positive feedback loops from proprioceptors involved in leg movements of the locust

J Comp Physiol A (1988) 163:425-440 Journal of Sensory, Comparative.--,, and

�9 Springer-Verlag 1988

Positive feedback loops from proprioceptors involved in leg movements of the locust

M. Burrows 1 and H.J. Pfliiger 2. 1 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom 2 Fakult/it f/Jr Biologie der Universit/it, D-7750 Konstanz, Federal Republic of Germany

Accepted March 24, 1988

Summary. 1. Two campaniform sensilla (CS) on the proximal tibia of a hindleg monitor strains set up when a locust prepares to kick, or when a resistance is met during locomotion. The connections made by these afferents with interneurones and leg motor neurones have been investigated and correlated with their role in locomotion.

2. When flexor and extensor tibiae muscles co- contract before a kick afferents from both campaniform sensilla spike at frequencies up to 650 Hz. They do not spike when the tibia is extended actively or passively unless it encounters a resistance. The fast extensor tibiae motor neurone (FETi) then produces a sequence of spikes in a thrusting response with feedback from the CS afferents maintaining the excitation. Destroying the two campaniform sensilla abolishes the re-excitation of FETi.

3. Mechanical stimulation of a single sensillum excites extensor and flexor tibiae motor neurones. The single afferent from either CS evokes EPSPs in the fast extensor motor neurone and in certain fast flexor tibiae motor neurones which follow each sensory spike with a central latency of 1.6 ms that suggests direct connections. The input from one receptor is powerful enough to evoke spikes in FETi. The slow extensor motor neurone does not receive a direct input, although it is excited and slow flexor tibiae motor neurones are unaffected.

4. Some nonspiking interneurones receive direct connections from both afferents in parallel with the motor neurones. One of these interneurones excites the slow and fast extensor tibiae too-

Abbreviations: CS campaniform sensilla; FETi fast extensor tibiae motor neurone; SETi slow extensor tibiae motor neurone

* Permanent address : Fachbereich Biologie, WE5, Neurobiolo- gie, Freie Universit/it, K6nigin-Luise-Strasse 28-30, D-1000 Berlin 33

tor neurones probably by disinhibition. Hyperpo- larization of this interneurone abolishes the excitatory effect of the CS on the slow extensor motor neurone and reduces the excitation of the fast. The disinhibitory pathway may involve a second nonspiking interneurone with direct inhibitory connections to both extensor motor neurones. Other nonspiking interneurones distribute the effects of the CS afferents to motor neurones of other joints.

5. The branches of the afferents from the campaniform sensilla and those of the motor neurones and interneurones in which they evoke EPSPs project to the same regions of neuropil in the metathoracic ganglion.

6. The pathways described will ensure that more force is generated by the extensor muscle when the tibia is extended against a resistance. The excitatory feedback to the extensor and flexor motor neurones will also contribute to their co-contraction when generating the force necessary for a kick.

Introduction

The stresses set up in the exoskeleton of insects during locomotion are monitored by small, strateg- ically placed campaniform sensilla. They are em- bedded in the cuticle and are excited by compres- sion perpendicular to their long axis (Spinola and Chapman 1975). On the tibia of cockroach legs, for example, are two groups of these campaniform sensilla oriented at right angles to each other (Pringle 1938). One group, stimulated by forces generated by the flexor tibiae muscle, excites extensor tibiae motor neurones, whilst the other group stimulated by activity of the extensor muscle excites flexor motor neurones (Zill and Moran 1981 ; Zill et al. 1981). They could therefore influence the

426 M. Burrows and H.J. Pflfiger : Positive feedback from proprioceptors

power output of these muscles by negative feedback, but the organization of the central reflex pathways is unknown. The tibiae of the hindlegs of locusts are used in movements where large forces may be generated. For example, they are used in walking, climbing, in defensive thrusting and in providing the propulsive force in jumping, kicking and swimming (Brown 1967; Godden 1975; Heitler and Burrows 1977a; Pfl/iger and Burrows 1978). The tibia is moved by a powerful extensor muscle and by a much weaker flexor muscle which can act antagonistically during walking, but which contract together to generate the high forces required for jumping and kicking. The con- tractions of these muscles set up stresses in the proximal tibia which could be monitored by two campaniform sensilla. These individual sensilla are distinct from the one prominent group on the tibia of a hindleg, and the groups of sensilla that occur on the tibia of the other legs of the locust and the cockroach. We have determined when these receptors are excited in voluntary movements, the reflex effects they exert, and the connections that their afferents make with neurones in the central nervous system. We show that they excite both extensor and flexor tibiae motor neurones to increase the extensor force if resistance is met during walking, and to enhance the co-contraction of both muscles when preparing for a kick.

Material and methods

Adult locusts, Schistocerca gregaria (Forskfil) were obtained from our cultures in Konstanz or Cambridge.

Physiology. A locust was mounted ventral surface uppermost with the femur of the left hindleg firmly fixed. The metathoracic ganglion was exposed and supported on a wax-coated steel platform to isolate it from movements of the thorax. Before recording began, the sheath of the ganglion was treated with a 0.1% solution (wt/vol) of Protease (Sigma Type XIV) in sa- line. Microelectrodes filled with 2 M potassium acetate and with DC resistance of 50-80 M ~ were driven across the sheath into the cell bodies of motor neurones or into the neuropilar processes of local interneurones. Motor neurones were character- ized by correlating their spikes with potentials recorded extracellularly from particular muscles and by observing the movements of a hindleg evoked when current was injected through the microelectrode. Nonspiking interneurones were character- ized according to established criteria (see Burrows and Siegler 1978) and by their effects on known motor neurones when depolarized with current.

Spikes of a campaniform sensillum (CS) were recorded extracellularly with 100 gm diameter hook electrodes from nerves in the femur. N5B1, which contains the axon of the afferent from the anterior CS, was exposed by removing a small piece of cuticle from the anterior face of the femur just proximal and ventral to the black semi-lunar process (Fig. 1). N5B2, which contains the axon of the afferent from the posterior CS, was exposed by a similar dissection from the posterior face of the femur. Each CS could be stimulated mechanically by

pressure applied with a size 0.1 minuten pin held in a microman- ipulator, and whose tip was bent to avoid piercing the soft dome of the sensillum.

The activity of muscles that move the tibia was recorded with pairs of 50 ~tm steel wires inserted through the cuticle and into the body of the muscles. Tibial movements were recorded with a photocell. All recordings were stored on FM magnetic tape for later analysis and display on a Gould ESI000 recorder, or on an XY plotter linked to a digital oscilloscope. The results are based on successful dual recordings from afferents and central neurones in 36 locusts.

Anatomy. The morphology of the two campaniform sensilla was examined in a Philips 505 scanning electron microscope either with or without fixation in 2.5 glutaraldehyde in 0.05 M phosphate buffer pH 7.4, followed by dehydration, critical point drying and coating with a 75 nm layer of gold. The pe- ripheral pattern of innervation in the proximal tibia was revealed by placing the nerves exposed in the distal femur in a pool of 3-6% hexammine cobaltic chloride surrounded by vaseline. Diffusion was allowed for 24-36 h at 6 ~ The stained afferent cell bodies of the receptors were viewed through the intact but cleared cuticle. Three successful stains of each nerve were obtained.

To reveal the central arborizations of the CS afferents 3rd or 4th instar larvae were used because the nerves linking the receptors with the metathoracic ganglion are shorter. The ar- rangement of the sensilla in these juveniles appears to be the same as in the adult. A drop of distilled water was placed over the dome of the sensillum for 5 rain before it was punc- tured with a minuten pin. The water was replaced with a 1.5% solution of hexammine cobaltic chloride, sealed with vaseline and diffusion allowed for 4 days at 6 ~ The metathoracic ganglion was then dissected, the cobalt developed (Pitman et al. 1972) and intensified with silver (Bacon and Altman 1977). The stained afferent was drawn first from a wholemount of the ganglion and then from sections cut in soft Durcupan (Fluka Chemie, FRG) at 14 gin. Two successful stains of each CS were obtained. The projections of adjacent hairs were some- times stained by leakage of the cobalt but could easily be distin- guished by their ventral position.

Motor neurones were stained after physiological character- ization by the intracellular injection of cobalt using electrodes filled with 0.1 M hexammine cobaltic chloride (Brogan and Pit- man 1981) and with the subsequent procedure as for the CS afferents. Structures within the ganglion are named according to Tyrer and Gregory (1982), and Pfliiger et al. (in press). A list of abbreviations used is given in the legend to Fig. 12.

Results

Strains in the proximal tibia of a hindleg are monitored by campaniform sensilla (CS). An anterior CS, toward the anterior face of the tibia and a posterior CS form the subject of this paper (Fig. 1A, B). The anterior CS is typically the more prominent because of its black colour and its larger size (Fig. 1B), but both are orientated so that the long axis of their oval domes is aligned approximately with the long axis o f the tibia. More distally is a group of 5-7 (n=6 locusts) CS toward the posterior face and a single CS at the same level toward the anterior face. These also monitor cuti-

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Fig. 1A-D. The morphology and innervation of two campaniform sensilla on the proximal tibia of a hindleg. A A right hindleg viewed anteriorly with a box around the region of the femoro-tibial joint that is shown in more detail in B-D. B The fem0ro-tibial joint viewed dorsally with anterior to the right. All visible trichoid and basiconic sensilla on the proximal tibia are drawn. The anterior and posterior CS are indicated by the large arrows and are shown in greater detail in the scanning electron micrographs. The average diameter of the pit of the anterior CS is 15.8 gm (n =7, range 11.5-17.1 gm) and that of the posterior CS 12.9 ~tm (n= 5, range 112.3 13.8). The anterior CS deviates from the long axis of the tibia by an average angle of 19 ~ (n=7, range 17-22 ~ and the posterior CS by 4 ~ (n= 5, range 0-9~ More distal CS are indicated by smaller arrows and the open arrow indicates the buckling region. C Innervation by N5B1 of the anterior CS, other sensilla on the anterior face of the tibia and the subgenual organ. D Innervation by N5B2 of the posterior CS and some of the other sensilla on the posterior face of the tibia. Cobalt was introduced into the cut end of the nerves in the femur and allowed to diffuse distally

cular strain but mediate other motor effects that are not described in this paper.

N5B1 innervates the anterior CS (Fig. 1C), and trichoid and basiconic sensilla on the anterior face of the tibia proximal to the subgenual organ. It

does not innervate any other campaniform sensilla or any muscles. N5B2 innervates the posterior CS (Fig. 1D), trichoid and basiconic sensilla on the posterior face of the tibia and the more distal campaniform sensilla. It also innervates tarsal muscles

428 M. Burrows and H.J. Pfliiger: Positive feedback from proprioceptors

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Fig, 2A-D. Action of a campaniform sensillum during movements of a hindleg. A, B Identification of the afferent spike from the anterior CS. Upper traces are DC recordings from a blunt microelectrode pushed into the pit of the CS, and the lower traces are AC recordings made with hook electrodes from N5B1 4 mm away in the distal femur. In B four traces are superimposed. C Action during thrusting. The hind tibia is forcibly flexed and spikes in the fast extensor tibiae (FETi) motor neurone (myogram on lower trace) resist the imposed movement. Each motor spike is followed by a high frequency burst of afferent spikes from the anterior CS recorded in N5B1 (upper trace). D Action during a kick. The anterior CS produces a high frequency sequence of spikes (first trace) during the co-contraction phase of the kick. Myograms from the flexor (second trace) and extensor (third trace) tibiae muscles are shown. Movement of the tibia was monitored with a photocell (not shown), with the arrow indicating when extension of the tibia occurred. Calibration: A 800 ms; B 3.3 ms; C 100 ms; D 50 ms

and receptors on the more distal tibia and on the tarsus.

When an individual CS is stimulated selectively by mechanical pressure, bursts of spikes in a single afferent can be recorded from the appropriate nerve in the femur. These spikes can be traced to a particular CS by placing a blunt microelectrode into the pit of the receptor. For example, spikes recorded by an electrode in the pit of the anterior CS consistently correlate with spikes recorded extracellularly from N5B1 in the femur (Fig. 2A, B). Cutting N5B1 distal to the CS eliminates input from the subgenual organ and leaves the CS afferent as the only large spike in this nerve during leg movements. Identification of the spike of the CS afferent is therefore unambiguous even when other receptors innervated by this nerve are active. The conduction velocity of the afferent, measured either between electrodes on N5BI in the distal femur and on N5 near the metathoracic ganglion, or by an electrode at the sensillum and a second on N5B1 (Fig. 2B), averages 2.3 ms (n=6 for each afferent, range 1.9-2.9). The spike will thus take approximately 17 ms to travel from the receptor to the metathoracic ganglion.

Action of the campaniform sensilla during leg movements The two CS afferents do not spike when the tibia is extended passively, or when a hindleg, which

is not load bearing, extends under the control of either the slow or fast extensor tibiae motor neurones (SETi and FETi). If, however, an active extension meets with resistance the CS afferents then spike and a sequence of spikes by FETi leading to a powerful thrusting movement of the tibia may follow (Fig. 2 C). After each FETi spike the afferent from the anterior CS, for example, produces a burst of spikes with instantaneous frequencies as high as 500 Hz and continues to spike until the strain ceases (Fig. 2 C).

The CS afferents also spike rapidly during the preparation for a kick and give a reliable indication of the preparedness of the locust (Fig. 2D). The anterior CS begins to spike when the tibia is fully flexed about the femur and when the co-contraction of flexor and extensor tibiae muscles has be- gun (Fig. 2 D). The frequency of spikes rapidly increases to 250-300 Hz during the co-contraction of the flexor and extensor muscles and just before the kick occurs groups of spikes may occur at instantaneous frequencies of 650 Hz (Fig. 2D). As soon as the tibia is extended the afferent spikes stop, though more may occur if the rapidly extend- ing tibia encounters any resistance (Fig. 2 D).

These observations of voluntary movements show that when the cuticle of the tibia is under strain, the two CS afferents spike rapidly and both flexor and extensor motor neurones may be excited. A causal link between the afferent spikes

M. Burrows and H.J. Pflfiger: Positive feedback from proprioceptors 429

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Fig. 3A-D. The two campaniform sensilla mediate re-excitation of the fast extensor tibiae motor neurone when an extension of a tibia meets resistance. An antidromic spike was evoked in FETi by electrical stimulation with a pair of electrodes implanted in the extensor tibiae muscle. A The tibia is free to move, and spikes in N5B1 from the subgenual organ have no effect on FETi. B The tibia is restrained and spikes from the anterior CS precede a wave of depolarization in FETi. C The tibia of another locust is restrained and spikes from the posterior CS in N5B2 (arrow) precede a depolarization in FETi. The large spikes are from a tarsal motor neurone. D The effect of sequentially cutting N5B1 and N5B2 on the re-excitation of FETi. Each sweep represents the average of 16 antidromic FETi spikes. When both nerves are cut the re-excitation is abolished

and the motor response is not yet established because other receptors in the vicinity could also be involved. To demonstrate a role for the CS in excit- ing the motor neurones the following sequence of ablation experiments was undertaken, each com- plete sequence being performed in one locust.

A single electrical stimulus to the extensor tibiae muscle evokes an antidromic spike in FETi, a twitch contraction of the extensor muscle and a rapid movement of the tibia, the force and amplitude of which depend on the femoro-tibial angle (Burrows and Horridge 1974). If the tibia is allowed to move freely, spikes do not occur in either of the CS afferents and the antidromic spike recorded in the soma of FETi is followed only by a small hyperpolarization (Fig. 3A). Nevertheless, the evoked movement excites many other receptors both at the joint itself and in the proximal tibia including, for example, afferents from the subgenual organ (Fig. 3 A). If the tibia is restrained, there- by simulating any increased resistance that might be met during a voluntary extension, the stimulus now evokes a burst of spikes in both the anterior and posterior CS and the antidromic spike in FETi is followed by a wave of depolarizing potentials (Fig. 3 B, C) that may be sufficient to evoke a further spike. The depolarization of FETi is not im- paired by sequentially cutting the apodeme of the femoral chordotonal organ, the strand of the strand receptor, the lateral nerve, which contains

the axons of joint receptors, or cutting N5BI distal to the CS so that subgenual afferents are removed. If N5B1 is now cut in the distal femur so that input from the anterior CS is removed the amplitude of the depolarization in FETi is approximately halved (Fig. 3D). Cutting N5B2 distal to the posterior CS has no further effect, but finally cutting it proximal to the CS abolishes the depolarization (Fig. 3 D). The same result is obtained if N5B2 is cut before N5B1. Therefore, the depolarizing re- excitation of FETi depends on afferents in the tibia proximal to the subgenual organ and innervated by both N5B1 and N5B2. Destroying the two campaniform sensilla by mechanical damage or cautery with a minuten pin apparently leaves adjacent receptors unimpaired but abolishes the re-excitation of FETi. Both the anterior and posterior CS must therefore mediate the re-excitation of FETi. The pathways by which this and other motor effects of the two CS are mediated were therefore ex- plored. The strategy was first to determine the connections made by the anterior CS and then to con- firm that the connections made by the posterior CS were similar.

Connections of the anterior CS with motor neurones

Pressing on the anterior CS evokes bursts of spikes exclusively in its afferent which are associated with depolarizations of FETi (Fig. 4). The pattern and

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Fig. 4A-E. The afferent from the anterior CS excites the fast extensor tibiae motor neurone (FETi). A Pressing on the anterior CS (arrows) evokes a burst of afferent spikes 0ower trace, recorded from NSB] in the femur) and a corresponding depolarization of the soma of FETi (upper trace). B The same experiment is repeated in a~other locust with a recording from a neuropilar branch of FETi and with a lower frequency of afferent spikes. In this particular recording the evoked depolaTization is small. C Each afferent spike is followed by an EPSP recorded in the soma of FETi. D Superimposed sweeps triggered by the afferent spike in N5BI (third trace, 30.3 mm from the metathoracic ganglion) show the afferent spike recorded in N5 (second trace, 5.3 mm from the gangtiort) and a consistent EPSP at a constant latency in FETi (first trace). E Pressing on the CS (second trace) depolarizes FETi (first trace) and evokes a spike and a twitch contraction of the extensor tibiae muscle (myogram on third trace). The tibia is unable to extend and the resulting strain excites the CS (and other receptors) leading to a re-excitation and spikes of FETi Recordings in A, B and E are each from a different locust, those in C and D from the same locust. Calibration: vertical A, C 4 m V ; B 2 m V ; D l m V ; E 1 0 m V ; horizontal A 500 ms; B, C~ E 250 ms; D 8 ms

frequency of afferent spikes is reflected closely in the waveform of the depolarization in the motor neurone (Fig. 4A, B). As recorded in the soma of FETi (Fig. 4A) the depolarization is somewhat smoothed relative to that recorded in one of its neuropilar processes (Fig. 4B), so that individual potentials may be less easy to discern. Favourable somatic recordings, however, show that a depolarizing potential follows each afferent spike (Fig. 4 C). No failures occur even when frequencies exceed 300 Hz. Recording the afferent spike at two places so that its conduction velocity ca:~ be measured, and superimposing sweeps of the oscilloscope show that the depolarizing potentials occur with a constant central latency of 1.6 ms (Fig. 4D). This central delay includes the time taken by the afferent spike to travel from its entry to the recta- thoracic ganglion to the synaptic sites and for synaptic transmission itself, and suggests that only one synapse is involved. Hyperpolarizing FETi with a steady current applied through the recording microelectrode enhances the amplitude of the depolarization evoked by the CS afferent. Moreover, pressing on the CS can often depolarize the FETi

sufficiently to evoke a spike (Fig. 4E). The force generated by an evoked motor spike will, if the tibia is unable to move, call forth further afferent spikes of the two campaniform sensilla. These in turn will depolarize the FETi and evoke a second spike, in a process that will continue, by virtue of the positive feedback, until one wave of depolarization fails to trigger a spike (Fig. 4E). The result is a powerful thrusting movement of the tibia. The evidence therefore indicates that the afferent of the anterior CS synapses directly on the FETi motor neurone and evokes a chemically mediated EPSP.

The slow extensor tibiae motor neurone (SETi) is also depolarized at the same time as FETi when the anterior CS is mechanically stimulated (Fig 5A). No EPSPs in SETi, however, can be correlated with individual CS spikes and signal averaging fails to reveal a linked EPSP, although one is clearly shown in FETi recorded simulta- neously (Fig. 5 B). Depolarizing SETi with current to bring it close to spike threshold, ensures that each stimulus to the CS evokes spikes (Fig. 5C), but hyperpolarizing it with a steady current does not enhance its response (Fig. 5D), It is concluded

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Fig. 5A-D. The afferent from tlae anterior CS connects directly with the fast but not with the slow extensor tibiae motor neurone. A Presaing an the CS (arrowa) evokes a burst <of afferent spikes (third trace) and a dep~alarilation of SETi (fiest trace) and FETi (~e~artd trace). Both ra,atoe neurones are at ttteu nnrlnal membrane l~atential. B 5tgaat averages (256 ~wee~ps each tra~e} triggexev~ by 1he a{~eren'~ sp)kes show a ] i n k ~ EPSP ~ PE"fi but ~v~ i~ gETi. C S1s ~ clepe, larized wilh ~ DO e ~ r e n t and each stiraulu~ to the CS now eve, keg a group of metc, r ~pikes. D SET] ~s hyperpolarized bul ~hc response to th~ Cg stimulation is not enhanced. The diagram m this and subsequent figures shows the connections revealed. The filled triangle indicates a direct excitatory connection, the dotted line an indirect effect. Calibratiorl: vertical A, C, D SETi 10 mV, FETi 2 mV; horizontal A, C, D 500 ms; B 18 ms

that the CS afferent 4oea ~ot synapse dtrectly on the slow exter~gor molot nemone but eavi~e~ it by more complex pathways (see below and Fag. 14).

A fast motor neurone to the flexor tibiae muscle is depolarized by spikes of the CS at the same time as the FETi (Fig. 6A). If the stimulus to the CS is aufficient to evoke a spike in FETt, then the depotarizaticm ~f the flexor i~ augmented h~ the cenlral pathway wh]ch exists between lhe~e two motor neurones (Hoyle and Burrows 197~) and by feedback from the femoral chordotonal organ (Burrows 1987a) (Fig. 6B). In most somatic recording~ (ram fast flexors, individual EPSPs are too smelt t~ correlate with anterior CS ~ptkes Nev- ertheless. ~ignal averaging shows a clea~ EPSP in two fast flexors recorded sequentially in lhe game locust that occurs with the same latency as the EPSP in I:'ETi (Fig. 6 C, D). By contrast, slow flexors show no change in membrane potential when the CS ts stimulated and no synaptic potentials

linked to its afferent spikes can be demonstrated wilh signal averaging (Pig. 6E). 11 is concluded thai the afferent from the CS synapses directly on some fast but not on the slow flexor tibiae motor neurones studied so far.

We have also recorded from the following motor neurones but have not found direct connections w noticea~.e, ea~ato~~ effects f~vra ~bc Cg: t?ne single levator tarsi, Ia~t and slow dep~eg+or tarsi, retractor unguis and common inhibitors i-3.

Connections with nonapiking interneurones

The afferent from tb.e anterior CS also s~napse~ wilh certain nonspiking mterneurones (Fig. 7). A stimulus to the CS evokes depolarizing synaptic potentials in such an interneurone and a simultaneous depolarization of FETi (Fig. 7 A), Repeating the stimuli with the irtterneurone held hyperpolarized by a steady DC current show~ that each affer-


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Fig. 6A-E, The afferent from the anterior CS excites fast but not slow flexor tibiae motor neurones. A Pressing on the CS (arrows) evokes a burst of afferent spikes (third trace) and a simultaneous depolarization of a fast flexor (first trace) and the fast extensor (second trace) tibiae motor neurones. B The afferent input depolarizes FETi sufficiently to evoke a spike, and as a result the depolarization of the fast flexor motor neurone is augmented by the central connection between these two neurones and by sensory feedback. C-E Signal averages (256 sweeps each trace) triggered by the afferent spike reveal an EPSP with the same latency in FETi and in two fast (C, D), but not in a slow flexor tibiae motor neurone (E) recorded sequentially in the same locust. Recordings in A are from one locust, those in B from a second and those in C-E from a third locust. Calibration: vertical A flexor 4 mV, FETi 2 mV; B both 10 mV; horizontal A 500 ms; B 125 ms; C-E 18 ms

ent spike is followed without failure by an enlarged EPSP (Fig. 7 B). The EPSPs occur consistently and with a constant latency that is the same as to the EPSP in the FETi motor neurone (Fig. 7C, D). The connection to this nonspiking interneurone would therefore appear to be direct and chemically mediated.

If a pulse of depolarizing current is injected into this interneurone, the slow extensor tibiae motor neurone (SETi) is slowly depolarized and spikes (Fig. 8 A). Gradually increasing the current evokes further spikes in SETi (Fig. 8 B, C). With currents greater than 2 nA, however, the number and frequency of evoked motor spikes does not increase even though their frequency is well below the maxi- mum of which this motor neurone is capable. The saturation with the low currents observed here cou- pled with a failure to demonstrate a conductance increase suggests that the excitatory effect results from disinhibition. The impaled nonspiking inter-

neurone might inhibit a second nonspiking interneurone which does not receive a direct input from the CS but which has a tonic inhibitory effect on the slow extensor motor neurone (Fig. 8 D). When the tonic inhibition produced by the second nonspiking interneurone is removed the motor neurone would be allowed to depolarize�9 A similar effect, also explicable by this pathway, is exerted on FETi, but the evoked depolarization is only about 1 mV as recorded in the soma and has never been observed to evoke a spike.

Is this disinhibitory pathway able to explain the depolarization of the SETi motor neurone when the CS is stimulated? To test this, the membrane potential of the nonspiking interneurone receiving a direct afferent input was manipulated experimentally. With the interneurone at its normal membrane potential, stimulation of the CS evokes a depolarization and a spike in SETi (Fig. 9A). If the interneurone is hyperpolarized the response

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Fig. 7A-D. The afferent from the anterior CS excites a nonspiking interneurone. A Pressing on the CS (arrows) evokes a burst of afferent spikes (third trace) followed by depolarizing potentials in the nonspiking interneurone (first trace) and a simultaneous depolarization of FETi (second trace). B The nonspiking interneurone is hyperpolarized with a DC current and the stimuli repeated. Enlarged EPSPs in the interneurone now clearly correspond with the afferent spikes, and the first stimulus now evokes activity in the flexor tibiae myogram (fourth trace). Multiple sweeps (C) or signal averages (128 sweeps) (D) triggered by the afferent spikes show EPSPs with the same latency in the interneurone and in FETi. Calibration: vertical interneurone A, B 10 mV; C 2 mV; FETi A, B 10 mV; C 0.3 mV; horizontal A, B 250 ms; C 8 ms; D 17 ms

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Fig. 8A-D. The nonspiking interneurone excited by the anterior CS in Fig. 7 excites the SETi motor neurone. A-C Current of increasing strength (fourth traces) is injected into the nonspiking interneurone (first traces) using a partially balanced bridge circuit while recordings are made intracellularly from the soma of SETi (second traces), and extracellularly from the extensor tibiae muscle (third traces). The injected current evokes a depolarization of SETi, and spikes whose number increases with the current used. D Diagram to show a pathway that could explain these observations. The triangle indicates an excitatory connection and the filled circles inhibitory connections. The recordings were made from interneurone 1. Calibration: vertical, interneurone 10 mV, SETi 4 mV, current 16 nA


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Fig. 9A-F, Manipulating the membrane potential of the nonspiking interneurone shown in Figs. 7, 8 alters the response of extensor and flexor tibiae motor neurones to stimulation of the anterior CS. A The interneurone is at its resting membrane potential (first trace). Pressing on the CS (arrows) evokes spikes in its afferent (third trace) and a depolarization of SETi (second trace). Only the one spike of SETi is recorded in a myogram from the femoral muscles (fourth trace). B The interneurone is hyperpolarized by a DC current of 2.5 nA and SETi now fails to respond to the stimuli, but spikes in flexor tibiae motor neurones are recorded in the myogram (fourth trace). C The interneurone is depolarized by a DC current of 1.5 nA and SETi now spikes when the CS is pressed but flexor spikes are abser~t. D, E The interneurone is now recorded with FETi (second traces). D With the interneurone at its normal membrane potential a large depolarization of FETi results. E With the interneurone hyperpolarized the response of FETi is reduced and flexor tibiae motor neurones now spike (myogram on fourth trace). F Diagram to show the connections that could explain these observations. The recordings are from interneurone 1. Calibration: vertical, interneurone 10 mV, SETi 4 mV, FETi 2 mV; horizontal A-C 250 ms; D, E 500 ms

of SETi is abolished (Fig. 9 B). If the interneurone is depolarized, stimulation of the CS now elicits an enhanced response with several spikes in SETi (Fig. 9 C).

Changing the membrane potential of this interneurone also alters the response of FETi and some flexor tibiae motor neurones. When the interneu-

tone is at its norma] membrane potential, stimulation of the CS evokes a large depolarization of FETi (Fig. 9 D). Hyperpolarizing the interneurone can reduce the evoked depolarization to the same stimulus by as much as 40% (Fig. 9E). Flexor tibiae motor neurones are also affected. Stimulation of the CS with the interneurone at its resting poten-


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Fig. 10A-E. The afferent from the posterior CS also synapses with fast motor neurones innervating extensor and flexor tibiae muscles. A Pressing on the posterior CS evokes afferent spikes in N5B2 and EPSPs in a fast flexor tibiae motor neurone. B These EPSPs occur with a constant latency. C A depolarization of FETi occurs whenever a burst of afferent spikes from the CS occurs in N5B2. D SETi is also depolarized but does not receive a direct input. E The EPSPs in FETi and a fast flexor occur with the same latency as revealed by signal averaging (128 sweeps). The afferent spike is recorded from N5B2 29 mm from the metathoracic ganglion and from N5 3 mm from the ganglion. Calibration: vertical A 4 mV; B 3 mV; C 2 mV; D FETi 2 mV, SETi 4 mV; horizontal A, C, D 250 ms; B 7 ms; E 18 ms

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Fig. l t A - D . Afferents from the anterior and posterior campaniform sensilla converge onto a nonspiking interneurone that excites depressor trochanteris motor neurones. A Pressing on the anterior CS evokes spikes in N5B1 and a corresponding depolarization of the interneurone. B Superimposed sweeps triggered by the afferent spikes show EPSPs at a constant latency in the interneurone. C An interneurone with the same motor effects in another locust. Pressing on the posterior CS evokes spikes in N5B2 (the sporadic large spikes are from the levator tarsi motor neurone) and EPSPs in the interneurone. D Superimposed sweeps show that the EPSPs occur with a constant latency. Calibration: vertical A, C 4 mV; B 2 .4mV; D 1.2 mV; horizontal A, C 250 ms; B , D 8 ms

tial or when depolarized does not evoke spikes in the flexor motor neurones monitored by a myogram (Fig. 9A, C and D). If the interneurone is hyperpolarized, however, stimulation of the CS often evokes flexor spikes (Figs. 7B, 9B, E). This

effect can be explained by an inhibitory connection of the nonspiking interneurone with flexor motor neurones (Fig. 9F). When the interneurone is hyperpolarized and unable to exert its inhibitory effect, the direct afferent connection with the flexor


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Fig. 12A-D. The projections of the anterior CS in the metathoracic ganglion of a 4th instar locust. A Drawing of a wholemount showing the axon stained with cobalt entering in N5 and branching in one half of the metathoracic neuromere. Dashed line indicates the approximate boundary of the neuropil, anterior is to the top and lateral nerves 1-3, 5 and 6 are numbered. B-D Transverse sections at the planes indicated in A show the branches relative to known features of the ganglion. Abbreviations, based on Tyrer and Gregory (1982) and Pflfiger et al. (1988) are: DC L IV, V dorsal commissures I, IV, V; DIT dorsal intermediate tract; D M T dorsal median tract; LAC, a, p anterior, posterior lateral association centre; L D T lateral dorsal tract; M D T median dorsal tract; M V T median ventral tract; SMC supramedian commissure; T3 neuromere of third thoracic segment; TT T-tract; VAC, a, l, v anterior, lateral, ventralmost, ventral association centre; VACdm, vm dorsal and ventral parts of the medial ventral association centre

motor neurones is able to make some of them spike. It may be the same motor neurones that receive the direct afferent input and the proposed inhibitory input from a nonspiking interneurone, but the flexor muscle is innervated by at least 9 motor neurones (Phillips 2981) and identification of all but a few is difficult.

Parallel connections of the posterior CS

All recordings indicate that the posterior CS makes the same pattern of connections as the anterior

CS within the central nervous system (Figs. 10 and 11). Each spike of this afferent is followed by a clear EPSP in a fast flexor tibiae motor neurone (Fig. 10A). The EPSP occurs consistently and with a constant latency (Fig. 10 B). Similarly, bursts of spikes in the afferent evoke depolarizations of FETi whose amplitude and duration are related to the number and frequency of afferent spikes (Fig. 10C). SETi is also depolarized and may spike as a result of the CS spikes (Fig. 10 D). Signal averaging shows that an EPSP is evoked in a fast flexor and FETi with the same latency (Fig. 10E), but


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fails to reveal a connec t ion with SETi. The central la tency to the EPSP is 1.5 ms, es t imated by recording the afferent spike at two places, and suggests tha t the connec t ion to certain fast f lexors and to FETi is direct.

The convergence of inputs f r o m the pos te r io r and an te r ior CS can also be d e m o n s t r a t e d on to nonsp ik ing interneurones . The in te rneurone in Fig. 11 excites m o t o r neurones o f the depressor t rochanter is muscle when depolar ized with a pulse of current . Each spike in the an te r ior CS con-

sistently evokes an EPSP with a shor t and cons tan t latency tha t suggests a direct connec t ion (Fig. 1 IA, B). Similarly each spike o f the pos te r io r CS evokes an EPSP that also appea r s to result f r o m a direct connec t ion (Fig. 11C, D).

The CS afferents and the motor neurones project to the same regions of neuropil

I f connect ions between the CS afferents and m o t o r or nonsp ik ing in terneurones are direct, then

438 M. Burrows and H,J. Pfliiger : Positive feedback from proprioceptors

branches of these neurones must project to the same region of neuropil. The single afferent from the anterior CS branches extensively in one half of the metathoracic ganglion, but has no branches which leave this neuromere (Fig. 12A). Its axon enters the neuropil in root 5iii of nerve 5 and imme- diately forms one set of branches that run anteriorly to end in the dorso-lateral margin of aLAC (Fig. 12A, C) and another set posteriorly (Fig. 12A, D). The main axon then continues anteriorly and medially forming a few small side branches before ending as an array of fine branches toward the midline. The most anterior of these branches end close to MVT and VIT (Fig. 12 B) whilst the more posterior ones end more ventrally near VIT or close to the dorsal edge of 1VAC (Fig. 12 C).

The branches of the FETi or a fast flexor tibiae motor neurone are far more numerous (Fig. 13 A, C). Branches of both extend near DIT and VIT at the same antero-posterior level as those of the CS afferent (Fig. 13 B, D). Moreover; branches of FETi extend into aLAC where there are also branches of the afferent (Figs. 12C, 13D). The morphology of the nonspiking interneurones has been described previously (Watkins et al. 1985) and have been shown to have branches in the same regions of neuropil as the afferents.

Discussion

The anterior and the posterior campaniform sensilla on the proximal tibia of a hindleg are excited by the strains set up when the tibia tries to extend against a resistance, and when preparing for a kick. The afferents from these two sensilla reflexly excite both extensor and flexor tibiae motor neurones to increase the force produced by both muscles. From the evidence presented, the properties and roles of the two sensilla appear equivalent: both directly excite FETi, certain flexor motor neurones and some nonspiking interneurones and indirectly excite SETi (Fig. 14). The afferent from the posterior CS, however, evokes an EPSP in certain fast flexor tibiae motor neurones that is considerably larger than that evoked by the anterior CS. This may indicate that the posterior CS provides the more effective input, or may merely reflect the different disposition of the input synapses relative to the recording site in the soma.

Behavioural role of the CS

The receptors spike only when there is strain in the cuticle of the proximal tibia. They do not spike,

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feedback Fig. 14. Diagram of the feedback pathways from the anterior CS revealed in this paper. The posterior CS makes the same set of connections. A single fexor tibiae motor neurone is shown, but the afferents are known to connect with at least three. The receptors are excited when either the flexor or extensor muscles contract and strain the cuticle in the proximal tibia. Further details of the pathways in the text

therefore, when the leg moves actively or passively throughout its entire range of velocities if the leg is bearing no load. We do not know if they would spike during walking when the leg is bearing load during its stance phase. If the CS afferents do spike then FETi should be excited, but myograms from locusts walking horizontally show no FETi spikes (Burns and Usherwood 1979). FETi still does not spike even when the locust is walking up a steep incline and load is maximal, though the frequency of SETi spikes may then exceed 200 Hz (Pfliiger, unpublished observations). It is inferred, therefore, that feedback from the CS may contribute to the excitation of SETi, but is insufficient to evoke spikes in FETi.

The efficacy of the feedback from the campaniform sensilla is readily seen if the tibia encounters a resistance. Then the afferents from the two campaniform sensilla spike rapidly and depolarize FETi so that it spikes repeatedly. The result is a powerful thrusting movement of the tibia. The positive feedback to FETi which causes it to be re- excitated after each spike is provided by the two campaniform sensilla. Destroying them, or cutting their nerves, abolishes the re-excitation. Further- more, mechanical stimulation of a single CS can depolarize FETi sufficiently to evoke a sequence of spikes (Fig. 4 E) whose pattern is similar to that

M. Burrows and H.J. Pfl/iger : Positive feedback from proprioceptors 439

in normal thrusting movements. The synaptic potentials underlying the re-excitation of FETi match those recorded at the same time in SETi (Burrows and Horridge 1974). The CS afferents, however, do not synapse directly on SETi but exert their effects through nonspiking local interneurones. This highlights the caution necessary in inferring connections from common patterns of synaptic potentials.

Heitler and Burrows (1977 b) also localized the source of re-excitation of FETi to receptors in the proximal tibia, but being unaware of the campaniform sensilla thought that the nearby subgenual organ was responsible. Receptors of this organ, in recordings made here, are seen to respond to substrate-borne vibrations and to both active and passive movements of the tibia, but not to stimuli that increase the strain in the cuticle.

The significance of the excitatory feedback to extensor and flexor motor neurones becomes ap- parent during a kick. The necessary power for these movements can only be produced by a slow isometric contraction of the extensor muscle, during which force is stored by strained cuticular ele- ments (Bennet-Clark 1975). Before a kick can occur the flexor and extensor muscles must co-contract (Godden 1975; Heitler and Burrows 1977a) and it is the inhibition of the flexor motor neurones that allows the stored force to be released explosi- vely. During the co-contraction phase the CS afferents spike at frequencies up to 650 Hz so that their input should contribute to the excitation of fast extensor and flexor motor neurones. For example, in quiescent locusts the frequencies of afferent spikes that are attained during co-contraction are sufficient to evoke spikes in FETi (compare Figs. 2 D, 4 E and 6 B). Nevertheless, destroying the two CS did not prevent the locust from kicking. More subtle effects might have been missed, however, because no measurements of the force or frequency of motor spikes have been made after such operations. These observations disclose the multi- plicity of pathways that contribute to complex movements such as kicking and show that the loss of one set of receptors does not forestall a movement.

Reflex pathways

The CS afferents appear to make direct connections with the fast extensor and with some fast flexor tibiae motor neurones and with particular nonspiking local interneurones (Fig. 14). Afferents from the femoral chordotonal organ, a propriocep- tot at the femoro-tibial joint, also synapse on too-

tor neurones, nonspiking local interneurones (Bur- rows et al., in press), spiking local interneurones (Burrows 1987a), and intersegmental interneurones (Laurent and Burrows, in press). Moreover, a receptor at the base of a movable spur on the tibia, which may be a modified CS, synapses on motor neurones (Laurent and Hustert, in press) and on spiking local interneurones (Burrows and Pflfiger 1986).

Other motor effects of the CS are mediated by connections with nonspiking interneurones, but as our survey of leg motor neurones has not been exhaustive, other direct connections may exist. One nonspiking interneurone receiving a direct input from the CS afferents excites depressor trochanteris motor neurones. Excitation of the slow extensor motor neurone can be explained by a pathway involving two nonspiking interneurones and a process of disinhibition (Fig. 14). Therefore, of the two excitatory motor neurones innervating the extensor tibiae muscle, one receives a direct afferent input whilst the other does not. No functional ex- planation can be proffered as yet for this pattern of connections, but pathways involving local interneurones should allow greater scope for modifica- tion. For example, inputs to a nonspiking interneurone could gate out the afferent effects on a motor neurone, as can be demonstrated experimentally by hyperpolarizing an interneurone (see Fig. 9 B). Furthermore, the effects of a nonspiking interneu- tone can be complex and widespread by virtue of the interactions that occur between the local interneurones (Burrows 1979, 1987b). Consider the two nonspiking interneurones whose interactions could explain the excitation of SETi by the CS afferents. Hyperpolarizing the first nonspiking interneurone while the CS receptors are stimulated not only de- creases the excitation of SETi in a graded fashion, but also increases the probability that flexor motor neurones will spike (Fig. 14). Some of the flexor motor neurones therefore receive a direct excitatory input from the CS afferents and an inhibitory input from a nonspiking interneurone (Fig. 14). Can these effects be placed in a behavioural con- text? During a co-contraction, the direct afferent input to the first nonspiking interneurone will gradually depolarize it. If at some point this input exceeds a threshold so that the inhibitory effect of the nonspiking interneurone will be expressed, the excitation of the flexors will be suppressed but the disinhibitory effects on FETi and SETi will be at their greatest. This is precisely the sequence of events that is observed at the end of the co- contraction phase (Heitler and Burrows 1977a). It is possible therefore that the feedback from the


campaniform sensilla can first lead to excitation of both sets of antagonist motor neurones, followed by inhibition of flexors. They could therefore contribute to the motor programme of a kick as one of many parallel channels.

Acknowledgements. This collaboration was made possible by a Twinning grant from the EEC Commission. M. Burrows was supported by a grant from SERC(UK) and by a grant from the Wellcome Foundation. H.J. Pflfiger was supported by grant PF128/4-1,5-1 from the DFG. We thank B.L. Watkins for the electron microscopy, and our Cambridge colleagues for their constructive comments on the manuscript.

References

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positive feedback loops from proprioceptors involved in leg movements of the locust

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