Gary T. Patterson

MAJOR PHYSIOLOGIC AND HISTOCHEMICAL CHARACTERISTICS OF INHERITED DYSTROPHY OF THE CHICKEN

Inherited muscular dystrophy of the chicken was first described more than 20 years ago.’ Since then. several genetic lines have been developed, some have even become extinct, and many studies have been undertaken of the morphologic, physiologic, and biochemical properties of muscles of birds with the disorder. Early research on the abnormality has been reviewed by Julian and Asmundson2 and Asmundson et ~ 1 . ~ This paper discusses some of the major characteristics of the dystrophic chicken that occur in the several lines developed and maintained at the University of California, at Davis, and describes how these properties have been investigated in our laboratory.

Distribution of acetylcholinesterase activity in normal, dystrophic, and denervated muscles of the chicken

Abstract One distinctive property of denervated and genetically dystrophic muscles of chickens is high acetylcholinesterase activity found in the fibers. The distribution of AChE activity in single fibers from these muscles was studied by using fresh frozen serial sections, a specific histochemical stain, and photodensitometry. The results confirm the findings of a previous report (22) that high AChE activity occurs only around the neuromuscular junction region in fibers from biceps muscles of 6-week-old dystrophic chickens. In contrast, both normal and dystrophic biceps muscle of 6-week old chickens that had been denervated for 5 days exhibited high AChE activity throughout the length of the fibers. These results suggest that changes in AChE activity due to denervation are superimposed on the activity already present in dystrophy. The data support the idea that inherited muscular dystrophy in the chicken involves a specific defect in the regulation of AChE and perhaps other molecules around the neuromuscular junction.

Righting ability and skeletal muscle properties of phenytoin-treated dystrophic chickens.

Abstract Chickens with an inherited muscular dystrophy (line 413) and genetically related normal controls (line 412) were treated with phenytoin (diphenylhydantoin, DPH) on Days 1 through 40 ex ovo . DPH (20 mg/kg, intraperitoneally, b.i.d.) and a combination of DPH and intermittent “exercise” (twice-daily testing of righting ability) beneficially affected muscle function, morphology, and biochemistry. DPH dramatically improved the righting ability of dystrophic chicks, and the exhaustion scores of many treated birds were still as high as those of normal chicks after 30 days of treatment. Muscles from chicks killed after 27 to 40 days of treatment had decreased fiber diameters, increased lactic dehydrogenase (LDH) activity, and reduced acetylcholinesterase and butyrylcholinesterase activities. The effects of DPH were more pronounced in the posterior latissimus dorsi than in the pectoral muscle and were potentiated by exercise. Exercise alone produced a transient increase in righting ability and increased LDH activity of dystrophic muscles. The abnormal, rounded appearance of dystrophic muscle fibers was not altered by treatments. Research reported elsewhere showed that DPH partially corrected abnormal electromyographic activity of dystrophic chicken muscles. The data presented here show that other symptoms of avian dystrophy are also alleviated by DPH and suggest that abnormal membrane activity plays an important role in this myopathy.

Distribution of extrajunctional acetylcholinesterase in muscle of normal and dystrophic chickens

Abstract A specific histochemical staining of serial cross sections of frozen muscle samples for AChE activity was performed to investigate the distribution of sarcoplasmic AChE activity and its relationship to the motor end plate in individual muscle fibers of 1-, 2-, and 6-week-old normal and dystrophic chickens. A photographic cytophotometric technique was used to determine AChE activity. There were no differences between normal and dystrophic muscle fibers in the distribution or level of AChE activity at both 1 and 2 weeks of age. By 6 weeks, AChE activity had spread to either side of the motor end plates for approximately five times the distance found in normal fibers. In addition, the level of AChE activity had almost tripled in dystrophic fibers in comparison to normal fibers. These findings suggest that dystrophic chicken muscle develops similarly to normal muscle with respect to AChE localization and level of activity for at least 2 weeks following hatching, and then AChE spreads along the muscle fiber from the motor end plate. The data are consistent with the idea that there is a myogenic defect in the maturation of AChE regulation associated with the motor end plate of dystrophic chickens.

Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles

Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.

Phenytoin, methysergide, and penicillamine in hereditary muscular dystrophy of the chicken

Abstract Chickens with an inherited muscular dystrophy (line 413) were treated from days 1 through 90 ex ovo with either phenytoin, methysergide, or d -penicillamine. Under the conditions of the study, phenytoin and methysergide were equally effective in improving the righting ability of dystrophic birds, but penicillamine had little or no effect. Phenytoin and penicillamine decreased the abnormally high creatine kinase activity in dystrophic plasma, but only phenytoin reduced the high acetylcholinesterase activity in plasma and fast-twitch muscles of dystrophic birds. Beneficial effects of methysergide and phenytoin were also shown in trials as short as 25 days. None of the treatments, improved the abnormal histological appearance of dystrophic muscles.

Baclofen, procainamide, verapamil, and prenylamine in hereditary muscular dystrophy of the chicken

Abstract Two “myotonia antagonists” (baclofen and procainamide) and two “calcium antagonists” (verapamil and prenylamine) were evaluated for their effects on hereditary muscular dystrophy of the chicken. Righting ability of dystrophic chicks was improved only by baclofen and procainamide. Plasma creatine kinase activity of dystrophic chicks was reduced 45% after chronic administration of baclofen, but was still nearly nine-fold greater than activity in plasma of normal chicks. Baclofen and verapamil both reduced acetylcholinesterase activity in pectoralis major muscles of dystrophic chicks by about 40%, but these values were still significantly greater than those measured in muscles of normal chicks. The data provide further support for the concept that impaired righting ability of young dystrophic chickens is associated with the presence of myotonia in affected muscles, but do not rule out the possibility that certain of the biochemical features of the avian dystrophy may involve calcium.

Serum regulation of acetylcholinesterase in cultured myotubes

A large (20S) collagen-tailed form of acetylcholinesterase associated with the neuromuscular junction appears in cultures of chick embryo muscle cells when horse serum is withdrawn from the medium. In this report, 10-day-old cultures were incubated 2 days in serum-free medium or in medium containing either horse, bovine, fetal calf, chicken, heat-treated horse or chicken serum, low (less than 100K) or high (less than 100K) molecular weight fractions of horse serum, or fibronectin. Total acetylcholinesterase activity and activity of the 20S form increased in medium without serum, with fetal calf serum and with the low-molecular-weight fraction of horse serum. The largest increase occurred with fibronectin. The results suggest that a factor(s) greater than 100K in adult sera inhibits total acetylcholinesterase production and formation of the 20S form of the enzyme.

Water deprivation: Beneficial effect on muscular dystrophy in chickens

Chicks affected with hereditary muscular dystrophy were deprived of water for 1 to 4 days at ages to 37 days ex ovo. Water deprivation partially alleviated impaired righting ability and reduced the typically elevated plasma creatine kinase activity by as much as 90%. Muscles from water-deprived chicks showed several qualitative histologic improvements, including decreased sarcoplasmic staining for acetylcholinesterase activity, reduced fiber diameters, and a decreased incidence of abnormally large rounded fibers, but retained the high degree of fiber diameter variability characteristic of dystrophic muscles. Feed deprivation reduced body weight to a similar extent as water deprivation but had lesser effects on creatine kinase activity and did not improve righting ability or muscle histology. Although the mechanism of the improvements is unknown, the magnitude and scope of the effects suggest that water deprivation beneficially alters a major abnormality in dystrophic chickens.