in firing rate as the


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Fig. 8. Responses of phasic units at different velocities of joint movement. The discharge frequencies(ordinate) of two units, one flexion sensitive (upper), the other extension sensitive (lower) are plottedat different velocities of joint movement (abscissa) over an arc of 12° starting at a joint angle of 30°.Both units show linear increases in firing frequency at low velocities of joint movement but saturationat higher velocities. Each point is mean discharge + S.D. over 25 consecutive cycles.

tibia was moved away from the mid-range of joint angle and decreases upon return(Fig. 9B). During rapid ramp or sinusoidal movements complete patterning ofactivity occurred with spiking during movements in one direction, and completeinhibition upon return (Fig. 9C).

The majority of tonic units were identical to phasic units in their directionallysensitive distribution within the organ and their dendritic orientation. Thus, unitstonically active in joint flexion were found in the dorsal group of receptors and haddendrites orientated parallel to the leg axis. Units active in joint extension were foundin the ventrally located group and had dendrites that were angled with respect to theleg axis. Three units however were noted to be exceptional. These units were active inextreme extension (140-160°) and were penetrated close to the attachment of theorgan. Units close to the attachment were extremely difficult to penetrate and hold inintracellular recordings; however, a Lucifer fill of one such unit showed a dendriticorientation characteristic of the dorsal group.

Lucifer fills of tonic units showed cell bodies that were always smaller than thoseseen in phasic units (maximum diameter, 60 /im) and often had substantially shorterdendrites (maximum length, 100/im).

450 S. N. ZILL

Effects of direct displacement of ligaments upon unit responses. To further evaluatethe relative contributions of the two major insertions of the chordotonal organ,responses of individual receptors were examined to both displacement of the tibia andlifting or pulling of the ligaments. Units of the dorsal group that responded to jointflexion also fired in response to stretch of the main ligament. Receptors of the ventralgroup that fired in response to joint extension also responded to stretch of the flexorligament. The responses of some phasic units in each group were exceptional. Inranges of joint extension, some phasic receptors of the dorsal group, that showedactivity in response to joint flexion and to stretch of the main ligament, also respondedto release of stretch of the flexor ligament. Other receptors were also located in theventral group that responded to both joint flexions and release of the main ligament.

Ai

Stim.

CO

CO- JUUUUL

Fig. 9. Tonic unit responses. Individual tonic units show irregular baseline levels of activity thatdepend upon joint position, increasing as the joint is moved away from the median range (Ai, 40°; ii,30°). Similar increases are seen during step displacements of the leg (iii, 5°; iv, 10°). Largedisplacements (v, IS °) produce an intense phasic discharge that gradually adapts to a lower tonic level.Return movements are followed by an inhibition of background activity paralleling the hysteresisshown by the organ as a whole. (B) Tonic units respond directionally to joint movements (CO).During very rapid repeated displacements (C) tonic activity shows discrete patterning with firing onlyduring movement away from the median range and complete inhibition upon return. Calibration:vertical, 60mV; horizontal, Ai-iv, 0-5s; v, 10a; B, 0-5s; C, 0-2s.


Thus, it appears that some receptors in each group can respond to relaxation as well asstretch of the ligaments, as has been found for the propus-dactyl organ of crustaceans(Mill & Lowe, 1972). For all receptors, however, pulling or releasing the ligamentsmimicked their responses to joint movement.

Action potential form and the effects ofsoma hyperpolarization. Neither phasic nortonic units showed large shifts in soma membrane potential during joint movementcomparable with generator potentials seen in other receptors, such as the crustaceanstretch receptor (Eyzaguirre & Kuffler, 1955). This finding is in agreement withstudies of other bipolar neurones such as those of the PD organ of the crab leg(Mendelson, 1963, 1966) and the pit receptors of the crayfish abdomen (Mellon &Kennedy, 1964). In those receptors, however, action potentials often arise abruptlyfrom the baseline, without initial inflections, and are thought to originate in the distal,dendritic process of the cell. In the present study, all stable recordings showed actionpotentials with distinct initial inflections (Fig. 10). Further experiments are plannedto examine the possibility that these pre-potentials represent residues of generatorpotentials that spread into the soma.

Effects of injected current. Phasic and tonic units differed with respect to the effectsof depolarizing current injected into the soma. Phasic units responded to currentinjection with a brief intense discharge that rapidly adapted, even at high levels ofcurrent, and abruptly ceased (Fig. 11A). In contrast, tonic units showed sustainedincreases in discharge rate even to low levels of current (Fig. UBi). Higher levels ofinjected current produced intense, sustained discharges that were often followed byperiods of inhibition of background activity (Fig. UBii). This was particularlypronounced when current was injected over longer periods (Fig. 1 IBiii). Some adap-tation occurred during prolonged constant current injection but sustained increases intonic activity were followed by periods of inhibition lasting for up to 30 s aftercessation of current injection.

The effects of depolarizing current injected into the somata of tonic units oftenmatched the effects of step displacements of the tibia. Fig. 12 plots the firing of a singletonic unit to displacements of two different amplitudes and current injection at twodifferent levels. Large displacements (Fig. 12Ai) and high levels of injected current(Fig. 12Bi) produced increases of activity showing an initial phasic discharge andsubsequent sustained elevation of firing rate. Return to the initial position andcessation of current both produced an inhibition of background activity. Smalldisplacements (Fig. 12Aii) and low levels of current injection (Fig. IZBii) werefollowed by a rapid recovery to baseline levels of activity.

Recordings from axons of chordotonal sensilla

To examine more completely the range of responsiveness of individual chordotonalsensilla, intracellular recordings (N=9 animals) were taken from receptor axons closeto the metathoracic ganglion. These axons were identified by their intense, shortlatency firing in response to lift of either the main or flexor ligaments of the organ, andconsistent responses to subsequent changes in the position of the tibia. It can be notedthat all the chordotonal receptor axons were invariably located as a group in a well

452 S. N. ZlLL

Joint angle

"V \_

CO

J JV L

Fig. 10. Effects of soma hyperpolarization. (A) A phasic unit fires a single action potential to stepdisplacement of the tibia. Progressive injection of hyperpolarizing current (2, 4, 5 nA) producesblocking of the action potential (bridge circuit out of balance to record the response). (B) A tonic unit,held hyperpolarized (3 nA) shows no sustained shift in baseline upon tibial displacement. Calibration:vertical, 20mV; horizontal, A, 50ms, B, 250 ms.

Ai

CO-

I

Aii

-1 I—

Bi

LBiii

Fig. 11. Effects of soma depolarization. (A) Phasic units show only transient firing to all levels ofdepolarizing current. (B) Tonic units show sustained activity to injected current (i,ii). Higher levelsof current produce a discharge that adapts slowly over time and is followed by inhibition ofbackground activity (iii). Calibration: vertical, current: A, 3; B, 2nA; voltage: A,B, 40mV;horizontal A.Bi.ii, 0-3 s, Biii, 0-4s.


Displacement

9 18 21 24 27 30

9 18 21 24 27 30

9 18 21

Time (s)24 27 30

Fig. 12. Comparison of effects of current and displacement. The number of spikes in each successivesecond are plotted for one tonic unit during displacement and depolarizations at different leveb. (A)Large displacements of the tibia (i) produce a sustained elevation in firing frequency that is followedby inhibition of background activity upon return. Returns after small displacements (ii) show littleinhibition. (B) High levels of current injection (i) produce sustained activity that adapts more slowlybut is also followed by inhibition of tonic activity. Low levels of current injection (ii) produce onlyslight rebound inhibition.

454 S. N. Znxdefined area of nerve 5 (mid-ventral). This agrees with previous findings of anorderly, somatotopic projection of afferent axons in insect peripheral nerves (Zill,Underwood, Rowley & Moran, 1980). The identification of axons as afferentprojections was also confirmed by Lucifer dye injection.

Individual phasic and tonic receptors responded only in a defined portion of therange of femoro-tibial joint angle, that is, the organ showed range fractionation (Fig.13). Phasic units were active in ranges of either flexion or extension but not both.Different phasic units showed considerable variety in the ranges of joint angle withinwhich they responded (maximum, 103°; minimum, 22°), although in generalresponse ranges were quite large. Axons of tonic units proved to be difficult topenetrate individually, presumably due to their small size. All tonic units showedresponses in either flexion or extension and increased their discharge as the joint anglewas moved away from the rest position, confirming observations from soma re-cordings. Response ranges of tonic units were smaller than those of phasic units, theminimum being 15° for a tonic unit active only at nearly full flexion. This latter typecould be of considerable importance in signalling preparedness for the jump.

PHASIC

Respond to iflexion jj

iii

Respond to iextension jj

0 20 40 60 80 100 120 140 160

20-, B TONIC

1 .OH~»

\

\I >

I \ fA /

20 40 60 80 100 120 140 160Joint angle (degrees)

Fig. 13. Range fractionation. The response ranges of five phasic and three tonic units recordedintracellularly from a single animal are plotted on the abscissa (0° = full flexion, 170° = fullextension). Phasic units had large response ranges. Tonic units had smaller response ranges andincreased their response frequency (plotted on the ordinate) in the extreme ranges of joint angle.


DISCUSSION

Responses of chordotonal sensilla

The present study has shown that the femoral chordotonal organ contains fourtypes of receptors, all directionally sensitive: tonic receptors, responding in ranges ofeither joint flexion or extension, and phasic receptors that fire only in response toflexion or extension movements. These basic types of sensilla have been found inevery chordotonal organ that has been studied physiologically (Wiersma & Boettiger,1959; Bush, 1965). Individual receptors in joint chordotonal organs thus invariablyindicate direction as well as joint movement.

Certain phasic receptors responded to all velocities of joint movements with onlysingle action potentials. These units are similar to phasic receptors found in otherchordotonal organs (Wiersma & Boettiger, 1959) that apparently function asextremely sensitive movement detectors. Other phasic receptors showed an increasein firing frequency that was linearly related to the velocity of joint movement butshowed saturation during very rapid movements. Similar saturation of responsivenesshas been found in units of the propus-dactyl (PD) chordotonal organ of the crableg (Mill & Lowe, 1972) that may show a maximum response of only 24-25 Hz atvelocities as low as 5 mm s~'. Other chordotonal organs of the crab leg show saturationat higher response frequencies (Bush, 1965) similar to those found for the locustfemoral chordotonal organ in the present study. It can be noted that, in the locust,limits to response frequency are not set by refractoriness of the spike-transmittingcapacities of the soma as direct depolarization of the cell bodies of chordotonal sensillaproduced much higher firing frequencies than did mechanical stimulation. Rather,as postulated by Wiersma (in Mendelson, 1963), these limitations in frequency areprobably due to properties of the mechano-electric transducer membrane of thereceptor dendrite.

All tonic units of the femoral chordotonal organ progressively increased their firingrate in positions away from the median range of joint angle and reached maximumactivity in the extremes of tibial position. Similar types of responses have been foundin other arthropod chordotonal organs (Cohen, 1963; Bush, 1965). This type ofresponse provides the maximum changes in frequency in ranges farthest from theresting position of the joint. Tonic units of chordotonal organs of other arthropodscan, however, show more complex changes in firing frequency than those found in thelocust (U. Bassler, personal communication). Tonic units of the femoral chordotonalorgan also showed some phasic discharges to joint movement, similar to the'intermediate' type units found in organs of crab legs (Wiersma & Boettiger, 1959;Clarac, 1968), but those organs also contain some pure tonic units that do not possessphasic properties, a type not found in the locust. It should be noted that many tonicreceptors had quite small somata so it is possible that such units are present in theorgan but remained undetected. If so, they probably form only a small percentage ofthe total receptors of the organ.

456 S. N. ZILL

In sum, the characteristics of the sensilla of the locust femoral chordotonal organfound in the present study are clearly within the spectrum of types shown by otherarthropod chordotonal organs. The specific responses of the receptors in the locust,however, may reflect the specialized functions of the organ (see below).

Grouping of receptors

The sensilla of the metathoracic femoral chordotonal organ were found to beconsistently organized into two groups, based upon their dendritic and scolopaleorientations and their directional sensitivity. Spatial separation according to directionof response has been shown for other leg chordotonal organs. For example, in the PDorgan of the crab leg flexion- and extension-sensitive cells are found on opposite sidesof the receptor strand (Wiersma & Boettiger, 1959; Hartman & Boettiger, 1967).Anatomical grouping of receptors according to dendritic orientation has been found inother insect leg chordotonal organs (Young, 1970) and is a prominent feature ofsensilla associated with auditory receptors (Doolan & Young, 1981). In those organs,it has been postulated that anatomical orientation determines the specific range ofresponsiveness, although this has not yet been specifically examined.

Adequate stimuli for chordotonal sensilla

The present study has shown that each of the two ligaments of the metathoracicfemoral chordotonal organ selectively produce tonic responses in different ranges ofjoint angle: the main ligament produces tonic afferent discharges in ranges of jointflexion (0-80°) while the flexor ligament mediates responses in ranges of jointextension. As the receptors of the organ also show a consistent separation according totheir directional sensitivity and range of responsiveness it would appear that each ofthese ligaments selectively acts upon different groups of sensilla: stretch of the mainligament produces tonic activity in the dorsal group of sensilla, stretch of the flexorligament elicits responses from tonic units in the ventral group. These conclusionsare supported by those experiments in which ligaments were directly stretched,producing selective activation of tonic receptors. Further, as each of these groups ofreceptors shows a consistent dendritic and scolopale orientation, the adequatemechanical stimuli for the tonic receptors would appear to be as follows: the dorsalgroup of receptors are clearly orientated so as to be stretched by pulling of the mainligament; the ventral group of receptors, whose dendrites terminate close to the flexorligament, should be both stretched and bent by this attachment. Thus both stretchingand bending of the dendrite would seem to be adequate stimuli for tonic receptors.

The effects of the chordotonal ligaments upon phasic receptors may be morecomplex. Phasic units of the dorsal group that respond in ranges of joint flexion andthose receptors of the ventral group active in ranges of extension, should respond tostretching and bending, respectively, as do the tonic units of those groups. However,the dorsal group of receptors also contains phasic units responding to flexionmovements at angles greater than 80° while the ventral group possesses receptorsactive at joint angles less than 80°. These receptors may respond to ligament


relaxation rather than stretch as shown by those experiments in which responses wererecorded while the insertions of the organ were directly manipulated. Relaxation-sensitive receptors have been conclusively identified in the PD organ of the crab (Mill& Lowe, 1972, 1973), and have been shown to possess morphologically specializeddendritic terminations. However, in the locust there may also be some interaction ofstretch of each of the ligaments to affect a small number of receptors in both groups.For example, the flexor ligament may be responsible for discharges elicited in rangesof extreme extension from receptors close to the attachment of the organ. Also,experiments examining phasic responses to joint displacements when individualligaments had been severed produced equivocal results due to unavoidable slightmovements of the cut ligaments. For the majority of receptors of the organ, however,individual pulls to the main or flexor ligaments effectively mimicked the effects ofjoint displacements.

Unfortunately, there is no clear agreement in the literature about the nature ofsensory transduction in chordotonal organs to permit a simple analysis of themechanical forces that affect these receptors. Wiersma & Boettiger (1959) firstproposed that stretch of the dendrites was the effective stimulus for receptors of thecrab PD organ, a view that has subsequently been adopted for the myochordotonalorgan (Cohen, 1963) and for crustacean antennal organs (Wyse & Maynard, 1965). Incontrast, Mendelson (1963), Bush (1965) and Taylor (1967) have suggested thatflexion or bending of the dendrite is the adequate stimulus. Further, as noted above,individual units may respond to relaxation as well as stretch (Mill & Lowe, 1972). Theresults of the present study suggest that each of these views may be correct and that,depending upon the mechanical arrangement of sensilla, individual receptors mayrespond to stretching, bending or relaxation. Direct observation of dye-filled recep-tors might aid in understanding the anatomical basis for differential responses inreceptors of the femoral chordotonal organ.

Interpretation of behavioural experiments

The results of the present study clarify the specific effects of the behaviouralexperiments of Bassler (1968, 1979) in which the ligaments of the organ were ablatedor their insertions moved. First, Bassler (1968) showed that jumping could not beelicited after cutting the main ligament of the organ. The present study has shown thatthe effect of this operation is to eliminate responses in ranges of joint flexion. Thedischarge of the organ, however, does not indicate full tibial extension when the jointis flexed, as assumed by Heitler & Burrows (1977), but remains equivalent to that seenat a joint angle of 80°. Ablation of the main ligament also eliminates the specific inputof the chordotonal organ during the co-contraction phase prior to the jump. Thisfinding supports the view (Pearson et al. 1980) that the femoral chordotonal organprovides a substantial contribution to triggering of the jump.

Bassler (1979) also showed that switching the insertion of the main ligament of thechordotonal organ, so that it was stretched by joint extension rather than flexion, hasdiscrete effects upon posture and locomotion. After this operation, animals would

458 S. N. ZILL

often hold the tibia in full extension, even while walking. The present study suggeststhat the effect of such alteration of the main ligament would be to reverse the inputfrom tonic units in the dorsal group of receptors, exciting these sensilla upon jointextension rather than joint flexion. Any slight extension movements would then beperceived as joint flexions by the animal. The reflex compensatory response toperceived flexion would be joint extension that would then further excite the dorsalreceptors. The compensatory system would now be caught in continuous positivefeedback which would generate complete joint extension. It should be noted that theresponses of the ventral group of receptors should remain unaltered. The animal is,thus, apparently incapable of overcoming erroneous input from one group ofreceptors with that provided by another. The next paper in this series also shows thatpotent reflex effects can be elicited by excitation of only one of the groups of receptorsof the chordotonal organ (Zill, 1985).

Cellular properties of individual sensilla

One finding of the present study differs from previous accounts of responses ofbipolar neurones (Mendelson, 1963, 1966; Mellon & Kennedy, 1964). Moderatelevels of depolarizing current injection into the somata of tonic units produced asustained increase in firing rate that was followed by an inhibition of backgroundactivity. While the effects of soma depolarization were not extensively examined,Mellon & Kennedy found only phasic discharges in response to extracellular stimula-tion of the pit receptors, although it was not determined whether these units respondtonically to mechanical stimuli or phasically to small vibrations (Mellon, 1963).Mendelson (1963, 1966) used current injection in studies of the PD receptors of thecrab leg, but does not mention any post-excitatory inhibition, although he did notsystematically classify units as phasic or tonic. These differences are explicable if it isassumed that current passed into the soma of tonic units of the locust chordotonalorgan spreads and directly affects the distal spike initiating zone. It should-again benoted that the dendrites of tonic units were often quite short (often less than 75 Jim).In crustaceans, dendrites of bipolar receptors are quite long, commonly exceeding200 /im. In those receptors, high levels of injected current would be required to affectthe distal dendrite. The proximity of the dendritic end to the soma in locustchordotonal sensilla may prove useful for studying the mechanisms underlyingtransduction in these receptors.

Hysteresis

The tonic units of the femoral chordotonal organ showed pronounced hysteresis:leg movements towards the median range of joint angle were accompanied by asubstantial inhibition of activity followed only slowly by recovery to normal back-ground firing. Other insect receptors show similar directional hysteresis, such as themesothoracic femoral chordotonal organ (Burns, 1974) and the multipolar recep-tors of the metathoracic joint (Coillot & Boistel, 1969). The cellular mechanismsunderlying this phenomenon are unknown. Burns (1974) speculated that in the


mesothoracic organ, the mechanical link to the flexor tibia muscle produced hysteresisdue to viscosity in the muscle fibres. This is apparently not the case for themetathoracic organ as there is no direct ligament to flexor muscle fibres. Rather, theresults of soma depolarization suggest that refractoriness of the spike-initiatingmechanism may contribute to hysteresis. Intense firing produced by either currentinjection or large joint movements was followed by periods of inhibition of back-ground activity upon return. Unfortunately, it was not possible to study the effects ofvery large joint movements so that a strict comparison between firing rate andsubsequent inhibition for both movement and current injection could not beobtained. It should be noted that other mechanisms may also contribute to hysteresis,such as the time course of mechanical recovery of dendritic deformation or theproperties of the mechano-transducer membrane itself.

Potential functions ofsensilla of the femoral chordotonal organ

The characteristics of receptors of the locust femoral chordotonal organ may beconsidered adaptations for the specialized uses and properties of the metathoracic legand suggest the following functions for this receptor.

(1) Detection of joint movement. All the sensilla of the chordotonal organ are highlysensitive to joint movement. Some phasic receptors, with large somata and axons,apparently only detect joint movement but these can rapidly inform the nervoussystem of changes in joint angle. As succeeding studies in this series will show, thissensitivity permits the locust rapidly to respond to imposed movements either bycompensation or by withdrawal of the leg when compensation is undesirable.

(2) Detection of readiness for the jump. One clear specialization of the organ, that isa consequence of the anatomy of the femoro-tibial joint, is its response to tibialbending produced by contractions of the extensor muscle when the leg is fully flexed.Further, because of the fractionation of response range, some individual sensilla mayselectively provide the nervous system with precise information as to whethersufficient energy is stored in deformation of the cuticle to execute successfully a jumpor defensive kick.

(3) Assisting in load compensation. As will be shown in the next study in this series,one of the key functions of the chordotonal organ is to produce reflex discharges in legmotoneurones to compensate for changes in load. There are several characteristics ofindividual receptors of the organ that precisely attune it to this function. First, alltonic and most phasic units show discharges that are sensitive to the velocity of jointmovement. Purely tonic receptors, that would respond only to joint angle, were notfound in the organ. By increasing frequency to increasing velocity of movement, theorgan provides an input to motoneurones that signals the appropriate level of activityneeded for compensation. Further, all tonic units increase their discharge rate inranges furthest from the median joint angle. These units also show the greatesthysteresis at these extremes. It can be noted that these characteristics provide thegreatest change in afferent frequency in joint positions that require the largestchanges in motoneurone firing rate for compensation due to the declining mechanicaladvantage of the tibial muscles (Burrows & Horridge, 1974).

460 S. N. ZILL

In sum, the properties of individual sensilla of the femoral chordotonal organsuggest that these receptors are specialized to fulfill discrete functions in the locust'sbehaviour. Many limb proprioceptive sense organs may also be particularly adapted tomatch the anatomical and physiological characteristics of the joints and muscles theyinnervate.

This work was supported by NSF Grant SPI-7914916 and NIH Grant5F32NS06373. I thank Graham Hoyle and Eric Schabtach for helpful comments onthe manuscript and Suzanne Royer for assisting with histological preparations.

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