Function of Peripheral Inhibitory Axons in Insects. Department of Physiology, University of Alberta, Edmonton, Canada

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1 AMER. ZOOL., 13: (1973). Function of Peripheral Inhibitory Axons in Insects K. G. PEARSON Department of Physiology, University of Alberta, Edmonton, Canada SYNOPSIS. There are now many examples in insects of axons which elicit hyperpolarizing junctional potentials in the muscle fibers they innervate. With the muscles bathed in haemolymph, electrical stimulation of these axons causes a decrease in the magnitude of slow contractions. This property allows them to be defined as inhibitory. Although inhibitory axons have the ability to regulate the magnitude of maintained slow contractions, there is little evidence that this is their normal function. The inhibitory axons supplying at least three insect muscles function to increase the rate o relaxations following each contraction of a rhythmic sequence. Moreover, when the haemolymph potassium concentration is high, some inhibitory axons probably ensure complete relaxation between rhythmic contractions by preventing potassium contractions in tonic muscle fibers. There is no convincing evidence that inhibitory axons can facilitate muscular contractions by becoming active immediately before the excitatory input. INTRODUCTION The existence of peripheral inhibitory axons in crustaceans has been known for many years (see reviews by Wiersma, 1961; Atwood, 1968), but only quite recently has the widespread existence of these axons in insects become apparent. With the exception of the specific inhibitory axon to the claw opener muscle (Wilson and Davis, 1965), the function of both specific and common inhibitory axons in crustaceans is uncertain, although a number of possibilities have been suggested (Atwood, 1968). This lack of knowledge regarding the function of peripheral inhibitory axons in crustaceans is surprising, given the period over which their existence has been known. By comparison we now have a reasonable understanding of the normal functions of inhibitory axons innervating three insect muscles. In this article the experimental data on these and other possible functions are reviewed. OCCURRENCE OF PERIPHERAL INHIBITORY AXONS IN INSECTS There are now many examples in insects of muscles supplied by an axon which elic- I would like to thank R. B. Stein, A. S. French, and R. A. DiCaprio for their helpful discussions and criticisms during the preparation of this paper. its hyperpolarizing junctional potentials in the fibers it innervates (Table 1). Seven species of insect have so far been found to contain this type of motor axon and there is little reason for doubting their existence in many other insect species. Moreover, these axons innervate muscles involved in a variety of motor acts; namely, walking, flight, respiration, and abdominal movement. Thus, they do not appear to be specialized to function during one particular type of behavior, and again we can be reasonably confident that these axons function in a wide variety of motor acts in addition to those mentioned above. Conventionally, axons giving rise to hyperpolarizing junctional potentials have been termed "inhibitory" and indeed, in a number of studies it has been shown that axons producing hyperpolarizing junctional potentials can, when electrically stimulated, cause a marked reduction of spontaneous and evoked slow contractions (Table 2). There have, however, been reports of a lack of any mechanical inhibitory influence in the locust extensor tibiae and anterior coxal adductor muscles (Hoyle, 1955, 1966). In subsequent studies where there has been more careful control of the activity in the "inhibitory" axons and the muscle environment, strong mechanical inhibition has been consistently observed in 321

2 322 K. G. PEARSON 1 TABLE 1. Muscles in which hyperpolarizing junctional potentials have been recorded from single fibers. Animal Muscle Author (s) Locust Extensor tibiae Hoyle (1955) Usherwood and Grundfest (1965) Anterior coxal adductor Hoyle (1966) Usherwood (1968) Bemotor coxae Kutseh ajid Usherwood (1970) Grasshopper Extensor tibiae Usherwood and Grundfest (1965) Anterior coxal adductor Hoyle (1966) Abdominal intersegmental Tyrer (1971) Cockroach Bee Beetle Stick Insect Moth Extensor tibiae First spiracle Posterior coxal levator Coxal depressor StDrnal remotor Tergal remotor Anterior coxal adductor Posterior eoxal adductor Episternal promotor Subalar Ventral longitudinal Dorsoventral fibrillar Basalar fibrillar Anterior coxal adductor Sterno-pedal Usherwood and Grundfest (1965) Atwood et al. (1969) Miller (1969) Pearson and Bergman (1969) Pearson and lies (1971) Bergman and Pearson (1968) Bergman and Pearson (1968) Ikeda and Boettiger (1965a) Ikeda and Boettiger (19656) both muscles (Usherwood and Grundfest, 1965; Usherwood, 1968). At present it appears very likely that all axons giving hyperpolarizing junctional potentials have the ability to reduce the magnitude of tonic contractions produced by excitatory motor axons innervating the same muscle fibers. This conclusion, therefore, justifies our general use of the term inhibitory when referring to axons giving hyperpolarizing junctional potentials. However, this term can be misleading when considering the function of these axons in intact animals because, apart from possibly functioning to reduce the magnitude of slow contractions, they do, and could, have other functions (see below). Despite this shortcoming, the term inhibitory will be used throughout this article. Peripheral inhibitory neurons may be classified as either common or specific depending on whether they innervate a number of functionally different muscles or innervate a single muscle (or group of functionally identical muscles) respectively. Currently there is no conclusive evidence TABLE 2. Reports of mechanical inhibition in insect muscle. Animal Muscle Author (s) Locust and Grasshopper Cockroach Levator tarsus Extensor tibiae Anterior coxal adductor Coxal depressor 135b Coxal depressor 135d, e Extensor tibiae First spiracle Posterior coxal levator Coxal depressor 177d, e Eipley and Ewer (1951) "Usherwood and Grundfest (1965) 5 Hoylo (1966) "Usherwood (1968) Becht and Dresden (1956) Becht (1959) Guthrie (1967) ' Miller (1969) "Pearson and Bergman (1969) "Pearson and lies (1971) " Study in. which hyperpolarizing junctional potentials were recorded from single muscle fibers.

3 PERIPHERAL INHIBITION IN INSECTS 323 for the existence of specific inhibitory axons to any leg muscles in insects. The inhibitory axons supplying the cockroach coxal levator and depressor muscles and the locust extensor tibiae and anterior coxal adductor muscles have all been shown to be branches of common inhibitory neurons (Pearson and Bergman, 1969; Pearson and lies, 1971), while it is probable that the inhibitory innervation of the cockroach extensor tibiae muscle is also from common inhibitors (Pearson and lies, 1971). By comparison many leg muscles of crustaceans are innervated by specific inhibitory axons (Wiersma, 1961). The only specific inhibitory neuron so far identified in insects is the inhibitory axon to the first spiracle muscle of the cockroach (P. L. Miller, personal communication). More extensive investigations are required before the inhibitory axon to the flight, thoracic, and abdominal muscles can be classified as either common or specific. The inhibitory innervation of insect and crustacean muscle also differs in the maximum number of inhibitory axons innervating a single muscle. No crustacean muscle has been found to have more than dual inhibitory innervation (a specific inhibitor and one branch of a common inhibitor), whereas parts of the coxal levator and depressor muscles of the cockroach are innervated by branches of three different common inhibitory neurons (Pearson and lies, 1971). Many single fibers in these muscles receive all three inhibitory axons. The flexor tibiae muscle of the cockroach may receive an even greater number of inhibitory axons since at least five distinct increments in a hyperpolarizing response have been recorded in a single fiber as the stimulus strength to nerve 3B was increased (T. Smyth, personal communication). The purpose of multiple inhibitory innervation of single insect muscle fibers is unknown. FUNCTION OF PERIPHERAL INHIBITORY AXONS There have been only a small number of investigations specifically designed to determine the function of peripheral inhibitory axons in insects (Hoyle, 1966; Usherwood and Runion, 1970; Miller, 1969; lies and Pearson, 1971). Even so, these studies have given us information on the likely function of these axons in a few muscles, while other investigations have suggested further possibilities. In the following sections the evidence for the various functions which have been proposed is reviewed and assessed. Increased rate of relaxation Cockroach coxal depressor muscles. During walking in the cockroach, extension movements of the femur in the meso- and metathoracic legs are produced by contractions in the coxal depressor muscles. These muscles are innervated by five motor axons which have been classified as one fast, one slow, and three inhibitory (Pearson and lies, 1971). For stepping movements of less than about 10/sec, the fast axon is not active, and contractions are produced by bursts of activity in the single slow motor axon (Pearson, 1972). The discharge patterns of the inhibitory axons have not been recorded in walking animals, but in restrained preparations all three discharge at high rates during bursts of activity in excitatory axons to the antagonistic levator muscles (Pearson and lies, 1970; lies and Pearson, 1971). Since the characteristics of the burst activity of the excitatory motor axons to the coxal levator and depressor muscles are very similar to those seen in normal walking animals, it seems reasonable to assume that during walking die inhibitory axons become maximally active in the intervals between the bursts of activity in the slow axon. This type of discharge pattern suggests that the function of the inhibitory axons is to increase the rate of relaxation of contractions produced by bursts of activity in the slow axon. The need for an increased rate of relaxation is indicated by the fact that the relaxation time of almost 1 sec following an isometric slow contraction (Usherwood, 1962; lies and Pearson, 1971) is considerably longer than the time for the femur flexion movement (< 150 msec) during walking (Delcomyn, 1971).

4 324 K. G. PEARSON Electrical stimulation of the three inhibitory axons immediately after the selective activation of the slow axon causes a marked increase in the rate of relaxation (lies and Pearson, 1971). To test whether this increased rate of relaxation could be functionally important, the slow and inhibitory axons were stimulated with a pattern similar to that occurring during walking, that is, alternating bursts of activity in the excitatory and inhibitory axons (Fig. 1) (lies and Pearson, 1971). With the inhibitory input, there was almost complete relaxation of the muscle between contractions, whereas without inhibition there was a decrease in the rate of relaxation and, as a result, the muscle only partially relaxed between contractions. Thus, in a walking animal the inhibitory axons will function to produce a faster relaxation of the coxal depressor muscles and minimize the resistance to shortening of the antagonistic coxal levator muscles. A larger and more rapid femur flexion movement will then occur during the stepping phase of walking. However, this is unlikely to be the only function of the three inhibitory motoneurons innervating the coxal depressor muscles, since these same motoneurons innervate the posterior coxal levator O-5q stim D, 2 sec FIG. 1. Simulation of tension in the coxal depressor muscles of a walking cockroach showing the effect of removing the inhibitory input on rhythmic contractions. A single slow axon, D a, and an inhibitory axon, c.i., were stimulated alternately and the tension recorded in muscles 177D, E (top). The inhibitory axon was a branch of the common inhibitory neuron described by Pearson and Bergman (1969). The excitatory and inhibitory bursts lasted 500 and 200 msec respectively, and the stimulus rates within the bursts were 100 and 80 impulses/second respectively. With inhibition there were large fluctuations in tension and almost complete relaxation between contractions. With no inhibition there was a decrease in the rate of relaxation, and as a result the muscle only partially relaxed between contractions. The stimulus pattern is shown in the bottom traces. muscle and at least one of them innervate many other leg muscles (Pearson and Bergman, 1969; Pearson and lies, 1971). Locust extensor tibiae muscle. The metathoracic extensor tibiae muscle of the locust is innervated by one fast, one slow, and one inhibitory axon (Hoyle, 1965; Usherwood and Grundfest, 1965). During normal walking the fast axon remains inactive and rhythmic contractions of the muscle are produced by bursts of activity in the slow motor axon. The inhibitory neuron has been found to discharge one to four impulses after each burst in the slow axon and just before the flexion movement of the tibia (Usherwood and Runion, 1970). A simulation of the mechanical responses of the metathoracic extensor tibiae muscle of the free walking animal was obtained by stimulating the slow excitatory and inhibitory axons in a restrained animal with the patterns of activity recorded in a free walking animal. These simulations clearly demonstrated that the relaxation of the metathoracic extensor tibiae muscle is accelerated by the activity in the inhibitory neuron. "This inhibitory action presumably enables the flexor tibiae muscle to propel the tibia forward to the flexed position without having first to overcome the residue tension in the metathoracic extensor tibiae muscle" (Usherwood and Runion, 1970). Spiracle muscle in the cockroach. The closer muscle of the first spiracle in the thorax of the cockroach (Blaberus) is innervated by two excitatory motor axons and one inhibitory neuron (Miller, 1969). The excitatory and inhibitory motor axons run in different nerves which allows the inhibitory nerve to be transected without damage to the excitatory innervation. Using the opposite unoperated site as a control, Miller (1969) demonstrated that removal of the inhibitory input led to a marked slowing of the opening movement, that is, a decrease in the rate of relaxation of the closer muscle. Miller also reported that recording from the small nerve trunk containing the inhibitory neuron showed that bursts of motor impulses began just before and continued throughout

5 PERIPHERAL INHIBITION IN INSECTS 325 each valve opening. These motor impulses probably arose from the inhibitory nerve. Thus, one function of the inhibitory axon to the spiracle muscle is similar to that of the inhibitory axons to the leg depressor muscles, that is, to produce a faster relaxation after a slow contraction. Anterior coxal adductor muscle in the locust. In an isolated nerve muscle preparation of the locust, Hoyle (1966) showed that activity in the inhibitory axon to the anterior coxal adductor muscle, immediately after activity in an excitatory motor axon to this muscle, could increase the rate of relaxation. This, then, is a fourth example in insects where activity in an inhibitory axon can produce a faster relaxation. Unfortunately, in this case the discharge patterns of the excitatory and inhibitory axons have not been recorded during normal walking or during any other behavior. Thus, it is not known whether the inhibitory axon discharges at the end of the excitatory activity, which would have to occur if the inhibitory axon was to function to increase the rate of relaxation. Mechanism for increased rate of relaxation. At present we can only speculate on how the inhibitory axons increase the rate of relaxation. In the locust extensor tibiae and cockroach coxal depi'essor muscles, the long relaxation time following a slow contraction is unlikely to be due to a slow repolarization of the external membrane of any muscle fibers. The relaxation time in both of these muscles can be as high as 1 sec while the time for repolarization of the external membrane of single fibers is less than 100 msec (Usherwood and Grundfest, 1965; Pearson and lies, 1971; lies and Pearson, 1971). Thus, in these muscles it is probable that slow repolarization of the transverse tubular system is responsible for the low rate of relaxation. A hyperpolarization of the external membrane due to activity in an inhibitory axon immediately after a slow contraction would electrotonically spread into the transverse tubules, increase the rate of repolarization of the tubular membrane, and thus increase the rate of relaxation (see Dudel, 1970). Facilitation of contractions Locust extensor tibiae muscle. In the initial study on the properties of the axons innervating the metathoracic extensor tibiae muscle of the locust, Hoyle (1955) observed that activity in the axon which was subsequently demonstrated to be inhibitory (Usherwood and Grundfest, 1965), could potentiate the amplitude of the twitch contractions initiated by impulses in the single fast axon to this muscle. In contrast to this, Usherwood and Grundfest (1965) later reported that inhibitory stimulation had no effect on the twitch contractions. Moreover, Usherwood (1968) has argued that it is difficult to understand how the inhibitory axon could affect the fast responses since most of the fibers innervated by the fast axon do not receive the inhibitory axon. However, this argument is not overwhelming because it is conceivable that the inhibitory input could indirectly affect the fast response by altering the properties of the passively shortening muscle fibers not innervated by the fast axon (see below). In the absence of any other studies it can not be confidently concluded that the fast responses are always unaffected by inhibitory activity. In fact, the possibility that inhibitory input could potentiate fast responses is appealing because in the extensor tibiae muscles of the pro- and mesothoracic legs of a walking animal the inhibitory axon is maximally active just before bursts of activity in the fast axon (M. Burns, personal communication). Locust anterior coxal adductor muscle. The properties of the inhibitory axon to the anterior coxal adductor muscle of the locust have been examined by Hoyle (1966) and critically re-examined by Usherwood (1968). Hoyle isolated the muscle, bathed it in saline, and found in about one-third of the preparations that an increase in activity in the inhibitory axon caused an increase in the amplitude of the mechanical response to single impulses in an excitatory axon to the muscle. He also observed that the inhibitory axon elicited depolarizing junctional potentials in many fibers of these preparations and suggested

6 326 K. G. PEARSON that these depolarizing potentials were responsible for the tension enhancement. If this is the reason for facilitation, then facilitation probably does not occur in normal animals because depolarizing junctional potentials are not elicited by the inhibitory axon when the muscle is bathed in haemolymph (Usherwood, 1968; Pearson and Bergman, 1969). Usherwood (1968) never observed facilitatory effects in haemolymph bathed muscles, whereas earlier, Hoyle (1966) had reported that twitches preceded by inhibitory activity were larger than twitches occurring alone even when the muscles were bathed in haemolymph. Unfortunately, this effect is not convincingly displayed in any of Hoyle's published records (see Figs. 12, 13, and 14 in Hoyle, 1966). In fact the opposite effect, that is, a decrease in twitch amplitude when preceded by inhibition, can be seen in two of these records (far right of lower two sets in Fig. 14 of Hoyle, 1966). Thus, it is extremely doubtful whether the inhibitory axon to the anterior coxal adductor muscle has the ability to facilitate contractions in the intact animal. Mechanisms for facilitation. On the basis of the present experimental data, the evidence that inhibitory axons function to facilitate muscular contractions must be regarded as very weak. Nevertheless, this is definitely a possibility, and it is therefore worth considering mechanisms for producing facilitation. When the inhibitory axons innervate the fibers which are not actively contracting, these fibers will passively shorten and could produce a force to resist shortening. Inhibitory input to the passively shortening fibers during active contractions of the other fibers would ensure uncoupling of the myofilaments and hence would minimize the resistance to shortening. The overall effect of inhibition would be the development of a greater external force by the muscle. One muscle where this mechanism could operate is the locust extensor tibiae muscle. At least two mechanisms could lead to facilitation when the actively tontracting fillers are innervated by the inhibitor)' axons. The first of these is that a brief hyperpolarizing potential due to inhibitory activity just prior to the activating depolarization would increase the magnitude of the depolarization. The most obvious way this could occur is that the initial hyperpolarization decreases the inactivation of a sodium conductance system (Hodgkin and Huxley, 1952). A second method for potentiation has been suggested by Atwood and Walcott (1965). These workers recorded potentials from leg muscles in walking crabs and found in two muscles that the inhibitory axons were highly active during the initial part of the burst of activity in single excitatory axons to these muscles. This type of inhibition would prevent any development of tension but allow the full development of facilitation of the excitatory junctional potentials. Thus, at the termination of inhibitory activity there may be a more rapid increase of tension because of the facilitated amplitude of the excitatory junctional potentials. Regulation of the magnitude of maintained tonic contractions There are now a number of examples in insects where tonic contractions can be reduced by electrical stimulation of an inhibitory axon to the muscle (Table 2). However, there is little evidence to suggest that inhibitory axons normally function in this manner. The slow and inhibitory axons to the extensor tibiae muscle of the locust are often both active in standing animals, and during postural adjustments there are parallel changes in the activity of the two axons (Runion and Usherwood, 1968). Obviously, the inhibitory axon in part determines the output tension, but it is not clear that a change in the activity of the inhibitory axon alone is a mechanism used by the animal to initiate a change in posture. Occasionally, the slow axon is not active in a standing animal, which has led Usherwood (1968) to speculate that the magnitude of tonic contractions due to elevated haemolymph potassium could be controlled by changes in the activity of the inhibitory axon alone.

7 PERIPHERAL INHIBITION IN INSECTS 327 In the cockroach, many fibers in the posterior coxal levator muscle are innervated by one inhibitory axon and only one very slow excitatory axon. This very slow axon contributes little to the shortening of this muscle during walking (Pearson and lies, 1971) and is therefore probably involved in controlling the animal's posture. The close association of the very slow excitatory and the inhibitory innervation suggests that the inhibitory axon is also involved in tonic control of leg position. Of further interest in this regard is that the inhibitory axon is one branch of a common inhibitory neuron, other branches of which innervate many different muscles in different parts of the leg (Pearson and Bergman, 1969). The appealing possibility is immediately suggested that generalized changes in posture, for example, elevating the body from the ground, are brought about by changes in activity in this single common inhibitory neuron rather than by changes in many slow excitatory axons to different muscles in the leg. At present we require a greater knowledge of the discharge patterns of the inhibitory and slow motoneurons to different muscles during postural adjustments before we can assess this possibility. Reduction of the effects of fluctuations in haemolymph-potassium The potassium concentration in the haemolymph of insects can vary over a wide range depending on the diet of the animal (Hoyle, 1954; Usherwood, 1968; Pichon, 1970). In adult locusts and cockroaches this concentration has been reported to vary from about 10 to 30 niaf (Hoyle, 1954; Pichon, 1970), but such a large variation was not found in subsequent studies on locusts (Usherwood, 1968). Many tonic muscle fibers in insects are partially contracted in the absence of any excitatory input (Usherwood, 1967, 1968) so any variation in external potassium, and hence membrane potential, would be expected to alter the degree of resting contracture. Since all tonic muscle fibers in insects appear to receive inhibitory innervation (Usherwood, 1967; Cochrane et al., 1972; Miller, 1969; Pearson and Bergman, 1969; Pearson and lies, 1971; Tyrer, 1971), a possible function for the inhibitory input is to antagonize the effects of any changes in external potassium concentration. The membrane potential of tonic muscle fibers would be stabilized if increased external potassium lead to an increase in the discharge rate of the inhibitory neurons. The existence of common inhibitory neurons possessing a widespread innervation of many muscles makes this possibility even more attractive because the membrane potential in tonic fibers of many muscles would be stabilized by changes in activity in a single neuron. At present, however, the little data available do not support this notion. The activity in the common inhibitory neuron described by Pearson and Bergman (1969) was recorded in cockroaches deprived of food and water for 5 days. In animals starved in this manner, the concentration of external potassium is elevated (Pichon, 1970). Thus, if the common inhibitor functions to counteract fluctuations in haemolymphpotassium, its activity would be expected to increase. The opposite result was obtained. In none of the food and water deprived animals was the common inhibitory neuron as active as in animals given distilled water for five days after a period of starvation (Pearson, unpublished). In the latter group of animals, the external potassium concentration was probably lower than normal (Pichon, 1970). A further indication in resting animals that the common inhibitory neuron does not reduce the effects of changes in external potassium concentration is the low level of activity in this neuron in molting cockroaches (Pearson, unpublished). In these animals, the external potassium concentration is probably quite high, since in the locust the haemolymph potassium concentration is markedly elevated during molting (Hoyle, 1956). Although it appears unlikely that in resting animals the inhibitory neurons antagonize the effects of changes in haemolymphpotassium, they could function in this man-

8 328 K. G. PEARSON ner during rhythmic motor activity. Miller (1969) reported that in the absence of all input to the first spiracle closer muscle of starved cockroaches the muscle was not completely relaxed and the spiracle valve only partially opened. Incomplete relaxation of the closer muscle was probably due to an elevated external potassium concentration. Complete relaxation was observed when the excitatory and inhibitory inputs were normal, while with only the inhibitory innervation intact there were rhythmic relaxations of the closer muscle. Thus, continuous activity in the inhibitory axon to the closer muscle during the opening phase ensures complete relaxation of this muscle despite any variations in external potassium. Similarly, in the coxal depressor muscles of the cockroach, the inhibitory axons discharge throughout the contractions in the antagonistic levator muscles and may also function to ensure complete relaxation of depressor muscles during leg levation when the haemolymph potassium concentration is high. CONCLUSIONS Although peripheral inhibitory axons are widespread in insects (Table 1), we understand their normal function in only three instances. The function in all three is the same, namely to increase the rate of muscle relaxation during a sequence of rhythmic contractions. It is quite clear that this is not the function of all peripheral inhibitory axons; for example, the inhibitory axons to the posterior coxal levator muscle in the cockroach do not have the appropriate patterns of activity to function to increase the rate of relaxation. Unfortunately, none of the experimental data so far allows us to make any confident assertion about other functions. Nevertheless, these data are suggestive and the most likely functions, apart from increasing the rate of relaxation, are to ensure complete relaxation between rhythmic contractions, to regulate the magnitude of maintained tonic contractions, and to facilitate brief muscular contractions (see above). This list is obviously not exhaustive and other possible functions may become apparent with the accumulation of further data. Some inhibitory axons may have more than one function. For instance, the inhibitory axon to the first spiracle muscle of die cockroach produces a faster relaxation of the closer muscle as well as probably preventing potassium contractions in tonic muscle fibers throughout the relaxation phase (Miller, 1969). Another example is the inhibitory axon to the locust extensor tibiae muscle which produces a faster relaxation during walking (Usherwood and Runion, 1970), but may also function to i - egulate the magnitude of slow contractions in the standing animal (Runion and Usherwood, 1968) and to facilitate fast contractions (Hoyle, 1955). It is possible, of course, that some axons giving hyperpolarizing junctional potentials are functionless and may be "evolutionary relics" (Hoyle, 1966). There is no substantial reason for this belief at the moment, and clearly this can not be accepted until all functional possibilities have been thoroughly tested and rejected. There are two possible approaches for gaining further information about the function (or functions) of any particular peripheral inhibitory axon. The first of these is to selectively remove the inhibitory input and observe the subsequent behavior. In some situations it may be possible to remove the inhibitory input simply by transecting the inhibitory axon but leaving the excitatory axons intact (see Miller, 1969). Another technique, which has not yet been explored, is to inject drugs to selectively block inhibitory transmission (for example, picrotoxin or bicuculline). The second approach is to record the activity in inhibitory and excitatory axons to the muscle during various behavioral acts (and in animals in which the haemolymph ionic concentrations have been altered), then simulate the mechanical events in the same muscle of a restrained preparation (see Usherwood and Runion, 1970). An important parameter which may also have to be considered in these simulations is how the concomitant contractions and relaxations in agonist and antagonist

9 PERIPHERAL INHIBITION IN INSECTS 329 muscles in the normal animal affect the external load on the muscle under study. In addition, changes in load due to external forces may have to be considered. Apart from attempting to gain an understanding of the function of individual inhibitory axons, we must also discover the functional significance of common inhibitory neurons and the reasons for multiple inhibitory innervation of single muscles. REFERENCES Atwood, H. L Peripheral inhibition in crustacean muscle. Experientia 24: Atwood, H. L., T. Smyth, and H. S. Johnston Xeuromuscular synapses in the cockroach extensor tibiae muscle. J. Insect Physiol. 15: Atwood, H. L., and B. Walcott Recording of electrical activity and movement from legs of walking crabs. Can. J. Zool. 43: Bccht, G Studies on insect muscles. Bijdr. Dierk. 29:1-36. Bccht, C, and D. Dresden Physiology of the locomolory muscles in the cockroach. Nature (London) 177: Bergman, S. J., and K. C. Pearson Inhibition in cockroach muscle. J. Physiol. 195:22P. Cochrane, D. C, H. V. Elder, and P. N. R. Usherwood Physiology and ultrasiruclure of phasic and tonic skeletal muscle fibers in the locust, Schistocerca gregaria. J. Cell Sci. 10: Dclcomyn, F The locomotion of the cockroach, Periplaneta umericana. J. Exp. Biol. 54: Dudel, J Acceleration of relaxation by hyperpolarization of the crayfish muscle fibre membrane. Pfliigcrs Arch. 320: Giithrie, D. M The regeneration of motor axons in an insect. J. Insect Physiol. 13: Hodgkin, A. L., and A. V. Huxley A quantitative description of membrane current arid its application to conduction and excitation of nerve. ). Physiol. 117: Hoyle, G Changes in the blood potassium concentration of the African migratory locust (Locusta migratoria migratorioides R & F) during food deprivation, and the effect on neuromuscular activity. J. Exp. Biol. 31: Hoyle, G Neuromuscular mechanisms of a locust skeletal muscle. Proc. Roy. Soc. London B 143: Hoyle, C Sodium and potassium changes occurring in the haemolymph of insects at the time of moulting and their physiological consequences. Nature (London) 178: Hoyle, G Functioning of the inhibitory-conditioning axon innervating insect muscles. J. Exp. Biol. 44: Ikeda, K., and E. G. Boettiger. 1965a. Studies of the flight mechanisms of insects. II. The innervation and the electrical activity of the fibrillar muscles of the bumble bee, Bombus. J. Insect Physiol. 11: Ikeda, K., and E. G. Boettiger Studies of the flight mechanisms of insects. III. The innervation and the electrical activity of the basalar fibrillar flight muscles of the beetle, Oryctes rhinoceros. J. Insect Physiol. 11: lies, J. F., and K. G. Pearson Coxal depressor muscles of the cockroach and the role of peripheral inhibition. J. Exp. Biol. 55: Kutsch, W., and P. N. R. Usherwood Studies on the innervation and electrical activity of flight muscles in the locust, Shistocerca gregaria. J. Exp. Biol. 52: Miller, P. L Inhibitory nerves to insect spiracles. Nature (London) 221: Pearson, K. G Central programming and reflex control of walking in the cockroach. J. Exp. Biol. 56: Pearson, K. G., and S. J. Bergman Common inhibitory motoneurones in insects. J. Exp. Biol. 50: Pearson, K. G., and J. F. lies Discharge patterns of coxal levator and depressor motoneurones of the cockroach, Periplaneta americana. J. Exp. Biol. 52: Pearson, K. G., and J. F. lies Innervation patterns of the coxal depressor muscles of the cockroach, Periplaneta americana. J. Exp. Biol. 54: Pichon, Y Ionic content of haemolymph in the cockroach, Periplaneta americana. J. Exp. Biol. 53: Piek, T., and P. Mantel A study of the different types of action potentials and miniature potentials in insect muscles. Comp. Biochem. Physiol. 34: Ripley, S. H., and D. W. Ewer Peripheral inhibition in the locust. Nature (London) 167: Runion, H. I., and P. N. R. Usherwood Tarsal receptors and leg reflexes in the locust. J. Exp. Biol. 49: Tyrer, N. M Innervation of the abdominal intersegmental muscles in the grasshopper. II. Physiological analysis. J. Exp. Biol. 55: Usherwood, P. N. R The nature of "fast" and "slow" contractions in the coxal muscles of the cockroach. J. Insect Physiol. 8: Usherwood, P. N. R Insect neuromuscular mechanisms. Amer. Zool. 7: Usherwood, P. N. R A critical study of the evidence for peripheral inhibitory axons in insects. J. Exp. Biol. 49: Usherwood, P. N. R., and H. Grundfest Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28: Usherwood, P. N. R., and H. I. Runion Analysis of the mechanical responses of metathor-

10 330 K. G. PEARSON acic extensor tibiae muscles of free-walking lo- Press, New York. casts. J. Exp. Biol. 52: Wilson, D. M., and W. J. Davis Nerve im- Wiersma, C. A. G The neuromuscular sys- pulse patterns and reflex control in the motor tem, p In The physiology of cms- system of the crayfish claw. J. Exp. Biol. 43:193- taceans. T. H. Waterman [ed.], Vol. 2. Academic 210.