J. Cell Sci. 10, 667-682 (1972) 667

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EFFECTS OF DENERVATION ON THE

ULTRASTRUCTURE OF INSECT MUSCLE

D. REES* AND P. N. R. USHERWOODDepartment of Zoology, University of Glasgow, Glasgow, Scotland

SUMMARY

The structure of normal and denervated muscle fibres in the metathoracic retractor unguismuscle of the locust (Schistocerca gregaria) has been examined. Section of the 2 motor neuronswhich innervate this muscle results initially in muscle hypertrophy but this is followed about4 days post neurotomy by progressive atrophy. Atrophy of the retractor unguis muscle ischaracterized by a decrease in muscle volume and degeneration of muscle organelles, e.g.mitochondria, sarcoplasmic reticulum, transverse tubular system, protein filaments, etc. Duringits later stages the degeneration of the denervated muscle is possibly assisted by the phagocyticaction of haemocytes which invade the muscle.

INTRODUCTION

When the motor nerve supplying a vertebrate skeletal muscle is transected themuscle initially hypertrophies and then rapidly atrophies (Hogenhuis & Engel, 1965;Miledi & Slater, 1969; Muscatello, Margreth & Aloisi, 1965; Pellegrino & Franzini,

Hypertrophy and atrophy of a vertebrate muscle are due to changes in the relationbetween synthesis and breakdown of proteins in that muscle. Hypertrophy is related toincreased utilization of amino acids for protein synthesis (e.g. Gutmann et al. 1966).Muscle atrophy is accompanied by a decrease in size and an increase in the number ofmuscle mitochondria, loss of myofUaments, fragmentation of the muscle plasmamembrane, sarcoplasmic reticulum and Z-disks, dilation of the lateral cisternae of thetriads, and lipid deposition within the necrotic fibres. A similar necrosis of musclealso occurs in dystrophic myopathies (e.g. Caulfield, 1966; Banker, 1968; Johnson &Pearse, 1968; Stolk, 1962) and after treatment of muscle with certain myotoxic agents(e.g. D'Agostino, 1963; Price, Pease & Pearson, 1962). The structural changes invertebrate muscle which follow denervation are accompanied by certain biochemicalchanges, for example, reductions in muscle ATP and glycogen (Hogenhuis & Engel,1965) and mitochondrial ATP (Michalazzi, Mor & Dianzani, 1957), and in bothdenervated (Bass & Hudlicka, i960) and dystrophic (Lin, Hudson & Strickland, 1969)vertebrate muscle there are indications that the muscle mitochondria are unable tooxidize pyruvic and fatty acids, with the consequential build-up and deposition of lipidbodies within the necrotic muscle fibres. These structural and biochemical changes

• Present address: Department of Entomology, University of California, Riverside,California 92502, U.S.A.

43-2

668 D. Rees and P. N. R. Usherwood

have been cited as indirect evidence for the so-called 'trophic' influence of nerve overmuscle (see reviews by Guth, 1968; Gutmann, 1962).

Investigations into the effects of motor nerve section on insect muscle are few andhave been mainly physiological rather than structural. In this paper, in which theeffects of motor nerve section on the ultrastructural properties of a locust skeletalmuscle are described, it will be demonstrated that there are many similarities betweendenervated insect muscle and denervated vertebrate muscle.

MATERIALS AND METHODSThe ultrastructure of normal and denervated metathoracic retractor unguis muscles of the

locust Schistocerca gregaria were examined. The techniques for denervating the retractor unguismuscle and for preparing sections of this muscle for electron microscopy were identical to thosedescribed by Rees & Usherwood (1972). Denervated and contralateral control muscles werefixed at various times after denervation, up to a maximum of 68 days post nerve section, andexamined. The cross-sectional areas of these muscles were also measured. Transverse sectionsof denervated and contralateral control muscles, taken from the centres of these muscles, werephotographed under a light microscope and the photographic negatives projected on to a grid.The sum total cross-sectional area of the muscle fibres in the denervated and control sectionswere determined and compared. In this manner it was possible to obtain an idea of the changesin muscle volume which follow section of the retractor unguis nerve fibres.

The histochemical techniques of Gomori (1950) and Holt & Hicks (1961) were used to locateany acid phosphatase in the denervated and control muscles.

RESULTS

Normal muscle

The general morphology of the locust metathoracic retractor unguis muscle has beendescribed elsewhere (Usherwood, 1967; Cochrane, Elder & Usherwood, 1972). Basicallythe muscle is composed of 2 bundles of fibres: a bundle of ' red' fibres and a bundle of'white' fibres (Usherwood, 1967). The white fibres (Fig. 1) contain fewer mitochondriathan the red fibres (Fig. 2) although the mitochondria in both types of fibre contain' whorled' cristae amidst an electron-dense matrix. The mitochondria lie on either sideof the Z-disk, where they appear to encircle the myofibrils. The plasma membrane ofthe retractor unguis muscle fibre is invaginated in the vicinity of the Z-disks and op-posite the regions of overlap of the actin and myosin filaments, to form the transversetubular system. In transverse section the myofibrils appear either polygonal or rec-tangular and each myofibril is surrounded by a system of anastomosing vesicularelements which constitute the sarcoplasmic reticulum (Fig. 3). In the regions of over-lap of the actin and myosin filaments the sarcoplasmic reticulum is expanded into flat,elliptically shaped cisternae on to which abut similarly shaped dilations of the trans-verse tubular system. These intimate associations of transverse tubular system andsarcoplasmic reticulum constitute 'dyads' (Bennett & Porter, 1953; Smith, 1962). Thedilations of the transverse tubular system are usually more electron-dense than the restof the transverse tubular system and the sarcoplasmic reticulum, due to the presenceof what has been interpreted as granular material (Hoyle, 1965). Similar material isalso found in the sarcoplasmic reticulum but it is less densely distributed here.

Denervated locust muscle 669

Denervated muscle

During the first 9 days after section of the nerve supply to the retractor unguismuscle there was little change in the fine structure of the muscle. However, the musclefibres hypertrophied by about 15% during the first 4 post-operative days and thishypertrophy was subsequently followed by a progressive atrophy. The first change inthe cyto-architecture of the muscle occurred about 11 days post-operatively when thetransverse tubular system became dilated and highly granulated in appearance. By thetwentieth day the cross-sectional area of the denervated muscle had decreased to about35 % normal; the muscle mitochondria were noticeably atrophied at this time and thenumber of lipid inclusions in the muscle had also increased, indicating an apparentdecrease in mitochondrial-linked fatty-acid oxidation (Lin et al. 1969). By the twenty-fourth and twenty-fifth post-operative days the cross-sectional area of the myofibrilshad significantly decreased, mainly due to loss of myofilaments from their peripheralaspects (Fig. 4), a change comparable to that seen in denervated rat (Pellegrino &Franzini, 1963) and frog (Muscatello et al. 1965) muscle. At this time the dyadiccomponents of the transverse tubular system in the peripheral parts of the fibresappeared as dark granulated sacs (Fig. 5) and in the centre of the fibres the transversetubular system was either swollen, disrupted or absent altogether. On the other hand,the cisternal components of the sarcoplasmic reticulum were quite normal in appear-ance, although the sarcoplasmic reticulum around each myofibril was noticeablyreduced in quantity. Further atrophy of the muscle during subsequent post-operativedays was accompanied by a progressive disorganization of the components of the musclefibres (Figs. 6, 7) and by the sixty-eighth day the denervated muscle had atrophiedby about 80%. The muscle mitochondria which remained at this time were dilatedand frequently disrupted and in the peripheral aspects of some fibres there was apreponderance of' whorled' tubules, which seemingly originated from the sarcoplasmicreticulum (Fig. 8). It is unlikely that the 'whorled' tubules originated from the trans-verse tubular system since they have a thin (about 5-6 nm) apparently single, mem-brane like the sarcoplasmic reticulum (Smith, 1962). The membrane of the transversetubular system is much thicker (about 10 nm) and apparently 'double'. Furthermorethe granular appearance of the 'whorled' tubules is due to the presence of numerousribosomal-like particles (about 16 nm diameter). Such structures are unlikely to befound in the transverse tubular system. Unfortunately, we have not been able to followthe development of these tubules and cannot therefore be categoric concerning theirorigin from sarcoplasmic reticulum. Similar structures seen in developing skeletalmuscle fibres grown in culture (Ishikawa, 1968) and degenerating frog (Muscatelloet al. 1965) and rat (Miledi & Slater, 1969) muscle are considered to originate fromthe transverse tubular system in the case of the developing muscle and from sarco-plasmic reticulum in the case of the degenerating muscles.

Significantly, little hydrolytic (acid phosphatase) activity was found in the denerva-ted preparations until about the twentieth day following neurotomy, when intense acidphosphatase activity was demonstrated around the periphery of the muscle fibres.Much of this was localized within haemocytes which had become associated with the

670 D. Rees and P. N. R. Usherwood

necrotic muscle fibres. Locust haemocytes are well provided with endoplasmic reti-culum and Golgi bodies, and the numerous dense granules which are found in as-sociation with these organelles are considered to be lysosomal in origin and to containacid phosphatase (Crossley, 1964, 1965). It is perhaps significant that haemocytes wererarely found on the surface of control muscle fibres.

The aposynaptic apparatus of the nerve-muscle synapses on the retractor unguismuscle which contains various granules, vesicles and microtubules disappears about5 days after section of the retractor unguis nerve fibres; that is, at about the same timethat the entire muscle fibre membrane becomes measurably sens itive to glutamate(Usherwood, 1969).

DISCUSSION

Following 'denervation' 3 specific and characteristic physiological changes occur invertebrate skeletal muscle which do not occur during disuse atrophy (Eccles, 1941;Gutmann, 1962): an increased sensitivity of the non-synaptic muscle membrane toacetylcholine (e.g. Axelsson & Thesleff, 1957); fibrillation (e.g. Li, Shy & Wells, 1957);and changes in the electrical properties of the muscle membrane (e.g. Hubbard, 1963).Similar changes also occur in insect muscle following denervation. For example,sensitivity to glutamate, which is thought to be a transmitter at insect excitatory nerve-muscle synapses (Usherwood & Machili, 1968), is normally restricted to the excitatorypost-synaptic membrane, but following denervation it spreads over the entire surfaceof the muscle fibre (Usherwood, 1969). Voskrensenskaya (1945) described spontaneoustwitching in the extremities of the locust leg 24 h after section of the nerves to the legmuscles. Fibrillation and other spontaneous mechanical activities have also beenobserved in other neurotomized insect muscles (Beranek, 1959; Beranek & Novotny,1958; Case, 1956; Jacklet & Cohen, 1967; Usherwood, 19636) and in the denervatedretractor unguis muscle. In our experience these fibrillations cease about 15 days postdenervation and the muscle becomes very inexcitable, although it will give a veryprolonged contraction in response to intense electrical stimulation. The electricalproperties of insect muscles also change following denervation. For example, Usher-wood (1963 a) has reported a decline in the membrane potential of locust muscle fibresimmediately following synaptic transmission failure as a result of section of the motorneurons to these fibres. It can be seen, therefore, that the phenomena which serve tocharacterize denervated vertebrate muscles are also properties of denervated insectmuscles.

The hypertrophy of locust muscle fibres which follows motor nerve transectionoccurs at a time when the nerve-muscle synapses are still intact. It seems reasonable toassume that this hypertrophy is similar to that which occurs in neurotomized ratdiaphragm muscles where it appears that the actin and myosin filaments increase innumber. Miledi & Slater (1969) have suggested that there is normally a turnover ofcontractile filaments in muscles and that the increase in their numbers during ' dener-vation' hypertrophy could be due either to an increase in their rate of synthesis or adecrease in their rate of decay. The observation by Gutmann et al. (1966) that there is

Denervated locust muscle 671

an increase in the incorporation of amino acids into total muscle protein of rat dia-phragm muscle following section of the phrenic nerve seemingly adds some weight tothis idea.

Atrophy of the denervated retractor unguis muscle ofthe locust appears to be causedby the loss of peripherally situated myosin filaments within the myofibrils. It is perhapssignificant that the only acid phosphatase activity found in denervated preparations27 days after neurotomy was located in the haemocytes, which were at this time closelyassociated with the periphery of the degenerating muscle fibres. Other types of hydro-lytic enzymes, e.g. cathepsin, alkaline phosphatase, acid ribonuclease, etc., may, ofcourse, have been present. Acid phosphatase has been detected within the lysosomesof haemocytes in other insects where these structures were actively engaged in removaland/or absorption of histolysing muscle tissues (Van Rees, 1888; Crossley, 1964, 1965;Whitten, 1964). In histolysing tissues of the blowfly, Calliphora erythrocephala,haemocytes bearing pseudopodia have been reported to be actively engaged in theabsorption of the necrotic muscle fibres, but, as in the locust, this occurs only duringthe later stages of muscle degeneration and changes in the sarcolemma and in theappearance of the muscle fibres precedes haemocyte invasion (Crossley, 1965). Theregressive changes in denervated insect muscle which precede haemocyte invasionindicate that the initial signal for muscle atrophy has its origin, not in the haemocytes,but in the muscle itself. In denervated vertebrate muscles (Pellegrino & Franzini,1963) and some dystrophic myopathies (Johnson & Pearse, 1968; Pennington, 1961;Stolk, 1962) increases in both the lysosomal content of the muscles and in diffuselyoccurring acid phosphatase have been reported. It has been suggested that thesehydrolytic agents are mainly responsible for the regressive changes in vertebrateskeletal muscle which follow neurotomy and it is perhaps interesting to note thatMiledi & Slater (1969) have suggested that these agents arise from satellite cells.

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BANKER, B. Q. (1968). A phase and electron microscopic study of dystrophic muscle. J. Neuro-path, exp. Neurol. 27, 183-209.

BASS, A. & HUDLICKA, O. (i960). Utilization of oxygen, glucose, unesterified fatty acids, carbondioxide and lactic acid in normal and denervated muscle 'in situ'. Physiol. bohemoslov. 9,401—407.

BENNETT, H. S.& PORTER, K. R. (1953). An electron microscope study of sectioned breast muscleof the domestic fowl. Am. J. Anat. 93, 61-106.

BERXNEK, R. (1959). Spontaneous electrical activity and excitability of the denervated insectmuscle. Physiol. bohemoslov. 8, 87.

BERANEK, R. & NOVOTNY, I. (1958). Spontaneous electrical activity in the denervated muscle ofthe cockroach, Periplaneta americana. Nature, Land. 182, 957-958.

CASE, J. F. (1956). Spontaneous activity in denervated insect muscle. Science, N.Y. 124, 1079-1080.

CAULFIELD, J. B. (1966). Electron microscopic observations of the dystrophic hamster muscle.Arm. N.Y. Acad. Sci. 138, 151-159.

COCHRANE, D. G., ELDER, H. Y. & USHERWOOD, P. N. R. (1972). Physiology and ultrastructureof phasic and tonic skeletal muscle fibres in the locust, Schistocerca gregaria. J. Cell Sci. 10,419-441.

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CROSSLEY, A. C. S. (1964). An experimental analysis of the origins and physiology of haemo-cytes in the blue blow-fly Calliphora erythrocepliala. J. exp. Zool. 157, 375-398.

CROSSLEY, A. C. S. (1965). Transformations in the abdominal muscles of the blue blow-flyCalliphora erythrocephala (Meig.) during metamorphosis.^. Embryo!, exp. Morph. 14, 80-110.

D'AGOSTINO, A. N. (1963). An electron microscope study of skeletal and cardiac muscle of therat poisoned by Plasmocid. Lab. Invest. 12, 1060-1070.

ECCLES, J. C. (1941). Disuse atrophy of skeletal muscle. Med. J. Aust. 2, 160-164.GOMORI, G. (1950). An improved histochemical technique for acid phosphatase. Stain Technol.

25, 81-85.GUTH, L. (1968). 'Trophic ' influences of nerve on muscle. Pliysiol. Rev. 48, 645-687.GUTMANN, E. (1962). Denervation and disuse atrophy in cross-striated muscle. Revue can. Biol.

21, 353-365-GUTMANN, E., HANfaovA, M., HAJEK, I., KLICPERA, M. & SYROVY, I. (1966). The postdener-

vation hypertrophy of the diaphragm. Physio!. bohemoslov. 15, 508-524.HAGOPIAN, M. & SPIRO, D. (1967). The sarcoplasmic reticulum and its association with the

T-system in an insect. J. Cell Biol. 32, 535-545.HOGENHUIS, L. A. H. & ENGEL, W. K. (1965). Histochemistry and cytochemistry of experi-

mentally denervated guinea pig muscle. Acta anat. 60, 39—65.HOLT, S. J. & HICKS, R. M. (1961). The localization of acid phosphatase in rat liver cells as

revealed by combined cytochemical staining and electron microscopy. J. biopiiys. biochem.Cytol. 11, 47-66.

HOYLE, G. (1965). Nature of the excitatory sarcoplasmic reticular junction. Science, N. Y. 149,70-72.

HUBBARD, S. J. (1963). The electrical constants and the component conductances of frogskeletal muscle after denervation. J. Physiol., Lond. 165, 443-456.

ISHIKAVVA, M. (1968). Formation of elaborate networks of T-system tubules in cultured skeletalmuscle with special reference to the T-system formation. J. Cell Biol. 38, 51-66.

JACKLET, J. W. & COHEN, M. T. (1967). Nerve regeneration: correlation of electrical, histolo-gical and behavioural events. Science, N.Y. 156, 1640-1643.

JOHNSON, M. & PEARSE, A. G. E. (1968). The cytochemistry and electron microscopy of con-genital muscular dystrophy in the hamster. In Research in Muscular Dystropliy. Proc. Symp.Muse. Dystr. iv, pp. 171-187.

Li, C , SHY, C. M. & WELLS, J. (1957). Some properties of mammalian skeletal muscle fibreswith particular reference to fibrillation potentials.^. Pliysiol., Lond. 135, 522-535.

LIN, C. H., HUDSON, A. J. & STRICKLAND, K. P. (1969). Fatty acid metabolism in dystrophicmuscle in vitro. Life Sci. 8, 21—26.

MICHALAZZI, L., MOR, M. A. & DIANZANI, M. U. (1957). Adenosine triphosphate content inguinea pig muscle after denervation. Experientia 13, 117.

MILEDI, R. & SLATER, C. R. (1969). Electron-microscope structure of denervated skeletalmuscle. Proc. R. Soc. B 174, 253-269.

MUSCATELLO, V., MARGRETH, A. & ALOISI, M. (1965). On the differential response of sarcoplasmand myoplasm to denervation in frog muscle. J. Cell Biol. 27, 1-24.

PELLEGRINO, C. & FRANZINI, C. (1963). An electron microscope study of denervation atrophy inred and white skeletal muscle fibres. J. Cell Biol. 17, 327-349.

PENNINGTON, R. J. (1961). Biochemistry and dystrophic muscle. Biocliem. J. 80, 649-653.PRICE, A. M., PEASE, D. C. & PEARSON, C. M. (1962). Selective actin filament and Z-band

degeneration induced by Plasmocid. Lab. Invest, n , 549-561.REES, D. & USHERWOOD, P. N. R. (1972). Fine structure of normal and degenerating motor

axons and nerve-muscle synapses in the locust, Schistocerca gregaria. Comp. Physiol. Biochem.(in the Press).

SMITH, D. S. (1962). Cytological studies on some insect muscles. Revue can. Biol. 21, 279-301.STOLK, A. (1962). Muscular dystrophy in lower vertebrates. Revue can. Biol. 21, 445-455.USHERWOOD, P. N. R. (1963 a). Response of insect muscle to denervation. 1. Resting potential

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Denervated locust muscle 673USHERWOOD, P. N. R. (1969). Glutamate sensitivity of denervated insect muscle fibres. Nature,

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{Received 23 September 1971)

ABBREVIATIONS ON PLATES

AdInitnsc

A-banddyadI-bandmitochondrionnucleussarcolemma

srtttsivtZ

sarcoplasmic reticulumtracheoletransverse tubular system'whorled' tubulesZ-disk

674 J)- R^ and P- N. R. Usherzvood

Fig. i. Longitudinal section of a control white muscle fibre. Note how the sarcoplasmicreticulum surrounds the fibrils. A nucleus lies at the periphery of the muscle fibre.Fixed after soaking for 2 h in locust saline, x 7370.Fig. 2. Longitudinal section of a red muscle fibre in a normal metathoracic retractorunguis muscle. The electron micrograph shows the normal arrangement of the fibrilswithin the muscle fibre. The sarcoplasmic reticulum lies between the fibrils. Darkelliptical components of the transverse tubular system form dyads with the sarcoplas-mic reticulum in the region of overlap of the actin and myosin filaments. The sarcomerelength of the muscle fibre is that region between opposite Z-disks. Fixed direct fromhaemolymph. x 14000.

(WeDenervated locust muscle

676 D. Rees and P. N. R. Usherwood

Fig. 3. Transverse section of a normal white muscle fibre, illustrating the large mito-chondria characteristic of this type of muscle fibre and the arrangement of the sarco-plasmic reticulum and transverse tubular system. Note that the mitochondria arepredominately located near the Z-disk. x 27400.

Denervated locust muscle 677

678 D. Rees and P. N. R. Usherwood

Fig. 4. Transverse section of a white muscle fibre 25 days post nerve section. The trans-verse tubular system is greatly distended and frequently densely granular in appearance.The muscle mitochondria are swollen and somewhat fragmented, x 30000.Fig. 5. Higher magnification of 25-day denervated muscle fibre, illustrating denselygranulated dilation of TTS. The dyadic component of the SR membrane is charac-terized by the electron-dense bodies which lie on its outer surface (Hagopian & Spiro,1967). x 100000.

Denervated locust muscle 679

:?» .tts

sr

w

•!••• • •:-:-^. *'•. V '**••':•*'•:'•.£

• 4 t > J > v > 1 ^ - -

68o D. Rees and P. N. R. Usherwood

Fig. 6. Longitudinal section of a white muscle fibre 41 days post nerve section. Thegeneral organization of this muscle has been markedly affected by atrophy. The Z-disks are irregularly arranged and there are few muscle mitochondria. Glycogenparticles are distributed between the myofibrils. x 18000.Fig. 7. Longitudinal section of a white muscle fibre 68 days post nerve section. Theswelling of parts of the TTS seen in earlier stages of atrophy is still apparent, theswollen regions appearing densely granulated. The mitochondria are also verydistended, x 49000.

^enervated locust muscle 681

682 D. Rees and P. N. R. Usherwood

Fig. 8. Transverse section of periphery of a white muscle fibre 68 days after nervesection. A complex system of 'whorled' tubules, apparently derived from thesarcoplasmic reticulum, is present below the sarcolemma. x 24200.