Control of muscle degeneration following autotomy of a hindleg in the grasshopper, Barytettix humphreysii

Journal of Insect Physiology 48 (2002) 91–102 www.elsevier.com/locate/jinsphys

Control of muscle degeneration following autotomy of a hindleg in the grasshopper, Barytettix humphreysii K.E. Personius a

a, b,*

, R.F. Chapman

a

Interdisciplinary Program in Physiological Sciences Graduate Program and ARL Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA b Department of Physical Therapy, Exercise and Nutrition Sciences, University at Buffalo School of Health Related Professions, 405 Kimball Tower, Buffalo, NY 14214, USA Received 2 August 2001; accepted 1 October 2001

Abstract When the grasshopper, Barrytettix humphreysii, sheds a hindlimb during autotomy, certain thoracic muscles degenerate although they are neither directly damaged nor denervated. Muscle degeneration is induced when a leg nerve (N5) that does not innervate the thoracic muscles is severed. Together these results suggest that transneuronal mechanisms influence muscle survival. To further characterize this autotomy-induced process, we studied the degeneration of a thoracic tergotrochanteral muscle (M#133b,c) following autotomy or experimental manipulation in adult animals. Its degeneration is correlated with reduced activity of its neural input and occurs by programmed cell death (PCD). PCD onset is variable between individual muscle fibers, indicating that the trigger of degeneration is fiber specific. Muscle degeneration appears to be triggered by the loss of proprioceptive input from the autotomized limb, since severing of axons from proprioceptive organs, but not exteroceptive chemo- or mechanoreceptors, leads to muscle degeneration. Muscle disuse, neuronal degeneration, or changes in juvenile hormone titer do not appear to play a role in autotomyinduced degeneration. We propose that the loss of proprioceptive input from proximal campaniform sensilla on the tibia deafferents the thoracic muscle motor neurons and leads to a decrease in their activity. Muscle degeneration is ultimately triggered by the loss of normal neural activity.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Programmed cell death; Transneuronal; Activity-dependent

1. Introduction The process of autotomy, spontaneously shedding a part of the body, often to avoid capture, is relatively common in insects and other arthropods, and frequently involves loss of a leg (Buck and Edwards, 1990; Carlberg, 1986; Formanowicz, 1990; Hopkins et al., 1999; Moore and Tabashnik, 1989; Tanaka et al., 1987). In insect legs, the break occurs between the trochanter and femur. No muscles cross the line of fracture but nerves carrying information to and from the leg are broken. Loss of the hindleg, in grasshoppers, is followed by degeneration of six muscles inserted into the coxa and trochanter that are responsible for movements of the * Corresponding author, at the University of Buffalo School of Health Related Professions. Tel.: +1-716-829-2941-258; fax: +1-716829-2428. E-mail address: [email protected] (K.E. Personius).

whole leg in the intact insect (Clinton and Arbas, 1994). These muscles are not damaged directly during autotomy, nor is their nerve supply damaged, since they are innervated by nerves intrinsic to the thorax (nerves N3 and N4). The process of degeneration extends over a period of days during which the muscles progressively lose their function (Personius and Arbas, 1998) and the extent of degeneration varies between muscles (Clinton and Arbas, 1995). In this paper, we address two questions: how is the process of degeneration triggered, and how is the subsequent degeneration controlled? Degeneration of muscles at metamorphosis involves the decline of the molting hormone, 20-hydroxydecdysterone (20-HE), and is sometimes associated with eclosion hormone (Schwartz and Truman, 1984; Kimura and Truman, 1990; Kobayashi and Ishikawa, 1993). 20-HE is mainly produced by the prothoracic glands during development, but in grasshoppers these glands break down soon after

0022-1910/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 1 4 9 - 4

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the adult molt. In some insects abdominal oenocytes produce small quantities of 20-HE, but this is not true for locust (Locusta migratoria; reviewed by Rees, 1985). 20-HE is also produced by the ovaries of adult female grasshoppers (Hagedorn, 1983). Since muscle degeneration following autotomy occurs during earlier stages of development as well as in mature adults of both sexes, 20-HE is unlikely to be involved. Muscle degeneration after dispersal by flight may be triggered by a rise in juvenile hormone titer subsequent to feeding upon host plants after migration (Rankin and Riddiford, 1977; Kobayashi and Ishikawa 1993, 1994). In addition, Meusers and Pflu¨ ger (1998) have found that one section of the flight muscle (M#114) of Locusta migratoria degenerates following the imaginal molt and this degeneration is triggered by a decline in juvenile hormone titer. Mordue (1977) showed that antennectomy of the desert locust, Schistocerca gregaria, leads to changes consistent with an increase in juvenile hormone titer so that, arguably, any injury that removes an appendage, such as autotomy, could produce a similar effect. An alternative to hormonal triggering of degeneration is a direct neural effect. Nerve 5, the only nerve severed during autotomy, carries motor axons from the metathoracic ganglion to the distal hindlimb and hundreds of sensory axons from the chemoreceptors and exteroreceptive and proprioceptive mechanoreceptors of the hindlimb to the ganglion (Burrows, 1992). The motor neurons within N5 have been shown to survive following the axotomy (deliberate cutting) of their axons (Horridge and Burrows, 1974). In contrast, the sensory axons of N5 degenerate soon after axotomy (Horridge and Burrows, 1974; Zill et al., 1980). This loss of sensory input has been shown to decrease excitatory input to metathoracic motor neurons (Horridge and Burrows, 1974). Hence, the severing of N5 sensory axons during autotomy may deafferent the motor neurons innervating the affected thoracic muscles and lead to a relatively quiescent neuromuscular junction. The subsequent loss of normal motor neuron input may trigger thoracic muscle degeneration. Examples of muscle degeneration induced by the loss of motor neuron input are well documented in other animals (Ashby et al., 1993a,b). For these reasons, the possibility that sensory input and/or juvenile hormone act as triggers for muscle degeneration following autotomy has been investigated. The effects of sensory inputs on muscle degeneration were examined by partial deafferentation of the hindleg. The importance of motor output to the degenerating muscles was determined by cutting the nerve (N3c) supplying them and also by recording activity from the nerve at intervals after autotomy. Juvenile hormone production was eliminated using precocene. The process of muscle degeneration, once initiated, could proceed in an uncontrolled manner by wasting, or

by a controlled process, programmed cell death (PCD). Wasting, in vertebrates, is thought to occur following muscle disuse. Protein synthesis is decreased and protein degradation increased, but without the occurrence of a specific genetically controlled program (Goldspink et al., 1983). Degeneration of muscles after metamorphosis or flight in insects has been shown to result from PCD (Lockshin, 1981; Schwartz and Truman, 1984; Schwartz, 1986; Kimura and Truman, 1990), a genetically controlled process involving the de novo synthesis of protein. The possibility that this is also involved in degeneration of muscles following autotomy is investigated here by examination of ubiquitin levels, together with the abundance of pyknotic nuclei and DNA fragmentation in the muscles. In summary, spontaneous ensemble activity of N3c, which innervates four of the affected thoracic muscles, is significantly reduced following autotomy. Muscle degeneration via PCD appears to be triggered by the loss of proprioceptive input from proximal campaniform sensilla on the tibia, deafferenting the thoracic muscle motor neurons and leading to a decrease in activity. Juvenile hormone does not appear to play a role in muscle degeneration following autotomy. Thus, severe muscle degeneration via PCD can be induced in an adult grasshopper by reduced motor neuron activity.

2. Materials and methods 2.1. Experimental animals Adult grasshoppers, Barytettix humphreysii, from a laboratory colony were used in all experiments. Animals were reared on a 16:8 light/dark cycle at room air temperature, approximately 24 °C. Radiant heat from 100 W tungsten light bulbs allowed the insects to regulate their body temperature by changing their position within the cage during the photophase. Animals were fed romaine lettuce with wheat germ/bran mix ad libitum. As necessary, animals were induced to autotomize a single hindlimb by holding the grasshopper by the hindlimb and gently shaking. Only two muscles, the tergotrochanteral depressor muscle (M#133b,c) and the tergal promoter/elevator (M#118), of the group of degenerating thoracic muscles were examined in this study. 2.2. Experiments on neural activity 2.2.1. Partial deafferentation To determine whether deafferentation triggered muscle degeneration, sections of one hindlimb were removed at precise locations. Amputations were made between the tibia and tarsus (tarsus cut), between the first and second spine of the tibia (mid-tibial cut), or through the

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very proximal tibia (prox-tibial cut; see Fig. 5A). The extent of degeneration was determined by measuring protein content of M#133b,c and M#118 at 15 days following experimental ablation. Partial deafferentation was not well tolerated and more that 50% of the grasshoppers spontaneously autotomized the experimental hindlimb. Spontaneous autotomy was more common for the two more proximal amputation than the distal amputation and these insects were not used for further analysis. Electrophysiological changes in M#133b,c were assayed 10 days following the proximal tibial cut as described in Personius and Arbas (1998). 2.2.2. Severing of nerve 3 To determine whether direct denervation of M#133b,c also leads to degeneration with a similar time course to that following autotomy, B. humphresysii were cold-anesthetized at 0 °C for 10–15 min and pinned out on a dissecting dish ventral side up. After cleaning with acetone and 95% ethyl alcohol, a section of the ventral cuticle was removed and one side of the metathoracic ganglion and its nerves was exposed. N3 was identified and a section of the nerve just distal to the ganglion was removed. The ventral cuticle was replaced and sealed in place with paraffin wax (paraplast). The ventral side of the insect was cleaned again with 95% ethyl alcohol and the insect warmed to room temperature before returning to its cage. Fifteen days following experimental axotomy, animals were again cold-anesthetized and M#133b,c was removed from both sides of the insect for protein content analysis. The unoperated side of M#133b,c served as an internal control. To test whether the denervation was successful, the distal branch of N3 which innervates M#133b,c was stimulated by suction electrode. Only preparations in which stimulation did not lead to muscle contraction were used. Reinnervation did occur in a few insects. In two animals, sham operations were performed by opening the ventral cuticle but not severing N3. Protein content of M#133b,c was determined for all animals 15 days following the operation. To reduce mortality following axotomy, all grasshoppers used in these experiments, including controls, were 14 days past their molt to adulthood at the time of the operation. 2.2.3. Changes in nerve 3c activity To determine whether autotomy led to long-term changes in motor neuron activity, grasshoppers were induced to autotomize one hindlimb on the first day following their molt to adulthood (day 0). Bilateral extracellular recordings from N3c (Fig. 2B) were obtained from adult animals 1, 3, 5, 10 and 15 days post-autotomy. Insects were first cold-anesthetized at 0 °C for 15 min and pinned out on a dissecting dish ventral side up. After cleaning with 95% ethyl alcohol, the ventral cuticle was removed to expose the metathoracic ganglion

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along with N3c bilaterally. N3c innervates the thoracic muscles, M#121, M#130, M#133d and M#133b,c, all of which degenerate following autotomy. While superfusing with grasshopper saline (Personius and Arbas, 1998), suction electrode recordings from N3c were obtained using a differential AC amplifier (A-M systems). Extracellular signals were amplified 1000-fold and filtered (60 cycle notch filter, 10 Hz low cut-off, 10 kHz high cutoff) and later analyzed with Datapac III software (RUN Technologies). 2.2.4. Structural changes in nerve 3c To determine whether structural changes occurred in N3c following autotomy, the metathoracic ganglion with N3c intact bilaterally was partially dissected from insects 15 and 25 days after autotomy and fixed in situ with 2.5% glutaraldehyde, 0.5% paraformaldehyde, 0.18 M CaCl2, 0.58 mM sucrose, and 0.1 M phosphate buffer for 1 h (Tolbert and Hildebrand, 1981). After 1 h, the metathoracic ganglion with N3c was removed and fixed in the same solution overnight at 4 °C. The ganglion was then cut in half and each half was prepared for microscopy by secondary fixation in 0.05% osmium tetroxide, followed by rinsing in buffer, dehydration through graded ethanols, and embedded in Epon/Araldite. Cross-sections were cut through N3c at several positions along its length. One-micrometer-thick sections were cut on an Ultracut E (Reichert-Jung) and stained with toluidine blue. 2.2.5. Protein content The protein content of M#133b,c and/or M#118 was determined by the Bradford method (Bradford, 1976). After removing M#133b,c and M#118, the entire muscle was dissolved in 100 µl of 0.5 M NaOH overnight at 56 °C. Unknown assays contained 30 or 40 µl of muscle sample, 70 or 60 µl of 0.5 M NaOH and 1000 µl of BioRad protein assay reagent, respectively. Absorbance was measured at 595 nm by a BioSpec-1601 (Shimadzu) and protein concentration (µg protein per ml solution) was determined from a standard curve. Protein content of M#133b,c or M#118 was then calculated. Spectrophotometric measurements were made 10–30 min after addition of Bio-Rad reagent. 2.3. Muscle changes 2.3.1. Muscle preparation Dissections were performed in low Ca2+ saline to reduce muscle contractions. M#133b,c was removed with the cuticle at its origin and insertion intact. Muscles samples used for immunohistochemistry were prepared for cryostat cross-sections. The muscles were placed in carboxymethylcellulose and frozen in isopentane cooled by liquid nitrogen. Muscle preparations were mounted with Tissue TEK on a cold chuck (⫺20 °C) of a cry-

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otome (B/K instruments) and serial cross-sections (20 µm) were made and placed on dry, glycerin-subbed cover slips. Whole-muscle samples for longitudinal sections were fixed in situ in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight, dissected out in PBS, embedded in paraffin (paraplast), and serially sectioned (6 µm). 2.3.2. Ubiquitin immunohistochemistry and filamentous actin labeling To determine whether ubiquitin was upregulated in degenerating muscle, frozen cross-sections were double labeled for ubiquitin-immunoreactivity and filamentous actin. Cross-sections were fixed in 4% paraformaldehyde for 2 h and permeabilized in PBS-Tx (1% Triton-100 in PBS) several times. Nonspecific activity was blocked by immersing the sections in a solution of 30 mg powdered milk per 1 ml buffer, 0.25% bovine serum albumin and 2% normal goat serum in Tris buffer (0.05 M Tris, 0.85% NaCl and 0.25% Triton-100) for 1 h (Dr N. D. Davis, personal communication). Samples were labeled for the primary antibody (rabbit anti-ubiquitin, Chemicon) diluted 1:1000 in blocking solution overnight at 4 °C. Following washing in PBS, samples were labeled for the secondary antibody (Rhodamine conjugated goat anti-rabbit, Jackson Immunoresearch, Inc.) diluted 1:100 in blocking solution for 1 h. Samples were again washed in PBS and incubated in 66 nM bodipy-phallacidin (Molecular Probes, Inc.) in PBS for 2 h to label filamentous actin. Preparations were mounted in 80% glycerol/0.05 M carbonate–bicarbonate. 2.3.3. Labeling of nuclei Since chromatin condensation is a common feature of PCD, paraffin-embedded longitudinal muscle sections were labeled with propidium iodide (Sigma). After deparaffinization and rehydration, sections were incubated in 25 µM propidium iodide in PBS for 3 min. Some sections were later stained with hematoxylin–eosin to better visualize the relationship between fiber cytoplasm and pyknotic nuclei. These samples, in addition to propidium iodide labeled sections, were used to determine the percent of pyknotic nuclei at 10 and 15 days post-autotomy. The number of pyknotic nuclei versus the total nuclei number was determined for three similar serial sections that were at least 60 µm apart and then averaged to give the percent of pyknotic nuclei for that muscle. Muscle samples from the autotomized and internal control sides of three insects were used for both time periods 2.3.4. TUNEL (TdT-mediated dUTP nick end labeling) TUNEL labeling was used to determine whether DNA fragmentation occurs in muscle nuclei following autotomy. After deparaffinization and rehydration, longitudinal muscle sections from both the autotomized and internal control sides of the insect were washed in PBS,

permeabilized in proteinase K (10 µg/ml in Tris–HCl buffer), rinsed in the TdT mixture plus nucleotide mixture in reaction buffer (experimental preparations), or the nucleotide mixture in reaction buffer alone for negative control. Sections were washed in PBS and mounted in 80% glycerol/0.05 carbonate–bicarbonate. 2.3.5. Viewing of preparations Preparations were viewed with a confocal microscope (MRC-600 with a Nikon Optipho-2 microscope and a krypton/argon laser light source; BioRad). Two shambling channels and dichromatic cubes were used (BioRad K1 and K2: excitation wavelengths of 488 and 568 nM, respectively), optical sections were recorded for one or two dyes. If two dyes were used, the images were merged by using different pseudo-color (red for propidium iodide or rhodamine and green for bodipy-phallacidin or FITC-dUTP). Hematoxylin–eosin preparations, nuclear counts of propidium iodide stained preparations, and nerve cross-sections were viewed with a Leitz microscope (Laborlux S). 2.4. Blockade of juvenile hormone production Precocene II (Sigma-Aldrich) is a plant-derived compound which has been shown to cause severe atrophy of the corpora allata and inhibit juvenile hormone production in the locust, Locusta migratoria (Pener et al., 1978). We used this compound to determine whether changes in juvenile hormone titer trigger autotomyinduced muscle degeneration. Test insects received a single injection of 100 µg of precocene II in 2 µl of acetone, while controls received an equal amount of acetone only. Injections were made in the abdomen on the first day following the animal’s molt to adulthood. Two weeks following the injections, animals were induced to autotomize a single hindlimb. Protein content of M#133b,c was determined bilaterally 15 days following autotomy. To assure that precocene II was active in destroying the corpora allata of B. humphresysii, two insects were sacrificed 2 weeks following injection. As Pener et al. (1978) consistently found, following this dose of precocene II, the corpora allata were completely ablated. 2.5. Statistics A one-way analysis of variance (ANOVA) with Bonferroni correction was used to compare the changes in the ratio of N3c ensemble activity (experimental vs. control), and to compare changes in protein content following autotomy, severing of N3 and partial deafferentation. Percentage data for pyknotic nuclei following autotomy was normalized by arcsin transformation with single-tailed unpaired t-test.

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3. Results 3.1. Protein content of muscle following autotomy, denervation or partial deafferentation The extent of muscle degeneration, as measured by the ratio of protein content of M#133b,c on the experimental to control side, was similar 15 days following autotomy and direct denervation (Fig. 1B; auto- and axo-, respectively). Sham operated animals demonstrated no degeneration of M#133b,c 15 days after surgery, since a ratio of 1 indicates the muscle protein content on the experimental and control side were the same (Fig. 1B, sham-). Fig. 1A shows the location of the various experimental amputations along with the proprioceptive sensilla of the tibia and tarsus. Insects deafferentated by severing the hindlimb between the tibia and tarsus (tarsus) or between the first and second tibial spines (mid-tibia) demonstrated no degeneration of M#133b,c 15 days post-amputation (Fig. 1B). Amputation through the proximal tibia (prox-tibia), however, resulted in significant muscle degeneration when compared to the tarsus and mid-tibia sections (Fig. 1B; ANOVA with Bonferroni correction; pⱕ0.05; n=3–10 insects at each time period). The extent of degeneration following the proximal tibia amputation was less than that following autotomy or denervation (Fig. 1B; one-way ANOVA with Bonferroni correction; p⬍0.05) suggesting that the proximal amputation did not fully activate the degenerative response. However, the extent of muscle degeneration was significant since the ratio of M#133b,c protein content was similar 15 days following proximal amputation to that seen 10 day after autotomy alone (Fig. 1B; 70±10% vs. 77±6%; proximal amputation vs. autotomy). Electrophysiological changes similar to those found following autotomy (Personius and Arbas, 1998) also occurred following the proximal tibial amputations, but not more distal amputations. For example, the average muscle fiber resting membrane potential was significantly reduced 10 days following tibial sections when compared to internal-control fibers (50.7±2.5 vs. 60.8±1.4 mV; autotomized side vs. internal control; mean±SE; p⬍0.05; one-tailed paired t-test; 20 muscle fibers per side; n=3 animals). Additionally, post-inhibitory rebound was found in about 50% of the muscle fibers on the severed side, although they are not present on the internal-control side (data not shown). M#118, a hindlimb elevator and promoter, also showed reductions in protein content following autotomy and proximal tibial sections, although the changes were not as extreme. Fifteen days post-autotomy or proximal tibial amputation, for M#118 the ratio of protein content was reduced to 0.554±0.113 and 0.758±0.045 of control, respectively (data not shown).

Fig. 1. Direct denervation and progressive loss of sensory input lead to muscle degneration. (A) The proprioceptive organs of the hind tibia and tarsus of a melanopline grasshopper showing the number of neurons in each unit and the positions of the three experimental amputations (bold arrows; based on McFarlane, 1953, and unpublished data). (B) The relative protein content of muscle #133b,c following various experimental manipulations is shown. Axotomy of N3 (axo-) led to similar degree of muscle degeneration as autotomy (auto-) 15 days post-manipulation. No degeneration was seen following sham axotomy (sham-). Significant muscle degeneration occurred 15 days after proximal tibial amputation (prox-tibia) compared to sections through the middle tibia (mid-tibia) or sections that removed the tarsus (tarsus) which produced no degeneration (*p⬍0.05, one-way ANOVA with Bonferroni correction, prox-tibia n=10, mid-tibia n=3, tarsus n=2). A similar degree of muscle degeneration occurred 15 days after proximal tibial amputation and 10 days following autotomy-alone (10 day auto-), when significant electophysiolgical and histological changes are evident. Thus, the proximal tibial amputation, which damaged the subgenual and proximal campaniform sensilla and decreases normal proprioceptive input, partially mimics autotomy-induced muscle degeneration.

3.2. Changes in nerve 3c activity Fig. 2A shows the location of N3c and N5 of the metathoracic ganglion, as well as the possible neuronal interactions affected by autotomy. The resting ensemble activity of N3c on the side of autotomy was unchanged

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Fig. 2. Nerve 3c ensemble activity is reduced following autotomy. (A) A schematic drawing of the metathoracic ganglion and possible neuronal interactions affected by autotomy. Autotomy severs the leg nerve (N5) which carries both motor axons to the distal hindlimb muscles and sensory axons from the leg afferents. The multiple leg afferents terminate on metathoracic interneurons (open circle) and motoneurons (filled circles). The thoracic muscles that degenerate are innervated by nerves 3 and 4. Nerve 3c (N3c) which innervates four of the degenerative muscles is shown. (B) The ensemble activity of N3c was significantly reduced 10 and 15 days following autotomy in the grasshopper, B. humphresysii (*p⬍0.05, one-way ANOVA with Bonferroni correction, n=5 insects for each time period, mean±SE). Records were obtained from both sides of an animal and activity on the side of autotomy was expressed relative to that on the contralateral side (log scale). One-minute records were made 1 to 2 h following ice anesthesia. Using Datapac III (Run Technologies), a threshold was set so that spikes of all sizes above the baseline noise within the record were counted. The average number of spikes per recording was 300±35 (mean±SE). Superposition of spikes was rare due to the small number of axons in N3c. (C) A sample record from an animal 10 days postautotomy. The frequency of N3c activity is decreased on the autotomized side compared to the internal-control side.

3.3. Cross-section of N3c N3c contains 10 motor axons and a few sensory axons. By 15 days post-autotomy, M#133b,c has undergone severe degeneration; however, the axons of N3c, which innervate this muscle, do not show degenerative changes (Fig. 3A). No degenerative changes are seen in the contralateral-control side 25 days post-autotomy (Fig. 3B). Changes in nerve histology, including misshapen axons, abnormal separation of neural and glial tissue, and dense staining of the neural lamella (arrows) are, however, present on the experimental side by 25 days post-autotomy (Fig. 3C; n=3 insects for each time period). 3.4. Prococene II injections

in B. humphresysii 1, 3 and 5 days following autotomy, when compared to the firing frequency of the contralateral N3c (Fig. 2B; note log scale). By 10 days and 15 days post-autotomy, however, the ratio of ensemble N3c activity between the two sides of the animal was significantly reduced (ANOVA with Bonferroni correction; pⱕ0.05; n=5 insects for each time period). A sample record of bilateral N3c activity is shown in Fig. 2C. The decreased frequency of activity on the autotomized side is clearly seen. Thus, changes in N3c activity are correlated with the onset of rapid muscle degeneration.

Insects injected with prococene II plus solvent or solvent alone both demonstrated degeneration of M#133b,c 15 days post-autotomy. The percent of degeneration relative to control in both cases was similar to that found following autotomy alone (0.353±0.031 and 0.248±0.054 vs. 0.339±0.075; injections of prococene II and solvent alone vs. autotomy; Fig. 4; n=4–7 insects respectively). Thus, the corpora allata were not necessary for the induction of muscle degeneration following autotomy. 3.5. Ubiquitin-immunoreactivity is increased following autotomy Muscle sections from the unautotomized side continued to exhibit filamentous actin labeling with only background ubiquitin-immunoreactivity 15 days after autotomy (Fig. 5A,B). Sections from the autotomized side showed upregulation of ubiquitin-immunoreactivity

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Fig. 4. Precocene II injections did not affect the degree of muscle degeneration. Experimental animals were injected with precocene II and solvent, while control animals were injected with solvent only. Insects received injections 14 days prior to autotomy. Open bars show protein content of M#133b,c on the unautotomized side, filled bars show the autotomized side 15 days post-autotomy (n=4 control animals and 7 experimental animals; data are mean±SE). Precocene II, which ablated the corpora allata and thus inhibits juvenile hormone production, had no effect on the extent of muscle degeneration following autotomy.

fibers by 3 days post-autotomy (Fig. 5C; arrow) but no loss in muscle fiber number, since the fibers that showed increased ubiquitin-immunoreactivity continued to lightly label for filamentous actin (Fig. 5C,D; arrows). By 10 days post-autotomy muscle fiber loss was evident in all preparations, with areas of heavy ubiquitin-immunoreactivity labeling showing no labeling for filamentous actin (Fig. 5E,F; arrows). Further muscle fiber loss was seen on day 15, and large areas of muscle tissue that were unlabeled for filamentous actin were heavily labeled for ubiquitin-immunoreactivity (Fig. 5G,H). Even within individual fibers, areas of low filamentous actin labeling showed strong ubiquitin-immunoreactivity (Fig. 5G,H; arrows; n=3 insects at each time period). 3.6. Chromatin consolidation is present following autotomy

Fig. 3. Nerve 3c atrophy occurs after degeneration of M#133b,c is complete. (A) Cross-sections of nerve 3c from contained 10 motor axons (large profiles) and a few small sensory axons and no degeneration was evident 15 days post-autotomy. (B) Axons on the unautotomized side remained normal 25 days post-autotomy when degeneration of M#133b,c is complete; however, (C) degenerative changes were seen on the side of autotomy. These changes include misshapen axons, abnormal separation of neural and glial tissue, and dense staining of the neural lamella (n=3 animals for each time period; arrows; scale bar=25 µm).

Muscle sections stained with propidium iodide or hematoxylin–eosin were used to determine the percent of pyknotic nuclei following autotomy. Pyknotic nuclei are clearly evident as dark round nuclei in the fibers in these sections (arrows). The onset of PCD was non-uniform throughout a muscle resulting in a relatively low percent of pyknotic nuclei. Fig. 6A shows a single muscle fiber in which all nuclei were pyknotic, while other fibers within the same muscle contain only normal nuclei. Furthermore, the onset of PCD was not uniform even within a single muscle fiber, since some fibers were found to contain normal nuclei at one end of the fiber and only pyknotic nuclei at further points along the fiber (Fig. 6B). Note that the fiber diameter is decreased in the region with pyknotic nuclei. The percent of pyknotic

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Fig. 5. Ubiquitin-immunoreactivity is upregulated following autotomy, while F-actin is reduced in the same fibers. Internal-control muscle 15 days post-autotomy double labeled for F-actin (A) and ubiquitin-immunoreactivity (B). Ubiquitin-immunoreactivity is increased in a few muscle fibers 3 days post-autotomy (D). Labeling for F-actin is also reduced in these same muscle fibers (C; arrows). Muscle fiber loss is present 10 days post-autotomy and areas with no F-actin labeling (E) show heavy ubiquitin-immunoreactivity (F; arrows). Increased ubiquitin-immunoreactivity is seen in most muscle fibers 15 days post-autotomy (H), while these same fibers show no F-actin labeling (G). Even within individual fibers, areas without F-actin labeling show heavier ubiquitin-immunoreactivity (arrows, bar=125 µm).

nuclei (filled circles) was increased over internal control samples (open circles) in all animals 10 and 15 days post-autotomy (Fig. 6C). Significant increases were found for each time period (day 10, 5.6±1.9 vs. 0.3±0.2; day 15, 3.6±1.0 vs. 0.3±0.2; autotomized side vs. internal-control; mean±SE; one-tailed t-test; pⱕ0.05; n=3 insects at 10 and 15 days). Between 580 and 1500 nuclei were counted per group. For example, 21 pyknotic nuclei were counted in M#133b,c preparations 15 days post-autotomy out of a total of 580 nuclei.

3.7. TUNEL labeling for DNA fragmentation is present following autotomy Positive TUNEL labeling was found in a few muscle nuclei 3 days post-autotomy (Fig. 7B; green nuclei; arrows) even though little muscle degeneration was evident (note presence of fiber striations and absence of pyknotic nuclei). Muscle sections from internal-control fibers showed no TUNEL positive nuclei, only normal nuclei labeled with propidium iodide (red) was seen (day

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Fig. 6. The onset of programmed cell death is not uniform within M#133b,c. (A) Pyknotic nuclei, evident as dark round circles (arrows), are present in longitudinal sections of M#133b,c stained with hematoxylin–eosin. Ten days post-autotomy, only one muscle fiber from this section contains pyknotic nuclei while all other fibers, some of which are striated, contain normal nuclei indicating that the onset of PCD is fiber specific. Thus, the % of pyknotic nuclei was relatively low. (B) The onset of PCD is variable within individual muscle fibers, as shown by one fiber which appears normal at one end, but suddenly becomes narrower with pyknotic nuclei at the other end (scale bar=250 µm). (C) The % of pyknotic nuclei is significantly increased in muscle #133b,c sections from the side of autotomy (filled circles) compared with sections from the internal-control side (open circles) in all animals at both 10 and 15 days post-autotomy (single-tailed unpaired t-test; n=3 for both time periods; p⬍0.05).

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Fig. 7. Positive TdT-mediated dUTP nick end labeling (TUNEL) is present in muscle #133b,c following autotomy. (A) Muscle sections from fibers on the internal-control side did not demonstrate TUNEL labeling, only normal propridium iodide labeled nuclei (gray) are present. (B) By 3 days post-autotomy, little muscle degeneration has occurred (note fiber striations) although positive TUNEL labeling (white nuclei; arrows) is already present. Pyknotic nuclei are not seen. (C) By 15 days post-autotomy, condensed rounded pyknotic nuclei are present and some of these nuclei are positive for TUNEL labeling (white nuclei; arrows). Scale bar=250 µm.

15; Fig. 7A). By 15 days post-autotomy, severe degeneration was present and positive TUNEL labeling was usually found in round pyknotic nuclei (Fig. 7C; green nuclei; arrows; n=3 insects for each time period).

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4. Discussion 4.1. Evidence for a neuronal trigger The trigger for muscle degeneration following autotomy appears to be the loss of normal proprioceptive input to the motor neurons innervating the affected thoracic muscles. Arbas and Weidner (1991) showed that muscle degeneration associated with autotomy was the result of severing N5. Here, we demonstrate that distal amputations of the tibia do not induce muscle degeneration, but amputations through the proximal tibia lead to muscle degeneration of thoracic musculature. Since N5 is the only nerve in the leg which is damaged during autotomy and it carries both efferent and afferent information to/from the distal parts of the leg, this suggests that the more proximal cut removes or damages some particular features innervated by N5 that are necessary to induce muscle degeneration. Grasshoppers have no muscles in the tarsus. The tibia, however, has four, the tarsal depressor and levator muscles and two parts of the retractor unguis (pretarsal depressor) muscle that attaches to a long apodeme. The mid-tibial amputation removed three of these units, while the proximal cut commonly damaged the more proximal unit of the retractor unguis. In Schistocerca gregaria, the tarsal levator muscle is innervated by a single motor neuron, the tarsal depressor probably by three and the retractor unguis by two motor neurons presumably supplying all three units of the muscle (the third is in the femur). All the muscles are innervated by the same two inhibitory neurons (common inhibitors 2 and 3; Burrows, 1996). It is very likely that the muscles in the hindleg of B. humphreysii have a similar innervation. Thus, both the long and short tibial cuts damage the same small number (probably 8) of efferent neurons making it unlikely that damage to the efferent supply to the legs provides the trigger for muscle degeneration, since only the proximal amputation led to muscle degeneration. The afferent axons in N5 come from exteroceptors and proprioceptors on the leg. In Schistocerca gregaria, and probably in all grasshoppers, there are three types of exteroceptors on the hind tarsus: trichoid mechanoreceptors each with a single neuron, canal sensilla on the tarsal pads with a single neuron and probably also mechanoreceptors, and small uniporous sensilla, usually with five neurons, with a combined mechanical and chemical function (Kendall, 1970). On the third-stage larva of S. gregaria, roughly equivalent in size to an adult B. humphreysii, there are about 230 sensilla on the fore tarsus with a smaller number on the hind tarsus (Kendall, 1970). Based on Kendall’s data, perhaps 40 would be expected to be uniporous sensilla so that the overall exterosensory input from the hind tarsus would derive from about 300 neurons. Smaller numbers of

trichoid and uniporous sensilla are present on the tibia, becoming less abundant towards the proximal end. Thus the progressively more proximal cuts made in our experiments would have involved removal of progressively greater amounts of exterosensory input, but with no qualitative difference in inputs from the proximal and mid-cuts of the tibia. This is also the case with proprioceptive inputs. Fig. 1A shows the distribution of proprioceptive sensilla on the hindleg of a melanopline grasshopper base on data from Melanoplus sanguinipes (McFarlane, 1953) and our own unpublished data on B. humphreysii. Two types of proprioceptive sensilla are present: chordotonal organs within the leg and campaniform sensilla in the cuticle. In the distal tarsomere, there are two small chordotonal organs with a total of five sensory neurons; distally in the tibia is a single organ with three neurons, while proximally the two parts of the subgenual organ contain between 15 and 22 sensory neurons. There is one campaniform sensillum on the basitarsus, another distally on the tibia and a total of six at the proximal end of the tibia. The tarsal cut has removed the input from six proprioceptive neurons and the mid-tibia cut added a further four. The proximal tibia amputation, however, removed or damaged the subgenual organ and five of the proximal campaniform sensilla. The differences between the proximal and mid-tibial cuts are that the latter removes the input from a somewhat larger number of mechanical and chemosensory exteroceptive neurons and some additional proprioceptive neurons. Whereas the mid-tibial amputation severs the axons of 10 proprioceptive neurons, the proximal amputation probably increases this number to at least 25 and includes loss or severe damage to the subgenual organ, an important proprioceptor in most insects. These proprioceptors, unlike the exteroceptors, are more likely to have monosynaptic connections with the motor neurons to the affected thoracic muscles (reviewed by Burrows, 1992), and we suggest that the loss of inputs from proprioceptors below the knee joint provides the trigger for muscle degeneration. The extent of degeneration 15 days following the proximal tibial amputation was less than that following autotomy. However, the degree of muscle degeneration following proximal amputation was significant, similar to that seen 10 days after autotomy-alone when M#133b,c cross-sectional area is reduced by 80% (Personius and Arbas, 1998) and when we have shown that multiple markers of PCD are present. Furthermore, significant electrophysiological changes similar to those seen 10 days after autotomy-alone were seen. Thus, the rate of muscle degeneration may be slower following proximal tibial amputation, or perhaps only the first phase of muscle degeneration (muscle atrophy) is activated and the final degenerative phase, involving PCD, requires further ablation of sensory inputs. Autotomy

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severs N5 at the coxo-trochanteral joint while the nerve cuts made by Arbas and Weidner (1991) were proximal to this. These more proximal cuts would have involved considerable increases in removal of sensory inputs, but perhaps most notably would remove input from the large chordotonal organ in the femur and Brunner’s organ. Perhaps this more extensive removal of proprioceptors is responsible for initiating a full degenerative response. 4.1.1. Role of neuronal activity in muscle degeneration If loss of proprioceptive input is the trigger for muscle degeneration, it must presumably act via the motor neurons supplying the muscles. In vertebrate neuromuscular systems both muscle contraction itself and activitydependent trophic factor release have been shown to regulate muscle degeneration. For example, blocking excitation–contraction coupling prevents the maintenance of primary muscle fibers (Ashby et al., 1993b), while glial growth factor 2, a member of the neuregulin family of trophic factors, has been shown to increase muscle fiber survival. Release of neuregulin at the neuromuscular junction is thought to be activity dependent (Trachtenberg, 1998). Survival of muscle fibers in the grasshopper following autotomy also appears to be dependent upon adequate neuronal activity, since we found that the reduction in N3c ensemble activity is correlated with multiple markers of PCD (Figs 2, 5–7). Surprisingly, the degree of muscle degeneration follow direct denervation of M#133b,c and autotomy was similar, even though neuromuscular junction efficacy is intact follow autotomy (Fig. 1B; Personius and Arbas, 1998). This suggests that neural activity is more important than nerve contact in the maintenance of muscle in this system. Whether muscle contraction itself or activity-dependent trophic factors release is ultimately responsible for the maintenance of muscle following autotomy is unclear. Evidence for trophic factors acting as modulators of neuromuscular junction plasticity in Drosophila is well documented (Davis and Goodman, 1998; Davis, 2000; Paradis et al., 2001), but their role in maintenance remains unclear. Neuronal degeneration, however, is not involved in triggering autotomy-induced muscle degeneration. We show here that cross-sections of N3c are normal in appearance 15 days post-autotomy when M#133b,c has undergone significant degeneration. Changes in N3c histology were only seen 25 days post-autotomy when muscle degeneration is complete (Fig. 3). In addition, synaptic failure is only seen in highly degenerated muscle fibers (Personius and Arbas, 1998). Thus, as seen in M. sexta, neural degeneration follows the degeneration of muscle (Hegstrom and Truman, 1996). Muscle degeneration following autotomy is not triggered by increases in juvenile hormone titer. Precocene II injections, which ablate the corpora allata, have no effect on the degree

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of muscle degeneration following autotomy. The corpora allata are the only known source of juvenile hormone. Thus, the trigger for muscle degeneration following autotomy is unlike that producing muscle degeneration after metamorphosis or flight where humoral changes are involved. This is in keeping with the fact that these other instances of degeneration are components of normal development occurring at specific times when morphogenetic hormones are active. Autotomy, by contrast, may occur at any time during development or adult life. 4.2. Evidence that muscle degeneration occurs by PCD The experimental sections through the mid- and proximal tibia unloaded M#133b,c, a depressor of the hindlimb, since the insects no longer took weight on the hindlimb following these amputations. Muscle degeneration, therefore, could have been a result of disuse. Our experimental results, however, argue against this possibility. First, M#118 was found to degenerate following severing of the proximal tibia even though this muscle was not unloaded since it elevates and promotes the hindlimb. Second, disuse atrophy is not associated with electrophysiological changes (Atwood et al., 1978), but M#133b,c demonstrated electrophysiological changes after the proximal tibia was severed. Third, even though both sections through the tibia would unload M#133b,c, only the most proximal section induced muscle degeneration. These results agree with those of Arbas and Weidner (1991) who showed that experimental reweighting of the hindlimb did not prevent muscle degeneration. Multiple markers for PCD (i.e. upregulation of ubiquitinimmunoreactivity, positive TUNEL labeling and chromatin condensation) demonstrate that muscle degeneration occurs by PCD (Figs. 5–7). These indicators of PCD peaked at the time of muscle fiber loss and rapid degeneration that occurs about 10–15 days post-autotomy (Personius and Arbas, 1998). The onset of M#133b,c degeneration is protracted over several days, since some fibers contained only pyknotic nuclei while other fibers within the same muscle contained only normal nuclei (Fig. 6). Furthermore, positive TUNEL labeling and ubiquitin-immunoreactivity is seen over a course of several days, unlike muscle degeneration following eclosion in Manduca sexta, which is tightly controlled by ecdysone receptor expression and steroid titer (Hegstrom et al., 1998). Since the occurrence of autotomy is unpredictable, it might be supposed that the associated muscle degeneration would occur in an uncontrolled manner as a result of wastage following disuse. Degeneration of insect muscle, in general, is presumed to contribute to the nutrient pool available to the insect. Perhaps programmed cell death, which occurs following autotomy as well as in other regular instances of muscle degeneration, makes the resources of the muscle available in a form more readily utilizable than would otherwise be the case.

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Acknowledgements This work is dedicated to Dr E.A. Arbas. The authors would like to thank Drs A.S. Clinton, J. Belanger, R.B. Levine, D.G. Stuart and A.J. Yool for reading earlier versions of this manuscript. P. Jansma provided excellent technical assistance. This work was supported in part by an APTA Neurology Section Scholarship to K.E.P.

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