Transneuronal Induction of Muscle Atrophy in Grasshoppers Edmund A. Arbas'* and Mark H. Weidner2 'Division of Neurobiology, Arizona Research Laboratories and Department of Physiology, University of Arizona, Tucson, Arizona 85721; and *Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
SUMMARY Autotomy is a process in grasshoppers whereby one or both hindlimbs can be shed to escape a predator or can be abandoned if damaged. It occurs between the trochanter and the femur (second and third leg segments) and once lost, the legs never regenerate. Autotomy severs branches of the leg nerve (N5 j but damages no muscles since none span the autotomy plane. We find, however, that undamaged muscles intrinsic to the thorax of grasshoppers, Barytettixpsolus, atrophy to 115%of their normal mass after autotomy of a hindlimb. These muscles operate the coxa and trochanter (first and second leg segments j and are innervated by branches of nerves 3 and 4; nerve branches that are not damaged by autotomy.
Atrophy is localized to the side and body segment where autotomy occurs. Atrophy is evident 7-10 days after loss of a limb, is complete by about 30 days, and follows a similar time courqe whether induced in young adult, or sexually mature grasshoppers. During autotomy, leg nerve 5 is severed distal to the trochanter, the thoracic muscles lose their normal static and dynamic load, and these muscles are subsequently no longer used to support the weight of the insect during posture and locomotion. Experimental loading and unloading of the affected muscles, and cutting of nerves indicated that it is the severing of leg nerve 5 during autotomy that transneuronally induces muscle atrophy.
INTRODUCTION
control of muscle properties by neurons is influenced to a greater or lesser degree in different muscles by the genetic makeup of the muscle itself (see reviews by Edgerton, Martin, Bodine. and Roy, 1985; Sanes, 1987). Properties of mature motor neurons seem also to be regulated by activity of their target muscles (Czeh, Gallego, Kudo, and Kuno, 1978; Brown and Lunn, 1988; Henderson, 1988; Oppenheim and Haverkamp, 1988). Thus, the normal development and functioning of neuromuscular systems shows a significant dependence on trophic relations between neurons and their targets. Another manifestation of this type of dependence is evident in that neurons may atrophy or degenerate following the death of their afferents or targets (Cowan, 1970; Payne, Pearson, and Cornwell, 1984; Benshalom and White, 1988; Oppenheim and Haverkamp, 1988; Deitch and Rubel, I989a,b). Thus, the effects of localized injury may radiate long distances along synaptically connected pathways. The tendency for retrograde and antero-
Neurons depend on their interactions with each other for development and maintenance of their mature form and function (Bittner, 1977; Purves and Lichtman, 1985; Murphey, 1986). Muscle cells similarly depend on their inputs. Cross-reinnervation studies of denervated muscles have shown that muscle contractile properties ( Buller, Eccles, and Eccles, 1960), motor unit properties (Chan et al., 1982; Foehring, Sypert, and Munson, 1987a,b), and biochemical properties (Buller, Mommaerts, and Seraydarian, 1969; Salmons and Sreter, 1976) are governed, at least in part, by the identity of the neurons that innervate them. The Received December 20, 1990; accepted February 20, 1991 Journal of Neurobiology, Vol. 22, No. 5 , pp. 536-546 (199 I ) 0 I99 1 John Wiley & Sons, lnc. CCC 0022-3034/91/050536- I 1$04.00 * To whom correspondence should be addressed.
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Atrophy ofinsect Muscle
grade regression following deafferentation or damage varies markedly in different parts of the nervous system and across species (Cowan. 1970), though its occurrence is well documented in vertebrates as well as invertebrates (Guth, 1968, 1969; Cowan, 1970; Bittner, 1977; Finlayson, 1975). We report here that atrophy of certain thoracic muscles in grasshoppers is induced by hindlimb autotomy without direct damage either to the muscles or to the nerves that innervate them. We present the results of experiments designed to test the influence of unloading, altered use, and nerve damage on the thoracic muscles. The results demonstrate that damage to leg nerves that occurs during hindlimb autotomy transneuronally induces atrophy of these thoracic muscles. Some of this work has been presented previously in abstract form ( Weidner and Arbas, 1985) .
METHODS Insects used in this study were the flightless grasshoppers Barytetlix psolus (Cohn and Cantrall, 1974) raised in laboratory colonies from wild-caught individuals. All insects were fed Romaine lettuce daily, provided with bran ud lib, and kept on a lh:8 h light/dark cycle at 25-30°C. Experimental groups consisted of male and female grasshoppers within 2-3 days of the same age. Except where noted otherwise, all insects of an experimental group were induced to autotomize a hindlimb, or were surgically treated within the first 4 days after their final moult. Autotomy of a hindlimb was induced by pinching the femur with forceps until the leg was released. Nerve section or tenotomy was performed on grasshoppers anesthetized by cooling or with CO,. For nerve section, a window was opened in the cuticle of the metathoracic sternum, and leg nerve 5 (N5) was located and cut proximal to the coxa. Sham operations included all of the procedures except nerve section. For tenotomy. a window was opened in the dorsal cuticle of the femur just proximal to the femoro-tibia1joint. and the tendon of the tibia1 extensor muscle was cut. In each case, the cuticle was resealed with cyanoacrylate glue. Care was taken to avoid damage to tracheae, muscles. or nerve trunks as the case required. Muscles were reloaded following autotomy by reattaching the autotomized legs to the trochanter in their normal orientation with cyanoacrylate glue. Detached hindlimbs were reattached about 1 h after autotomy to permit the wound to seal and blood to clot. Care was taken to ensure free movement of the coxal joint. To compensate for dehydration of the autotomized limb over time, a piece of wire of known weight was glued to the femur. With practice, it was possible to estimate the size of the wire such that, 30 days after leg reattachment,
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the mass ofthe loaded. dehydrated leg was within 1070of that of the hydrated contralateral leg. For measurements of muscle mass, each grasshopper was anesthetized by cooling, then decapitated, and its abdomen and gut was removed. The thorax was bisected along the midline. and each hemithorax was pinned out in saline. Six muscles were studied in detail (Figs. 1, 2). These were the tergal and pleural promoters of the coxa (no. 1 18 and 128), tergal and pleural remoters of the coxa ( 1 19, 120, and 129) and the depressor of the trochanter ( 133 b,c) (numbering as per Snodgrass, 1929; Arbas. 1983). For some studies, the mesothoracic counterparts of these muscles were also examined. Each muscle was dissected from the hemithorax after removal of superficial fat, tracheae, and nerve branches, and was maintained in saline solution until weighing. Immediately prior to weighing, excess saline was blotted from the muscles and they were weighed together to the nearest 0.01 mg on an analytical balance. Differences between muscle masses measured under different experimental conditions were evaluated using the two-tailed or one-tailed Mann-Whitney U (MWU) test or the onetailed Wilcoxon Signed Ranks (WSR) test with a significance level of p I 0.05.
RESULTS Autotomy and Atrophy of Muscle
Autotomy is a process that has evolved in a number of vertebrate and invertebrate animals whereby nonessential parts of the body can be shed for defensive purposes, or damaged extremities may be abandoned (Edmunds, 1974; McVean, 1975; Findlay and McVean, 1977). Juvenile and adult grasshoppers readily autotomize their hindlimbs with the autotomy plane occuning between the trochanter and the femur (Fig. 2). After the leg is detached. a membrane closes the wound, minimizing bleeding. The limb does not regenerate. In the process of autotomy, metathoracic nerves 5B1 and 5B2, which innervate musculature and sensory structures of the hindlimb, are severed. No muscles are damaged, however, as none span the joint between the trochanter and femur. Thus, proximal leg muscles intrinsic to the trochanter, coxa, and thorax are left intact and fully innervated (Fig. 2; Table 1). Six muscles that operate the proximal leg joints and are retained following autotomy were examined in the flightless grasshopper, B. psolus. These were the anterior and posterior tergocoxal muscles (no. 118, 119, 120), the anterior and posterior pleurocoxal muscles ( 128, 129), and the tergotro-
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Figure 1 Photomicrograph of thoracic musculature in the right hemithorax of a normal mature adult female Barytettixpsolus ( A ) , and a female of the same age that had been induced to autotomize a right hindleg 6 weeks earlier (B). Meta- but not mesothoracic muscles are much smaller after autotomy. Anterior is to the left. rnera-metathoracic segment: tym-inner view of the tympanum. Metathoracic muscles no.: 1 19, 120, and 133 are labelled for reference; 118, 128, and 129 lie lateral to these and are hidden from view; 89 and 90 are also labelled.
Atrophj, oflnsecl Muscle
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B.
/ / Figure 2 Schematic drawing ofthe grasshopper leg, certain thoracic muscles, and their innervation by the metathordcic ganglion. ( A ) Drawing of the metathoracic leg with six thoracic muscles and their coxal insertions. Muscle 133 b,c passes through the coxa and inserts on the trochanter. The autotomy plane (between the trochanter and the femur) is indicated by a dashed line. ( R ) Drawing of the innervation of the coxal muscles and Icg by nerves from the metathoracic ganglion. Numbers refer to nerve roots. Not drawn to scale.
chanteral muscle ( i 3 3 b,c). Since B. psolus lack the large indirect flight muscles found in locusts (Arbas, 1983) these are the largest muscles on either side of the pterothorax [Fig. 1 (A)]. As might be expected, the combined mass of these six muscles does not differ significantly from one side of the thorax to the other in normal insects ( p = 0.55, metathoracic comparison; p = 0.9 1, mesothoracic comparison with a two-tailed MWU test) (Fig. 3 ) . Similarly, there was no significant difference between the mass of the six metathorack muscles and their six mesothoracic counter-
parts (i.e., muscle no. 89, 90, 91, 98, 99, and 103 b,c) (Arbas, 1983) in normal insects ( p = 0.46 comparing all sets of metathoracic muscles with all sets of mesothoracic muscles with a two-tailed MWU test) (Fig. 3 ) . Comparing these muscles 6 weeks or longer after autotomy of a single hindlimb however, showed significant atrophy ( p < 0.0 1 ) on the side and in the segment where autotomy had occurred [Figs. 1 (B), 41. The six metathoracic muscles ipsilateral to the autotomy site had a combined mass ranging from 8%-21% of normal in each of 10 insects (Fig. 4). Although the
Arbas and Wcidner
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Table 1 Innervation of Thoracic Muscles Affected by Hindlimb Autotomy
Muscle No.
118 1 I9
120 128 129 133 b, c
Nerve Providing Innervation
Function Tergal promotor coxae Tcrgal remoter coxae Second tergal remoter coxae Promoter coxae Remoter coxae Depressor trochanter
N4D, N4D2
N3A2 N4D, N3C,
Left
Right
14 13 12 11 In
2
10
9
4;
q 4 3
2 1 n Metothoracic
3
Right Normal Left
Mesothorokic
Figure 3 Comparisons of thoracic muscles on left and right sides of normal insects. Scale bars = the mean (+S.E.M.) of the combined mass of the six muscles studied (see Methods) on either side of the metathoracic and mesothoracic segments of 10 normal grasshoppers. The muscles did not differ significantly either across a given segment or between segments.
Right
9
P Y
8 7
I
6 5
4 3
2 1 0
Metothoracic
Borytettix psolus Meto. Legs Intact Right
16-1 15 14 13 12 11 10
N3A,
loss of a hindlimb changes the distribution of the load that must be supported by other muscles (a hindleg can weigh as much as 160 mg in a 1.8-g female B. psolus), no compensatory enlargement of muscles in the contralateral metathoracic segment, or on either side of the mesothoracic segment was evident (Fig. 4 ) . Thus, autotomy of a hindleg in B. psolus results in the loss of muscles that operate the jettisoned distal leg segments and atrophy of thoracic muscles that operate the two proximal leg segments that are retained. Because metathoracic muscles contralateral to the autotomy site were not different from those of normal insects, we used the contralateral muscles as internal controls for subsequent experiments.
Left
Barytettix psolus Left Meta. Leg Autotomized
Mesothoracic
Figure 4 Comparison of muscle groups in 10 grasshoppers 6 weeks after autotomy of a left metathoracic leg with those of normal insects. Control values (“Normal”) are the means of the combined values for left and right sides for each body segment shown in Figure 3. Muscles were significantly smaller ( p 5 0.05) on the side of the segment where hindlimb autotomy occurred. Neither the mass of the contralateral metathoracic muscles, nor the mass of mesothoracic muscles of either side were significantly different from normal.
Do Muscles Atrophy or Simply Stop Growing?
Although young adult grasshoppers do not increase markedly in size as they mature, they do gain weight as their internal organs and muscles grow. Muscles ofyoung adult grasshoppersare considerably smaller than those of sexually mature adults. The low muscle mass observed on the side of autotomy 6 weeks after a young adult grasshopper loses a hindlimb might be caused by atrophy, or might simply represent the cessation of growth of immature muscles. We tested for this possibility by inducing 60 young adult B. psolus each to autotomize a hindlimb, and comparing the muscles on either side of the metathoracic segment in samples of five individuals taken from this group at 3-day intervals (Fig. 5 ). The combined mass of the muscles on either side of the metathorax was near 3 mg at the time that autotomy was induced (Fig. 5 , day 0). For 3 weeks following autotomy the contralateral muscles grew steadily to a mean value of 12.3 mg. The masses of the contralateral muscles seemed to level off after this 3-week growth period with considerable variability among individuals. For example, the masses of the contralateral muscles from different individuals ranged from 6 to 18 mg on day 30. The mass of muscles ipsilateral to the autotomy
Atrophy of’insect Muscle
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ture at the time autotomy was induced, and had completed their period of muscular growth. By 7 days after autotomy, the mean mass of muscles on the treated side was 10 k 0.93 mg compared to 10.8 k 1.1 mg on the control side, a difference essentially at criterion level for significance ( p = 0.053 one-tailed WSR test). By 10 days after autotomy, the atrophy was clearly significant ( p = 0.03), and atrophy appeared to be complete in about 45 days when the mean mass of these muscles fell to about 1.5 mg. It appears that the rate and extent of atrophy induced by autotomy is similar both in young adult and in sexually mature B. psolus.
12 15 18 21 24 27 30 33
DAYS POST-AUTOTOMY
Figure 5 Time course of changes in metathoracic coxal muscle mass of young adults. Insects that had undergone their terminal moult within 5 days of each other were all induced to autotomize a hindlimb on day 0. Filled circles represent the mean combined masses of the six muscles contralateral to the site of autotomy measured at different time intervals postautotomy. Open circles represent mean combined masses of muscles ipsilateral to the autotomy site. Five grasshoppers (two males, thrcc females) were sampled for each point.
site changed little for the first 9 days, then fell to about half of the initial mass by the third week after autotomy, and continued to decline measurably until about 30 days after autotomy. The first significant difference in muscle mass on the two sides was evident by day 6 after autotomy ( p = 0.03, onetailed WSR test).
Is Atrophy Unique to Muscles of Legs that Can Be Autotomized? Since the ability to autotomize a leg is an adaptation restricted to the metathoracic segment in grasshoppers,the question arises as to whether atrophy of the thoracic muscles is also an adaptation for autotomy. To test this notion. a single mesothoracic leg was removed by cutting at the level of the coxa in each of 10 young adult grasshoppers, and bilateral sets of tergocoxal and tergotrochanteral muscles were compared in both meso- and metathoracic segments 49 days after loss of the limb. As was found for autotomy, muscle atrophy was restricted to the side of the segment where a limb was lost. Mesothoracic muscles ipsilateral to the treated limb had a mass of 5.7 0.88 mg and were significantly smaller than their contralateral counterparts
*
Borytettix psolus Right Meto. Leg Autotomized
Is Muscle Atrophy Restricted to Immature Grasshoppers?
Developing systems are in many instances more prone to undergo widespread changes in structure and function following deafferentation or injury than are mature ones (Cowan, 1970; Deitch and Rubel, I989a). Since grasshoppersundergo considerable growth over the first several weeks of adult life and only become sexually mature after this growth period, young adults can be considered to be developing systems. To determinc whether the tendency for muscles to atrophy is a property of the immature system, 45 grasshoppers were each induced to autotomize one hindlimb 29-36 days after their terminal moult, and the muscles of both sides of the metathorax were compared in samples of five individuals taken from this group at 3-day intervals (Fig. 6 ) . These insects were sexually ma-
k
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!
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13
16
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DAYS POST-AUTOTOMY
Figure 6 Time course of changes in metathoracic coxal muscle mass of sexually mature adults expressed as mean (2S.E.M.) 5% of control (contralateral) muscles. Five grasshoppers were sampled for each point. Statistically significant atrophy was first evident on day 10.
542
Arbas and TVdnnCr
which had a mass of 14.1 t- 1.2 mg ( p < 0.01, two-tailed MWU test). Metathoracic muscles did not differ on the two sides ( p = 0.52, two-tailed MWU test).
Normal
d
8 lL
90 80
x
70
0
Possible Mechanisms Inducing Atrophy Zoss of Static Load. Autotomy of a hindlimb might induce muscle atrophy by a number ofpossible mechanisms. Loss of the large hindlimb removes a load that can comprise up to about 10%of the insect’s total body mass from the coxal and trochanteral muscles. The coxa permits rotation in all directions, so the load provided by the hindlimb affects the various thoracic muscles in different ways depending on whether the insect rests or locomotes on horizontal or vertical surfaces, or upside down underneath horizontal surfaces. It might be that removal of static load leads to wasting of muscle through a lack of “exercise” with all other factors being equal to the normal condition. To test this possibility, we loaded the coxal muscles after autotomy by reattaching the autotomized hindlimb in 10 insects in its normal orientation using cyanoacrylate glue (see Methods). Grasshoppers with reattached limbs carried them with abnormal posture, but were otherwise observed to assume all different orientations (vertical, horizontal, and upside down) as they moved around in their cages. Twenty-eight to 30 days after autotomy and reattachment of the leg, the muscles ipsilateral to the autotomy site were found to have a mean mass of 1.7 & 0.2 mg while the contralateral control muscles weighed 12.7 k 0.8 mg, that is, the ipsilateral muscles atrophied to 13.1 k 1.4% of the mass of their contralateral counterparts [Fig. 7 (“Reloaded”)]. The mass of these muscles was significantly different from contralateral controls or the mass of muscles in normal insects ( p < O.Ol), but was not significantly different from atrophied muscles in insects that had not had a limb reattached ( p = 0.82. two-tailed MWU test). Thus, reloading the muscles following autotomy did not prevent muscle atrophy.
Loss of Dynamic Load or Altered Use. Although reattachment of a weighted leg placed a load on the coxal and trochanteral muscles in the experiments described above, it might be that the muscles atrophied because the leg was not used to support the weight of the insect during posture and locomotion. To test the effect of altered usage, a small window was opened in the cuticle of the femur of anesthetized grasshoppers, and the tendon of the tibial extensor muscle was transected in 10 young adult grasshoppers. Tenotorny of the tibial extensor
2 v)
2 3 5:
2
Tendon cut
120, 110 100
60
50 40 30 20 10 0
Figure 7 Metathoracic muscle mass 30 days after autotomy, transection of N5. limb reattachment. or tcnotomy of the tibial extensor muscle compared to normal \dues. Each bar represents the mass of the muscles on the treated side as the mean (+S.E.M.) ‘70of control (contralateral) muscles in 10 grasshoppers. Normal bar represents the mean mass of control insects [Fig. 2(A)] expressed as a percent of left over right side.
muscle was shown in locusts to cause rapid degenerative effects in that muscle (Jahromi and Bloom, 1979), but was employed here to change the use of the limb. When the recovered insects first flexed the tibia during normal movements, they were unable subsequently to extend it actively again, and the leg was disabled as a load-bearing appendage. Recovery from tenotomy was not observed over the course of these experiments. Note that in this set of experiments, autotomy was never induced, thoracic muscles were never unloaded, and leg nerves remained undamaged. Thirty days after tenotomy of the tibial extensor muscle, thoracic muscles showed no signs of atrophy [Fig. 7 (“Tendon cut”)]. Thus, altered use ofthe leg, or its disuse in normal posture and locomotion did not induce atrophy of the thoracic muscles.
Nerve Damage. Another mechanism by which atrophy could be induced is by the severing of leg nerve 5 during autotomy. To explore the effects of nerve section without loss of a limb, branches of N5 were severed in the proximal femur through a small window opened in the cuticle in 24 grasshoppers. Every insect in this group autotomized the treated leg within 24 h. Sham-operated grasshoppers, in which the femoral cuticle was opened, but nerve branches were not cut. showed a much lower tendency to autotomize the treated leg, for example, 10%-20% of grasshoppers whose tibial extensor muscles were tenotomiaed typically autotomized the treated leg. This suggests that total denervation of the limb was responsible for the increased tendency to autotomize the leg. As an alternative to total denervation of the leg, we transected N5
iltropky of Insect Muscle
proximal to the coxa through a small window opened in the sternal cuticle. This procedure left the leg partially innervated via axons that run in N3B, which joins N5Bl within the coxa [Fig. 2(B)] (Hoyle, 1955). The tibia on the operated side was thus still capable of slow extension due to the firing of the “slow extensor tibiae” ( SETi) motorneuron. Transection of N5 in this location also caused abnormal postures of the leg. No recovery of normal function in the treated leg was evident in any of the grasshoppers from which muscles were weighed. When muscles of the two sides of the metathorax were compared 28-35 days following N5 transection, muscles ipsilateral to the transected nerve were found to have a mean mass of 1.9 k 0.2 mg compared to 12.2 k 0.7 mg for the contralateral muscles, that is, they atrophied to about 16% of the control muscles [Fig. 7 (“Nerve cut”)]. While again significantly different from control ( p < 0.01), this value was not significantly different from the mass of muscles that atrophied either with ( p = 0.29) or without ( p = 0.31. twotailed MWU test) a load. Since autotomy was never induced and the coxal muscles were left loaded by an attached, hydrated, and partially innervated limb in these experiments, the resultsindicate that it is neither the neural signal to autotomize nor unloading that induces atrophy. Rather, it is the transection of the leg nerve.
DISCUSSION Data presented here show that hindlimb autotomy in certain grasshoppers causes the atrophy of a set of undamaged, innervated muscles within the thorax. This is similar to the situation in crabs and crayfish where thoracic muscles proximal to the autotomy plane also lose up to about half their mass following loss of a limb (Moffett, 1987). Since crustaceans regenerate their limbs, this atrophy is reversible. This is not the case in grasshoppers where autotomized limbs do not regenerate. Although the six coxal and trochanteral muscles all atrophy following hindlimb autotomy, many other muscles within the thorax show no signs of atrophy following loss of a leg. Why is this so? Cowan ( 1970) has suggested that in “neuronally closed’ systems (that is, networks with strong synaptic interactions) there might be an increased sensitivity to transneuronally mediated degenerative influences. The “neuronally closed” system here is that which controls a complex multijointed limb. Movements across different joints of the legs of locusts and grasshoppers are coordinated, in part,
543
through the action of intraleg reflexes that result from the pattern of synaptic interactions between sensory and motor circuits of the leg (Burrows and Homdge, 1974). Autotomy separates the proximal two leg segments from the distal leg segments and, by breaking leg nerve 5. may remove a significant amount of input from receptors on the leg to the ganglionic circuitry controlling intersegmental reflexes that coordinate coxal and trochanteral movements with those of distal leg segments. Reflexes that integrate movements of leg segments distal to the femur have been studied intensively (Burrows and Horridge, 1974; Burrows and Pfluger, 1986; Pfluger and Burrows, 1987; Burrows, 1987; Burrows, Laurent, and Fields, 1988; Laurent and Burrows, 1988b; Laurent and Hustert, 1988), and significant recent advances have been made in understanding the cellular basis of interleg reflexes (Laurent, 1986, 1987, 1988; Laurent and Burrows, 1988a). Reflexes that integrate movements across the proximal segments of the leg have not been studied directly, but are also very likely to exist. Experiments describcd in this report indicate that damage to N5 causes the atrophy of the thoracic muscles. What might the actual cellular changes induced by the nerve damage be? Severing of N5 does not damage the motorneurons of the coxal and tergotrochanteral muscles directly, but causes the axotomy of large numbers of sensory fibers carrying information from the leg to the central nervous system (CNS) . It is known from nerve section experiments (Zill, Underwood, Rowley, and Moran, 1980) that the severed axonal stumps of sensory neurons with somata in the periphery degenerate by about 7 days. Degeneration of severed sensory axons might induce changes through deaflerentation of central neurons and among them. possibly, motorneurons to the muscles that atrophy. Experimental deafferentation is known to cause reduction of dendritic growth in developing insect nervous system (Murphey, Mendenhall, Palka, and Edwards, 1975), as well as to induce metabolic changes including reduced protein synthesis (Meyer and Edwards, 1982). Evidence of similar effects of deafferentation is available in some vertebrate systems as well (Oswald and Rubel. 1985; Deitch and Rubel, 1989a). Breakage of N5 during hindlimb autotomy also causes axotomy of motorneurons that innervate the leg muscles located in distal leg segments. Axotomy causes normally nonelectrogenic insect motorneuron somata to become electrically excitable (Pitman, Tweedle, and Cohen, 1972; Goodman and Heitler, 1979), undergo metabolic changes in-
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Arbas and Weidner
dicative of increased protein synthesis (Cohen and Jacklet, 1965; Pitman et al., 1972), and lose known physiological inputs (Homdge and Burrows. 1974). Other retrograde transneuronal influences of motorneuron axotomy have not been explored. Another possibility is that a multiaxonal neuron with axons in both N5 and the nerves to the muscles that atrophy would signal the damage to N5 directly. Multiaxonal neurons of this type are known in metathoracic ganglion, for example, the common inhibitor (CI) which branches in metathoracic nerves 3, 4, and 5 (Hale and Burrows, 1985), and some of the dorsal unpaired median (DUM) neurons (Hoyle, 1978). Some of the thoracic muscles that atrophy are indeed innervated by neurons of this type, for example, muscle no. I20 is innervated by the CI, and other muscles are innervated by DUM neurons. But other muscles that atrophy in B. psolzis receive innervation neither from the CI nor from DUM neurons with axons in leg nerve 5. Hence, their atrophy cannot be initiated directly by a neuron with one axon damaged in N5. For example, the innervation of muscle no. I33 b,c, the tergotrochanteral depressor muscle. has been studied by the backfilling of motor nerves in both locusts and B. psolus (Villalobos and Arbas. 1988). In both, muscle no. 133 b,c was found to be innervated by three motorneurons [confirming the lack of innervation by CI in locust reported by Hale and Burrows ( 1985)] and possibly by at least one DUM neuron. The DUM neuron innervating muscle no. 133 was found, however. to have axons in N3 and N4, but not in N5 (Villalobos and Arbas, 1988). During the development of holometabolous insects, muscle atrophy and degeneration has been shown to be controlled by hormones (Weeks and Truman 1986a,b), raising the possibility that some humoral factor might act as an intermediary to mediate atrophy of the thoracic muscles of grasshoppers following damage to N5 that occurs during autotomy. The observation that muscle atrophy is restricted to the side and segment ofautotomy eliminates this possibility, since grasshoppers, like other insects. have an open circulatory system and all thoracic muscles would have been bathed by the same substance. Finally, ample evidence exists that alterations in spiking activity of motorneurons can effect significant changes in the physiological and structural properties of arthropod muscle cells during development and in maturity (reviewed by Atwood and Lnenicka, 1987; Govind, Mellon, and Quigley,
1987). It is possible that some alteration of the physiology of motor neurons of the affected muscles leads to atrophy of their targets. Whether muscle atrophy is due to an alteration in the activity level or pattern of firing in muscle and/or motorneurons or to a transneuronal trophic effect is the subject of our ongoing research. Preliminary studies in this project were camed out in the laboratory of Dr. R . L. Calabrese at the Biological Laboratories, Harvard Univ. and we thank him for providing facilities and discussions on the project. We also thank Ms. Christine Iovino and Susanna Ma for technical assistance, Charles A. Hedgecock, R.B.P. for photographic assistance, and Drs. R. B. Levine and L. P. Tolbert for comments on the manuscript. This work was supported in part by NIH grants NS 07309,BRSG SO7 RR07002, the Whitehall Foundation, and the University of Arizona Foundation.
REFERENCES
ARBAS. E. A. ( 1983). Thoracic morphology of a flightless Mexican grasshopper, Barytetlix psolus: comparison with the locust, Schistocercu gregariu. J. Morphol. 176:I41-1 53. ATWOOD,H. L,. and LNENICKA. G. A. (1987).Role of activity in determining properties of the neuromuscular system in Crustaceans. Am. Zool. 27:977-989. BEXSHALOM,G. and WHITE: E. L. (1988). Dendritic spines are susceptible to structural alterations induced by degeneration of their presynaptic afferents. Brain Res. 443:377-382. BITTNER,G.D.( 1977). Trophic interactions of Crustacean neurons. In: IdentlJied Neurons and Behavior of ilrlhropods. G. Hoyle, Ed., Plenum Press, NY. pp.
507-532. BROWN,M. C.and LUNN,E.R. ( 1988).Mechanisms of interaction between motoneurons and muscles. In: Plusticitjj o f t h e Xeuromusculur System. A. J. Buller, Ed., Ciba Foundation Symp. 138,John Wiley & Sons, Inc. pp. 78-90. BULLER,A. J., ECCLES,J. C., and ECCLES,R. M. ( 1960). Interactions between motoneurones and muscles in respect of the characteristic speeds of their responses. J. Physiol. 150:399-416. BULLER,A. J., MOMMAERTS, W. F. M., and SERAYDARIAN, K. ( 1969). Enzymic properties of myosin in fast and slow twitch muscles of the cat following crossreinnervation. J. Physiol. 20558 1-597. BURROWS,M. ( 1987).Parallel processing of proprioceptive signals by spiking local interneurons and motor neurons in the locust. J . Neurosci. 7:1064-1080. BURROWS, M.and HORRIDGE, G. A. ( 1974).The organization of inputs to motoneurons ofthe locust metatho-
Atrophy of Insect Muscle racic leg. Philos. Truns. R . Soc. Lond. [Biol.]269:4994. BURROWS,M. and PFXJGER, H. J. (1986). Processing by local interneurons of mechanosensory signals involved in a leg reflex of the locust. J. Nezmwi. 6:2764-2777. BURROWS,M., LAURENT,G. J., and FIELD.L. H. ( 1988). Proprioceptive inputs to nonspiking local interneurons contribute to local reflexes of a locust hindleg. J. Neurosci. 8:3085-3093. CHAN,M., EDGERTON,V. R., GOSLOW,G. E.. KURATA, H., RASMUSSEN, S., and SPECTOR, S. A. ( 1982). Histochemical and physiological properties of cat motor units after self- and cross-reinnervation. J. f’hvsiol. 332:343-36 1. COWAN,W. M. (1970). Anterograde and retrograde trans-neuronal degeneration in the central and peripheral nervous system. In: Contemporary Research Methods in iVcuruanutom~:.J. W. H. Nauta and S. 0.E. Ebbesson, Eds., Springer Verlag, NY, pp. 2 17251. COHEN,M. J. and JACKLET:J. W. (1965). Neurons of insects: RNA changes during injury and regeneration. Science 148: 1237- 1 239. COHN, T. and CANTRALL,I. J . (1974). Variation and speciation in the grasshoppers of the Conalcaeini (Orthoptera: Acrididae: Melanoplinae): The lowland forms of western Mexico, the genus Baryfetti-x.Sun Diego Soc. LVutl.IIistory :\fern. 6: I - 13I . CZEH, G., GALLEGO,R., KUDO, N., and KUNO, M. ( 1978). Evidence for the maintenance of motoneurone properties by muscle activity. .I. Ph.v.yiol. 281:239-252. DEITCH,J. S . and RUBEL,E. W. ( 1989a). Rapid changes in ultrastructure during deafferentation-induced dendritic atrophy. J. Corny. Neural. 281:234-258. DEITCH,J. S. and RUBEL,E. W. (1989b). Changes in neuronal cell bodies in! \ I Laminaris during deafferentation-induced dendritic atrophy. J. Comp. Neurol. 281:259-268. EDGERTON? V. R., MARTIN,R. P., BODINE,S. C.. and ROY; R. R. ( 1985). How flexible i3 the neural control of muscle properties? J. E.xp. Hiol. 115393-402. EDMUNDS,M. ( 1974). Defense in Animals, Longman Group Ltd., Essex. 357 pp. FINDLAY,I. and MCVEAN,A. ( 1977). The nervous control of limb autotomy in the hermit crab Pagurus hernhardus and the role of the cuticular stress detector. CSDI. J . Exp. Bid. 70:93-104. FINLAYSON, L. H. ( 1975). Development and degeneration. In: Insect Muscle. P. N. R. Ushenvood, Ed., Academic Press, NY, pp. 75-150. FOEHRING. R. C., SYPERT,G. W., and MUNSON,J. 13. ( 1987a). Motor-unit properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles with medial gastrocnemius nerve. I. Influence of motoneurons on muscle. J. Neurophysiol. 57: 12 101226.
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FOEHRING,R. C., SYPERT,G. W., and MUNSON,J. B. ( 1987b). Motor-unit properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles with medial gastrocnemius nerve. 11. Influence of muscle on motoneurons. J. Neurophysiol. 57: 12271245. GOODMAN,C. S. and HEITLER,W. J. (1979). Electrical properties of insect neurones with spiking and nonspiking somata: normal, axotomized, and colchicinetreated neurones. J . Exp. Biol. 83:95-12 1. GOVIND,C. K., MELLON,DEF., and QUIGLEY.M. M. (1987). Muscle and muscle fiber type transformation in clawed crustaceans. Am. 2001.27:1079-1098. GUTH,L. ( 1968). “Trophic” influences of nerve on muscle. Physiol. Rev. 48:645-687. GUTH, L. ( 1969). “Trophic” effects of vertebrate neurons. Nezrrosci. Rex Prog. 7: 1-73. M. ( 1985). Innervation patHALE,J. P. and BURROWS. terns of inhibitory motor neurones in the thorax ofthe locust. J. Exp. Bid. 117:40 1-4 13. HENDERSON,C. E. (1988). The role of muscle in the dcvelopment and differentiation of spinal motoneurons: in vitro studies. In: Plasticity ofthc Neuromuscular System. A. J. Buller, Ed., Ciba Foundation Symp. 138, John Wiley & Sons, Inc.. pp. 172-185. HORRIDC;E, G. A. and BURROWS. M. (1974). Synapses upon motoneurons of locusts during retrograde degeneration. Philos. Trans. R. Soc. Lond. [Biol.] 269:95- 108. HOYLE,G. (1955). The anatomy and innervation ofloc u t skeletal muscle. Pror. R. Soc. I,ond. [Biol.] 14328 1-292. HOYLE,G. (197X). The dorsal, unpaired, median neurons of the locust metathoracic ganglion. J. h’eurobiol. 9:43-57. JAHROMI,S. S. and BLOOM,J. W. (1979). Structural changes in locust leg muscle fibres in response to enotomy and joint immobilization. J . fnsect Physiol. 25:767-780. LAURENT,G. ( 1986). Thoracic intersegmental interneurones in the locust with mechanoreceptive inputs from a leg. J. C,’un7p. Physiol. [ A ] . 159:171-186. G. ( 1987). Parallel effects of joint receptors LAURENT, on motor neurones and intersegmental intcrneurones in the locust. J. Comp. Physiol. [ A ] .160:341-353. LA~JRENT, G. ( 1988). Local circuits underlying excitation and inhibition of intersegmental intcrneurones in the locust. .I. Cornp. Phy.siol. [ A ] 162:145-157. LAURENT,G. and BURROWS, M. ( 1988a). A population of ascending intersegmental interneurones in the locust with mechanosensory inputs from a hind leg. J. Comp. Ncurol. 275: 1- 12. G. and BURROWS, M. ( 198%). Direct excitaLAURENT, tion of nonspiking local interneurones by exteroceptors underlies tactile reflexes in the locust. J. Comp. Physiol. [ A ] .162563-572. LAURENT,G. and HUSTERT.R. ( 19x8). Motor neuronal receptive fields delimit patterns of motor activity dur-
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.4rbas und W'cidner
ing locomotion of the locust. .I. Neurosci. 8:43494366. MCVEAN,A. R. ( 1975). Autotomy: mini review. Comp. Biochem. Phy.siol. [A] 51:479-505. MEYER;M. R. and EDWARDS, J. S. (1982). Metabolic changes in dcafferented central neurons of an insect, Aclzeta domc,rticus I. Effects upon amino acid uptake and incorporation. J . Neurosci. 2: 165 1-1659. MEYER,M. R. and EDWARDS,J. S. (1982). Metabolic changes in deafferented central neurons of an insect, Achrta dornesticiis.J. Ncwnsci. 2:165 1-1659. MOFFETT, S. ( 1987). Muscles proximal to the fracture plane atrophy after limb autotomy in decapod crustaceans. J . Exp. Zool. 244:485-490. MURPHEY.R. K. ( 1986). Thc myth of the inflexible invertebrate: competition and synaptic remodelling in the development of invertebrate nervous systems. J. Nrurohiol. 17585-59 1. MURPHEY. R. K., MENDENHALL, B., PALKA,J., and GDWARDS, J . S. ( 1975). Deafferentation slows the growth of specific dendrites of identificd g a n t interncurons. .I. C u t p . h'c.iirol. 159:407-4 18. OPPENHEIM,R. W. and HAVERKAMP, L. J. (1988). Neurotrophic interactions in the development of spinal cord motoneurons. In: l~laslicityc f ~ Neziromush ~ crdar .S~:stern.A. J . Buller, Ed., Ciba Foundation Symp. 138. John Wiley & Sons. Inc.. pp. 152-165. OSWALD.S. and RUBEL.E. W. (1985). Afferent influences on brain stem auditory nuclei of the chicken: cessation of amino acid incorporation as a n antcccdent to age-dependent trans-neuronal degeneration. J. Comp. Nezrrol. 23I :385-3 9 5. PAYNE.B. R., PEARSON,H. E., and CORNWELL,P. ( 1984). Transneuronal degeneration of beta retinal ganglion cells in the cat. Proc. R. Soc. Cond. [Biol.] 222: 15-32. PITMAN, R. M., TWEEDLE,c. D., and COHEN. M. J.
( 1972). Electrical responses of insect central neurons:
augmentation by nerve section or colchicine. Science 178507-509. PFLUGER,H. J. and BURROWS,M. ( 1987). A strand receptor with a central cell body synapses upon spiking local interneurones in the locust. J. Cump. Physiol. [A] 160:295-304. PURVES.D. and LICHTMAN, J. W. ( 1985). Principles of ~VPUI-CZI Development. Sinaucr Associates Inc., Sunderland, MA, 433 pp. SALMONS. S. and SRETER,F. A. ( 1976). Significance of impulse activity in the transformation of skcletal muscle type. Nuture 263:30-34. SANES,J. R. ( 1987). Cell lineage and the origin of muscle fiber types. TEcncls Nmrosri. 10:2 19-22 1. SNODGRASS, R. E. ( 1929). The thoracic mechanism of a grasshoppcr and its antccedents. Smithsoniun Misc. Coll. 82: 1 -I 1 I . VILLALOBOS, A. R. and ARBAS.E. A. (1988). Structure of motorneurons to muscles that atrophy following hindlimb autotomy in grasshoppers. Soc. Seurosci. Abstr. V. 34:687. WEEKS,J. C. and TRUMAN, J . W. (1986a). Steroid control of neuron and muscle development during the metamorphosis of an insect. '1. Neurohiol. 17:249267. WEEKS,J. C. and TRUMAN, J. W. ( 1986b). Hormonally mediated reprogramming of muscles and motoneurones during the larval-pupal transformation of the tobacco hornworm, Munduc'a sexta. J. Exp. Biol. 125: 1-1 3. WEIDNER.M. and ARBAS.E. A. (1985). Transneuronal induction of muscle atrophy in grasshoppers. Soc. Ncurosci. Abstr. I I:479. ZILL. S. N.. UNDERWOOD, M. A., ROWLEY,J. C.. and MORAN.D. T. ( 1980). A somatotopic organization of groups of afferents in insect peripheral nerves. Brain RPS.I98 :25 3-2 69.
SUMMARY Autotomy is a process in grasshoppers whereby one or both hindlimbs can be shed to escape a predator or can be abandoned if damaged. It occurs between the trochanter and the femur (second and third leg segments) and once lost, the legs never regenerate. Autotomy severs branches of the leg nerve (N5 j but damages no muscles since none span the autotomy plane. We find, however, that undamaged muscles intrinsic to the thorax of grasshoppers, Barytettixpsolus, atrophy to 115%of their normal mass after autotomy of a hindlimb. These muscles operate the coxa and trochanter (first and second leg segments j and are innervated by branches of nerves 3 and 4; nerve branches that are not damaged by autotomy.
Atrophy is localized to the side and body segment where autotomy occurs. Atrophy is evident 7-10 days after loss of a limb, is complete by about 30 days, and follows a similar time courqe whether induced in young adult, or sexually mature grasshoppers. During autotomy, leg nerve 5 is severed distal to the trochanter, the thoracic muscles lose their normal static and dynamic load, and these muscles are subsequently no longer used to support the weight of the insect during posture and locomotion. Experimental loading and unloading of the affected muscles, and cutting of nerves indicated that it is the severing of leg nerve 5 during autotomy that transneuronally induces muscle atrophy.
INTRODUCTION
control of muscle properties by neurons is influenced to a greater or lesser degree in different muscles by the genetic makeup of the muscle itself (see reviews by Edgerton, Martin, Bodine. and Roy, 1985; Sanes, 1987). Properties of mature motor neurons seem also to be regulated by activity of their target muscles (Czeh, Gallego, Kudo, and Kuno, 1978; Brown and Lunn, 1988; Henderson, 1988; Oppenheim and Haverkamp, 1988). Thus, the normal development and functioning of neuromuscular systems shows a significant dependence on trophic relations between neurons and their targets. Another manifestation of this type of dependence is evident in that neurons may atrophy or degenerate following the death of their afferents or targets (Cowan, 1970; Payne, Pearson, and Cornwell, 1984; Benshalom and White, 1988; Oppenheim and Haverkamp, 1988; Deitch and Rubel, I989a,b). Thus, the effects of localized injury may radiate long distances along synaptically connected pathways. The tendency for retrograde and antero-
Neurons depend on their interactions with each other for development and maintenance of their mature form and function (Bittner, 1977; Purves and Lichtman, 1985; Murphey, 1986). Muscle cells similarly depend on their inputs. Cross-reinnervation studies of denervated muscles have shown that muscle contractile properties ( Buller, Eccles, and Eccles, 1960), motor unit properties (Chan et al., 1982; Foehring, Sypert, and Munson, 1987a,b), and biochemical properties (Buller, Mommaerts, and Seraydarian, 1969; Salmons and Sreter, 1976) are governed, at least in part, by the identity of the neurons that innervate them. The Received December 20, 1990; accepted February 20, 1991 Journal of Neurobiology, Vol. 22, No. 5 , pp. 536-546 (199 I ) 0 I99 1 John Wiley & Sons, lnc. CCC 0022-3034/91/050536- I 1$04.00 * To whom correspondence should be addressed.
536
Atrophy ofinsect Muscle
grade regression following deafferentation or damage varies markedly in different parts of the nervous system and across species (Cowan. 1970), though its occurrence is well documented in vertebrates as well as invertebrates (Guth, 1968, 1969; Cowan, 1970; Bittner, 1977; Finlayson, 1975). We report here that atrophy of certain thoracic muscles in grasshoppers is induced by hindlimb autotomy without direct damage either to the muscles or to the nerves that innervate them. We present the results of experiments designed to test the influence of unloading, altered use, and nerve damage on the thoracic muscles. The results demonstrate that damage to leg nerves that occurs during hindlimb autotomy transneuronally induces atrophy of these thoracic muscles. Some of this work has been presented previously in abstract form ( Weidner and Arbas, 1985) .
METHODS Insects used in this study were the flightless grasshoppers Barytetlix psolus (Cohn and Cantrall, 1974) raised in laboratory colonies from wild-caught individuals. All insects were fed Romaine lettuce daily, provided with bran ud lib, and kept on a lh:8 h light/dark cycle at 25-30°C. Experimental groups consisted of male and female grasshoppers within 2-3 days of the same age. Except where noted otherwise, all insects of an experimental group were induced to autotomize a hindlimb, or were surgically treated within the first 4 days after their final moult. Autotomy of a hindlimb was induced by pinching the femur with forceps until the leg was released. Nerve section or tenotomy was performed on grasshoppers anesthetized by cooling or with CO,. For nerve section, a window was opened in the cuticle of the metathoracic sternum, and leg nerve 5 (N5) was located and cut proximal to the coxa. Sham operations included all of the procedures except nerve section. For tenotomy. a window was opened in the dorsal cuticle of the femur just proximal to the femoro-tibia1joint. and the tendon of the tibia1 extensor muscle was cut. In each case, the cuticle was resealed with cyanoacrylate glue. Care was taken to avoid damage to tracheae, muscles. or nerve trunks as the case required. Muscles were reloaded following autotomy by reattaching the autotomized legs to the trochanter in their normal orientation with cyanoacrylate glue. Detached hindlimbs were reattached about 1 h after autotomy to permit the wound to seal and blood to clot. Care was taken to ensure free movement of the coxal joint. To compensate for dehydration of the autotomized limb over time, a piece of wire of known weight was glued to the femur. With practice, it was possible to estimate the size of the wire such that, 30 days after leg reattachment,
537
the mass ofthe loaded. dehydrated leg was within 1070of that of the hydrated contralateral leg. For measurements of muscle mass, each grasshopper was anesthetized by cooling, then decapitated, and its abdomen and gut was removed. The thorax was bisected along the midline. and each hemithorax was pinned out in saline. Six muscles were studied in detail (Figs. 1, 2). These were the tergal and pleural promoters of the coxa (no. 1 18 and 128), tergal and pleural remoters of the coxa ( 1 19, 120, and 129) and the depressor of the trochanter ( 133 b,c) (numbering as per Snodgrass, 1929; Arbas. 1983). For some studies, the mesothoracic counterparts of these muscles were also examined. Each muscle was dissected from the hemithorax after removal of superficial fat, tracheae, and nerve branches, and was maintained in saline solution until weighing. Immediately prior to weighing, excess saline was blotted from the muscles and they were weighed together to the nearest 0.01 mg on an analytical balance. Differences between muscle masses measured under different experimental conditions were evaluated using the two-tailed or one-tailed Mann-Whitney U (MWU) test or the onetailed Wilcoxon Signed Ranks (WSR) test with a significance level of p I 0.05.
RESULTS Autotomy and Atrophy of Muscle
Autotomy is a process that has evolved in a number of vertebrate and invertebrate animals whereby nonessential parts of the body can be shed for defensive purposes, or damaged extremities may be abandoned (Edmunds, 1974; McVean, 1975; Findlay and McVean, 1977). Juvenile and adult grasshoppers readily autotomize their hindlimbs with the autotomy plane occuning between the trochanter and the femur (Fig. 2). After the leg is detached. a membrane closes the wound, minimizing bleeding. The limb does not regenerate. In the process of autotomy, metathoracic nerves 5B1 and 5B2, which innervate musculature and sensory structures of the hindlimb, are severed. No muscles are damaged, however, as none span the joint between the trochanter and femur. Thus, proximal leg muscles intrinsic to the trochanter, coxa, and thorax are left intact and fully innervated (Fig. 2; Table 1). Six muscles that operate the proximal leg joints and are retained following autotomy were examined in the flightless grasshopper, B. psolus. These were the anterior and posterior tergocoxal muscles (no. 118, 119, 120), the anterior and posterior pleurocoxal muscles ( 128, 129), and the tergotro-
538
ilrbas and Weidner
Figure 1 Photomicrograph of thoracic musculature in the right hemithorax of a normal mature adult female Barytettixpsolus ( A ) , and a female of the same age that had been induced to autotomize a right hindleg 6 weeks earlier (B). Meta- but not mesothoracic muscles are much smaller after autotomy. Anterior is to the left. rnera-metathoracic segment: tym-inner view of the tympanum. Metathoracic muscles no.: 1 19, 120, and 133 are labelled for reference; 118, 128, and 129 lie lateral to these and are hidden from view; 89 and 90 are also labelled.
Atrophj, oflnsecl Muscle
539
B.
/ / Figure 2 Schematic drawing ofthe grasshopper leg, certain thoracic muscles, and their innervation by the metathordcic ganglion. ( A ) Drawing of the metathoracic leg with six thoracic muscles and their coxal insertions. Muscle 133 b,c passes through the coxa and inserts on the trochanter. The autotomy plane (between the trochanter and the femur) is indicated by a dashed line. ( R ) Drawing of the innervation of the coxal muscles and Icg by nerves from the metathoracic ganglion. Numbers refer to nerve roots. Not drawn to scale.
chanteral muscle ( i 3 3 b,c). Since B. psolus lack the large indirect flight muscles found in locusts (Arbas, 1983) these are the largest muscles on either side of the pterothorax [Fig. 1 (A)]. As might be expected, the combined mass of these six muscles does not differ significantly from one side of the thorax to the other in normal insects ( p = 0.55, metathoracic comparison; p = 0.9 1, mesothoracic comparison with a two-tailed MWU test) (Fig. 3 ) . Similarly, there was no significant difference between the mass of the six metathorack muscles and their six mesothoracic counter-
parts (i.e., muscle no. 89, 90, 91, 98, 99, and 103 b,c) (Arbas, 1983) in normal insects ( p = 0.46 comparing all sets of metathoracic muscles with all sets of mesothoracic muscles with a two-tailed MWU test) (Fig. 3 ) . Comparing these muscles 6 weeks or longer after autotomy of a single hindlimb however, showed significant atrophy ( p < 0.0 1 ) on the side and in the segment where autotomy had occurred [Figs. 1 (B), 41. The six metathoracic muscles ipsilateral to the autotomy site had a combined mass ranging from 8%-21% of normal in each of 10 insects (Fig. 4). Although the
Arbas and Wcidner
540
Table 1 Innervation of Thoracic Muscles Affected by Hindlimb Autotomy
Muscle No.
118 1 I9
120 128 129 133 b, c
Nerve Providing Innervation
Function Tergal promotor coxae Tcrgal remoter coxae Second tergal remoter coxae Promoter coxae Remoter coxae Depressor trochanter
N4D, N4D2
N3A2 N4D, N3C,
Left
Right
14 13 12 11 In
2
10
9
4;
q 4 3
2 1 n Metothoracic
3
Right Normal Left
Mesothorokic
Figure 3 Comparisons of thoracic muscles on left and right sides of normal insects. Scale bars = the mean (+S.E.M.) of the combined mass of the six muscles studied (see Methods) on either side of the metathoracic and mesothoracic segments of 10 normal grasshoppers. The muscles did not differ significantly either across a given segment or between segments.
Right
9
P Y
8 7
I
6 5
4 3
2 1 0
Metothoracic
Borytettix psolus Meto. Legs Intact Right
16-1 15 14 13 12 11 10
N3A,
loss of a hindlimb changes the distribution of the load that must be supported by other muscles (a hindleg can weigh as much as 160 mg in a 1.8-g female B. psolus), no compensatory enlargement of muscles in the contralateral metathoracic segment, or on either side of the mesothoracic segment was evident (Fig. 4 ) . Thus, autotomy of a hindleg in B. psolus results in the loss of muscles that operate the jettisoned distal leg segments and atrophy of thoracic muscles that operate the two proximal leg segments that are retained. Because metathoracic muscles contralateral to the autotomy site were not different from those of normal insects, we used the contralateral muscles as internal controls for subsequent experiments.
Left
Barytettix psolus Left Meta. Leg Autotomized
Mesothoracic
Figure 4 Comparison of muscle groups in 10 grasshoppers 6 weeks after autotomy of a left metathoracic leg with those of normal insects. Control values (“Normal”) are the means of the combined values for left and right sides for each body segment shown in Figure 3. Muscles were significantly smaller ( p 5 0.05) on the side of the segment where hindlimb autotomy occurred. Neither the mass of the contralateral metathoracic muscles, nor the mass of mesothoracic muscles of either side were significantly different from normal.
Do Muscles Atrophy or Simply Stop Growing?
Although young adult grasshoppers do not increase markedly in size as they mature, they do gain weight as their internal organs and muscles grow. Muscles ofyoung adult grasshoppersare considerably smaller than those of sexually mature adults. The low muscle mass observed on the side of autotomy 6 weeks after a young adult grasshopper loses a hindlimb might be caused by atrophy, or might simply represent the cessation of growth of immature muscles. We tested for this possibility by inducing 60 young adult B. psolus each to autotomize a hindlimb, and comparing the muscles on either side of the metathoracic segment in samples of five individuals taken from this group at 3-day intervals (Fig. 5 ). The combined mass of the muscles on either side of the metathorax was near 3 mg at the time that autotomy was induced (Fig. 5 , day 0). For 3 weeks following autotomy the contralateral muscles grew steadily to a mean value of 12.3 mg. The masses of the contralateral muscles seemed to level off after this 3-week growth period with considerable variability among individuals. For example, the masses of the contralateral muscles from different individuals ranged from 6 to 18 mg on day 30. The mass of muscles ipsilateral to the autotomy
Atrophy of’insect Muscle
Borytettix psolus Right Meto. Leg Autotomized
-
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Left
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~i
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6
9
541
ture at the time autotomy was induced, and had completed their period of muscular growth. By 7 days after autotomy, the mean mass of muscles on the treated side was 10 k 0.93 mg compared to 10.8 k 1.1 mg on the control side, a difference essentially at criterion level for significance ( p = 0.053 one-tailed WSR test). By 10 days after autotomy, the atrophy was clearly significant ( p = 0.03), and atrophy appeared to be complete in about 45 days when the mean mass of these muscles fell to about 1.5 mg. It appears that the rate and extent of atrophy induced by autotomy is similar both in young adult and in sexually mature B. psolus.
12 15 18 21 24 27 30 33
DAYS POST-AUTOTOMY
Figure 5 Time course of changes in metathoracic coxal muscle mass of young adults. Insects that had undergone their terminal moult within 5 days of each other were all induced to autotomize a hindlimb on day 0. Filled circles represent the mean combined masses of the six muscles contralateral to the site of autotomy measured at different time intervals postautotomy. Open circles represent mean combined masses of muscles ipsilateral to the autotomy site. Five grasshoppers (two males, thrcc females) were sampled for each point.
site changed little for the first 9 days, then fell to about half of the initial mass by the third week after autotomy, and continued to decline measurably until about 30 days after autotomy. The first significant difference in muscle mass on the two sides was evident by day 6 after autotomy ( p = 0.03, onetailed WSR test).
Is Atrophy Unique to Muscles of Legs that Can Be Autotomized? Since the ability to autotomize a leg is an adaptation restricted to the metathoracic segment in grasshoppers,the question arises as to whether atrophy of the thoracic muscles is also an adaptation for autotomy. To test this notion. a single mesothoracic leg was removed by cutting at the level of the coxa in each of 10 young adult grasshoppers, and bilateral sets of tergocoxal and tergotrochanteral muscles were compared in both meso- and metathoracic segments 49 days after loss of the limb. As was found for autotomy, muscle atrophy was restricted to the side of the segment where a limb was lost. Mesothoracic muscles ipsilateral to the treated limb had a mass of 5.7 0.88 mg and were significantly smaller than their contralateral counterparts
*
Borytettix psolus Right Meto. Leg Autotomized
Is Muscle Atrophy Restricted to Immature Grasshoppers?
Developing systems are in many instances more prone to undergo widespread changes in structure and function following deafferentation or injury than are mature ones (Cowan, 1970; Deitch and Rubel, I989a). Since grasshoppersundergo considerable growth over the first several weeks of adult life and only become sexually mature after this growth period, young adults can be considered to be developing systems. To determinc whether the tendency for muscles to atrophy is a property of the immature system, 45 grasshoppers were each induced to autotomize one hindlimb 29-36 days after their terminal moult, and the muscles of both sides of the metathorax were compared in samples of five individuals taken from this group at 3-day intervals (Fig. 6 ) . These insects were sexually ma-
k
*
\
70.-
60-
\ T
50-
y
I
g
2
*
403020--
‘0 L\T
t
10.-
0
4
; 4
I
I
!
I
I
I
;
7
10
13
16
19
22
25
:
:
I
46
DAYS POST-AUTOTOMY
Figure 6 Time course of changes in metathoracic coxal muscle mass of sexually mature adults expressed as mean (2S.E.M.) 5% of control (contralateral) muscles. Five grasshoppers were sampled for each point. Statistically significant atrophy was first evident on day 10.
542
Arbas and TVdnnCr
which had a mass of 14.1 t- 1.2 mg ( p < 0.01, two-tailed MWU test). Metathoracic muscles did not differ on the two sides ( p = 0.52, two-tailed MWU test).
Normal
d
8 lL
90 80
x
70
0
Possible Mechanisms Inducing Atrophy Zoss of Static Load. Autotomy of a hindlimb might induce muscle atrophy by a number ofpossible mechanisms. Loss of the large hindlimb removes a load that can comprise up to about 10%of the insect’s total body mass from the coxal and trochanteral muscles. The coxa permits rotation in all directions, so the load provided by the hindlimb affects the various thoracic muscles in different ways depending on whether the insect rests or locomotes on horizontal or vertical surfaces, or upside down underneath horizontal surfaces. It might be that removal of static load leads to wasting of muscle through a lack of “exercise” with all other factors being equal to the normal condition. To test this possibility, we loaded the coxal muscles after autotomy by reattaching the autotomized hindlimb in 10 insects in its normal orientation using cyanoacrylate glue (see Methods). Grasshoppers with reattached limbs carried them with abnormal posture, but were otherwise observed to assume all different orientations (vertical, horizontal, and upside down) as they moved around in their cages. Twenty-eight to 30 days after autotomy and reattachment of the leg, the muscles ipsilateral to the autotomy site were found to have a mean mass of 1.7 & 0.2 mg while the contralateral control muscles weighed 12.7 k 0.8 mg, that is, the ipsilateral muscles atrophied to 13.1 k 1.4% of the mass of their contralateral counterparts [Fig. 7 (“Reloaded”)]. The mass of these muscles was significantly different from contralateral controls or the mass of muscles in normal insects ( p < O.Ol), but was not significantly different from atrophied muscles in insects that had not had a limb reattached ( p = 0.82. two-tailed MWU test). Thus, reloading the muscles following autotomy did not prevent muscle atrophy.
Loss of Dynamic Load or Altered Use. Although reattachment of a weighted leg placed a load on the coxal and trochanteral muscles in the experiments described above, it might be that the muscles atrophied because the leg was not used to support the weight of the insect during posture and locomotion. To test the effect of altered usage, a small window was opened in the cuticle of the femur of anesthetized grasshoppers, and the tendon of the tibial extensor muscle was transected in 10 young adult grasshoppers. Tenotorny of the tibial extensor
2 v)
2 3 5:
2
Tendon cut
120, 110 100
60
50 40 30 20 10 0
Figure 7 Metathoracic muscle mass 30 days after autotomy, transection of N5. limb reattachment. or tcnotomy of the tibial extensor muscle compared to normal \dues. Each bar represents the mass of the muscles on the treated side as the mean (+S.E.M.) ‘70of control (contralateral) muscles in 10 grasshoppers. Normal bar represents the mean mass of control insects [Fig. 2(A)] expressed as a percent of left over right side.
muscle was shown in locusts to cause rapid degenerative effects in that muscle (Jahromi and Bloom, 1979), but was employed here to change the use of the limb. When the recovered insects first flexed the tibia during normal movements, they were unable subsequently to extend it actively again, and the leg was disabled as a load-bearing appendage. Recovery from tenotomy was not observed over the course of these experiments. Note that in this set of experiments, autotomy was never induced, thoracic muscles were never unloaded, and leg nerves remained undamaged. Thirty days after tenotomy of the tibial extensor muscle, thoracic muscles showed no signs of atrophy [Fig. 7 (“Tendon cut”)]. Thus, altered use ofthe leg, or its disuse in normal posture and locomotion did not induce atrophy of the thoracic muscles.
Nerve Damage. Another mechanism by which atrophy could be induced is by the severing of leg nerve 5 during autotomy. To explore the effects of nerve section without loss of a limb, branches of N5 were severed in the proximal femur through a small window opened in the cuticle in 24 grasshoppers. Every insect in this group autotomized the treated leg within 24 h. Sham-operated grasshoppers, in which the femoral cuticle was opened, but nerve branches were not cut. showed a much lower tendency to autotomize the treated leg, for example, 10%-20% of grasshoppers whose tibial extensor muscles were tenotomiaed typically autotomized the treated leg. This suggests that total denervation of the limb was responsible for the increased tendency to autotomize the leg. As an alternative to total denervation of the leg, we transected N5
iltropky of Insect Muscle
proximal to the coxa through a small window opened in the sternal cuticle. This procedure left the leg partially innervated via axons that run in N3B, which joins N5Bl within the coxa [Fig. 2(B)] (Hoyle, 1955). The tibia on the operated side was thus still capable of slow extension due to the firing of the “slow extensor tibiae” ( SETi) motorneuron. Transection of N5 in this location also caused abnormal postures of the leg. No recovery of normal function in the treated leg was evident in any of the grasshoppers from which muscles were weighed. When muscles of the two sides of the metathorax were compared 28-35 days following N5 transection, muscles ipsilateral to the transected nerve were found to have a mean mass of 1.9 k 0.2 mg compared to 12.2 k 0.7 mg for the contralateral muscles, that is, they atrophied to about 16% of the control muscles [Fig. 7 (“Nerve cut”)]. While again significantly different from control ( p < 0.01), this value was not significantly different from the mass of muscles that atrophied either with ( p = 0.29) or without ( p = 0.31. twotailed MWU test) a load. Since autotomy was never induced and the coxal muscles were left loaded by an attached, hydrated, and partially innervated limb in these experiments, the resultsindicate that it is neither the neural signal to autotomize nor unloading that induces atrophy. Rather, it is the transection of the leg nerve.
DISCUSSION Data presented here show that hindlimb autotomy in certain grasshoppers causes the atrophy of a set of undamaged, innervated muscles within the thorax. This is similar to the situation in crabs and crayfish where thoracic muscles proximal to the autotomy plane also lose up to about half their mass following loss of a limb (Moffett, 1987). Since crustaceans regenerate their limbs, this atrophy is reversible. This is not the case in grasshoppers where autotomized limbs do not regenerate. Although the six coxal and trochanteral muscles all atrophy following hindlimb autotomy, many other muscles within the thorax show no signs of atrophy following loss of a leg. Why is this so? Cowan ( 1970) has suggested that in “neuronally closed’ systems (that is, networks with strong synaptic interactions) there might be an increased sensitivity to transneuronally mediated degenerative influences. The “neuronally closed” system here is that which controls a complex multijointed limb. Movements across different joints of the legs of locusts and grasshoppers are coordinated, in part,
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through the action of intraleg reflexes that result from the pattern of synaptic interactions between sensory and motor circuits of the leg (Burrows and Homdge, 1974). Autotomy separates the proximal two leg segments from the distal leg segments and, by breaking leg nerve 5. may remove a significant amount of input from receptors on the leg to the ganglionic circuitry controlling intersegmental reflexes that coordinate coxal and trochanteral movements with those of distal leg segments. Reflexes that integrate movements of leg segments distal to the femur have been studied intensively (Burrows and Horridge, 1974; Burrows and Pfluger, 1986; Pfluger and Burrows, 1987; Burrows, 1987; Burrows, Laurent, and Fields, 1988; Laurent and Burrows, 1988b; Laurent and Hustert, 1988), and significant recent advances have been made in understanding the cellular basis of interleg reflexes (Laurent, 1986, 1987, 1988; Laurent and Burrows, 1988a). Reflexes that integrate movements across the proximal segments of the leg have not been studied directly, but are also very likely to exist. Experiments describcd in this report indicate that damage to N5 causes the atrophy of the thoracic muscles. What might the actual cellular changes induced by the nerve damage be? Severing of N5 does not damage the motorneurons of the coxal and tergotrochanteral muscles directly, but causes the axotomy of large numbers of sensory fibers carrying information from the leg to the central nervous system (CNS) . It is known from nerve section experiments (Zill, Underwood, Rowley, and Moran, 1980) that the severed axonal stumps of sensory neurons with somata in the periphery degenerate by about 7 days. Degeneration of severed sensory axons might induce changes through deaflerentation of central neurons and among them. possibly, motorneurons to the muscles that atrophy. Experimental deafferentation is known to cause reduction of dendritic growth in developing insect nervous system (Murphey, Mendenhall, Palka, and Edwards, 1975), as well as to induce metabolic changes including reduced protein synthesis (Meyer and Edwards, 1982). Evidence of similar effects of deafferentation is available in some vertebrate systems as well (Oswald and Rubel. 1985; Deitch and Rubel, 1989a). Breakage of N5 during hindlimb autotomy also causes axotomy of motorneurons that innervate the leg muscles located in distal leg segments. Axotomy causes normally nonelectrogenic insect motorneuron somata to become electrically excitable (Pitman, Tweedle, and Cohen, 1972; Goodman and Heitler, 1979), undergo metabolic changes in-
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dicative of increased protein synthesis (Cohen and Jacklet, 1965; Pitman et al., 1972), and lose known physiological inputs (Homdge and Burrows. 1974). Other retrograde transneuronal influences of motorneuron axotomy have not been explored. Another possibility is that a multiaxonal neuron with axons in both N5 and the nerves to the muscles that atrophy would signal the damage to N5 directly. Multiaxonal neurons of this type are known in metathoracic ganglion, for example, the common inhibitor (CI) which branches in metathoracic nerves 3, 4, and 5 (Hale and Burrows, 1985), and some of the dorsal unpaired median (DUM) neurons (Hoyle, 1978). Some of the thoracic muscles that atrophy are indeed innervated by neurons of this type, for example, muscle no. I20 is innervated by the CI, and other muscles are innervated by DUM neurons. But other muscles that atrophy in B. psolzis receive innervation neither from the CI nor from DUM neurons with axons in leg nerve 5. Hence, their atrophy cannot be initiated directly by a neuron with one axon damaged in N5. For example, the innervation of muscle no. I33 b,c, the tergotrochanteral depressor muscle. has been studied by the backfilling of motor nerves in both locusts and B. psolus (Villalobos and Arbas. 1988). In both, muscle no. 133 b,c was found to be innervated by three motorneurons [confirming the lack of innervation by CI in locust reported by Hale and Burrows ( 1985)] and possibly by at least one DUM neuron. The DUM neuron innervating muscle no. 133 was found, however. to have axons in N3 and N4, but not in N5 (Villalobos and Arbas, 1988). During the development of holometabolous insects, muscle atrophy and degeneration has been shown to be controlled by hormones (Weeks and Truman 1986a,b), raising the possibility that some humoral factor might act as an intermediary to mediate atrophy of the thoracic muscles of grasshoppers following damage to N5 that occurs during autotomy. The observation that muscle atrophy is restricted to the side and segment ofautotomy eliminates this possibility, since grasshoppers, like other insects. have an open circulatory system and all thoracic muscles would have been bathed by the same substance. Finally, ample evidence exists that alterations in spiking activity of motorneurons can effect significant changes in the physiological and structural properties of arthropod muscle cells during development and in maturity (reviewed by Atwood and Lnenicka, 1987; Govind, Mellon, and Quigley,
1987). It is possible that some alteration of the physiology of motor neurons of the affected muscles leads to atrophy of their targets. Whether muscle atrophy is due to an alteration in the activity level or pattern of firing in muscle and/or motorneurons or to a transneuronal trophic effect is the subject of our ongoing research. Preliminary studies in this project were camed out in the laboratory of Dr. R . L. Calabrese at the Biological Laboratories, Harvard Univ. and we thank him for providing facilities and discussions on the project. We also thank Ms. Christine Iovino and Susanna Ma for technical assistance, Charles A. Hedgecock, R.B.P. for photographic assistance, and Drs. R. B. Levine and L. P. Tolbert for comments on the manuscript. This work was supported in part by NIH grants NS 07309,BRSG SO7 RR07002, the Whitehall Foundation, and the University of Arizona Foundation.
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