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A comparative morphological study of muscle spindles in the avian anterior and posterior latissimus dorsi… Hatfield, Linda Jean 1981

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A COMPARATIVE MORPHOLOGICAL STUDY OF MUSCLE SPINDLES IN THE AVIAN ANTERIOR AND POSTERIOR LATISSIMUS DORSI MUSCLES by LINDA JEAN HATFIELD B.Sc.R(P.T.), The Univ e r s i t y of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of ANATOMY) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1981 (c) Linda Jean H a t f i e l d , 1981 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 11 /IQ^ ABSTRACT A study of muscle spindles in two s y n e r g i s t i c avian muscles was undertaken to determine whether morphological or q u a n t i t a t i v e differences existed between muscle spindles r e s i d i n g i n a slow-red (tonic) muscle and a fast-white (twitch) muscle. The avian a n t e r i o r (ALD) and p o s t e r i o r (PLD) latissimus d o r s i muscles were chosen since they are unique among vertebrates as paradigms of a slow-red and a fast-white muscle r e s p e c t i v e l y . S e r i a l frozen sections of muscle were stained with Haematoxylin and Eosin or Gomori triehrome and muscle spindles r e s i d i n g in the ALD were assessed and compared with those i n the PLD with regard to organization, d i s t r i b u t i o n and density, Contents of muscle spindles were examined for i n t r a f u s a l f i b r e s i z e , number and morphology. Attention was also d i r e c t e d to the r e l a t i o n s h i p between muscle spindles and the surrounding extrafusal muscle i n which they were located. Differences were found between muscle spindles r e s i d i n g in the two muscles. In the slow ALD, muscle spindles were r e l a t i v e l y evenly d i s t r i b u t e d , whereas in the fast PLD, they were concentrated around the single nerve entry point into the muscle. The ALD muscle spindle index was the highest yet published for chicken muscle and was 2.3 times higher than that of i t s fast counterpart. A bimodal trend in i n t r a f u s a l f i b r e diameter was noted i n the ALD, and a trimodal trend was found i n the PLD. The former had 42% fewer i n t r a f u s a l f i b r e s than the l a t t e r . Muscle spindles were shorter i n the ALD, with an average length of 1.9mm compared with 2.3mm i n the PLD. An i n t e r e s t i n g feature of the slow muscle was the monofibril muscle spindle, containing a s i n g l e i n t r a f u s a l f i b r e . With a few exceptions, ALD muscle spindles were located within the i n t e r f a s c i c u l a r perimysium close to a neurovascular trunk. PLD muscle spindles were r a r e l y seen i n these areas but were frequently found within a muscle f a s c i c l e , surrounded by c l o s e l y apposed extrafusal f i b r e s . Moreover, neurovascular trunks were less frequently seen i n the PLD. As an adjunct to t h i s study, three ALD-PLD p a i r s from the Storrs Connecticut s t r a i n o f muscular dystrophic chickens were also examined to compare muscle spindles i n these muscles with those of normal animals. In the PLD, which i s known to e x h i b i t early and progressive pathological change, muscle spindles appeared r e l a t i v e l y normal u n t i l marked extrafusal f i b r e degeneration had occured. By t h i s time evidence of muscle spindle involvement included capsular hypertrophy and i n t r a f u s a l f i b r e s p l i t t i n g . Whereas the slow ALD has been reported to r e t a i n apparent normalcy i n muscular dystrophy, subtle changes were seen i n some of the muscle spindles examined. These included an increase i n number of i n t r a f u s a l f i b r e s per muscle spindle compared with those i n the normal. iv TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF ILLUSTRATIONS ACKNOWLEDGEMENTS INTRODUCTION SCOPE AND AIMS OF THE PRESENT INVESTIGATION MATERIALS AND METHODS RESULTS TABLES ILLUSTRATIONS DISCUSSION REFERENCES LIST OF TABLES Tables 1 Number and density of muscle spindles i n s i x normal ALD-PLD muscle pa i r s 2 Range of i n t r a f u s a l f i b r e s per muscle spindle i n s i x normal ALD-PLD muscle pa i r s 3 Comparison between dystrophic and normal ALD and PLD muscles v i LIST OF ILLUSTRATIONS Figures P a g e 1 Frozen sections of normal ALD and PLD muscles 28 2 Longitudinal reconstruction of an ALD and a PLD 30 3 Histograms of muscle spindles per muscle cross-section 31 4 Transverse reconstruction of an ALD and a PLD 32 5 Frozen sections of polar regions of ALD and PLD muscle spindles 33 6 Frozen sections of juxta-equatorial regions of ALD and PLD 35 muscle spindles 7 Frozen sections of equatorial regions of ALD and PLD muscle 37 spindles 8 Frozen sections of conjunctive and monofibril muscle spindles 39 i n the ALD •9 Graph depicting number of i n t r a f u s a l f i b r e s per ALD and PLD 41 muscle spindle 10 Size histogram of ALD and PLD i n t r a f u s a l f i b r e s 42 11 Size histogram of ALD and PLD extrafusal f i b r e s 43 12 Frozen sections of equatorial regions of ALD and PLD muscle 44 spindles 13 P a r a f f i n sections of ALD and PLD muscle spindles 46 14 Frozen sections of muscular dystrophic PLD including muscle 48 spindles 15 Frozen sections showing i n t r a f u s a l f i b r e abnormalities i n ' 50 dystrophic muscle spindles v i i ACKNOWLEDGEMENTS To Dr. W. K. Ovalle, my supervisor during t h i s period of study, I extend sincere thanks. His ongoing i n t e r e s t i n my research, and h i s provision of guidance and encouragement were greatly appreciated. The advice and assistance given by the other members of my committee, Dr. P. R. Dow and Dr. K. Donnelly, are also appreciated. My thanks are extended to Ms Susan Shinn for continued technical assistance throughout. Her willingness to share her time and expertise were important i n the completion of t h i s research. My thanks are also extended to the s t a f f of the Anatomy Department, e s p e c i a l l y to Mr. B. Cox, during h i s t o l o g i c a l preparations. F i n a l l y , the support and technical help provided by my husband James B. H a t f i e l d were instrumental i n the completion of t h i s study. 1 INTRODUCTION Muscle spindles are complex sensory receptors situated between or within f a s c i c u l i of vertebrate s k e l e t a l muscles. They contain several components: small s p e c i a l i z e d i n t r a f u s a l f i b r e s enclosed by an outer and inner capsule, and both a sensory and a motor innervation. The sensory nerve endings supply the equatorial and juxta-equatorial regions of the muscle spindle, s p e c i f i c a l l y the i n t r a f u s a l f i b r e s , and the motor nerves supply the c o n t r a c t i l e poles of these muscle c e l l s . In the equatorial region, the capsule i s separated from the i n t r a f u s a l f i b r e s and t h e i r sensory nerve endings by a f l u i d - f i l l e d p e r i a x i a l space. The structure and function of mammalian muscle spindles have been studied i n d e t a i l . Notable reviews have been published concerning the r e l a t i o n s h i p between the cen t r a l nervous system and the muscle spindle (Granit, 1970; Matthews, 1972), the components of the muscle spindle and t h e i r functions (Barker, 1974), c h a r a c t e r i s t i c s of i n t r a f u s a l f i b r e s and t h e i r innervation (Boyd, 1962) and the functional morphology of i n t r a f u s a l f i b r e s (Smith and Ovalle, 1972). The majority of studies on muscle spindle morphology and function have been conducted on mammalian muscles, p a r t i c u l a r l y i n the cat. Some information i s a v a i l a b l e on amphibian and r e p t i l i a n species (Barker, 1974), but there i s r e l a t i v e l y l i t t l e on avian muscle spindles, and the degree to which they resemble or d i f f e r from those of other vertebrates. In addition, there i s a s c a r c i t y of data on whether muscle spindles r e f l e c t c h a r a c t e r i s t i c s of 2 the surrounding extrafusal muscle. In a l i g h t microscopic study of the chicken gastrocnemius muscle, De Anda and Rebollo (1967) found that avian muscle spindles were s i m i l a r i n o v e r a l l structure to those i n other species, containing a capsule, a p e r i a x i a l space, i n t r a f u s a l f i b r e s i n varying number, c a p i l l a r i e s and nerve endings. Moreover, p o s i t i o n r e l a t i v e to the surrounding extrafusal f i b r e s was found to be p a r a l l e l , as i n mammalian and amphibian species. Three types of i n t r a f u s a l f i b r e s were observed i n t h i s muscle, based on f i b r e s i z e , arrangement of m y o f i b r i l s as i d e n t i f i e d with ferric-haematoxylin, and nuclear configuration i n the equatorial regions. Large-diameter i n t r a f u s a l f i b r e s extended beyond the capsule, and frequently inserted on the perimysium of an adjacent extrafusal f i b r e . Small-diameter f i b r e s frequently ended within the capsule. An intermediate s i z e of i n t r a f u s a l f i b r e s was also i d e n t i f i e d , however not a l l groups were always seen i n each muscle spindle. One or two types of sensory nerve reached the spindle u n i t s . Those having a large diameter gave o r i g i n to the primary endings on large and intermediate i n t r a f u s a l f i b r e s , and t h i n sensory nerve f i b r e s gave o r i g i n to the primary endings on small i n t r a f u s a l f i b r e s . These workers found no evidence of annulo-spiral endings, but described primary endings with wide synaptic contact, and s i m i l a r but less complex and extensive endings to the small i n t r a f u s a l f i b r e s . No secondary endings were found innervating these muscle spindles. Two types of motor nerves were observed; thick f i b r e s supplied large-diameter i n t r a f u s a l f i b r e s at the polar regions, with endings c h a r a c t e r i s t i c of motor end-plates, and thinner nerve f i b r e s to small i n t r a f u s a l f i b r e s , with simpler endings. 3 A histochemical analysis of the same muscle (Rebollo and De Anda, 1967) further confirmed the presence of three kinds of avian i n t r a f u s a l f i b r e s . Small-diameter f i b r e s contained large amounts of glycogen i n the polar regions, and large-diameter f i b r e s had intense phosphorylase a c t i v i t y and v a r i a b l e glycogen content. Intermediate f i b r e s were negative f o r phosphorylase with v a r i a b l e glycogen content. A l l i n t r a f u s a l f i b r e s showed increased metabolic a c t i v i t y i n the equatorial region, associated with t h e i r primary sensory innervation and more dense nucleation. Comparisons between muscle spindles i n the s a r t o r i u s and adductor profundus muscles of four avian species by Maier and Eldred (1971) showed s i m i l a r i t i e s i n nuclear aggregation pattern i n i n t r a f u s a l f i b r e s . The "bag" arrangement of n u c l e i present i n mammalian muscle spindles, and described i n the b i r d by De Anda and Rebollo (1967), was not observed by these workers. They found no bimodal or trimodal d i s t r i b u t i o n of i n t r a f u s a l f i b r e diameters, nor a consistent pattern of nuclear aggregation. In a subsequent study on the muscle spindles i n the f l e x o r carpi u l n a r i s of the pidgeon (Maier, 1977), a c l a s s i f i c a t i o n of i n t r a f u s a l f i b r e s by comparing siz e with i n t e n s i t y of the m y o f i b r i l l a r ATPase reaction was attempted. No constant s i z e r e l a t i o n s h i p was found to correspond with i n t e n s i t y of s t a i n i n g and i t was suggested that f i b r e measurement was of l i m i t e d value i n c l a s s i f y i n g i n t r a f u s a l f i b r e s of avian muscle spindles. In an electron microscopic study of the budgerigar s a r t o r i u s muscle, James and Meek (1973) confirmed previous l i g h t microscopic studies on the avian muscle spindle, i n d i c a t i n g that t h e i r o v e r a l l structure was s i m i l a r to those of mammalian species. No u l t r a s t r u c t u r a l features were 4 found to suggest the presence of more than one type of i n t r a f u s a l f i b r e , nor were nuclear bags seen. These findings were i n accord with those of Maier and Eldred (1971) and i t was suggested that the degree of overlap of myonuclei r e f l e c t e d d i f f e r e n t f i x a t i o n techniques. The outer capsule of the muscle spindle was considerably thinner than that of small mammals, but marked development of the inner capsule was observed. Each i n t r a f u s a l f i b r e was surrounded by layers of f l a t t e n e d e p i t h e l i a l c e l l s . C a p i l l a r i e s were occa s i o n a l l y observed within the p e r i a x i a l space at i t s maximum diameter, and were separated from the i n t r a f u s a l f i b r e s by inner capsule c e l l s . No fenestrations were seen i n the c a p i l l a r y endothelia.Irregular Z l i n e s of the m y o f i b r i l s and t h e i r lack of M bands, together with a r e l a t i v e l y sparse T system suggested that they undergo either slow-twitch or even tonic contractions. This would i n d i c a t e that the i n t r a f u s a l f i b r e s of these avian muscle spindles resembled mammalian nuclear bag f i b r e s more than nuclear chain f i b r e s as evidence pointed to a slower rate of contraction i n the former than the l a t t e r . In an u l t r a s t r u c t u r a l study of avian muscle spindles i n the f l e x o r and extensor h a l l u c i s longus, Adal (1973) i d e n t i f i e d three kinds of i n t r a f u s a l f i b r e s based on myofilament arrangements , siz e and number of mitochondria, and amount of sarcoplasmic reticulum. While no segregation of i n t r a f u s a l f i b r e s into "bag" and "chain" types was seen, i t was suggested that these muscle c e l l s might be c l a s s i f i e d according to the degree to which t h e i r f i n e structure resembled the bag and chain f i b r e s of mammalian muscle spindles. No morphological d i f f e r e n c e was demonstrated between i n t r a f u s a l f i b r e s i n the f l e x o r from those i n the 5 extensor h a l l u c i s longus muscles. The monofibril muscle spindle, containing only one i n t r a f u s a l f i b r e , was not found i n e i t h e r muscle, but was mentioned by Maier and Eldred (1971) as being found frequently i n avian muscles. Muscle spindles of snakes and l i z a r d s also contain only one i n t r a f u s a l f i b r e (Fukami, 1970; P a l l o t and Taberner, 1973). The f i n e structure of the muscle spindle capsule i n the a n t e r i o r and p o s t e r i o r latissimus d o r s i muscles of the chicken (Ovalle, 1976) was found to consist of an outer portion, composed of multilayered f l a t t e n e d e p i t h e l i a l c e l l s d i r e c t l y continuous with the perineurium, as found i n other species (Shantha and Bourne, 1968). It has been suggested that the capsule plays a r o l e as a metabolically a c t i v e d i f f u s i o n b a r r i e r to the entrance of substances from the external milieu. The inner capsule was implicated i n the active synthesis of the amorphous and f i b r i l l a r material occupying the p e r i a x i a l space, i n addition to the r o l e of p r o t e c t i n g the sensory nerve endings on the i n t r a f u s a l f i b r e s (Ovalle, 1978). Studies on vertebrate muscles have indicated a c o r r e l a t i o n between muscle spindle density, muscle f i b r e type and muscle function. Richmond and Abrahams (1975a; 1975b) found higher muscle spindle indices i n muscles histochemically i d e n t i f i e d as slow-contracting than i n those found to be f a s t - c o n t r a c t i n g . In addition, muscle spindles were more numerous i n i n regions r i c h i n slow extrafusal f i b r e s than i n areas containing mainly f a s t f i b r e s . In a subsequent study on the proprioceptive components of the cat neck, Richmond and Abrahams (1979) discovered high muscle spindle d e n s i t i e s i n the muscles subserving head and neck movements. These workers also found that muscle spindles were 6 even more numerous i n the small p e r i v e r t e b r a l muscles, where they were frequently oriented i n chains between intramuscular tendons, or clustered i n large complexes of 3-10 spindles. Muscle spindles were also found i n large numbers associated with myotendon junctions. (Richmond and Abrahams, 1979), Bridgman et a l (1962) and Lennartsson (1979) have also shown that mammalian muscles performing finely-co-ordinated movements, muscles of j o i n t s t a b i l i z a t i o n and those containing predominantly slow f i b r e s have a r e l a t i v e l y high muscle spindle index. Adal and Chew Cheng (1980) r e c e n t l y examined the number, d i s t r i b u t i o n and density of muscle spindles i n two dorsal wing muscles of the domestic duck. Although t h e i r sample was.small, a r e l a t i v e l y high spindle index was found i n both muscles, supporting the observations of Maier and Eldred (1971) and James and Meek (1973) that avian muscles frequently have a higher spindle index than i s found i n mammalian species. There has been a growing i n t e r e s t i n the avian a n t e r i o r (ALD) and p o s t e r i o r (PLD) latissimus d o r s i muscles during the l a s t twenty years, as they are unique paradigms of vertebrate s k e l e t a l muscles. It i s known that- these two muscles d i f f e r s t r i k i n g l y i n t h e i r development (Syrovy and Gutmann, 1967; Wilson et a l , 1973; Zelena and Sobotkova, 1973), p h y s i o l o g i c a l properties (Ginsborg, 1960a; 1960b; Hnik et a l , 1967; Fedde, 1969; Srihara and Vrbova, 1978; Vrbova et a l , 1978), morphology (Hess, 1961; Page, 1969; Hess, 1970) and histochemical and biochemical features (Reasons and Hikida, 1973; Wilson et a l , 1973; Ovalle, 1978), Moreover, the unique pattern of innervation of the slow, tonic ALD and the fa s t , twitch PLD has also been demonstrated (Ginsborg, 1960a; 1960b; 7 Hess, 1961). The c o n t r a c t i l e properties of the ALD and PLD d i f f e r remarkably both i n speed and maintenance of contraction. At 13-16 days of incubation each muscle develops tension and relaxes slowly, but by the 17th day an increase i n PLD contraction speed i s detectable, and t h i s trend continues u n t i l hatching (Gordon and Vrbova, 1975). S e n s i t i v i t y to a c e t y l c h o l i n e i s also s i m i l a r for the two muscles during early embryonic development i n that the muscle f i b r e s are s e n s i t i v e over t h e i r whole surface. However a f t e r nerve-muscle contact the s e n s i t i v i t y of the PLD decreases while the ALD remains approximately the same (Vrbova et a l , 1978), A f t e r establishment of neuromuscular junctions the PLD r e t a i n s t h i s s e n s i t i v i t y only at the motor end-plate, whereas the ALD continues to be s e n s i t i v e along the plasma membrane to a v a r i e d extent (Fedde, 1969; Gordon and Vrbova, 1975). The avian ALD has a multiple innervation in the form of several small-diameter nerve f i b r e s which terminate i n "en grappe" endings on each muscle f i b r e (Hess, 1961). Slow muscles undergo contracture and do not normally give r i s e to action p o t e n t i a l s (Ginsborg, 1960b; Harris et a l , 1973), rather they conduct l o c a l p o t e n t i a l s between synapses decrementally (Vrbova et a l , 1978). The innervation i s reported to release i n s u f f i c i e n t transmitter to i n i t i a t e action p o t e n t i a l s , however Ginsborg (1960b) found small-amplitude action p o t e n t i a l s and twitches i n several multiply-innervated slow extrafusal f i b r e s i n the ALD. Furthermore, Hnik et a l (1967) demonstrated propagated action p o t e n t i a l s i n a small number of ALD muscle f i b r e s following reinnervation by the PLD nerve. S p e c i a l i z a t i o n of 8 the post-synaptic membrane i s poorly developed (Hess, 1967; Page, 1969). The fast-twitch extrafusal f i b r e s comprising the PLD are f o c a l l y -innervated by one large-diameter nerve f i b r e with a singlte end-plate (Hess, 1961). These "en plaque" end-plates are large, r e l a t i v e to "en grappe" endings on slow extrafusal f i b r e s , and unlike i n the ALD, have junctional f o l d s . Moreover, they s t a i n intensely f o r cholinesterase (Fedde, 1969). Motor endings i n the slow ALD appear as dispersed droplets, occupy a smaller area of the muscle f i b r e and s t a i n r e l a t i v e l y l i g h t l y for cholinesterase (Hess, 1961). F o c a l l y innervated muscle f i b r e s undergo a twitch, propagate an action p o t e n t i a l , and are capable of high tension development (Hess, 1961); however, they are unable to maintain tension f or long periods of time (Page and S l a t e r , 1965). The twitch time for the slow ALD was found by these workers to be 6-7 times slower than that of the PLD. Slow muscle f i b r e s of the ALD lack a r e g u l a r l y arranged transverse tubular system and sarcoplasmic reticulum such as that of f a s t muscle f i b r e s (Hess, 1961; Page, 1969; Shear and Goldspink, 1971; Hikida, 1972), and the area of contact between these elements i s r e l a t i v e l y small (Page, 1969). The ALD obtains i t s c h a r a c t e r i s t i c " f e l d e r s t r u k t u r " appearance during growth (Shear and Goldspink, 1971; Vrbova et a l , 1978). The slow rate of tension development attained by tonic muscle f i b r e s during growth was found to be s u f f i c i e n t to cause r i p s and deformation of the Z d i s c s , but i n s u f f i c i e n t to completely s p l i t the m y o f i b r i l s l o n g i t u d i n a l l y . P a r t i a l subdivision of the m y o f i b r i l s was thought to r e s u l t , leading to lack of extensive development of the sarcoplasmic reticulum (Shear and Goldspink, 9 1971). The f a s t PLD consists almost e n t i r e l y of twitch or " f i b r i l l e n s t r u k t u r " muscle f i b r e s (Hess, 1961; 1967). The transverse tubular system and sarcoplasmic reticulum were found by Page (1969) and Shear (1978) to be extensive and well-organized, factors a t t r i b u t e d to s p l i t t i n g of the m y o f i b r i l s into d i s c r e t e , regular shapes during growth (Shear and Goldspink, 1971). Because of the rapid tension development (Ginsborg, 1960a), the Z discs could rupture and tear, r e s u l t i n g i n l o n g i t u d i n a l s p l i t t i n g of myofilaments and hence, formation of d i s c r e t e m y o f i b r i l s . Most vertebrate s k e l e t a l muscles consist of a mixture of at l e a s t two or three f i b r e types. The slow ALD and f a s t PLD were considered to have almost exclusive extrafusal f i b r e homogeneity (Ginsborg, 1960a; Hess, 1970), however i n studies of the histochemical c h a r a c t e r i s t i c s of the ALD (Nene and Chinoy, 1965; Ashmore and Doerr, 1976; Ovalle, 1978) slow extrafusal f i b r e s appeared to be of two v a r i e t i e s . The presence of two morphological and histochemical types of slow f i b r e s occurs i n the metapatagialis muscle of the pidgeon (Hikida and Bock, 1974), and i n hindlimb muscles of amphibians (Smith and Ovalle, 1973). There.is l i t t l e information a v a i l a b l e on the r e l a t i o n s h i p between muscle spindles and the muscles in which they reside. For example, do i n t r a f u s a l f i b r e s r e f l e c t i n any way the c h a r a c t e r i s t i c s of the surrounding muscle ? Ovalle (1978) i d e n t i f i e d two d i s t i n c t populations of extrafusal and i n t r a f u s a l f i b r e s i n the avian ALD, and suggested that the histochemical d i s p a r i t y between them may r e f l e c t the d i r e c t influence of 10 the sensory innervation on the i n t r a f u s a l f i b r e s . It would thus be of i n t e r e s t to examine muscle spindles i n the avian ALD and PLD because of the predominance of one extrafusal f i b r e type i n each muscle. Because of the known s t r u c t u r a l , f u n c t i o n a l , histochemical and biochemical d i f f e r e n c between these two s y n e r g i s t i c muscles, i t i s possible that t h e i r muscle spindles are also d i f f e r e n t . The ALD and PLD have proved useful experimental models i n c o r r e l a t i o n of structure and function, Moreover, the d i f f e r e n t response of the two muscles to the expression of hereditary muscular dystrophy presents a unique opportunity to investigate factors involved i n disease expression and target s p e c i f i c i t y . The f a s t , white PLD shows early phenotypic expression of muscular dystrophy whereas the slow, red ALD i s spared the e f f e c t s of the disease (Mazliah et a l , 1976; Cosmos et a l , 1979a; 1979b; Mazliah and Cosmos, 1979). Hereditary muscular dystrophy i n chickens was f i r s t reported by Assmundson and J u l i a n (1956), and described i n d e t a i l with regard to v a r i a b i l i t y of disease expression i n d i f f e r e n t s t r a i n s of affected birds by Holliday et a l (1968). Several reports have demonstrated morphological s i m i l a r i t i e s of t h i s disease i n chickens with human muscular dystrophies (Jul i a n and Assmundson, 1963; Assmundson et a l , 1966), however diffe r e n c e of opinion exist as to the relevancy of the chicken as a model for the various forms of human muscular dystrophy. On the other hand, Harris and S l a t e r (1980) emphasize that the dystrophic c h a r a c t e r i s t i c s common to the various animal species would provide a useful approach to the study of the pathogenesis of the disease. 11 R e l a t i v e l y l i t t l e i s known about s p e c i f i c pathological changes i n muscle spindles since i t has been reported that they are disease-r e s i s t a n t i n many kinds of muscle pathology (Cooper, 1960; Smith and Ovalle, 1972). Recent attention has been drawn to the involvement of muscle spindles i n various mammalian neuromuscular diseases (Cazzato and Walton, 1968; Patel et a l , 1968; Meier, 1969; Swash and Fox, 1974; Y e l l i n , 1974), however d i f f i c u l t i e s were noted with regard to evaluating the d i s c r e t e pathological changes seen i n biopsied material. In a comprehensive study of muscle spindle pathology, Cazzato and Walton (1968) concluded that i n human progressive muscular dystrophy the most prominent a l t e r a t i o n s i n the muscle spindles were thickening of the outer capsule and some atrophy of the i n t r a f u s a l f i b r e s . Unlike i n congenital muscular dystrophy, these and other workers noted no d i r e c t r e l a t i o n s h i p between the degree of abnormality of the muscle spindle and the s e v e r i t y of extrafusal f i b r e involvement. There i s no information i n the l i t e r a t u r e on the e f f e c t s of hereditary muscular dystrophy on the avian muscle spindle. Because the slow-tonic ALD i s reported not to phenotypically express the disease, i t would seem l i k e l y that the muscle spindles would also be spared. Conversely i t i s p o s s i b l e that the muscle spindles i n the dystrophic PLD would undergo pathological change because of the degeneration that i s known to occur i n the extrafusal muscle. As an adjunct to t h i s study on muscle spindles r e s i d i n g i n the normal ALD and PLD muscles, the e f f e c t s of hereditary muscular dystrophy on the morphology of muscle spindles in the same two muscles w i l l also be considered. 12 Scope and aims of the present i n v e s t i g a t i o n To examine by l i g h t microscopic, quantitative, and s e r i a l reconstruction methods, muscle spindles r e s i d i n g i n two s y n e r g i s t i c vertebrate s k e l e t a l muscles; the avian a n t e r i o r (ALD) and p o s t e r i o r (PLD) latissimus d o r s i . To a s c e r t a i n whether morphological and/or quantitative differences e x i s t between muscle spindles r e s i d i n g i n the slow-red ALD and the fast-white PLD. To determine the morphological r e l a t i o n s h i p s e x i s t i n g between ALD and PLD muscle spindles and the surrounding extrafusal muscle, endomysial connective t i s s u e and neurovascular elements. To examine muscle spindles i n the ALD and the PLD of the chicken with hereditary muscular dystrophy and to compare these findings with the normal.. To ascertain whether muscle spindles and surrounding t i s s u e i n the muscular dystrophic ALD and PLD are affected morphologically i n the same or d i f f e r e n t manner. 13 MATERIALS AND METHODS Six male and female white leghorn chickens aged seven weeks, and three muscular dystrophic chickens of the Storrs Connecticut s t r a i n , aged nine weeks, were k i l l e d with an overdose of chloroform. Body weights were 550-600 grams i n the normal and 600-900 grams i n the dystrophic birds. The a n t e r i o r (ALD) and posterior (PLD) latissimus d o r s i muscles, used i n t h i s study, co n s t i t u t e the most s u p e r f i c i a l layer of dorsal musculature of the back. These two s k e l e t a l muscles are d i s t i n c t l y innervated and remain separate throughout t h e i r course, unlike i n humans where the two muscles have fused. The s t r a p - l i k e ALD i s dark pink i n colour. It a r i s e s from the neural spines of lower c e r v i c a l vertebrae and traverses the upper back h o r i z o n t a l l y to i n s e r t as a wide, fle s h y semi-tendinous band on the upper medial aspect of the humerus. The PLD i s pale i n colour and fusiform i n shape. It a r i s e s from the neural spines of lower thoracic vertebrae and adjacent lumbar f a s c i a , traverses the thorax diagonally and i n s e r t s on the humerus as a d i s c r e t e tendon, s l i g h t l y ventral to the ALD (George and Berger, 1966). The r i g h t and l e f t ALD and PLD were removed from each animal. Each muscle was trimmed of s u p e r f i c i a l f a s c i a and tendon, weighed, pinned to a corkboard i n a moderately stretched p o s i t i o n and moistened with p h y s i o l o g i c a l s a l i n e . The muscles taken from two normal birds were prepared for p a r a f f i n embedding, while the remainder were prepared for frozen sectioning. 14 The four muscles prepared for p a r a f f i n sectioning were immediately f i x e d i n formal s a l i n e f o r four hours, placed i n 70% alcohol for 16 hours then dehydrated through ascending alcohols. A modification of the P e t e r f i method of double embedding (Brown, 1969) was used as i t tended to promote easier sectioning. S e r i a l transverse sections were made of each muscle from o r i g i n to. i n s e r t i o n at a c a l i b r a t e d thickness of 12u, and every eighth section was mounted on a glass s l i d e . Conventional Haematoxylin and Eosin was used to s t a i n the sections ( L i l l i e , 1965). Six normal and three dystrophic ALD-PLD p a i r s were quick-frozen i n isopentane-liquid nitrogen and s e r i a l l y sectioned i n t h e i r e n t i r e t y i n a cryostat at -20° C, at a c a l i b r a t e d thickness of lOu. Every 10th section was mounted on a glass s l i d e and stained e i t h e r with conventional Haematoxylin and Eosin or modified Gomori trichrome ( L i l l i e , 1965). After examination of both p a r a f f i n and frozen material with a Leitz Orthoplan microscope, i t was found that the quick-freezing technique had preserved the ti s s u e with minimal a r t e f a c t . Muscle spindles were e a s i l y i d e n t i f i e d and t h e i r contents could be counted, assessed and measured. The p a r a f f i n technique involved considerably more t i s s u e d i s t o r t i o n and, as a r e s u l t , many muscle spindles were ei t h e r d i f f i c u l t to i d e n t i f y or t h e i r contents appeared clumped i n one area of the capsular space (Fig. 13). As t h i s prevented accurate counting and measurement of i n t r a f u s a l f i b r e s , i t was decided to use the p a r a f f i n sections only as an adjunct to the frozen material. Accordingly, an examination was made of six normal ALD-PLD p a i r s using the following protocol. Muscle spindles were located by microscopy at x400 magnification, 15 numbered, and each equatorial region was marked on a scale drawing of the muscle cross-section using the micrometer scale readings on the mechanical stage of the microscope. Repeat procedures were done to check for error. Spindle units were then marked on a scale drawing as they appeared i n the s e r i a l sections, and the approximate length of each was calculated by counting the number of cross-sections i n which the muscle spindle was seen, and mul t i p l y i n g t h i s by a fac t o r of 10. Extracapsular and intr a c a p s u l a r polar, juxta-equatorial and equatorial regions were also noted and t h e i r respective lengths c a l c u l a t e d . The muscle spindle index (number of muscle spindles per gram of muscle weight) was cal c u l a t e d f o r the six normal ALD-PLD p a i r s , and comparisons were made with the r e s u l t s c o l l e c t e d from published data on spindle indices from other avian muscles and from those of other species. Longitudinal scale drawings of one normal ALD-PLD p a i r were made from s e r i a l tracings (Gaunt and Gaunt, 1978) u t i l i s i n g the microscope equipped with a Le i t z camera l u c i d a drawing attatchment. Both muscles were s e r i a l l y reconstructed from the traced sections. Included in each t r a c i n g were out l i n e s of muscle spindle capsules, extracapsular polar regions, number of i n t r a f u s a l f i b r e s per spindle u n i t , and lo c a t i o n of prominent neurovascular elements. The l o n g i t u d i n a l reconstructions gave a f a i r l y accurate representation of muscle spindle s i z e , l o c a t i o n and length r e l a t i v e to myotendon junctions and neural elements. In addition, a transverse plane representation of the two muscles was made from tracings taken at 4mm i n t e r v a l s i n order to i l l u s t r a t e the muscle spindle p o s i t i o n s r e l a t i v e to v e n t r a l and dorsal surfaces. Histograms were constructed from tracings 16 taken at 2mm i n t e r v a l s to show the degree of uniformity of spindle d i s t r i b u t i o n i n the two muscles. Int r a f u s a l f i b r e s were counted from 100 ALD and 100 PLD muscle spindles and the range of f i b r e s per spindle unit were depicted i n histograms. A t o t a l of 250 of these i n t r a f u s a l f i b r e s from the ALD and PLD were also measured i n juxta-equatorial regions. Fibre s i z e (cross-sectional diameter) was calculated by taking the average of two readings at r i g h t -angles and these r e s u l t s were depicted i n companion histograms. A s i m i l a r process was used to demonstrate extrafusal f i b r e diameters i n the two muscles. A t o t a l of 150 extrafusal f i b r e s from the mid-belly region of the ALD and i t s PLD counterpart were measured, and the r e s u l t s shown i n a s i m i l a r manner as that undertaken for the i n t r a f u s a l f i b r e s . F i n a l l y , three dystrophic ALD-PLD pair s were examined and compared with the normal sample i n order to assess any detectable morphological a l t e r a t i o n s i n muscle spindles and t h e i r surrounding extrafusal muscle. One of the three p a i r s was examined i n d e t a i l for l o c a t i o n , d i s t r i b u t i o n and appearance of muscle spindles, number and s i z e of i n t r a f u s a l f i b r e s and muscle spindle index. Random sections from the o r i g i n , mid-belly and i n s e r t i o n of the remaining ALD-PLD pa i r s were used for a d d i t i o n a l morphological data. 17 RESULTS Comparative data from quantitative analysis of muscle spindles i n s i x normal ALD-PLD p a i r s , and of each muscle weight, are shown i n Table 1. The mean weight of the ALD was 0.279gm, co n s i s t e n t l y less than that of the PLD with a mean weight of 0.316gm. A t o t a l of 315 muscle spindles were counted and examined; 212 and 103 from the ALD arid PLD re s p e c t i v e l y . The range of muscle spindles per muscle was 32-40 i n the ALD with a mean of 35.33, and 16-20 i n the PLD with a mean of 17.16. Muscle spindle indices f o r the s i x ALD-PLD p a i r s i s shown i n Table 1. This important measurement indicates the number of muscle spindles per gram of muscle weight. It was s i g n i f i c a n t l y higher i n the ALD (mean = 130.33), than i n the PLD (mean = 56.14). The l i g h t microscopic appearance of muscle spindles observed i n t h i s study was s i m i l a r i n many respects to that described f o r other avian muscles (De Anda and Rebollo, 1967; Maier and Eldred, 1971). In the present study, however, several differences were observed between muscle spindles r e s i d i n g i n the ALD and those present i n the PLD. Figure 1 shows the o v e r a l l appearance of the two muscles i n transverse section and the t y p i c a l l o c a t i o n of muscle spiridles. The ALD (Fig.la) shows two muscle spindles located at the periphery of d i s c r e t e muscle f a s c i c l e s close to a neurovascular trunk. The PLD (Fig.lb) shows a muscle spindle p a r t l y enclosed i n a f a s c i c l e , some distance from neurovascular elements. Perimysium 18 i s less evident here than i n the ALD, with a r e l a t i v e l y larger number of extrafusal f i b r e s contained i n each f a s c i c l e . The d i s t r i b u t i o n and i n d i v i d u a l lengths of a l l muscle spindles i n an ALD-PLD p a i r i s shown diagramatically i n Figure 2. The ALD has a r e l a t i v e l y even d i s t r i b u t i o n of spindle units located from o r i g i n on the vert e b r a l column to i n s e r t i o n on the proximal end of the humerus. A s l i g h t increase i n spindle density appeared to occur i n the region of entry of each of the three branches of the median nerve into the muscle. On the other hand, a r e l a t i v e l y uneven d i s t r i b u t i o n of spindle units was noted i n the PLD with the majority concentrated i n the d i s t a l t h i r d of the muscle, around the entry point of the sing l e branch of the median nerve which supplied i t . In three of the si x PLD muscles used i n t h i s study, only one muscle spindle was found i n the proximal t h i r d of each muscle b e l l y . Muscle spindle lengths, including extracapsular polar regions, were shorter i n the ALD, with a range of 0.8-3.7mm (mean = 1.9mm) compared with a range of 1.1-4.8mm (mean = 2.3mm) i n the PLD. The majority of i n t r a f u s a l f i b r e s were seen to extend beyond the capsule i n both the ALD and PLD and these f i b r e s terminated within a short distance of each other. The small monofibril muscle spindles found only i n the ALD were r a r e l y longer than 1mm. The longest muscle spindles were usually found i n the PLD, frequently extending 1mm beyond the l i m i t of the capsule at each pole. Figures 3 and 4 show the density of muscle spindles per transverse section at 2mm and 4mm i n t e r v a l s , r e s p e c t i v e l y . In Figure 3, nerve entry points correspond approximately with i n t e r v a l s 4,6 and 9 i n the ALD and between i n t ervals 6 and 8 i n the PLD. In Figure 4 i t can be seen that 19 muscle spindles are more frequently located on the ventral aspect of each muscle. It was also noted that the nerve supply entered each muscle on the v e n t r a l aspect. Moreover, at l e a s t one muscle spindle was observed i n the region of the myotendon junction at the i n s e r t i o n of both muscles. Polar regions of muscle spindles from the two muscles are shown fo r comparison i n Figure 5. Intrafusal f i b r e s were usually scattered within the endomysium of surrounding extrafusal f i b r e s i n the extracapsular regions i n the ALD (Fig.5a) , while they were compactly arranged within the endomysium i n the PLD (Fig.5b). Moreover, ALD muscle spindles were usually i s o l a t e d from surrounding muscle by prominent connective t i s s u e (Fig. 5c) •, while those i n the PLD were more intimately r e l a t e d with adjacent extrafusal muscle f i b r e s (Fig . 5 d ) . Juxta-equatorial muscle spindles i n the two muscles are seen i n Figure 6. A s i m i l a r r e l a t i o n s h i p between connective t i s s u e and the capsule as found i n the intracapsular polar spindles i s present i n the ALD (Fig.6a) . An intimate r e l a t i o n s h i p between capsule and extrafusal f i b r e s i s again seen i n the PLD (Fig.6b). Inner capsule c e l l s and a f l u i d -f i l l e d p e r i a x i a l space are present i n the ALD and PLD muscle spindles with a more d a r k l y - s t a i n i n g e x t r a c e l l u l a r material present i n the p e r i a x i a l space of the l a t t e r . This d i f f e r e n c e i n s t a i n i n g i n t e n s i t y can be seen i n examples of equatorial regions of ALD and PLD muscle spindles i n Figure 7. As can be seen, the ALD contains several i n t r a f u s a l f i b r e s and inner capsule c e l l s l y i n g within a l i g h t l y stained p e r i a x i a l space, surrounded by an attenuated outer capsule. In contrast, the PLD has a marked abundance of inner capsule c e l l s and a more darkly stained p e r i a x i a l space. Whereas the r e l a t i o n s h i p between the capsule of the PLD muscle spindle and 20 surrounding extrafusal f i b r e s remains approximately the same i n Figures 5, 6 and 7, the ALD counterpart shows a decrease in.connective t i s s u e towards the equatorial region. In Figure 7, therefore, the r e l a t i o n s h i p between ALD and PLD muscle spindle capsules and surrounding extrafusal muscle appears s i m i l a r . The lack of noticeable increase i n nucleation of i n t r a f u s a l f i b r e s i s apparent i n both examples, thus confirming previous observations i n other avian muscle spindles (Maier and Eldred, 1971; Adal, 1973; Maier, 1977). The primary sensory innervation, which enters the equatorial region, can be seen surrounding several i n t r a f u s a l f i b r e s (Fig.7). Conjunctive forms of muscle spindles include a l l those which either share elements or which l i e i n close proximity with other units (Richmond and Abrahams, 1975b).Only one tandem or series linkage was seen i n t h i s study, and i t occured i n the d i s t a l t h i r d of a PLD (Fig.2). Paired muscle spindles have some form of mechanical contact without sharing i n t r a f u s a l f i b r e s (Barker and l p , 1961). Several examples of these were found i n the ALD sample. Figure 8a shows two adjacent and p a r a l l e l muscle spindles with adherent but separate external capsules. The smaller of the two units i s m o n o f i b r i l , i n that i t contains only one i n t r a f u s a l f i b r e . Figure 8b shows such a monofibril muscle spindle at higher magnification, This section through the equatorial region i l l u s t r a t e s the presence of a p e r i a x i a l space, inner capsule c e l l s and a sensory innervation to the si n g l e i n t r a f u s a l f i b r e . Another muscle spindle p a i r with p a r a l l e l external capsules l y i n g adjacent to each other i s found i n Figure 8c. Attempts to c l a s s i f y avian i n t r a f u s a l f i b r e s according to si z e 21 have usually been unsuccessful (Maier and Eldred, 1971; Adal, 1973; Maier, 1977). In the present study, however, measurement of these muscle c e l l s i n the ALD and PLD showed diff e r e n c e s i n number of i n t r a f u s a l f i b r e s per muscle spindle and i n i n t r a f u s a l f i b r e diameter. Table 2 shows the range of i n t r a f u s a l f i b r e number measured i n 212 ALD and 103 PLD muscle spindles. From the ALD sample the range of i n t r a f u s a l f i b r e s per spindle unit was 1-8 with an o v e r a l l mean of 3.45. In the PLD sample the i n t r a f u s a l f i b r e range was 2 - 9 with an o v e r a l l mean of 4.91. A t o t a l of 24 monofibril muscle spindles was found i n the ALD sample, with a minimum of three and a maximum of eight per muscle. From a t o t a l of 100 ALD and 100 PLD muscle spindles, the number of i n t r a f u s a l f i b r e s per muscle spindle i s shown gr a p h i c a l l y i n Figure 9. Note that the maximum number of ALD muscle spindles (n = 21) contained three i n t r a f u s a l f i b r e s , compared with f i v e i n t r a f u s a l f i b r e s i n the maximum number of PLD spindles (n = 26). Measurement of i n t r a f u s a l f i b r e diameters was undertaken i n juxta-equatorial regions to avoid incorporating the primary sensory innervation into the reading,(Boyd, 1962). Measurements were taken from a t o t a l of 250 ALD and 250 PLD i n t r a f u s a l f i b r e s and t h e i r d i s t r i b u t i o n i s g r a p h i c a l l y depicted i n Figure 10. Intr a f u s a l f i b r e diameters i n the ALD ranged from 5 to 16.5JJ (mean = 10. l i p ) compared with a range of 4.5 - 18.Sp: (mean = 10.71u) i n the PLD. A bimodal d i s t r i b u t i o n i n i n t r a f u s a l f i b r e s i z e i n the ALD with peak values of 8IJ and IOJI, and a trimodal trend i n the PLD with peak d i s t r i b u t i o n between 7 - 8JJ, 9 - I O J J and 11 - 12u ind i c a t e the p o s s i b i l i t y of two types of i n t r a f u s a l f i b r e s i n the ALD and three i n the PLD. 22 Extrafusal f i b r e diameters were also measured from the mid-belly region of the two muscles and the r e s u l t s are shown i n Figure 11. The range of extrafusal f i b r e s i z e i n the ALD was 28 - 57u (mean = 43u) compared with a range of 16 - 43u i n the PLD (mean = 31u).It can also be seen i n Figures 1 and 12 that extrafusal f i b r e s i n the ALD a larger than those i n the PLD. In Figure 12, moreover, a prominent capsule surrounding the ALD muscle spindle and a less d i s t i n c t capsule of the PLD muscle spindle were also observed. P a r a f f i n material was also used as an adjunct to the frozen sections examined i n t h i s study. Figure 13 represents p a r a f f i n sedtions through d i f f e r e n t regions of ALD and PLD muscle spindles. It can be seen that extrafusal f i b r e s , external capsules and contents of muscle spindles are less well-defined i n the p a r a f f i n material than i n the frozen sections. One ALD-PLD p a i r was obtained from a chicken with muscular dystrophy, and analysed i n the same manner as the normal sample, except for the omission of the long i t u d i n a l and transverse reconstruction procedures. Whereas the l i g h t microscopic appearance of the majority of muscle spindles i n the dystrophic PLD was normal, some pathological changes were observed i n several spindle u n i t s . Widespread changes in PLD extrafusal f i b r e s were also found. In the dystrophic ALD counterpart subtle changes were seen i n muscle spindle c h a r a c t e r i s t i c s but the extrafusal muscle appeared normal. Figure 14a shows a dystrophic PLD with i r r e g u l a r l y arranged extrafusal f i b r e s , many of which are either considerably larger or 23 markedly smaller than the normal, Connective t i s s u e has i n f i l t r a t e d between many of the f i b r e s , giving the appearance of "more d i s c r e t e separation of the muscle f i b r e s into f a s c i c l e s . M y o f i b r i l l a r clumping, indicated by increased i n t e n s i t y of Eosin s t a i n , was also seen i n several muscle f i b r e s . Muscle spindles frequently appeared normal despite surrounding extrafusal f i b r e degeneration. Figure 14b shows such an example from the dystrophic PLD. The i n t r a f u s a l f i b r e s appear i n t a c t , however the external capsule has thickened. A t o t a l of 41 dystrophic muscle spindles were counted i n a muscle pa i r ; 28 i n the ALD and 13 i n the PLD. Table 3 compares data from t h i s dystrophic ALD-PLD p a i r with the r e s u l t s obtained i n the six normal muscle p a i r s . Although the mean weight of the normal sample was not d i r e c t l y compared with the dystrophic material because of a two-week age dif f e r e n c e , i t i s i n t e r e s t i n g to note that the r a t i o of the mean normal ALD-PLD weights was 0.9 : 1, compared with 1.57 : 1 i n the dystrophic sample. Data obtained from the two remaining dystrophic ALD-PLD pai r s also confirmed t h i s apparent weight increase i n the dystrophic ALD and marked weight loss i n i t s PLD counterpart. The muscle spindle index i n the dystrophic ALD (79.86) was notably lower than that seen i n the normal ALD sample (130.33) (Table 3). While the dystrophic PLD counterpart also contained fewer muscle spindles than that observed i n the normal sample, the spindle index was appreciably higher i n the dystrophic muscle (58.27) than i n the normal sample (Table 3). It i s known that the f u l l complement of muscle spindles i s attained by each muscle s h o r t l y a f t e r b i r t h (Zelena, 1957; 24 Shear and Goldspink, 1971; Barker, 1974), therefore a dystrophic muscle such as the PLD may show such an increase i n the muscle spindle index as atrophy and weight loss occur. A juxta-equatorial muscle spindle from the dystrophic PLD i s seen i n Figure 15a. Several abnormalities are present i n the form of extrafusal f i b r e atrophy, c e n t r a l l y placed n u c l e i , hypertrophied external capsule of the muscle spindle and apparent i n t r a f u s a l f i b r e s p l i t t i n g . Two adjacent i n t r a f u s a l f i b r e s are less than 3u i n diameter, approximately h a l f the diameter of the smallest i n t r a f u s a l f i b r e s i n the normal PLD sample, and i t i s tempting to speculate that they were o r i g i n a l l y a single f i b r e . The apparent normalcy of ALD extrafusal f i b r e s as reported previously (Cosmos et a l , 1979; Mazliah and Cosmos, 1979), i s confirmed i n the present study. However, some changes were noted i n the appearance of several dystrophic ALD spindles. There were no monofibril units observed. In addition, the number of i n t r a f u s a l f i b r e s per muscle spindle had apparently increased considerably (Table 3). This value was higher i n the dystrophic ALD than i n the PLD i n both range and mean. Figure 15b shows a dystrophic ALD equatorial muscle spindle containing 11 i n t r a f u s a l f i b r e s . Several other equally large units were found i n the dystrophic ALD material, whereas i n the normal sample only two or three spindles per muscle, with a maximum of 7 or 8 i n t r a f u s a l f i b r e s , were seen. The range of PLD i n t r a f u s a l f i b r e s per dystrophic muscle spindle (Table 3) was the same as that observed i n the normal sample, and the small increase i n the mean (5.00 compared with 4.91) may be due to some i n t r a f u s a l f i b r e s p l i t t i n g . 25 T a b l e A tabulation of the number and density of muscle spindles i n a t o t a l of 6 normal ALD-PLD p a i r s . Individual muscle weights are also indicated and were used to compute the muscle spindle index. Muscle Weight Muscle Spindle Muscle spindle i n grams content index ALD 0.183 32 175.05 PLD 0.212 17 80.14 ALD 0.282 32 113.33 PLD 0.344 17 47.05 ALD 0.272 36 131.37 PLD 0.342 16 46.88 ALD 0.363 40 110.26 PLD 0.401 20 49.85 ALD 0.322 40 124.30 PLD 0.399 16 47.14 ALD 0.251 32 127.49 PLD 0.258 17 65.82 ALD mean 0.279 35.33 130.33 PLD mean 0.316 17.16 56.14 ALD: PLD 1:1.133 2.06:1 2.32:1 ( r a t i o means) 26 e_2. Data i l l u s t r a t i n g the range of i n t r a f u s a l f i b r e s per muscle spindle in the s i x ALD-PLD p a i r s . The ALD-PLD r a t i o of means has been computed and i s indicated on the extreme r i g h t . Muscle Range Mean ALD:PLD ALD 1-8 3.14 1:1.54 PLD 3-7 4.83 ALD 1-7 3.13 1:1.57 PLD 3-7 4.90 ALD 1-8 •3.71 1:1.30 PLD 2-7 4.83 ALD 1-8 4.26 1:1.18 PLD 2-9 5.00 ALD 1-7 3.43 1:1.43 PLD 2-7 4.89 ALD 1-8 3.00 1:1.63 PLD 2-6 4.89 Total Mean 1:1.43 27 T a b l e 3. Summary of s a l i e n t data comparing dystrophic (n = 1) and normal (n = 6) ALD - PLD muscle p a i r s . The data indicated for normal muscles represents means. Dys. ALD Dys. PLD Normal Normal ALD PLD Muscle Weight 0.351 0.221 0.287 0.316 grams. Muscle Spindle 28 13 35.33 17.16 Number Muscle Spindle 79.86 58.27 130.33 56.14 Index I n t r a f u s a l range 2 - 1 1 2 - 9 1 - 8 2 - 9 f i b r e s per muscle mean 5.10 5.00 3.45 4.91 spindle 28 Figure 1 a. Transverse frozen section of the normal ALD. E x t r a f u s a l muscle f i b r e s are arranged into d i s c r e t e f a s c i c l e s . Two muscle spindles (arrows) are located in close proximity to prominent neurovascular elements. H and E s t a i n . X100 b. Transverse frozen section of the normal PLD. Whereas the extrafusal muscle f i b r e s are smaller than i n the ALD, here they are arranged into f a s c i c l e s with a larger c e l l u l a r population. A muscle spindle (arrow) i s seen at a further distance from neurovascular elements. H and E s t a i n . X100 29 30 Figure 2 Longitudinal reconstruction of an ALD and a PLD showing the d i s t r i b u t i o n and lengths of muscle spindles i n each muscle. Lengths of equatorial regions are indicated by thickened l i n e s Rostral ( r ) , caudal ( c ) , o r i g i n (o), i n s e r t i o n ( i ) . Scale: 1mm = lOOv. A L D c PLD r 31 Histogram i l l u s t r a t i n g the number of muscle spindles observed at 2 mm i n t e r v a l s from o r i g i n to i n s e r t i o n of one ALD ( l e f t ) and i t s companion PLD ( r i g h t ) . S l O "> 6 0) Z 0 ALD PLD 1 3 5 7 9 11 1 3 5 7 9 11 Interval 32 Figure H Transverse reconstruction showing the d i s t r i b u t i o n of muscle spindles i n one normal ALD-PLD pair from o r i g i n to i n s e r t i o n . Each dot in d i c a t e s a muscle spindle. Intervals between traced transverse sections are 1mm. Rostral ( r ) , caudal ( c ) , ventral (v), dorsal (d). A L D -^-Origin Insertion-** 33 Figure 5 Transverse frozen sections of polar regions of muscle spindles from the ALD (a, c) and the PLD (b, d). H and E s t a i n . XI,000 a. ALD. Several extracapsular i n t r a f u s a l f i b r e s are v i s i b l e between neighbouring e x t r a f u s a l f i b r e s . b. PLD. Several small extracapsular i n t r a f u s a l f i b r e s are more t i g h t l y arranged within the endomysium of the surrounding extrafusal f i b r e s . c. ALD. A capsule (arrow) intimately surrounds the i n t r a f u s a l f i b r e s in the polar region. Endomysial connective tissue associated with the capsule i s conspicuous. d. PLD. In t h i s muscle, a muscle spindle capsule (arrow), investing the i n t r a f u s a l f i b r e s , i s associated with minimal amounts of endomysial connective t i s s u e . 54 35 Figure 6 Transverse frozen sections of juxta-equatorial regions of muscle spindles i n the ALD (a) and the PLD (b) for comparison. H and E s t a i n xl,000. The ALD muscle spindle capsule (arrow) i s surrounded by an abundance of connective t i s s u e . The PLD e x h i b i t s less connective t i s s u e between i t s muscle spindle capsule (arrow) and the extrafusal f i b r e s . Two of the three i n t r a f u s a l f i b r e s i n the PLD appear larger than t h e i r counterparts i n the ALD. 36 37 Figure 7 Transverse frozen sections of equatorial regions of muscle spindles in the ALD (a) and PLD (b). H and E s t a i n . X900. In the ALD, note the sensory terminal (small arrow) on one of the f i v e i n t r a f u s a l f i b r e s , and the arrangement of inner capsule c e l l s (arrowhead). In the PLD, sensory terminals surrounding the i n t r a f u s a l f i b r e s are v i s i b l e (small arrows). Here, a large number of inner capsule c e l l s (arrowhead) are d i s t r i b u t e d throughout the p e r i a x i a l space. The outer capsules (curved arrows) and adjacent extrafusal f i b r e s (E) are indicated. 38 39 Figure 8 Transverse frozen sections of several ALD muscle spindles stained with H and E. a. Two p a r a l l e l muscle spindles are seen i n which t h e i r outer capsules (arrows) adhere but remain separate and d i s t i n c t . The equatorial muscle spindle contains f i v e i n t r a f u s a l f i b r e s and l i e s within the same section of endomysium as the mono-f i b r i l muscle spindle below. X750 b. High magnification view of the equatorial region of a mono-f i b r i l muscle spindle. A sensory terminal (arrow) on the i n t r a f u s a l f i b r e and an inner capsule c e l l (arrowhead) are v i s i b l e . XI,000 c. Section of two ALD muscle spindles situated on the periphery of adjacent f a s c i c l e s . The polar muscle spindle (above) contains f i v e i n t r a f u s a l f i b r e s , and the equatorial muscle spindle (below) contains four i n t r a f u s a l f i b r e s . X430 40 41 Figure 9 Graphic representation of the t o t a l number of i n t r a f u s a l f i b r e s per spindle e x i s t i n g i n a sample of 100 ALD and 100 PLD muscle spi n d l e s . 0 1 2 3 4 5 6 7 8 9 10 Total number of intrafusal fibres per muscle spindle 42 Figure 10 Size histograms of intrafusal fibres from selected muscle spindles in the ALD and the PLD. A total of 250 fibres from each muscle were measured, and this data was collected from six normal ALD-PLD pairs. Note the apparent bimodal distribution of cross-sectional diameters in the ALD, and a trimodal trend in the PLD. A L D P L D Intrafusal Fibre Size (/im) 43 Figure 11 Size histograms of extrafusal f i b r e s i n the two muscles. A t o t a l of 150 f i b r e s were selected randomly from the midbelly regions of each muscle. 4 4 2. • Tr3XL±v&v3z. f f o ' i e r . s e c t i o n . : c \ e ^ i i i - ; : - r i a i a u s c i t c ? i - d l e . G ' : i c r l '.ctvi^ Ojae .'- . $2?v« a.: . r i i ^ i i i f f b r e ? tmS ;-.heir ai-:'-;K.-'.f.t;e.' . s £/ «. - idft ir . . Nb_a t h e i i a Sfcl l - 'o .a f dned .-1 .:. ...x. i x « o f .v.... i. • th \ r . t h e _ r . t . . - i a ? . ,vrco. TI .rt . :• a p / r c a i u E - w cue-'. - caps'-.* . i«-psra i i^g t h e g i e p i ia .* r-x-. arfjse&jiu a x t r t d^s* . ! f i b r * 3. X750 i":* ijBVerBe /roznn secxi on of a ?Li/ equatorifei au-i-: l a spindle ' . . , -> . ' . ;r:. j i.romi stain, the lack ol a '"b^ g v pe" nuc?:-.r ... .. g u r a t i o o in j n y of era six iritr-.fusc .1 I i i r e s . s E s i s i l a r cc c'.-.'j:.. fet'o:: in ,.ie ALD. The oater cap -j.lt: i i l e s s prominent. 1 v/tfi .. l r o t ' e r t - . ; . . v x > sastl i . s ; c i PiJD e x . r s f u s a i fjHitea c o o p a i c . - -h tl :sa in ti A L D , X750 4 5 46 Figure 13 Transverse p a r a f f i n sections through d i f f e r e n t regions of muscle spindles stained with H and E. XI,000 a. Polar region of a PLD muscle spindle (arrow) containing f i v e , i n t r a f u s a l f i b r e s . Note the adjacent neurovascular trunk. b. Juxta-equatorial region of an ALD muscle spindle. Note the outer capsule, inner contents of the muscle spindle, and p e r i a x i a l space (asterisk) compared with those seen in the frozen material. c. Equatorial region of a PLD muscle spindle. Sensory terminals to two i n t r a f u s a l f i b r e s are indicated (arrows). Note also the abundance of inner capsule c e l l s and a greatly expanded p e r i a x i a l space ( a s t e r i s k ) . 47 48 Figure 14 a. Transverse frozen section of a muscular dystrophic PLD. Extr a f u s a l f i b r e s are of i r r e g u l a r s i z e . There i s evidence of m y o f i b r i l l a r clumping and connective tissue i n f i l t r a t i o n between these f i b r e s . A muscle spindle i s indicated (arrow). H and E s t a i n . X100 b. Transverse frozen section of a dystrophic PLD. Whereas the i n t r a f u s a l f i b r e s i n th i s polar muscle spindle appear r e l a t i v e l y normal, there i s thickening of i t s outer capsule. The surrounding extrafusal f i b r e s show some signs of degenera t i o n . Gomori trichrome s t a i n . X750 49 50 1 Figure 15 a. Transverse frozen section of a juxta-equatorial muscle spindle in the dystrophic PLD. The surrounding extrafusal f i b r e s are i r r e g u l a r in s i z e , and many contain c e n t r a l l y - l o c a t e d n u c l e i . There i s evidence of i n t r a f u s a l f i b r e s p l i t t i n g (arrows) within the muscle spindle. Some thickening of the outer capsule and i n f i l t r a t i o n of connective tissue between some extrafusal f i b r e s i s seen. H and E s t a i n . X750 b. Transverse frozen section of an equatorial muscle spindle i n the dystrophic ALD. Whereas the extrafusal f i b r e s appear unaltered, there i s an increase in the number of i n t r a f u s a l f i b r e s compared with that seen in normal muscles. Eleven i n t r a f u s a l f i b r e s are seen in t h i s section. Gomori trichrome s t a i n . X750 51 52 DISCUSSION The r e s u l t s of the present study demonstrate that muscle spindles i n the slow ALD are d i f f e r e n t from those i n the f a s t PLD, q u a n t i t a t i v e l y , morphologically arid i n t h e i r r e l a t i o n s h i p with the surrounding extrafusal muscle. When comparirig the features of the two muscles i t i s important to consider t h e i r respective functions. Both or i g i n a t e from neural spines of the vertebrae. The ALD arises from the low c e r v i c a l region and the PLD originates from the thoracic region. Both muscles i n s e r t on the upper medial aspect of the humerus. Their p r i n c i p a l action would appear to be adduction of the humerus. Functionally, the ALD supports the wing and prevents i t from drooping (Vrbova et a l , 1978), whereas the PLD demonstrates maximum isometric tension with the wing folded next to the body (Shear, 1978). It i s known that the slow ALD can maintain graded contractions for long periods of time, whereas the f a s t PLD can change length r a p i d l y , a t t a i n high tension, but cannot sustain prolonged contractions (Ginsborg, 1960a; 1960b). Amphibian muscles have been found to contain true slow and f a s t twitch f i b r e s s i m i l a r to those i n avian species, however the two f i b r e groups are intermingled within the same muscle (Kuf f l e r and Vaughan Williams, 1953). It i s thought that the slow f i b r e s maintain the p o s i t i o n developed by the fast f i b r e s and that the two f i b r e groups act as functional synergists. It i s p o s s i b l e that the avian ALD and PLD work i n a s i m i l a r manner to achieve and maintain 53 optimum wing p o s i t i o n , i n that the PLD acts as a prime mover i n adduction of the wing while the ALD s t a b i l i z e s the wing i n the appropriate p o s i t i o n attained by i t s synergist. The large d i s p a r i t y between density of muscle spindles i n the two muscles r e f l e c t s t h e i r d i f f e r e n t functions. SloW-twitch mammalian muscles (frequently r e f e r r e d to i n the l i t e r a t u r e as slow muscles), such as the soleus, and those muscles containing a majority of slow-twitch f i b r e s , are known to have s i g n i f i c a n t l y higher muscle spindle d e n s i t i e s than predominantly fast-twitch muscles (Swett and Eldred, 1960a; Richmond and Abrahams, 1975b). Small postural muscles of the cat neck, such as the intertransversarius,' contain up to 500 spindles per gram (Richmond and Abrahams, 1979). Larger neck muscles which maintain o v e r a l l head p o s i t i o n not only have a high muscle spindle density (46 - 106 per gram) but contain the majority i n regions of predominantly slow-twitch f i b r e s (Richmond and Abrahams, 1975b). Other mammalian muscles with c o n s i s t e n t l y high spindle d e n s i t i e s are those i n i t i a t i n g f i n e movements, such as those controllong d i s t a l extremity j o i n t s (Chin et a l , 1960). Muscles s t a b i l i z i n g important j o i n t s , such as masseter and temporalis have also been found to have high spindle d e n s i t i e s (Kubota and Masegi, 1977; Lennartsson, 1979). Muscles i n i t i a t i n g gross movement, including those operating over large proximal j o i n t s , have c o n s i s t e n t l y low spindle i n d i c e s . It i s i n t e r e s t i n g to note that the human latissimus d o r s i , a large muscle which adducts and medially rotates the humerus, has a spindle index of only 1.4 (Voss, 1956). This study confirms the findings of Adal and Chew Cheng (1980) 54 and Maier and Eldred (1971) that avian muscles tend to have a higher muscle spindle density than those of mammalian species. The slow ALD contains 130 spindles per gram, a higher density than previously published data on chicken muscle (Maier and Eldred, 1971). As i n mammalian slow-twitch muscles the higher spindle index of the ALD probably r e f l e c t s the necessity of accurate monitoring of small changes i n muscle length i n order to f a c i l i t a t e correct postural responses. Although the PLD has a low spindle index compared with i t s synergist, the highest values i n human muscles are no greater (Matthews, 1972), neither are some of the slow-twitch muscles of the cat neck (Richmond and Abrahams, 1975a). Because of the predominant fast-twitch f i b r e content of the PLD, a lower spindle index than was found i n t h i s study would l o g i c a l l y be expected, however the importance of the muscle as a functional synergist of the ALD may account for the r e l a t i v e l y high values obtained. ALD muscle spindles were normally located i n c l e f t s between muscle f a s c i c u l i and generally avoided the heavier planes of connective t i s s u e . In the PLD, with less connective t i s s u e separating the muscle f a s c i c u l i , muscle spindles were located either on the periphery, or surrounded by groups of extrafusal f i b r e s . Large slow-twitch postural muscles of the cat neck contain tendinous i n s c r i p t i o n s which e f f e c t i v e l y d ivide the muscle f i b r e s into c o n t r a c t i l e sub-units, each with an independent nerve supply from one or more c e r v i c a l segments (Richmond and Abrahams, 1975a). A s i m i l a r - s i t u a t i o n was observed i n the ALD which appeared to be divided, both l o n g i t u d i n a l l y and h o r i z o n t a l l y , by large 55 planes of connective t i s s u e . Furthermore, the nerve supply originated from three separate trunks of the median nerve, which entered the muscle at d i f f e r e n t points along the ventral surface. It i s proposed that the ALD functions i n a complex manner as a group of i n t e r - r e l a t e d sub-units. The lack of i n t e r f a s c i c u l a r perimysium and of connective t i s s u e i n s c r i p t i o n s i n the PLD i s not s u r p r i s i n g i n view of i t s rapid tension development following an action potential.(Ginsborg, 1960b). The presence of s t r u c t u r a l sub-units within t h i s muscle would tend to decrease the e f f i c i e n c y of muscle contraction. Muscle spindles usually follow the o v e r a l l pattern of muscle innervation, although they are not ne c e s s a r i l y located i n close proximity to the point of nerve entry into the muscle (Barker, 1974). This appeared to be true for the ALD-PLD sample examined i n the present study, p a r t i c u l a r l y i n the case of the l a t t e r , where a sing l e nerve trunk r a p i d l y divided on entering the d i s t a l t h i r d of the muscle. Approximately 5o% of the t o t a l number of spindles were found i n t h i s region. The ALD, on the other hand, contained muscle spindles throughout i t s length, which i s to be expected i n view of i t s function i n maintenance of wing p o s i t i o n and i n monitoring small changes i n muscle f i b r e length. The external capsule of the ALD muscle spindle appeared thicker than that of the PLD, although at the l i g h t microscopic l e v e l i t was sometimes d i f f i c u l t to d i s t i n g u i s h the capsule from the surrounding . i n t e r f a s c i c u l a r perimysium and connective t i s s u e i n s c r i p t i o n s . It can only be surmised that the contents of ALD spindles are more i s o l a t e d from the surrounding e x t r a c e l l u l a r space than those of the PLD, 56 p a r t i c u l a r l y in the polar region where the capsule i s well-developed (Ovalle, 1976). Bridgman and Eldred (1964) suggested that the outer capsule might function as a pressure sense organ, and that i n the expanded equatorial region the spindle contents would be subject to increased pressure during muscle contraction. In view of the more intimate r e l a t i o n s h i p apparent between PLD muscle spindles and the surrounding extrafusal f i b r e s than i n the ALD, i t i s suggested that muscle contraction would produce a greater amount of l a t e r a l pressure on the PLD spindle contents than on those of the ALD. There i s some evidence that slow-twitch mammalian muscles have longer muscle spindles than f a s t - t w i t c h muscles (Swett and Eldred, 1960b). In the present study, muscle spindles of the slow ALD were about 20% shorter than those of the f a s t PLD, however several contributing factors may account for t h i s d i f f e r e n c e . Monofibril spindles accounted for 11% of the t o t a l number i n the ALD. Their mean length r a r e l y exceded 50% of the mean spindle length, whereas the majority of spindles with long extracapsular portions were found i n the PLD. In addition, the ALD underwent a v a r i a b l e amount of contracture during d i s s e c t i o n and i s o l a t i o n , and was frozen at a smaller percentage of i t s true length than the PLD. A d i s p a r i t y i n muscle contracture was reported by Maier and Eldred (1971) when measuring muscle spindle lengths i n two avian hip muscles, one fixed i n f l e x i o n and the other i n extension. The extended muscle had 80% longer spindles than the muscle fixed i n f l e x i o n , and i t was suggested that t h i s f a c t had contributed to the r e s u l t s . 57 Other than the presence of short monofibril spindles i n the ALD sample, the main differ e n c e between spindle lengths i n the two muscles was seen i n muscle spindles containing large-diameter i n t r a f u s a l f i b r e s . Such f i b r e s frequently had an extracapsular course of 1mm or more at each pole, and were found i n spindles containing four or more i n t r a f u s a l f i b r e s , of which the majority were i n the PLD. Unlike the findings of Maier and Eldred (1971), considerable v a r i a b i l i t y i n spindle length was seen i n both the ALD and PLD sample. Inner capsule c e l l s were c o n s i s t e n t l y present i n the encapsulated portions of both ALD and PLD muscle spindles. In addition, they appeared to increase i n number with the development of the p e r i a x i a l space, p a r t i c u l a r l y i n the PLD. Ovalle (1976) noted that ALD and PLD i n t r a f u s a l f i b r e s were completely surrounded by cytoplasmic processes of inner capsule c e l l s but no di f f e r e n c e i n t h e i r arrangement i n the two muscles was noted. In attempting to f i n d reasons f o r the larger number of inner capsule c e l l s i n PLD spindles, the work of Cooper and Gladden (1974) on e l a s t i c f i b r e d i s t r i b u t i o n and function becomes s i g n i f i c a n t . They found that nuclear bag f i b r e s were surrounded by more e l a s t i c f i b r e s than were nuclear chain f i b r e s , and that i n addition, inner capsule c e l l s were associated with e l a s t i c f i b r e s . They suggested that these e l a s t i c f i b r e s functioned by compressing i n t r a f u s a l f i b r e s and t h e i r sensory terminals during s t r e t c h , r e s u l t i n g i n d i s t o r t i o n and dep o l a r i z a t i o n of sensory nerve endings. In the present study, large-diameter i n t r a f u s a l f i b r e s were more frequently seen i n the PLD spindles, and i t i s suggested that the dynamic response to i n t r a f u s a l f i b r e s t r e t c h 58 might be enhanced by the i n t e r p l a y between these components. At t h i s time there i s no histochemical confirmation of s p e c i f i c i n t r a f u s a l f i b r e types in the PLD, although two kinds of i n t r a f u s a l f i b r e s have been shown to occur i n the ALD (Ovalle, 1978). There i s no comparative quantitative data on i n t r a f u s a l f i b r e types in ALD and PLD muscle spindles, although studies on other avian muscles have reported either one or three types of f i b r e s . The r e s u l t s of the present study suggest the existence of two i n t r a f u s a l f i b r e populations i n the ALD and three i n the PLD. I f i n t r a f u s a l f i b r e c h a r a c t e r i s t i c s r e f l e c t those of surrounding extrafusal f i b r e s , the response of the muscle spindles would r e f l e c t muscle function. The ALD, a slow, tonic postural muscle with a c o n t r a c t i l e mechanism sui t a b l e f or prolonged, graded contractions, would seem to require an innervation with non-adaptive properties as found on mammalian nuclear chain f i b r e s (Matthews, 1972). Conversely, the PLD, a phasic muscle with twitch contractions would need an innervation with a dynamic component to stretch, as found supplying mammalian nuclear bag f i b r e s (Matthews, 1972). Whereas i t i s not suggested that f i b r e s i z e alone could determine i n t r a f u s a l f i b r e c h a r a c t e r i s t i c s , the dynamic component of PLD muscle spindles would be found in the upper h a l f of the i n t r a f u s a l f i b r e diameter range. It i s possible that some v a r i a t i o n i n i n t r a f u s a l f i b r e type may be influenced not only by the sensory and motor innervation, but by the presence or absence of a fusimotor grape-innervated component such as found i n frog muscle spindles (Brown, 1971). Chin (quoted by Barker, 1974) found a c o l l a t e r a l grape innervation to ALD 59 and both a grape and plate fusimotor innervation to PLD muscle spindles. There i s a lack of information on the s i g n i f i c a n c e of numbers of i n t r a f u s a l f i b r e s per muscle spindle, however two studies on the cat have indicated a lower number i n postural, slow-twitch muscles than i n f a s t - t w i t c h muscles (Swett and Eldred, 1960b; Richmond and Abrahams, 1975a). In the present study the slow ALD contained 40% fewer i n t r a f u s a l f i b r e s than the f a s t PLD and i t would be of i n t e r e s t to ascertain whether there i s any functional s i g n i f i c a n c e i n these r e s u l t s . One notable observation i n Richmond and Abrahams' (1975a) study i s the reduced number of nuclear bag f i b r e s i n the slow-twitch neck muscles, when compared with those of the hindlimb. Possibly, the spindles i n the slow-twitch muscles have less need for a dynamic component to st r e t c h than those located in the f a s t - t w i t c h muscles. An i n t e r e s t i n g feature of the ALD i s the monofibril muscle spindle, reminiscent of those found i n the snake and l i z a r d (Proske,1973; P a l l o t and Taberner, 1973). They have also been reported to occur i n the PLD (Chin, quoted by Barker, 1974). Two functional kinds of monofibril spindle are present i n the snake, one with a tonic response and the other with a phasic response to s t r e t c h or stimulation (Fukami, 1970). On morphological grounds, however, only one type of avian monofibril spindle was i d e n t i f i e d i n the present study. Each spindle i n the ALD monofibril sample had a well-developed external capsule, d i s t i n c t p e r i a x i a l space and a maximum of one nucleus per i n t r a f u s a l f i b r e cross-section. From the published descriptions of two types of snake spindles, the avian monofibril unit would seem to contain features s i m i l a r to both. 60 Extrafusal f i b r e diameter measurements taken from embryonic ALD and PLD muscles up to sexual maturity confirm the r e s u l t s of the present study, i n that the slow ALD has larger f i b r e s than the f a s t PLD. Embryonic ALD f i b r e s , which develop and function e a r l i e r than those of the PLD, were found by Gordon et a l (1974) to be 100% larger than embryonic PLD f i b r e s , and at age 29 days were 60% larger (Shear and Goldspink, 1973). A s i m i l a r trend was found by Ashurst and Vrbova (1979) i n muscles of 35 day old b i r d s . Hereditary muscular dystrophy in chickens i s s p e c i f i c a l l y expressed i n f a s t - t w i t c h , focally-innervated muscles such as the pectoral and the PLD, whereas slow, multiply-innervated muscles, such as the ALD, are spared disease phenotypes (Cosmos et a l , 1979a). These findings were confirmed in the present study, and i n addition some morphological changes were noted i n muscle spindles, p a r t i c u l a r l y those i n the PLD. The dystrophic PLD showed extrafusal f i b r e changes s i m i l a r to those described by Harris and S l a t e r (1980), which are most l i k e l y secondary accompaniments of the disease. These include increased v a r i a t i o n i n f i b r e diameter, increase i n number of myo-nuclei which tended towards central l o c a t i o n , f i b r o s i s and connective t i s s u e i n f i l t r a t i o n . M y o f i b r i l l a r clumping, a common u l t r a s t r u c t u r a l feature that can also be seen with the l i g h t microscope, occured randomly within areas of the muscle b e l l y where f i b r e degeneration was present. The amount of pathological change varied considerably within the muscle, with areas of normal f i b r e s i z e adjacent to those showing a l l the secondary signs 61 mentioned above. Hypertrophy of the muscle spindle capsule i s a common sequela of neuromuscular disease, and i s r e a d i l y v i s i b l e with the l i g h t microscope (Cazzato and Walton, 1968). Varying amounts of capsular thickening were seen i n the dystrophic PLD, within regions of muscle abnormality. It i s probable that i n t r i n s i c connective t i s s u e p r o l i f e r a t i o n i s taking place i n the capsule rather than deposition of collagenous material i n adjacent structures, because capsular changes were s i m i l a r whether the spindle was located i n i n t e r f a s c i c u l a r perimysium or surrounded by muscle f i b r e s . Functional changes i n the muscle spindle with hypertrophied capsules i s l i k e l y to be li m i t e d . The s e n s i t i v i t y of primary and secondary innervation could t h e o r e t i c a l l y be modified as a r e s u l t of increased cushioning of the i n t r a f u s a l f i b r e s from l a t e r a l pressure provided by contracting muscle, however other degenerative processes taking place within the muscle would make v e r i f i c a t i o n d i f f i c u l t . Oedematous swelling of the p e r i a x i a l space, reported by Cazzato and Walton (1968) to occur i n limb g i r d l e dystrophy, was not seen i n t h i s sample. Pathological changes i n PLD i n t r a f u s a l f i b r e s were d i f f i c u l t to evaluate because of the presence of p r o l i f e r a t i n g collagenous material within the p e r i a x i a l space. This tended to obscure the o u t l i n e of the f i b r e s and innervation. Fibre atrophy could not be i d e n t i f i e d with c e r t a i n t y , however several examples of i n t r a f u s a l f i b r e s p l i t t i n g were seen. In the dystrophic ALD, the mean number of i n t r a f u s a l f i b r e s increased by 40% over the normal, p a r t l y accounted for by the lack of 62 monofibril spindles. However some change of muscle function i s suggested as the reason for t h i s increase. The dystrophic PLD shows an abnormal calcium storage during the l a s t week of embryogenesis, around the time that twitch c h a r a c t e r i s t i c s are beginning to develop (Cosmos et a l , 1979b), while the ALD i s already functioning to hold the f o e t a l wing next to the body (Vrbova et a l , 1978). By 15 days of age the PLD can only a t t a i n 50% of i t s maximum twitch tension, and i t i s proposed that the ALD, i n the process of compensating for the loss of power i n i t s developing synergist, assumes part of i t s function. The increase i n ALD weight compared with the normal sample i s probably a r e s u l t of two f a c t o r s : f i r s t l y , the two week age d i f f e r e n c e between normal and dystrophic animals examined, and secondly, compensatory hypertrophy of the muscle. Modif i c a t i o n of dystrophic ALD muscle spindles during development might occur i f the muscle were forced to a l t e r i t s function. It i s suggested that the ALD not only maintains wing p o s i t i o n , but provides part of the i n i t i a t i n g force of wing adduction i n the absence of normal tension _ development i n the PLD. The l i m i t a t i o n s of t h i s study are r e a l i z e d , both regarding the small si z e of the normal and dystrophic samples. There i s need for a further series of experiments, p a r t i c u l a r l y on the histochemistry and f i n e structure of i n t r a f u s a l f i b r e s i n muscle spindles of the normal PLD. In addition, dystrophic ALD and PLD muscles from animals of various ages could be studied to more accurately assess changes i n muscle spindle morphology. 63 REFERENCES Adal, M.N. (1973). The f i n e structure of i n t r a f u s a l f i b r e s of muscle spindles in the domestic fowl. J . Anat., 115: 407-413. Adal, M.N., and Chew Cheng, S.B. (1980). Muscle spindles in two dorsal wing muscles of the domestic duck. J . Anat., 131: 543-550. 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