The so-called flexor strand of the femoral chordotonal organ of the locust jumping leg was found to be innervated by a single sensory neurone with its cell body in the metathoracic ganglion. The strand is therefore a new mechanoreceptor of the strand receptor class. It is sensitive to extension of the femoro-tibial joint. The innervation is rather unusual in the hind leg as well as in the fore and middle legs.
The morphology and physiology of the mechanoreceptors of insect legs have been well studied, particularly in the femora and tibiae of locusts. Thus we know that in all three locust leg pairs the femoro-tibial joint has one chordotonal organ (Slifer, 1935; Usherwood, Runion & Campbell, 1968; Burns, 1974), five multipolar sensory neurones arranged in three groups (Coillot & Boistel, 1968; Williamson & Burns, 1978) and, in the pro- and mesothoracic legs, one multipolar muscle tension receptor (Theophilidis & Burns, 1979).
In subsequent physiological investigation it has been assumed that additional sense organs are not present (Burrows & Horridge, 1974; Bässler, 1979; Field & Burrows, 1982). As will be shown here, one sense organ at least has so far been overlooked.
The present study was initiated by the discovery of a particular nerve during an investigation of coxal mechanoreceptors of the hind leg (Bräunig, 1982a). This nerve was not listed in the more detailed descriptions of locust leg innervation (Campbell, 1961; Coillot & Boistel, 1968; Heitler & Burrows, 1977); it had a rather small diameter and appeared to carry a single sensory axon towards the femoro-tibial joint. A previous study (Brâunig, 1982a) showed this axon to belong to a neurone of the strand receptor type, that is a sensory neurone, the cell body of which is located in the central nervous system (Bräunig & Hustert, 1980), but failed to reveal its peripheral structures. The present study describes the morphology of the peripheral end organs innervated by these neurones for all three leg pairs and investigates some physiological properties of metathoracic organ.
MATERIALS AND METHODS
Experiments were performed on adult locusts, Locusta migratoria migratorioides (R. & F.), obtained from a crowded laboratory culture.
Parts of the peripheral nervous system were stained with cobalt chloride (Pitman, Tweedie & Cohen, 1972) using essentially the method of Hustert (1978). Best results were obtained using isolated legs, cobalt chloride concentrations of 1·5–3% and filling times of 3–5 h at room temperature. To prevent desiccation of the extremely fine nerves under study (diameter frequently less than 5 μm) it was essential to perform all experimental steps with the preparation completely covered with locust saline (Clements & May, 1974).
Conventional extracellular recordings from sensory nerves were obtained with fine hook electrodes made out of sharpened stainless steel insect pins. Signals were preamplified, displayed on an oscilloscope screen and stored on tape for processing or transfer at 1/16 original speed to a pen recorder.
Sense organs were stimulated either by rotating the femoro-tibial joint by means of a feedback controlled d.c.-motor, or directly. In the latter case the strand of a sense organ was exposed and gripped with a pair of fine forceps forged from small insect pins mounted on a piezo-electric stimulator. Stimulating devices were carefully positioned with micromanipulators and were driven by function generators.
Connections between nerves 3B and 5B in the hind leg
There are two anastomoses between nerves 3B and 5B. One is found where the axons of the SETi- and CI-neurones join the anterior branch of the crural nerve (5B1) (Hoyle, 1955). A second anastomosis is formed by nerves 3B5c and 5B1c proximally in the femur (Bräunig, 1982a). Nerve 3B5c is one of the numerous fine coxal branches of nerve 3B, nerve 5B1c is the second femoral branch of nerve 5B1. Branch 5B1C, immediately after leaving the main nerve 5B1, fuses with nerve 3B5C and runs across the ventral surface of the most proximal anterior bundle of the extensor tibiae muscle, called the ‘fan’ by Hoyle (1978), towards the lateral (outer) femoral cuticle (Bräunig, 1982a). From here it travels distally between the cuticle and an underlying longitudinal trachea along the ventro-lateral ridge for about one-half the length of the femur (Fig. 1). Apart from the single axon of nerve 3B5C, nerve 5B1c contains axons from sensory hairs standing on that ridge and between adjacent muscle fascicoli (in the distal half of the femur this task is taken over by another branch of nerve 5B1).
To confirm these anatomical findings (Bräunig, 1982a) hook electrodes were placed on nerve 5BjC in the proximal half of the femur and on nerve 3B5 in the coxa. The recordings confirm a common unit in both nerves and show that it is sensory, since the spike is propagated towards the central nervous system (Fig. 3A).
Since the central projections of nerve 5B1c have revealed a single strand receptor neurone cell body in the metathoracic ganglion (Brâunig, 1982a) and the recording from that nerve showed a sensory unit sensitive to extension of the tibia, it was concluded that there must be a strand receptor associated with the joint between femur and tibia.
Search for the femoro-tibial strand receptor in the hind leg
Attempts were made to reveal the peripheral structure of the femoro-tibial strand receptor by staining nerve 5Bic distalwards with cobalt chloride starting from the proximal femoral region. These attempts failed so the axon was followed by physiological methods. A recording electrode was placed on nerve 5B1c proximally in the femur, the cuticle of the ventro-lateral femoral ridge carefully removed distally and fine nerve branches thus exposed were probed with a second electrode for the activity of the unit in question. In this fashion the axon could be traced as far as the femoral chordotonal organ.
Once a distal nerve branch was known to contain the axon it was stained with cobalt. These stainings showed a single axon travelling from the ventro-lateral ridge of the femur across the scoloparium of the femoral chordotonal organ into the so-called ‘flexor strand’ (Field & Burrows, 1982), a structure first described by Usherwood et al. (1968) as a ‘ligament’ connecting the chordotonal organ with the apodeme of the flexor tibiae muscle (Fig. 2A).
On this ‘ligament’ over a distance of about 500 μm the stained axon exhibits the peripheral branching pattern typical for strand receptor neurones (Bräunig, 1982a,b). A few first order branches were followed by numerous ramifications of higher order, becoming finer and finer. The region containing the terminal ramifications is located in the proximal quarter of the ‘ligament’ and appears slightly thickened.
Thus the so-called ‘flexor strand’ or ‘ligament’ of the femoral chordotonal organ is a separate mechanoreceptor of a different morphological class. It inserts on the chordotonal organ proximally and on the apodeme of the flexor tibiae muscle distally (Fig. 2A). In the following sections it will be called the femoro-tibial strand receptor, abbreviated fetiSR.
Afferent responses of the metathoracic femoro-tibial strand receptor
Extension of the tibia causes elongation of the flexor muscle apodeme which in turn causes elongation of the fetiSR receptor strand (compare Field & Burrows, 1982), and thereby stimulation of its receptor neurone (Fig. 3). Direct stimulation of fetiSR by pulling its strand (Fig. 3E) increases its discharge rate just as does extension of the tibia (Fig. 3B-D). All strand receptor neurones known so far are sensitive to elongation of their receptor strands (Bräunig & Hustert, 1980; Hustert, 1983; Bräunig, 1983).
The fetiSR neurone is active over almost the entire range of tibial positions (Fig. 3B). When the receptor has adapted after changing position, spiking is very regular (see small deviations of discharge rate in Fig. 3B,E). Only in the fully flexed position (0°, joint position defined as by Usherwood et al. 1968 and Siegler, 1981) is it completely silent in most preparations. It increases its discharge rate in proportion to joint angle almost linearly between 30° and 120° joint position and goes into saturation with further extension of the joint (Fig. 3B). At the fully extended position of the tibia (170°) fetiSR tends to give erratic responses like high frequency bursts interrupted by silent periods. This phenomenon is probably due to overstretching and is also frequently observed with coxal strand receptors (Bräunig, 1983).
The phasico-tonic response to ramp stimuli (Fig. 3C) shows that the receptor not only reacts in proportion to joint position, but also to its first derivation, i.e. velocity. This is also shown by the slight phase advance during sinusoidal stimuli (Fig. 3D), and the halt or decrease of the spike discharge rate at negative velocities during ramp or sinusoidal stimuli (Fig. 3C,D). Thus the fetiSR not only encodes joint position, but also provides the central nervous system with information about the velocity of active or passive movements of the femoro-tibial joint.
The femoro-tibial strand receptor in the fore and middle legs
The location of the receptor in the pro- and mesothoracic legs is homologous to that in the hind leg. The receptor strand lies parallel to the distalmost anterior bundle of the flexor tibiae muscle (Fig. 2B) which probably corresponds to the anterior accessory flexor of the metathoracic leg (muscle 136c in Fig. 2A). The innervation of the receptor, however, as well as its association with the femoral chordotonal organ, differs from that in the hind leg.
In the pro- and mesothoracic legs the fetiSR nerve leaves other branches of nerve 3B in the coxa (arrow in Fig. 2B) and, clinging to the cuticle, travels anterodorsally through the trochanter towards the distal scoloparium of the femoral chordotonal organ. From here it passes freely through the femoral lumen towards the receptor strand located distally in the femur. In contrast to the situation in the hind leg there is usually no anastomosis with a side branch of nerve 5B: in the pro- and mesothoracic leg the fetiSR nerve (3B5c) pursues its way through almost the entire length of the femur without joining other nerves and apparently not adhering to other structures such as tracheae or muscles (Fig. 2B).
In some preparations, nerve 3B5c fused with nerve 5B1e, a nerve supplying the muscle tension receptor and sensory hairs on the anterior distal wall of the femur (Theophilidis & Burns, 1979) with distal branches very close to the fetiSR receptor strand. This alternative route for the strand receptor axon is shown schematically in Fig. 4.
Implications from strand receptor innervation
A strand receptor is an insect mechanoreceptor consisting of a connective tissue strand that is innervated by one or more sensory neurones. The cell bodies of these neurones are not found in the periphery like those of the great majority of arthropod sensory cells, but are located in the central nervous system (Bräunig & Hustert, 1980). Furthermore the strand receptor neurones exhibit a typical morphology, and thereby can easily and unequivocally be distinguished from motor and other sensory neurones in the central projections of major peripheral nerves (Bräunig, 1982a,b). No neurones morphologically resembling strand receptor cells, in cobalt backfills, have been found in any peripheral nerves other than nerve 3B (Burrows, 1975; Bräunig, 1983).
So far, four strand receptor organs have been described for each locust leg: one in the subcoxal joint (Bräunig & Hustert, 1980; Bräunig, Hustert & Pflüger, 1981; Hustert, 1983), two in the coxo-trochanteral joint (Bräunig, 1982a,b) and the fetiSR in the present study. All of them are innervated by branches of nerve 3B.
The nerve branch innervating fetiSR is unusual in pursuing its own way through the leg. Although the fetiSR axon may use side branches of the main leg nerve (5B) for part of its way into the distal femoral region (nerve 5B1c in the hind leg and sometimes nerve 5B1e in the other legs), in the pro- and mesothorax it is normally able to find its peripheral target without making use of the leg nerve.
Nerve 3B, a major peripheral nerve in adult locusts and larvae, is established early during embryogenesis by a pioneer neurone growing out from the central nervous system into the leg bud (Bentley & Keshishian, 1982a,b; Ho & Goodman, 1982). It is believed that this pioneer neurone becomes the SETi motor neurone of the extensor tibiae muscle in the adult (C. S. Goodman, personal communication).
Another possibility is that the fetiSR neurone pioneers nerve 3B. This alternative interpretation is supported by the following: in backfills from embryonic limb buds Whitington & Seifert (1981) observed two cell bodies stained via nerve 3B, located anteriorly and ventrally in the thoracic ganglion. In the same region of the ganglion in the adult locust, the strand receptor neurones form a cluster of small cell bodies (including that of fetiSR) very close to the large cell body of the SETi motor neurone (Burrows, 1975; Brâunig, 1982b). Cobalt backfills of nerve 3B in newly hatched first instar hoppers, however, show SETi and strand receptor cell bodies of equal size (P. Bräunig, unpublished observations). That means that the motor neurone outgrows the sensory ones during postembryonic development, and that it is difficult to distinguish between fetiSR and SETi in embryos: there, both neurones have cell bodies of equal size and location, and their axons leave the ganglion via the same nerve towards the primordium of the extensor tibiae muscle (Bentley & Keshishian, 1982a,b). For all these reasons, it is possible that the neurone pioneering nerve 3B during embryogenesis of the leg nervous system is in fact fetiSR, not SETi.
Function of the femoro-tibial strand receptor
The functional role of the fetiSR is hard to assess at present. The multipolar neurones at the femoro-tibial joint (Coillot & Boistel, 1968, 1969; Williamson & Burns, 1978), the muscle tension receptor (Theophilidis & Burns, 1979) and many of the scolopidial cells of the femoral chordotonal organ (Usherwood et al. 1968; Burns, 1974) respond to extension of the tibia. Perhaps some parameter of tibial extension that they cannot encode is provided by the fetiSR.
Association of fetiSR and the femoral chordotonal organ
The direct insertion of the fetiSR strand on the femoral chordotonal organ in the hind leg as well as the connection of both receptors by a connective tissue ligament in the other two legs means that manipulation of one sense organ may influence the other and vice versa. It will be necessary to find out to what extent the techniques applied for dislocating the chordotonal organ apodeme (Bässler, 1979) impair the function of the strand receptor. The behavioural effects observed after these operations could at least partly be due to changed afferent responses of the fetiSR.
For the same reason we need comparative information for other insect species, where similar experiments have been performed (Graham & Bässler, 1981). The overall homology of mechanoreceptor numbers and location in the legs of insects (Bräunig, 1982a) make it very likely that the fetiSR is present in all insect legs, and that study should be made of its location, innervation and possible connection with other sense organs.
My thanks go to Professor Dr E. Florey, in whose laboratory this work was carried out, to Drs R. Hustert and G. Runhaar for critically reading and discussing the manuscript and to Dr D. Graham for polishing the English.