TH ESlS u—‘fi v! .- . . a...‘ Jan's. -1 LISE'EJR" 3.3:“.133- 3., 1‘ .11..“ Vl‘oi'. -- . ‘,,- L‘ U9, I ‘1 I ’ L‘s,,fi .v, RM“. 5 .IU-flt-‘h‘v ' fir???” Myra-,9- .,. 7" V ’1. ". ‘Wll-P§~*1“.prfi W. 351-05; .- f "a 0'.“ This is to certify that the dissertation entitled CHANGES IN MOTOR NERVE ENDINGS IN FAST- AND SLOW-TWITCH MUSCLES OF NORMAL AND ENDURANCE-EXERCISED RATS presented by Kenneth Ellis Stephens has been accepted towards fulfillment of the requirements for Ph. D. degreein Exercise Physiology hat/41 Major professor Date May 17, 1982 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .m—E RETURNING MATERIALS: Place in bodk drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. CHANGES IN MOTOR NERVE ENDINGS IN FAST- AND SLON-TWITCH MUSCLES OF NORMAL AND ENDURANCE-EXERCISED RATS By Kenneth Ellis Stephens A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health and Physical Education I982 INA ..§:FJI1I.UP {J ABSTRACT CHANGES IN MOTOR NERVE ENDINGS IN FAST- AND SLOW-TWITCH MUSCLES OF NORMAL AND ENDURANCE-EXERCISED RATS By Kenneth Ellis Stephens Morphological change in neuromuscular axonal terminals was examined using a combined silver-cholinesterase stain in 100 male Sprague-Dawley rats. Predominantly slow-twitch (adductor longus and soleus) and fast-twitch (gastrocnemius and rectus femoris) muscles were taken from sedentary animals at 6-28 weeks of age as well as from rats subjected to 4-16 weeks of intense endurance exercise. The exercise program initiated at 12 weeks of age was progressive in nature with the final group of rats running at 36 m/min for a total of 2 hours per day, 5 days per week. Motor nerve endings were categorized into defined morphological classes. In maturing animals the number of accessory end- ings increased significantly in slow-twitch muscles from 6 to l6 weeks, at which time it plateaued in the soleus and slightly decreased in the adductor longus. In fast-twitch muscles the increase ceased at 12 weeks of age, with a subsequent leveling off after that. Double endings also increased in the predominantly slow muscle up to between 20 and 24 weeks. Conversely, fast-twitch muscle displayed no such increase and, indeed, displayed less than l% double endings after puberty. The number of branched endings appeared to decrease post-puberty in all muscles to Kenneth Ellis Stephens below 3% where it remained for the duration of the study. Simple endings decreased significantly between 6 and 20 weeks in slow-twitch while in fast-twitch muscles the decrease was seen between 6 and l2 weeks of age. Examination of exercise data indicated few changes were brought about by functionally overloading the muscles. This study suggests that l) significant differences exist in the neuromuscular axonal terminals of post-pubertal fast-twitch and slow-twitch muscles; 2) with post-pubertal maturation an increase in the complexity of these terminal patterns develops in slow-twitch muscle which is not present in fast-twitch muscle; 3) exhaustive endurance exercise appears to have little effect on the modification of motor nerve endings. This indicates that normal matura- tional nerve ending changes are not due to alterations in neuromuscular activity. DEDICATION To Ellis, Margaret, and my Family--for their continuous encouragement and support. 11' ACKNOWLEDGEMENTS Sincere appreciation is expressed to Dr. C. D. TWeedle for his friendship, and for his continuous support and guidance both in the preparation of this dissertation and throughout my graduate program. Appreciation is also extended to Dr. N. D. Van Huss for his unending patience in directing my graduate committee and to Dr. J. R. Downs for his concern and many kindnesses over the last few years. Recognition is given to Dr. N. w. Heusner for his assistance in the preparation of this thesis. Gratitude is also extended to Jo Ann Janes and to David Anderson for the preparation of the tables and graphs included in this disserta- tion and to Dorthy Gendreau for its initial typing. Special thanks is extended to my parents and family; for without their love, patience, and understanding, this dissertation would not have been completed. iii TABLE OF CONTENTS CHAPTER Page I. THE PROBLEM .............................................. 1 Statement of the Problem .............................. 2 Significance .......................................... 3 Limitations of the Study .............................. 4 II. REVIEW OF RELATED LITERATURE ............................. 5 Introduction .......................................... 5 Initiation of Nerve-Muscle Connections ................ 5 Primitive Neuromuscular Junction ...................... 7 Polyneuronal Innervation .............................. 9 Elimination Mechanism .............................. 20 Mature Innervation Patterns ........................... 22 Specificity at the Neuromuscular Junction .......... 23 Morphological Age-Related Changes of Terminal Engings/Endplates ............................... 34 Forced Exercise Effects on Terminal Endings/ Endplates ....................................... 40 III. METHODS OF PROCEDURE ..................................... 43 Experimental Animals .................................. 43 Research Design and Treatment Groups .................. 43 Training Procedures ................................... 45 Animal Care ........................................... 47 Sacrifice Procedures .................................. 47 Tissue Analysis ....................................... 49 Right Leg .......................................... 49 Left Leg ........................................... 50 Analyses of Data ...................................... 51 IV. RESULTS AND DISCUSSION ................................... 55 Results ............................................... 55 Accessory Endings ..................................... 55 Ageing Effects ..................................... 55 Fiber Type Differences ............................. 59 Exercise Effects ................................... 59 iv CHAPTER Page Double Endings ........................................ 6O Ageing Effects ..................................... 60 Fiber Type Differences ............................. 60 Exercise Effects ................................... 62 Branched Endings ...................................... 62 Ageing Effects ..................................... 62 Fiber Type Differences ............................. 64 Exercise Effects ................................... 64 Simple Endings ........................................ 65 Ageing Effects ..................................... 65 Fiber Type Differences ............................. 65 Exercise Effects ................................... 67 Sprouts and Multiple Endings .......................... 67 Ageing Effects ..................................... 67 Fiber Type Differences ............................. 70 Exercise Effects ................................... 7O Complex Endings ....................................... 71 Ageing Effects ..................................... 71 Fiber Type Differences ............................. 71 Exercise Effects ................................... 73 Discussion ............................................ 73 Complexity at the Neuromuscular Junction ........... 73 Exercise Effects on the Neuromuscular Junctions.... 77 V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS ................ 78 Summary ............................................... 78 Conclusions ........................................... 79 Recommendations ....................................... 81 REFERENCES ...................................................... 82 APPENDICES A. TRAINING PROGRAM ......................................... 92 B. TRAINING, TREATMENT ENVIRONMENT, AND BODY WEIGHT VALUES.. 94 C. MUSCLE FIBER TYPE DATA ................................... 97 LIST OF TABLES TABLE Page 2.1 Morphological Specificity of Terminal Endings/Endplates-- Light Microscopic Results .................................. 24 2.2 Morphological Specificity of Terminal Endings/Endplates-- Electronmicroscopic Results ................................ 31 2.3 Age-Related Morphological Changes in Terminal Endings/ Endplates--Light Microscopic Results ....................... 35 2.4 Age-Related Morphological Changes in Terminal Endings/ Endplates--Electronmicroscopic Results ..................... 41 3.1 Research Design ............................................ 44 4.1 Means and Standard Deviations for Terminal Nerve Endings in Sedentary Animals ....................................... 57 4.2 Means and Standard Deviations for Terminal Nerve Endings in Exercised Animals ....................................... 58 5.1 Summary of Major Changes in Terminal Motor Endings ......... 80 vi LIST OF FIGURES FIGURE Page 3.1 Classifications of motor nerve terminals .................. 52 3.2 Cholinesterase-Bielchowsky stain for morphology of motor nerve endings ............................................. 53 4.1 Percentage accessory endings in selected muscles of sedentary and exercised rats. ...... . ...................... 56 4.2 Percentage double endings in selected muscles of sedentary and exercised rats .............................. 61 4.3 Percentage branched endings in selected muscles of sedentary and exercised rats .............................. 63 4.4 Percentage simple endings in selected muscles of sedentary and exercised rats .............................. 66 4.5 Percentage sprouts in selected muscles of sedentary and exercised rats ............................................ 68 4.6 Percentage multiple endings in selected muscles of sedentary and exercised rats .............................. 69 4.7 Percentage complex endings in selected muscles of sedentary and exercised rats .............................. 72 4.8 Changes in terminal nerve endings in specific muscles-- adductor longus and soleus ................................ 75 4.9 Changes in terminal nerve endings in specific muscles-- rectus femoris and gastrocnemius .......................... 76 vii CHAPTER I THE PROBLEM Using various histochemical techniques in conjunction with physio- logical, biochemical, and anatomical methods, three basic fiber types have been categorized in normal mammalian muscle (20,49,95). These fiber types are dynamic and capable of responding morphologically as well as metabolically to a wide variety of stimuli such as denervation (15,51, 61,63,75,77,98,119), cross-innervation (15,17,18,27,63,119), inactivity (9,12,119), exercise (25,50,69,70), or ageing (22,26,51,58,60,85,ll9). The neuromuscular junction has been shown to bear specific charac- teristics relative to muscle fiber type. Under light microscopic exami- nation varying degrees of ending/endplate complexity have been identified (6,30,31,43,56,66,79,90,94,115), and in some cases categories of terminal arborizations can be associated with specific fiber types (79,117,120). Examination of the post-synaptic portion of the endplate with the electron microscope has revealed distinct adaptations in terms of number and structure of junctional folds (23,40,42,87,9l,92,93,97, 109), endplate mitochondria (40,42,44,47,87,91,92,93), vesicle number or shape (44,47,52,87,92,97,109) or sole-plate sarcoplasmic development (87,91,92,93,97). Likewise, the morphology of the presynaptic portion of the junction (i.e., the terminal axon/ending) is closely linked to the muscle fiber type it innervates. Characteristics have been identified here in terms of terminal boutons (79,94,116), mitochondria (44,93), axoplasmic vesicles (44,52,93,lO9), collateral sprouting (6,43,116), or intra-endplate sprouting (94). Observations in normal animals have indicated that, like muscle, fiber types terminal endings/endplates are not static (33,49,71,84,94, 116,117). In the mammalian neonate, polyneuronal innervation (multiple axonal inputs into a single endplate) is characteristic (73,99,117,119). With maturation, polyneuronal innervation is lost by withdrawal of redun- dant terminal axons--1eaving an adult pattern of unineuronal innervation (73,99,117,1l9,125). It has been shown, however, that this adult pattern is subject to mutation during the normal ageing process either through ending/endplate degeneration and regeneration (33,71) or via continuous elaboration of terminal arborizations (116,117). Further, the neuro- muscular junction has been shown to be morphologically responsive to treatments such as denervation (34,63,119,125), reinnervation (34,63,84, 119,125), cross-innervation (33,37,84,119,125) and change in activity level (9,119,125). Statement of the Problem The overall objective of this investigation was to determine the effects of both maturation and an induced exhaustive endurance exercise program on the morphology of the motor endplate and its respective terminal axon in selected slow-twitch and fast-twitch muscles of male albino rats. The investigation sought to provide insight into the following questions: 1. What are the morphological differences in motor nerve endings between slow-twitch and fast-twitch muscles? 2. How does the motor ending structure, and consequently the innervation pattern, respond to chronic physical activity (endurance exercise) in these same muscles? 3. Are there changes in the motor ending structure in fast; and slow-twitch muscle which could be associated with maturation? Significance Information concerning synaptic plasticity under varied circum- stances is provided by the present study. Quantitative observations of various types of motor nerve endings obtained at selected points during maturation indicate the normal adaptive changes which occur and will be useful in differentiating these from pathological conditions of myoneural junctions. In addition, any such changes noted which are fiber-type specific should prove useful in the further understanding of normally and abnormally occurring nerve ending alterations. Finally, the specific quantitative evaluation of these endings in normal and exercised animals gives insight into adaptations in the terminal ramifi- cation brought about by increased workload. Limitations of the Study . The results of this study are restricted to normal male, Sprague-Dawley albino rats, and absolute values may prove to be species and/or strain specific. . The results are similarly limited to the muscles cited (soleus, adductor longus, gastrocnemius, and rectus femoris) and their respective fiber type populations. . The study was further restricted by limitations in histo- chemical techniques which preclude at present, the evaluation of quantitative enzyme concentrations in individual muscle fibers and the subsequent association of these fibers with specific motor ending structures. . While quantitative evaluation of the training program used was limited due to undetected deficiencies in the controlled- running wheels and the master control unit, it was established qualitatively that the exercise program was a very demanding endurance experience for the animals. . As four general sectioning and staining sessions were required to process all tissue, intersession variability may have influenced some of the results. CHAPTER II REVIEW OF RELATED LITERATURE Introduction The purpose of this investigation was to examine the morphological plasticity demonstrated at the myoneural junction of maturing and endurance-exercised rats. Terminal axons and their respective motor endings were demonstrated in selected fast- and slow-twitch hindlimb muscles and were subsequently categorized in accordance with a pre- defined criteria (117). The following review of literature related this examination with current research papers or theories providing insight into the differ- ences in motor ending morphology in specific muscle fiber types associ- ated with ageing or in conjunction with induced exercise. An attempt also has been made to include review of that literature which might explain the mechanisms for or functional significance of observed differences. Initiation of Nerve-Muscle Connections The initiation of nerve—muscle connection is characterized at the onset by the appearance in the myotomes of "exploratory fibers" or "pathfinder nerves" (64). While relatively few in number, these fine nerve outgrowths are thought to provide, for subsequent developing axons, a guidance mechanism or path along which they can course until a peri- pheral target organ is reached (64). Axonal growth, both primary and secondary, is readily evident among myoblasts in the form of growth cones (64.72). It is at this point, while the axon terminals are still quite separate from the myoblasts that primitive neuromuscular junction traits appear. Within the myoblasts, non-localized acetylcholine (ACh) receptors are being synthesized (45) and acetylcholinesterase (AChE) is present (110). Choline acetyltransferase (ChAc) has been detected at this time in the muscle mass (119). As ChAc has been proven to be localized to nerve terminals (65,119), this indicates the ability of the nerve terminal to synthesize ACh prior to formal contact with the muscle cell. The formal relationship between nerve and muscle thus first occurs between developing nerve terminals capable of releasing the neurotransmitter ACh and myotubes which have differentiated to the point at which they are capable of response to the nerve stimulus (i.e., they have increased membrane chemosensitivity to ACh) (266). The interdependence of nerve and muscle is established here for further existence or development. Motor axons failing to make permanent peripheral contacts are redundant and die back (72,119) while those which succeed in connecting with peripheral organs apparently mature further (73,119). Myotubes which remain functionally uninnervated cease to differentiate further, atrophy, and are replaced with fat (24,38,72,ll9). In this manner and due to the outgrowth of a large excess of motor axons full neurotization is normally ensured in the developing embryo. Primitive Neuromuscular Junction The earliest morphological demonstration of myoneural junctions in rats occurs at approximately 16 days in utgrg_(8,38,76,113,114), which coincides with the first signs of muscle contraction which can be elicited from rats by electrical stimulation (8,113). The appearance of visible myoneural junctions is obviously species specific as Hirano (68) and Bennett and Pettigrew (8) reported their presence between seven and nine days in 919 in chick embryos, whereas Fidzianska (46) reported nerve-muscle contacts in human fetal muscle between nine and 16 weeks. Teravainen's ultrastructural analysis (114) of the morphogenesis of the rat myoneural junction provided a basis for our current under- standing of the developmental stages. In it he suggests that initially, presynaptic axons with ACh-containing vesicles approach the developing myofibers, and that these terminals induce changes in the postsynaptic membrane which result in its thickening. At this stage the primary synaptic cleft is wide and apposition is irregular. With the narrowing of the cleft to roughly 500A, the first visible, primitive junction was noted to be characterized by "teloglial-covered axon terminals apposed to the thickened but unfolded plasma membrane of the muscle fiber." The presence at this junction of 1-8 axon terminals, side by side, con- taining terminal vesicles, mitochondria and occasionally agranular reticulum was reported. The formation of secondary synaptic clefts was seen to occur in the postsynaptic membrane between 20 days pre- and 4 days post-natal. These relatively immature, finger-like invaginations were found to develop to adult status (increased in number and depth) between five and ten days post-natal. Finally Teravainen noted in- creases in numbers of mitochondria and vesicles in axon terminals with age, as well as separation of terminals at the junction between the 10th and 16th post-natal day. Sequentially, these findings were in relative agreement with those of Kelly and lacks (76) for the rat, Hirano for birds (68) and Bennett and Pettigrew (8) for both. Post-natal examination of the maturation of junctions was carried out using kittens of various ages by Nystrbm (88). Using the classical definitions for "terminaisons en grappe" and "terminaisons en plaque" developed by Tschierau in 1879 he attempted to differentiate in "slow-red" and "fast-white" muscles between neuromuscular junctions. At no time were "en grappe" or grape-like clusters of endings seen; however, despite the fact that both soleus and gastrocnemius muscles displayed "en plaque" (plate-like) endings, there were distinct differences both in structure and development. During the first week the only difference noted was in size. The soleus, which had larger diameter muscle fibers, also had larger endings. This relationship between endplate size and muscle fiber diameter persisted into adulthood. At approximately two weeks of age differentiation of endplates from primitive disc-like structures was initiated in gastrocnemius from the center of the ending and in the soleus junctions from the periphery. By two months, the endplate of the "fast-white" muscle had developed distinct morphological characteristics which enabled its differentiation from the soleus terminal arborization: the former was well established and compact while the later appeared somewhat "fluted and wrinkled". In adult animals the gastrocnemius dis- played endings which were fairly wide spreading, long and smooth. This was in contrast to the shorter, more compact, wrinkled soleus terminal. Investigation of other “fast-white" muscles revealed endplates with traits similar to those found in gastrocnemius muscle. Other "slow-red" muscles showed the characteristic ending displayed by soleus muscle. Nystrbm failed to note any true mutli-innervation of endings at any stage of development. Polyneuronal Innervation Teravainen's observations (114) of 1-8 axons at endplate areas, and their subsequent separation from each other during development com- bined with the differences observed by Nystrbm (88) in endplate morph- ology and development in different fiber types is indicative of the plasticity of the myoneural junction during maturation. In the last decade much of the focus on plasticity in the neuromuscular junction has centered on polyneuronal innervation and its elimination. In 1970, Redfern (99) examined endplate potentials (e.p.p.s) in diaphragm muscles of neonatal rats during the first four weeks of life. By isolating intact the diaphragm and phrenic nerve in curarized mammalian Ringer's solution and subsequently stimulating systematically through the phrenic nerve at various strengths while recording e.p.p's intracellularly at the middle of the muscle fiber, Redfern was able to To observe the addition or subtraction of "units" to the e.p.p. and the resultant complex e.p.p. This he accomplished by examining recordings of step-wise increases in amplitude of e.p.p's with progressive stimulus strength increases. These step-wise increases were taken to indicate recruitment of axon terminals of varied thresholds. In several cases however, he surmised addition of units where abrupt jumps in e.p.p. amplitude were present. In this manner he concluded that there were "functional connections between more than one axon in the nerve trunk and a single neonatal muscle fiber and that these axons have different latent periods and thresholds to nerve stimulation." During the first week of existence, there were e.p.p's present with such multiple units, usually 2-4. Redfern noted a gradual shift to more single unit e.p.p's during the second week and indicated that by 16-18 days of age multiple units were rare. This decrease was thought, by him, to be representative of loss of polyneuronal innervation through elimination of superfluous nerve branches. Using kittens three days, two weeks, and six weeks of age, Bagust gt 31. (4) sought to establish whether Redfern's observations were attributable to true polyneuronal innervation or were, in fact, a reflec- tion of multiple innervation of one endplate by a single axon. Isometric contractions were elicited from the soleus and flexor hallicus longus muscles by stimulation of their respective ventral roots. The tetanic tension recorded by in 3939 nerve stimulation was significantly less than that attained by stimulating split (equal) nerve preparations in either muscle at either three days or two weeks. This was taken to be indicative of dual innervation of some fibers by more than one axon; ll i.e., polyneuronal innervation. The differences were initially large at three days, less at two weeks, and small in the flexor hallicus longus while absent in the soleus at six weeks. Apparently polyneuronal innerva- tion was removed by some mechanism during the first six post-natal weeks in the kitten, with the developing fast-twitch muscle lagging somewhat behind in its elimination. The authors suggested that through poly- neuronal innervation the adult pattern of innervation could be more pre- cisely linked to the demands of the nervous system through loss of less useful connections. In a subsequent report (122) the authors confirmed the preceding findings and indicated that reduction of polyneuronal innervation was not attributable to increased muscle fiber numbers. Total fiber counts at all ages considered were not significantly different in either muscle. Preliminary attempts at histological examination suggested the presence of multiple innervation of myofibers at two weeks but were unable to distinguish multi- from polyneuronal innervation. Axon coUnts, prelimin- ary also, indicated increases of approximately 15% between two and six weeks in both muscles which corresponded to those observed by Nystrbm (88). The increase in axon counts was thought in part, to be responsible for decreased motor unit sizes in maturing animals. It may also be con- sidered reflective of the extreme plasticity of the innervation pattern during this stage of development. In rat diaphragm examinations, Bennett and Pettigrew (8) confirmed the initial developmental sequence for innervation pattern using a com- bination of histochemical, ultrastructural, and electrophysiological 12 techniques. They established the presence of at least three synapses at a single site on developing myotubes by examining rise times for m.e.p.p's and also observing only one localized spot of ChE per myotube in association with several silver-stained axons. As with previous investigations, the number of nerve-muscle contacts was reduced to a unineuronal adult pattern between the second and fourth weeks. Bennett and Pettigrew also suggested a correlation between quantal content of the e.p.p. and the size of the nerve terminal, suggesting that with maturation more quanta are available for release on stimulation. Finally it was stated that the pattern of innervation in focally inner- vated muscle was established by the initial axon making contact with the myotube. While this initial contact was at random, subsequent contacts were made only at the initial endplate site in normal animals. It appeared to the authors that the first axon induced a change in surrounding myotube membrane which rendered it refractory to further innervation. This same report (8) provided insight into the species-specific nature of the nerve-muscle contact. A description of the sequence and development of chick anterior latissimus dorsi muscle indicated a dis- tributed-innervation pattern in which endplates were established along the length of the myotubes at distances greater than 170 um and at relatively regular intervals. These sites also were supposed to have been innervated by a single axon initially, followed by transitory multiple innervation, and bear an "en grappe" configuration for ChE deposits. Electrophysiological examination showed changes in stimulus 13 strength resulted in alteration of latency and rise times as well as amplitude of e.p.p's. The endplates apparently received a multiple and distributed innervation detectable at nine days incubation. Multiple innervation was generally removed by four weeks post hatching and the membrane areas between endplates appeared refractory to further axonal termination. In an effort to determine if membrane refractoriness is determined by nerve type, Bennett and Pettigrew (7) performed a series of experi- ments during development on the focally innervated rat tibialis anterior and on the avian posterior (focal innervation) and anterior (distributed innervation) latissimus dorsi muscles. Reinnervation of tibialis anterior muscle indicated original synapse sites as well as ectopic snyapses located in areas of new muscle growth (lengthened through addition of sarcomeres) were innervated during development. However, in muscles denervated at progressively longer times from birth, the percentage of these ectopic endplates was decreased. Consequently it was concluded that the muscle membrane present at denervation was still refractory to additional synapse formation for some time. The original synaptic site seemed the preferred junction as following the sixth post- natal week, almost all ectopic synapses were gone. In all cases "en plaque" endplates were characteristic of the area and extensive collateral sprouting was noted during reinnervation. Anterior latissimus dorsi muscles, normally of the "en grappe" type, also showed reinnerva- tion at original sites as well as at ectopic locations on muscle added during the denervation period. As in the original work, several hundred micrometers separated endplates. ,Cross-innervation between anterior and l4 posterior latissimus dorsi muscles produced myofibers with synapses which were characteristic of their new nerve. Thus the anterior latissimus dorsi became focally innervated with "en plaque" terminals, and electrical properties characteristic of fast nerve-muscle synapses. The posterior latissimus dorsi received "en grappe" terminals and a distributed-innervation pattern. These studies (7,8) indicated that the "nerve determines the pattern of synapses over an effector during development, and therefore the pattern of preferred sites of synapse formation in reinnervated and cross- innervated adult muscle." The mechanism by which the nerve establishes this pattern appears to be related to its ability to render the muscle membrane refractory during development. Reports by Brown gt al, (13,14) confirm results from previous investigations (4,7,8,99,ll4,122). They recognized genuine functional polyneuronal innervation in neonatal soleus muscles of rats at single synaptic sites using both electrophysiological techniques and histo- chemical ChE staining methods. Elimination of polyneuronal innervation was most apparent between the tenth and thirteenth day when fibers exhibiting multiple inputs decreased from 91% to 2.5%. As no decrease in total number of motor units was noted during this period electrophysio- logically, the reduction in number of terminals per site was attributed to a decrease in motor unit size. Furthermore, as there was a wide spread of these sizes which was considerably greater in young than adult animals, the authors concluded that rather than a synchronous loss by each motoneuron of a fixed percentage of its peripheral terminals, the elimination process acted predominantly on the terminals of the largest motor units. 15 In their latter report (14) the effects of partial denervation, denervation and cross-innervation during development were examined. Partial denervation of the soleus muscle on one side indicated consider- able slowing in the elimination of multiple terminals from those muscle fibers still associated with nerve supply. There was still evidence of polyneuronal innervation two weeks after the control side had developed its mature status. The presence of atrophied and hypertrophied fibers, and the apparent lack of axonal sprouting, as is visible in adult muscles (41,54,118), led to the conclusion that withdrawal of synapses can per- sist even while some myofibers become denervated. The existence of an upper size limit (i.e., a maximum number of fibers innervated) for developing motor units was advocated. Denervation during the first two days post-natal by nerve crush at the nerve entry point into the muscle resulted in reinnervation during the first two weeks in the general vicinity of the endplate band at both original and ectopic sites. Hyperinnervation was common and its removal proceeded at a similar rate to that in normal muscle, indicating neither age of terminals nor early muscle inactivity affected elimination of additional terminals. Cross- innervation experiments indicated no synapses could be formed by a foreign nerve in developing muscle unless the original nerve to the soleus was cut or crushed. In this event, foreign synapses were observed in both a distributed pattern about 1 mm apart (minority) and focally. In both cases, polyneuronal innervation was removed approxi- mately one week later than normal (i.e., during the third week), although in some cases dual synaptic sites were maintained (always more than 1 mm apart) indefinitely. In summarizing their results, Brownie; 21, (13) 16 indicated that survival of a specific terminal or a muscle fiber was highly dependent on its motor unit size, with those emanating from smaller units having an advantage. This competitive interaction among synapses was also thought to involve muscle-nerve communication of an unspecified nature. Bixby and VanEssen (10) using a variety of muscles in the rabbit again illustrated that elimination of polyneuronal innervation was not attributable to increases in myofiber numbers. Neither was there a loss of myelinated axons from the soleus nerve (237 at day 2 versus 233 at day 16). Elimination was attributed to retraction of synapses by individual motor nerves. No differences were detected in either rate or onset of elimination in muscles of varied contractile properties (soleus and extensor digitorum longus). Differences in onset of up to one week were noted when body position of the muscle was considered. Those located rostrally had earlier onset times. Using histochemical methods, Riley (103,104,105) corroborated the findings of the largely physiological studies preceding. In kitten soleus muscle he concluded that myofibers generally were innervated at a single endplate by 2 or more terminals in 72% of the cases examined in newborns. This percentage decreased to approximately 3% by 4 weeks and 1% by six weeks. The method of analysis employed indicated the terminals involved were from separate neurons, and consequently multiple terminals per endplate were deemed as anatomical substrate for poly- neuronal innervation. Findings in the soleus muscle of the rat paral- lelled the cat findings, although on a somewhat different time course. Seventy-three percent of the endplates in ll-day-old rats showed two or 17 more terminals. By the end of the second week, only 9% were poly- neuronally innervated. "Retraction bulbs", initially appearing as a swelling near the edge of the endplate and at the end of a fine axon, were seen. Advanced stages of these bulbs generally showed them as an oval swelling at the end of an axon and adjacent to the innervating axon. Riley suggested the final retraction stage to be complete resorption by the parent axon. As retraction bulbs were visible at various distances from the endplate at 11-15 days, during the period of greatest elimina- tion, a pattern of non-synchronous retraction of terminals was thought to reflect the mechanism for removal. Also observed however was evidence suggesting jn_§jtu_degeneration of redundant terminals, although retrac- tion seemed the preferred mode of removal. Riley subsequently reported that the reduction in polyneuronal innervation was not due to an increase in fiber number, as these remained constant after birth in his investigation (105). Furthermore, values for the rat soleus muscles used showed roughly 50% of the fibers were Type 11 between 11 and 15 days, indicating that fibers of both types were multi-innervated. Differentiation of fiber types apparently did not correlate with loss of polyneuronal innervation. Riley did indicate that more Type II fibers were multi-innervated, based on the relationship between fiber size, ATPase staining characteristics and terminal arbori- zation observations, and suggested activity might be an important factor in determining fiber types and innervation pattern. Subsequent work by Tweedle and Stephens (117) categorized endings during development as well as observing their morphogenesis. In two fast-twitch and two slow-twitch hindlimb rat muscles polyneuronal 18 innervation was seen to disappear leaving the unineuronal adult pattern between the second and third weeks. Retraction bulbs present during this period confirmed, in part, Riley's observations (104). Between the third and fourth weeks, a large number of branched endings indicated possible continued synaptic reorganization. After a relatively quiescent period between the third and fifth week unineuronally innervated end- plates became more complex, especially in slow-twitch muscles. This increase in complexity was thought possibly to reflect increases in myofiber size or workload. The mode of elimination of polyneuronal innervation was focused upon by four papers (11,80,101,106) using ultrastructural techniques. Reier and Hughes (101) thought that Wallerian-like degenerative changes, seen primarily between 7 and 14 days in the sciatic nerve (both myelinated and unmyelinated) fibers of post-natal mice and rats, could account for the spontaneous degeneration during peripheral nerve matura- tion. Rosenthal and Taraskevich (106) reporting a high incidence of degeneration in their terminals, generally agreed with the theory extoll- ing Wallerian-like degeneration as the mechanism of removal. Korneliussen and Jansen (80) found no evidence for degenerating intra- muscular axons or terminals in their study using rat soleus muscle. Reduction in the number of terminating axons at endplates was observed between the eighth and sixteenth days as was the presence of ridge-like extensions from muscle fibers between groups of terminals. Schwann cells both between terminating axons and, in some cases, investing terminations were visible also. Removal of redundant terminals was thought to be by retraction into the parent axon. 19 Bixby (11), using denervated rabbit diaphragm, characterized the stages of degeneration of terminals in developing muscle. After com- paring terminals and endplates in denervated with those in normal muscle, Bixby concluded that "no degenerating terminals were seen" in normal muscle, nor were there any signs of degeneration debris in Schwann cells at the endplates. He concurred strongly with the retraction theory. In examining the relationship between myelination and synapse elimination he found myelination lagged behind: terminals could be removed prior to myelination. While apparently not related to removal of redundant end- ings, irregular protrusions of terminals above the surface of the muscle fiber were seen, as were non-innervated areas of post-synaptic membrane. Both were taken to indicate the dynamic state of the developing endplate. Apart from denervation-reinnervation (7,11,13) or cross-innervation investigations (7) where the muscle and ending were dormant for various periods during development, the effects of increased or decreased activity on the genesis of the myoneural junction and the terminal axon (to the adult state) have been examined only slightly. No study using forced exercise or activity during this time period has been conducted. Stimulation of embryonic spinal cord produced a distributed pattern of innervation in normally, focally innervated chick posterior latissimus dorsi muscle (102) as well as the normal polyneuronal innervation pattern at each site. It was not known whether all sites and/or terminals were functional, however. Increase in activity resulting from electrical stimulation of the right sciatic nerve in rats produced a more rapid elimination of poly- neuronal innervation, detected electrophysiologically in both stimulated 20 muscle (soleus) and its contralateral counterpart (89). The reflex - activated, polyneuronal innervation reduction did occur later however than that on the stimulated side. Reducing neuromuscular activity with curare in the developing chick posterior and anterior latissimus dorsi muscles resulted in pro- longation of polyneuronal innervation as well as apparently increasing the number of terminals in contact with synaptic sites (112). By tenoto- mizing 4-day-old rat soleus muscle the mechanical activity of the muscle was suppressed (9). This decrease in activity resulted in a delay in the evolution of the adult pattern of innervation as polyneuronal inner- vation persisted well into the third week in tenotomized muscle. From these investigations and the observation that polyneuronal elimination coincides with a general increase in animal activity (99), it appeared that neuromuscular activity had an effect on innervation pattern and junctions. Elimination Mechanism The mechanism by which the neuromuscular system develops from a polyneuronal neonatal form to a unineuronal adult pattern is poorly understood. Generally, with no large scale death of motor neurons during the period of synapse disappearance and no apparent increase in number of muscle fibers, it would appear that the reduction in multiple inputs to synaptic sites is due to a reduction in motor unit size. Further, it appears this reduction is carried out through simple retraction of redundant terminals rather than via degenerative processes. Specifically the mechanism for determination of which terminal is to survive has eluded investigators. Jansen g_t_al_. (73) and Willshaw (124) 21 have indicated that random loss of terminals is an unacceptable mode as this would inevitably produce transient denervation of some developing fibers. Sufficient numbers of such denervated fibers, which survive for several weeks, are not visible during this period, nor is there a large amount of nerve sprouting which could explain their lack. Jansen £3 91, (73) further eliminated the possibility for competitive advantage of certain motor units, competitive or random selection by the muscle fiber, or selection by Schwann cells as accounting solely for the final scheme of innervation. They suggested that terminal elimination was related to the motor neuron's control of the ultimate number of synapses it made and that survival of specific endings was related to factors other than their level of activity or their simple ability to initiate contraction. Vrbova's group of investigators (89,119) has presented the only explicit proposal for elimination of polyneuronal innervation. Their theory is based on the actions of proteolytic enzymes released from the endplate in response to ACh. They propose that the multiple terminals at the endplates progressively secrete more ACh as the neonate becomes more active. This ACh causes 1yosomal enzymes to be released from the endplate region, or other cells in the vicinity, as well as providing a substrate for hydrolysis which ultimately will contribute to the forma- tion of the acidic environment necessary for enzyme action. These lysosomal enzymes then diffuse into the interstitial space and affect nerve terminal membranes or the connections of the terminals with the endplate. In order to survive Vrbova gt 31. suggest that a terminal must be replaced by its motor neuron and that these neurons apparently have a finite capacity to support endings. Consequently, some terminal 22 connections are lost as necessary replacement materials are unavailable, while others are maintained. Those lost are retracted once contact with the muscle fiber is broken. This then allows the parent neuron more replacement materials for its other branches and consequently facilitates its support of terminals at other endplates. A "feedback system" is advocated to insure the integrity of the surviving terminal. Should the terminal be being degraded faster than it can be regenerated, a decrease in ACh secretion will result in a decrease in the release, diffusion, and action of the lysosomal enzymes. The net result will be an equi- librium established between endplate and terminal which effectively guarantees maintenance of the unineuronal innervation pattern seen in the adult. Increases in activity as seen in the study by O'Brien gt 11. (89) would be reflected, according to the theory, in earlier removal of polyneuronal innervation while decreases in activity, as in the Benoit and Changeaux investigation (9), would result in delayed removal of super- numary endings. Such apparently was the case. Vrbova and her colleagues also suggest this "lysosomal theory" may be applicable during the initial phases of neuromuscular development. Finally they propose the ACh- produced release of lysosomal enzymes may play an important role in the dynamic state of mature nerve endings. Mature Innervation Patterns Numerous review articles (16,29,41,55,67,l15,118,119,123,125) pro- vide the historical basis for current research into the myonerual junction. 23 Much of this work carried on between 1840 and 1940 focused on mature endings and, while unique, contributes little to our current understand- ing of the plasticity and adaptability demonstrated at the neuromuscular junction or to the apparent muscle-specific morphology of the terminal. Consequently review here of these articles has been omitted. Likewise literature pertaining to generally accepted morphological characteristics of motor arborizations has been deleted. Comprehensive reviews of such traits have been compiled elsewhere (1,29,34,48,97,125,126). Specificity at the Neuromuscular Junction Histological/Histochemical Light-Microscopic Studies Morphological Characteristics (see Table 2.l)--Cole's initial investigations (30.31.32) emphasized the very specific nature of the terminal ending. His rather extensive 1955 publication (32) focused on endings in muscles of similar functions taken from normal vertebrates from elasmobranchii to mammalia classes. Using a gold chloride technique to identify terminals on teased fibers he reported a steady progression in ending characteristics from the tight, compact primitive ring of fish through the "terminaison en ligne" of amphibia and reptiles up to the "terminaison en plaque" or "grappe" of mammals. In addition, Cole reported both accessory endings and several instances of double endings, i.e., two motor endings on one muscle fiber. (From observation of the accompanying photographs, it appeared other categories of endings as described by Tweedle and Stephens (117) were also present including branched and multiples.) Cole attributed the differences in endings from varied classes to an adaptation to the particular functional needs of 24 .couauaunaaoc no: Augean. cancn Maugham: o: .m .soauaan no: one: nouaaacno ~H< .a .oaoano~o>ov ouuaouoi can mean Indooa saw: nouddneao can agenda oaducoahoann .m .uoauaacco aounloo egg-«n .Huuln a can uuvnuu on: .N accuuoom .noauuneno coachb no canoe sauna: uoaosoo< weaneduooau voodouanaoo .omuuu a can unusuu mean: .H occupapo gunman ecu-uncouauuu oases oa nwaa dunno .nmnueao summon» no: .hfldl can uuoagu Avouv spam .w .5505. :05an no: .393. on: :03» A333 aunt .H 5.90982 3 «won one: .voauonnsu acuaaunuud Mancuaunah .a .52. .9523 033d:- uau .boaoooou .0358 .m .uoaoasl omuvuonnd \Hdhsunon 1 soavdun no ago-«ucuauoa: Ocean .0 .uoaonal huouuuunuou undupoauo>uu¢m 1 cosvdna :0: and cannon» no nuonqdculuya: .n nadunoouovcn ..uo~oa:l aaolo>ol nsqloaoouuadc iuouauu- 1 gnawing no uncuuuauluous «cannon .d unuoaoh aspen: .mnnnsoam ladusnduo awaken amnquao uo none-«uouuuuuno acoumoaocnuo: .m muonuh condos no «caucuoaan 9005 Ann «antenuv .aao.oua unofiuaaua> .o«uoga¢ua=~ .H auuum ocuuoanu eaoo manage an: an pmaa oaoo .uolauohn acuaaonuod Macauaocsh .a .uoauusl novices nooaououuuv Hausaoshuu on .m .noou umcuvno khan-coon ted umununo vanaov .conadum no uaooqouqluoac .so:u«a :0 unooucculhoa: ouaunoouo>uudm .AaOGAloo and o-oodv :osaaan no ucouudcnluoa: .N uuonuh condos no Anamoaoao: . .aaououa accuacuu¢> convene .H madam ocuuoaau uaoo ho. uuauunquuHH ocuuu> mm meH ouoo nucuvauh vonuuuuuaoc concuaaoya can: caucus: mono: Hdlua< ooauuouom Anvuouumnvuo>nn engages «unaccouowz use"; .1 uoauagenu\uua«e=u Augustus co nauoauuooam Huouuoaoaguo: .H.~ canny 25 .ndnlnnd finanoanm1unoa Iona condo noaonna noawouIannu van» cannon» cannon and unannouon Mo condonounon hogan: obi: and .cNOHnloo: onol one cacao! nouubalao~n nu nunucno nono- Hunqanpa .oaonnl annual nu axounloo: cool and nmnunno uoaol Hana-Loy nno«aoom nononh no cocoa eonnnnao ounuouuonqaono iaxnsonoaonm canola» cocoon sundandum unoaom unmade uoaonun< Ann ommnonv one Homo unannoum pan on: «Access .ouun gonna ado-nu no oanannno and nanny» ununadan1oaaannno nooaaon noon«wo nunono«un~on on .nno«un«n¢> nudun cabana unonqu anonuuuun no can noun Hunqlnoa clan one u: vooo>nonnu nounnncnn .noaonunohn unununun nounn> o>¢n o» annoy one: nonunnl Han nu noaonncnu uncuaoom flung no ucddum canon-ac u<1nnu< on: .u< .mnu< asunom Ann om~1omav aux Hp apma nH .m ooh» can Achy sauna» voodoo-noanu .o .0 cube can Anchv unoauu one .9 .< oaha can Achy unooau mafia: .o .ouuuoonn onhu known can noaonncnfl .anun noon «nu an o «aha “no nan <9 .u .anan Hanonononsu one no < on»: as» ozone .an «a .o .n ooh» now anonn am mom .3 .< cube non haven-«nonnnu on: an on. new .o .ouuuoonn canon! annhuIOn can nounonoano mnuvnu .Ampv unsung-«c on» no» a. also on» no: manna-Lou uoaol no coda-awnowouno HaouuoHoanho: anonuh condoa no comm onenhaaox Ann coafluanpo 11.H.N Ganja 26 each class. His “terminaisons en ligne" were endings adapted to animals needing quick, violent muscular activities, while "terminaisons en grappe" were seen to reflect requirements for finely co-ordinated muscular activity. In this light Cole saw the latter as a primitive, less highly differentiated type of nerve terminal which was most related to tonic functions. In further restricting his observations Cole pointed out the high degree of specificity and functional adaptation of endings. His compari- son of neuromuscular contact areas suggested, for example, that in non- flight birds the value for this parameter was roughly one-half that of their flying counterparts. Apparently coincident with the loss of the capacity for the quick, forceful movements of flight was the loss of a significant amount of neuromuscular interaction area. Leg muscle com- parisons from both flight and non-flight birds to the back muscles of flight birds were not different--a fact which Cole surmised to be indica- tive of the lack of species alteration. He concluded that these and other similar observations were proof for functional adaptation of end- ings. Likewise the lack of structural differences of endplates taken from the selected muscles (muscles which "were closely related in" func- tion and histological traits) of each species was considered indicative of functional adaptation. In 1957 Cole (31) focused his investigation on differences which might exist between muscles of different functions taken from the same animal. 0n the basis of similar morphological characteristics of motor endings taken from seven muscles of animals of over 150 grams Cole was able to delineate 3 specific groups. Muscles capable of highly skilled 27 movements were found to have a constant pattern of compact "terminaisons en plaque" and narrow muscle fibers. The diaphragm, a relatively tonic- ally active respiratory muscle, was found to have a wide variety of end- ing types--both "terminaisons en grappe" and "en plaque" with the former appearing considerably more often. The postural and appendage muscles comprised the third grouping and featured large loose "terminaisons en plaque". Cole considered these findings supportive of his "functional adaptation" theory and further suggested that the "terminaisons en grappe" might "account for the absence of fatigue" seen in the diaphragm muscle. Hess (66) noted that in mammalian extraocular muscles fast (white) fibers had single endings of the "en plaque" type while the preponderance of terminals in slow (red) fibers was the "terminaison en grappe". Citing this and the lack of investigation of specificity of motor terminals as they relate to fiber types, Ogata (90) conducted a histo- chemical study with mice using a double staining method of succinic dehydrogenase (SDH) and cholinesterase (Ch). While differences in size and structure were noted, no marked differences in cholinesterase activity were seen. All endings were of the "en plaque" type for the gastrocnemius and adductor magnus muscles. Unfortunately, with Ogata's technique no indication of neural input was obtained. He did suggest that the differences in size and complexity might be attributed to physiological characteristics or muscle fiber size. Korneliussen and Waerhang (79) and Waerhang and Korneliussen (120) categorized terminal endings in diaphragm and hindlimb muscles of rats according to size and specific characteristics which these terminals dis- played within the endplate region (primarily intra-endplate branching 28 and bouton shape patterns). While the methylene blue stain used pre- sented problems, their results did indicate both muscle and fiber type specificity for endings. Type A terminals appeared related to "white" fibers, type B terminals were associated mainly with "intermediate" fibers, and type C arborizations innervated "red" fibers. Muscle fiber types were determined by a fat stain (78) and were well correlated with Ariano's classifications (3). Further the authors hypothesized that "each of the types of motor nerve terminals may innervate one of the three functional types of motor units defined by Burke gt 31. (20,21)". Finally, all endings were defined as "en plaque". As their categorization system and results appeared to be corrob- orated by earlier investigations (2,28,52,53), it was suggested that the pattern of innervation (size and number of terminal branches or swellings, and overall endplate size) was influential in both total amount of trans- mitter released and area available on the postsynaptic membrane for reception of transmitter. Presynaptic morphological traits were thought to possibly be involved with regard to the physiological differences of the various mammalian twitch fibers. Further examination of the postsynaptic membrane and endplate area by Ip (71) further emphasized the specific and dynamic nature of the postsynaptic ending. The finding of both pale- and strong-staining endplates in the same field, and in some cases associated with branches from the same terminal axon, suggested a non-uniform structure in normal muscle--one which was either related to specific fiber characteristics or one which was indicative of a transitional state. It was suggested that 29 a dynamic subneural apparatus could account for their differential staining characteristics and the possible fiber-type relationship. Comparison of prepubertal and postpubertal rat motor endings by Tweedle and Stephens (117) substantiated many of the previous findings. In mature animals terminal motor endings were categorized and it was found that primarily slow-twitch muscles had a different innervation pattern than those fast-twitch muscles examined. Generally slow-twitch muscles had terminals which were more complex in that they had either numerous branches entering one endplate region or several rather diffuse endplate areas associated with a branched terminal axon. This complexity was not visible in fast-twitch muscles nor in muscle taken from animals under 5 weeks of age. This elaboration of motor endings appeared, in some cases, to be related to activity level while in others, was surmised to be a function of a maturation or ageing process. Junction Size--As with morphological characteristics, endplates or terminal motor ending size nay be related to muscle fiber size or type. Numerous studies (2,53,56,59,7l,79,94,110,ll6) have reported a direct relationship between endplate diameter/length and muscle fiber diameter. Ogata (90), in addition, has reported a possible relationship between fiber type and endplate size. His white fibers had endplates on average 23p, while red and intermediate fibers had endplates measuring an average l4u and 20p respectively. Similarly, measurement of terminal ending diameters in the three types of endings from the work of Korneliussen and Waerhang (79,120) indicated A-type terminals innervated thicker fibers while B-type and C-type innervated intermediate and thin fibers respectively. As with 3O endplate data, that on endings appears to indicate that the size of the terminals is approximately proportional to the width of their muscle fibers. A correlation between fiber type and ending size was not directly available, though considered viable by the investigators. Dias (36) using intravital staining with methylene blue and a grid- area measure instead of diameter indicated an extremely wide range of ending areas was present in all muscles of the rabbit studied. He found the slow soleus had mean surface areas for motor endings which were signifi- cantly larger than either of the predominantly fast-twitch gastrocnemius or flexor digitorum longus muscles and consequently suggested a relation- ship existed between speed of contraction of a muscle and the size of its endings. In the soleus the large size of the motor ending was related to its function. It was proposed that in this postural, continuously active muscle the large size ending either facilitated the release of neurotransmitter or was involved in the trophic regulation of speed of contraction of the muscle. Electronmicroscopic Investigations (see Table 2.2)--The electron- microscopic investigations of myoneural junctions by Ogata's group (87,91,92) revealed the fine structural differences of motor endings and endplates taken from "red", "white", and "intermediate" muscle fibers. The differentiation of muscle fiber type by identification of the size and composition of mitochondrial chains among the interfibrillar spaces, deposition of mitochondria beneath the sarcolemma, and the size and shape of the bracelet-like mitochondria around the myofibril at each I-band enabled these investigators to firmly establish the specific characteristics for neuromuscular junctions of each fiber type. 31 .ononona ono: omnvnonooaul mnunononn mnoa uo anodaomonmmo obuonouxu .uwm on: ununonoup uo nauauonohm .onaou AI: H.0«m.o n swoon noon” nono~aonn and Am.u I onounaofl oaanonhn I: hon undouv nonou nae: noaodnuno no: oaonna nnoaom .N .nnonowaonoonu ouoa nunnnonooaax .Auma I nonononn Adonouunoanov nonou uo no«anonoua and «In H.o«o.o I dunno nool «m.~ I onnnnlol nuanonho onuaonooauooc AIS omv I: non ovuouv eonoaooov onol onoa noun: noanununo can onoonl unalonoonunoo .H onodom onno: o: cpma nonona nnmnoa .18 connoowv mwmu ovens: .anm.pwv goo: ono«>ona new: unoloonmo nu ono: oaflnoom .H Inocuuugn noonoaufl vac «a 6nd dunno .oonou on» nnnoao oououooo noun. uncuno> no: anonuu oaounolnounn .: .oovo nuanunnnunoanu on» on and onuou Hono«uon=n on» yo loauop on» no hano an: nononoob nodulao no: nnonuu cox .m .onuou on» no nonuo end .532 on» a. 2582 .338» 8.28.. 39:- .om.:: 3.. 2d. :25 32: .m 82 3.8 .Aumv duo: n=o«>oua new: anonoonuo nu onoa naunooz .A Hoanoonounu noes: pm one cannot .oouv-«nouoonono oaowvonnoanu uo ooaoaanno no: gonna ouounoauoann .m .oonoudooao ounlum .nonolouoond and unununnna nonql saw: .aoaaonn ono: canon .nno«aomonmmo ounnnonuouql nod: nonnuu ono: noun: among-no» and undo» noonaon nounnxo noono omnna .ocaou Honouuonnq nono~o>ov hanooa new: ooaoaneno Haul. non spongy no: .N .ounouoonno xoanloo .aou ono: loouaoouno nonpuuonna nu pwma unannonooaux .nonoloanono vno nonononp hdo>uonoano .mooo ouo: undo» .onuou Ala connoowv oaaom on: HonOuaonSa nacho-5n van noaoao>oc uao) new: ooanaacno omuou non unonuu oaqnz .H unaccouounm «on um ouno: .ooomo omnuvnuh conunouonoo coo: oaoon: mono: anaun< oononouoz Anvnoaomdano>nn monsoon anacononouanonuooam 11 novodnnnm\own«vnfl flung-hob no huaouuuoonm anoqmouonnho: .~.N oaona 32 .Hooon nuanooaoa onunnlol on» no unanaonnn nounonalonnon anmnoa Iznoanwnn noonouxo on: onouou on» nooaaop no>noono onoa noonononnnn o: .nOnuoonnnnuol nod hdoonunaon no onov Ana .ma.aa.bmv onOnaomnuno>nn nnon>oun no oooau nolnnnnOo huaunonom unannoz cannon announMna nonnouxu nnoaom Ann cannoom. as: mean noonon can anaonooum w; .naan .aaaunnnu .uaonnnnou ode-nu ounno non» nononno> gondola an: onol nuunoonn 1nnwno con and noun Hanno>o nu oaonvonuounn onoa canon-no» nonnn ouonvonnounm .uononoo> undo-Hooxn gondol- ans nnonnsn owned aqo>naouon new: oono Haono>o nn anomuou ono: nannnnnoa nopun no: .nuwnon onnnaaol announhoau0910a1u«wanna-ohm omnou o no: noaouneno nonnn owns: .noaonuo> unanonho nonon an: nomnon no: cannono ooona .hdanoonnnnmno eon can unoAnn nonao no o-ona non» nounolo haudnonom ono: nnonnn owns: no ownnnnu Adonunnnoasnv nnqlonoonunoc anonom .am oomnooa. can mean Hanna mod on. canon .couonvoluoonn: ono: noun-«nonconono nonoo .omnnnno onhuinoa«n nonuo no ouosa non» anon. nonuhon ans nonooc ono: onHo ovuon ”onenaonna .aoonoon and anomnoa one ono: canon-no» nonnn ouonnoluoanu .anooonn ono) unannonoovnl nonoao>ov Ado: no anopnsn omnuq .oannnnnoSI and nouns vdon nooaoon unooonn no: anoanonooon onflnoanoonno o>nonooxu .voooan huonnmonun ono: noun: ocdon Hananaonne nonon and vo>nno .unono nonouannc onon now-Hanna .nnncnonooanl nooouobon Ado: .nnoanonnunn on. sound-o» voxonn haooooa nan: HoonaAnnuo and Hadl- onoa umnncno nopnn no: .huno aoounoo nuances-onnon no ooono nooouoenoon aonxo unannonooanx 693% no: 03.520? on» one union on» no own: one noouaoa loonaoonom .unmndnan h~o>noonon nno noxoom haonoHo .mnoa ono: onuon .onaon Hananoonnn nnouolnn new: bonanza ono: ononnnnoa ooona nan: nouonoOnon noaonnnnu .onnnnonooann nonnoan end ooaonao> onlooaaoxo nosed: haugwna .nnonolnn nan: andn and mood ono: ononnn owns: no ounnvnfl .H Iuonnmonn Age onmuoomv on: Chad nounusuo mm and ousahddm amnnvnnn eonndononoo eon: oaonn: Hove! Holaa< oonononoz .ovnoaomnuoo>nn oozauanpo 11.N.N oanne 33 White fiber neuromuscular junctions were characterized as being the largest of the three types and the most complex. They displayed an extensive array of parallel, yet well-branched and anastomosed, junc- tional folds. These deep folds were separated from the myofibrils at their bases by scant sarcoplasm. Mitochondria at the junction were few and poorly developed. Red—fiber myoneural junctions, while smaller and simpler, displayed well-developed mitochondria both between folds and at their bases where, in company with nuclei, Golgi apparatus, and a large sarcoplasmic area, they provided a large space between the folds and the myofibrils. Intermediate-fiber synapses showed char- acteristics generally between those of the red and white junctions. The functional significance of these traits was thought by these investigators to be related to neuromuscular transmission. The wide junctional area, and characteristic fold pattern of the white fiber synapse was advocated as a mechanism for rapid transmission of impulses to the fast-contracting fiber. Such rapid transmission is not neces- sary in the slow-contracting fiber; consequently, the ending/endplate structure is smaller with less total surface area. Padykula and Gauthier (93) also suggested that the extensive surface area offered at the white neuromuscular junction could be related to supplying greater amounts of acetylcholine or some other trophic substance for in addition to the folding membrane network they found in white-muscle junctions significantly more sarcoplasmic vesicles, axoplasmic vesicles, and intramitochondrial granules. Santa and Engel (109) indicated that the postsynaptic receptor sites had a concentration approximately 20% higher in white and 34 intermediate fibers than in red. In addition the synaptic vesicle diameter was slightly smaller in red and intermediate fibers while the synaptic vesicle count per unit nerve terminal area was larger. Coupled with mean postsynaptic-to-presynaptic membrane length ratios which indicated white fiber endplates are more complex, this would appear to support the contention that the white fiber terminal and endplate are more suited to rapid, phasic impulse transmission. Morphological Age-Related Changes of Terminal Endings/Endplates Histological/Histochemical Light Microscopic Studies (see Table .2L§)r-Cole's (31) assertation that compact "terminaisons en plaque" were immature endings characteristic of young animals while loose "terminaisons en plaque" or "terminaisons en grappe" were more specific to older animals led to the assumption that there were age-related morphological variations in motor endings. Baker and Ip (6) investigated these age-related changes and the possible mechanism by which they were brought about. It was their hypothesis that terminals had "a limited life-span" and that they were "periodically replaced in normal muscle by collateral regeneration" once that life-span had expired. The mechanism they advocated for this turn- over was that either a new endplate innervated by a sprout from the degenerating endplate's terminal axon was formed on the muscle fiber or that the established endplate received a new ending from a sprout of the parent axon. The former case was thought to account for the presence of the observed "double endings" while both mechanisms accounted for the "accessory endings" which they saw. As an addendum they suggested that 35 .:n0nnono>nnon: an mnonnonnq aon no newnnnnon nonconcnn gonna annoxo noHHoam mnnnono Oman and onsonno wnn>nooon nonnannnm .m .ooaoaanno non ownnnno Hoanon mnoeo .=xoon mnnau: havnonoaan .oapnmn> onoa onoxo noaaozo one oouoaaono wnnuononowoa .: .nonndeo omnnnno Had no um.:~ manmnon mmnnnno dunno: and .na.w anono onnonno .nm.HH haovn Isnxonnnn an oooa ooHnOmoooon odomnn LOuosouoHoxn nH .m .:mwnnnno oannon: on hound Hanna and :nwnnnno hnommoooo: on oaoanno nonao nn noon onoa :nnnonnm: .N .030 nonuo on» no nonuno on nonno no ononnh condos Hoononom oonsa nonnnooo unnonno Honoz .oaomnn Honnon nu noon no nnoum no>Hnm nnooooononnm Ano>nm nonv mwaa ono: nononno nonnsnouonaan nno HonnEnouonq .Hnnoz .H onuooo on nonnnno: onoaom unannm .uoo m AH and noxnom .A.H.m oanoe oonv hononAnoa onoagnno mono: oanannn nonao nn oHnnomon no: unnonm oaoonn no nOnuonpnononnno .4 .onano onos onoa onnsonoonumoo and omnnvno no moan» and non oaoannn Anomnoav nonao .m unnoaom annoox .couonnaononn nmonnnono :ondoan no onoononnanou: noonaoo nno unooonn onoa noncononnn noon “anon ~h1m: :oanonm no ononnnnnsnou: on nannnno Annonov wnno» nm .N ononnn condos no onmnoe nno anon mm1am .nononnonoo nnonm onnnoHnu caoo aaonnonno>onom haouoanxonnqov 3 moo con: $329.8 23 on: 5 .882. confines» A :33 32:5: 333235 fix 8 RS 28 .oapnnn> onoa omnnvno hnOooooon and oapnoa .m .uonoonnnn on: own nun: ounu ouoannno nn oooononn no nwnonuan .Hdnnno o» unannn no oHoonE oa oaoonn Bonn ouoannno no omono no neon: nu nOnvonno> anoonnnnMnn on .m ononnm condos no :maoonoo unnno noon: Anzac m~1mm :33: 3:35 33m 8225 Soc 2.3 6% 6an 23392.33 93 239: as. nooaaon 82?... 3:20:32 oz .H hogan 3825: 338235 can on SE 38 ownnnnnh nonnannonou nondnnnooe non: modems: Honor Hoann< oonononom onnoaownvoo>nH ouasoom onnoooOLOn: unwnq 11 mononnnnm\mmnnnnm HonnEnoe nn mownonu Hnonwoaonnno: nouoaoz1ow< .m.m oHnnB 36 .Auonanool non gown nonnaooo anonasn nonnn odounl can nn undonooo < .a .annaunoo undo-on nonoldun unmannno on» .nouOIIHv nu uonuononn nonnn odounl one now and can on» nu .d "on: nun: .uonnloo noon and amnnnno now .3 .nnopnn Anouoluwov nomndn no nnnon on: and muddnnanoa nonnnoo and noaaono .nouon nun) nodadlu uoaoonaon cued ono: nonnaanno eon .d "nonconnnn uouonnl now and can mason no nanundnlao .anm on» n« noon onoa no nuoaoznnoa oudunvno nn nomndno ndnnlau own mnnond>vu nun: nondoand ononu onouna now nH .mnnnoomno onnduua onoa gonna cannulnoa no nanunononn nn ondononn n< .n .uoaonncno no nOnadnonomon Hanan anondnno nn onoonooo < .o .Ananxoanloo .o.nv nononno and uncanno :nnOnuoooo: nu oudononn n< .n .nwnnono soanlnu: nu ondonoon < .o ”own unnon¢>n¢ nan: vonuonnd anon» causal can nu .nonunanouuo no: loo-nu nodannnuomouao oooannno\wnnnnfl canons cannon no guano uo>Hnm cannon on nonnnuo: :03 .538 Aannv «unnna gunman noononom Rondo» an and .on .mn .on .w .n.ov «no man anon noonnse .unolnoao>oo wnnnsv ouonunononnnv on. oonononn noun. ouaannnu .0»: Anna gonna oluoon uoannnnno nunan> sauna-non nox< .uuuunaa one a. eouuononc and nodnmonnu no: nuns: ono on ononnl one no cause 6:000. and aunnn ona no cannonnn on» an nnoaann no» Imus nuanmon a loan nomnnnu nodao>honnn no nnoaaon one .no«u¢u«HoSoI> and nOHuounolwunn nannndnu .nOnnnuonon connonuonnnono ndasmonnu .mnnnnda- oada a» ounovv nonauunouuonono no haonnn> can: I 6030:. noun: ooaaannno can nauannd ado .unannonnn nonoao>ov duo: .Hnbo no: uudinnn wanna odds: .noauannno canon-la .nocnson voaonu ndnlnnd manor .mnnmd no“: nonnnooo conning navondnnd nonnonnnm .H ononuoom nononn no nudam coonouuonnu .ono uuoanuconun can canoes connnuox cacao» cannon flanonnmna nannouxfl .nnne own an. .ond .cuv «an nwou 29:35.: on and nnnflano nmnnvnnh nounndnonoo convunnooa non: noauunx Mono: Hulun< ounononoa Aavnoeomnaoo>nn confidanoo II.m.N canoe 37 .nqdnnnn nomnno» n« no nno~ on canon on on noon. onon canon“: Hangings .Auo~«mm I nanonnn «nnoanuaoo an: “Nana I nanonan annnlnouv noon nnn\ono: unnanonnn. o>nunoauo and nmnnnno Ibuoonooononnu anon. Hana-nouonn and .oonoc Iu>o annanonn- Honounauoo .uovounnno no nodunpnonnn onnnudnl an: Indiana ado nu noon onH< .Aoanun Icno non e.mv cannon nonnnn Hana-no» oanHAcnolnnunn son nun: nooanneco can-n. .ununu can adaannu guano .oonunvno non cannon nonnnn unnnlnoa oundnnnouonann m no>o can: noanunnno unomnnn on» no: andlnnd onnunz .Aoanunnno non m.~v cannon nondna Anna-non ovuanvnouananu son nan) uoanunnno onoanlun .ano~nnln on» no: nadlnnn mason .A nnOnuoom nononh no nndow no>Hnm onnnounonnaono “unto one and .03m .oom .ow. unouom an: own” cunnnuo nnn nnlnonun. an .uaoaaaon ananocnn eoanuuuonon nonannnooa con: caucus: Mono! Allun< ounononoz Aonnounmnauo>nn voanuanoo Il.m.w Danna 38 it was possible, though rare, for a new sole-plate structure to be formed on the muscle fiber by a sprout following abandonment of the old endplate. With continuous development and regeneration ongoing some sprouts were thought to end up redundant--these failed to form terminals. Tuffery (116), while in agreement with the finding of motor end- ings and endplates undergoing growth and degeneration in normal muscles, rejected the Barker and Ip "replacement hypothesis". She showed that few degenerating endplates received growth configurations and that those which did had growth configurations which were also degenerating. Collateral branching as evidence for replacement was also dismissed as there were insufficient increases in this measure when age was considered. Finally the Barker and Ip hypothesis failed to fully account for the increased complexity of ageing myoneural junctions. Tuffery proposed a theory of ending/endplate elaboration to account for the changes in innervation pattern seen in ageing animals. It was suggested that this elaboration was brought about by increasing workload which in young and maturing animals was seen as a reflection of increased body weight and animal activity, while in aged animals (senile) was attributable in part to a loss of muscle fibers with compensatory hyper— trophy of those remaining. With fiber hypertrophy and no visible sign of increase in endplate size in her study, Tuffery suggested that the elabora- tion process would provide a means for conveying increased nerve impulses or amounts of trophic substance to the synaptic area. As each "branch" provided additional input, a larger portion of the ending could be acti- vated to compensate for the relative decrease in neuromuscular contact area. The reduction of morphological variance between endings of 39 fast- and slow-twitch muscles with ageing was considered a further characteristic of the ageing myoneural junction. Gutmann and Hanzlikova (56) also showed myoneural junctions were somewhat transitory and reported a "dedifferentiation" of motor endplates with increasing age as well as some examples of degenerating endplates. The differences in fast- and slow-twitch muscle endplate sizes were seen to disappear with age although muscle fiber diameters remained discrete. In addition denervation-like changes were reported in the pattern of innervation where in animals of advanced age the distribution of endplates became somewhat more random with endplates being found in more distal parts of the muscle. These age-related change in endings/endplates were seen by the authors as closely related to a functional decrease in neuromuscular relations. These alterations were also thought to partly explain changes such as decreased fiber numbers (57,59), reduction of motor unit size (57.59.60), or denervation-like adaptations in aged muscle (56,57). The findings of Pestronk gt 11. (94) supports a theory for ending elaboration in mature animals and also Gutmann's "functional denervation" hypothesis for senile animals. The expansion of endplates and elaboration of intra-endplate terminals in their maturing animals and the relative decrease in these parameters in their older animals is indicative of the plasticity of the ageing neuromuscular junction, as is the increased sprouting observed by Fagg gt 31, (43). Further evidence for this mut- ability is presented pictorially by Pestronk gt El: (94) in the form of increased numbers of branched and double endings, and ultrastructurally by several authors (23,44,52). 4O Electronmicroscopic Investigations (see Table 2.4)--Fujisawa (47) has suggested that shrinkage, retraction, and eventual dissolution of terminals, as well as sprouting, accounts for synaptic reorganization in normal animals. As evidence he has identified, in the vicinity of totally or partially denervated endplates, preterminal nerve fibers which are normal in appearance. He also stated that there were insufficient mutated nerve fibers in normal muscle to account for degeneration as the prime mode of reorganization. Cardasis and Padykula (23) have suggested the stimulus for realign- ment during maturation and ageing was related to changes in speed of transmission of motor units, muscle fiber hypertrophy and/or natural conversion of muscle fiber types as explained by Kugelberg (81,82). They further emphasized the role that a rising workload imposed by con- tinuous body growth plays in muscle activity and synaptic reorganization. Forced Exercise Effects on Terminal Endings/ Endplates Scant information is available on the effects of exercise on motor ending/endplate structure. Reed (100) focused on the examination of motor endplate surface area in cholinesterase-succinic dehydrogenase stained sections taken from animals which were sedentary, involved in voluntary exercise, or subjected to a variety of forced exercise pro- grams. It was indicated that running, particularly high-intensity running, results in increased motor endplate surface area as well as accelerating the rate of area increase. It was noted that once the training program was allowed to plateau, endplate size was not maintained. Further, these changes were specific to the soleus muscle. Evaluation of 4] .onnoao unnnonAo cocoon: .v .monnaonnno oaoanvno and Honnanon nooaoon nonnno>noann HHoo nnonnom .o .unaon onunnnhanuon nono>oonn no noono and .snanonnon onsnnanonno naooao .ounoadannonnon .oodnpnuonona nu condononn .p nnmnoa Anson .oonn Hdnnnnonooowa nu ouoonoon one «odouuo> unannnho no moo; .n annOQHMna noononxm one find onv Hood unappom "nonono .oHooss nnoaou nu hannoannn .odonnanou oao .a onoaom onno: a: vno annon .nonnnunnadn on: non» noouuon nOnnno>noann Haoo nnounom oannooon nan: nouoanono one amnnono no nonnonnnoo annnonm .: .haunonoonn nooH swoon» noonn nuannno :ona onenaonza dunnonOhE onnEonoonuooo .m Aamonna .ooonu nono>nonnnn= non» noon condonnn Anuanonnuv unoxd Hanan «unannoooo .N Iona and .nnwnoa .oaaoo nnoanom announmna nOononxm nno onwana Hanna unocnnvon nun: nononoouod can madnnnnoa noun hp vono>oo .usnnonoonanoo Anzac ommiamv mea oanxanon non ono: noun: onaon HonOnnonnq no wound noaonn unanuonna anoaon no onnnu ono .H canny onoaom pom mm and unmocnou .mnnvno on» nnnana nonponnaonoo> .n .ouoannno can: oono woounoo no>nomno no nOnnoaoon onn omono no wood can: wnnuno no owoxnnnnm .o .A< OOMMaOOaV nonno uncann> no noaonoo> no oonomonn one .2 . . A 8:53 onnn mnnnno onn>nn nanoov onnnonon Hoanoaoxn noxn Hannanou no nOnunnnoonH .o Anzac mm» "hp nounnopunnnno ono: noun: onenoonnn modems: and «mm .mmmv mpma annnononn onus: end non nu haaonuo no>noono ono: oomnono o>nnononomoo .H swank Honoo: you b: ouoanenn omnnonnn nonnuononoc coo: nononnx Hone: Hoann< oonononom onnouownumo>nH opanoom anacomononenonaoodm Ii mononannw\mwnnvnm Honnnnoa nn momnnno Hoonononnno: connaomlow< .:.N oanoe 42 tibialis anterior muscles indicated that there was a trend toward decreased motor endplate size in all groups examined. A possible relation- ship between muscle fiber types, their conversion due to exercise, and motor endplate size was suggested. Owing to the relatively small number of endplates measured per treatment group the implications of these results should be carefully considered. Crockett gt 11. (35) considered the effects of endurance exercise on neuromuscular junction cholinesterase and choline acetyltransferase. While no differences were found in choline acetyltransferase which could be attributed to exercise, distinct differences did exist between the soleus, red vastus, and white vastus of the rat in this parameter suggest- ing fiber-type specificity for enzyme action. Cholinesterase changes were noted. In untrained animals it appeared that fast-twitch glycolytic fibers had l5% more cholinesterase than fast-twitch oxidative glycolytic fibers. Likewise the latter had 34% more activity than the slow-twitch oxidative fibers. Training affected only the white vastus (fast-twitch glycolytic fibers) muscle--here an increased level of cholinesterase was present. The apparent discrepancies in cholinesterase activity between specific fiber types were seen as compatible with the relative endplate size, degree of ending/endplate development, and the magnitude of the fiber-specific membrane potential. CHAPTER III METHODS OF PROCEDURE The purpose of the current investigation was to determine the effects of both maturation and an exhaustive, endurance-exercise program on the morphology of the motor nerve ending. Specific differences in the innervation pattern and terminal ending structure between selected fast- and slow-twitch muscles also were examined. Experimental Animals One hundred normal male albino rats (Sprague-Dawley strain) were obtained in two shipments (Table 3.1) from Hormone Assay, Inc., Chicago, Illinois. Animals were either 30 days or 72 days of age upon arrival. All were allowed a standard period of 12 days for adjustment to laboratory conditions prior to sacrifice or the onset of the respective treatments. Research Design and Treatment Groups In accord with the basic objectives of this investigation, treatment groups were designated by both type and duration of activity (Table 3.1). Each animal was randomly assigned to one of the following treatment groups: l) Control group (CON). The control group received no special treatment. These animals were housed in individual sedentary 43 44 un nan nm on nan m un can m un naz u ---- ---- mono aunnnnoam an 2a: nu un an: nu un an: nu un an: nu ---- ---- mpao na>nen< amnueaxu u on u on ---- ---- 2 un nan nu mn nan u un nan u un can m un nu: u an nu: u mama aunnnnuam un ca: nu un an: nu un za< mu un ca< mu un na< mu un na< mu mama na>naz< ngaoemuam m on on on on on 2 on u_ u e o o Amuaazv annnnmnn uu eu cu en un m Amuamzv oonmnnumm o< man nonuunzo cannon sonmomom .n.m onamn 45 cages (24cm x 18 cm x 18cm) throughout the investigation. 2) Endurance-running group (END). The endurance-running rats were housed in voluntary-activity cages for the initial lZ-day adjustment period and subsequently in sedentary cages for the duration of the experiment. The END animals were subjected to a previously standardized, progressive program of endurance running (Appendix A). Traininngrocedures The training program was administered once per day, between 1:00 P.M. and 5:00 P.M., five days per week (Monday through Friday). All training was conducted in individual controlled-running wheels (CRW) (121). Training was initiated when the animals were 84 days of age and was continued for 4, 8, 12, or 16 weeks. The folTowing data were collected during the training and main- tenance periods to monitor and document the study and to ensure the END animals were well trained: 1) Weekly records of the body weights of all animals were kept as were daily body weights (pre- and post-exercise) of all animals subjected to forced exercise. Onset-of-treatment and sacrifice weights also were noted (Appendix B). 2) Daily-records were kept of the temperature, barometric pressure, and relative humidity in both the training room and the animal housing quarters (Appendix B). 46 3) Daily records were kept of CRW master control unit settings used with the END group. These included acceleration time, work time, rest time, number of repetitions per bout, number of bouts, time between bouts, shock level, and expected running speed (Appendix A). 4) Dai1y records were kept of CRW results for each animal in the END group. These data included total number of revolutions run, percent of expected revolutions, cumulative duration of shock, and percent shock-free time. Data on total meters run (TMR) and percent expected meters (PEM) were calculated for each animal each day. Mean daily values of TMR and PEM for the END rats included in the study appear in Appendix B Calculation of TMR and PEM provided values which served as the chief criteria in the evaluation and comparison of training performances. Final inclusion of an END animal in the study was based on its ability to exceed 80% of the expected meters to be run and a subjective evalua- tion of the pattern of its training results. This was necessitated by several problems which arose during course of the study with the CRWs. The effect of "rocking" in the wheels (activation of the revolution counter without actually running a revolution) was thought in some cases to have slightly inflated the training values, specifically TMR and PEM. In addition, internal problems, on occasion, with the master control unit and individual problems with current flow in CRW grids negated the shock- related data. While these problems certainly limit the reproducibility of the study, it should be noted that the training program was very 47 exhaustive and intense. Those animals meeting the selection criteria were well-trained (endurance) subjects. Animal Care Throughout the experiment all animals received water and a commercial rat food (Wayne Laboratory Blox) §g_libitum. Animals in both CON and END groups were handled five days per week. The butcher's paper under the cages and running wheels was changed daily. Additional standardized pro- cedures for daily CRW cleaning were observed and all housing cages were steam cleaned every two weeks. The animals were maintained in a relatively constant environment with a controlled temperature and humidity. An automatically regulated light sequence was established so that the lights were off between 1:00 P.M. and 1:00 A.M. and on between 1:00 A.M. and 1:00 P.M. This lighting pattern enabled training of the animals during the active phase of their diurnal cycle. Sacrifice Procedures Four sacrifices were conducted; one each on the Monday approximately 72 hours following the last treatment of the exercised animals (Table 3.1). All tissues from the animals included in each sacrifice were processed immediately and then stored for simultaneous analysis following the last sacrifice. A standard sacrifice routine had been developed during the course of previous investigations and was continued without alteration in this 48 study. Likewise an experienced team was retained throughout all sacri- fices and tissue processing sessions. Immediately prior to sacrifices final body weights were recorded and a randomized sacrifice order was established. Subsequently, each animal was killed by a intraperitoneal injection (4 mg/lOO gm body weight) of a 6.48% sodium pentobarbital (Nembutal) solution. As soon as the animal was dead, the right hindlimb was skinned and the exposed super- ficial adductor longus muscle was removed. The superficial posterior crural muscles were exposed by reflecting the overlying tissue, and the gastrocnemius and soleus muscles were removed independently. Finally the rectus femoris muscle was removed. Immediately after removal from the animal a block of tissue from the belly area of each of the right hindlimb slow-twitch muscles and a similar tissue block from known areas of predominantly fast-twitch fibers in both the right hindlimb gastrocnemius and rectus femoris muscles was mounted on a cork strip using 5% gum tragacanth and subse- quently quick frozen in isopentane cooled with liquid nitrogen. Blocks were stored in a freezer at -80°F until sectioned. An identical procedure for removal of the left hindlimb muscles was carried out. However, prior to further processing the soleus and rectus femoris were utilized as part of an additional study to examine, jn_vitrg, the contractile characteristics of these maturing or exercised muscles. In addition, all excised left hindlimb muscles were weighed before place- ment for fixation in a 10% formal-saline solution with 0.5% dimethyl sulfoxide (DMSO). 49 This sequence of events (for removal of muscle) has been conducted routinely over the past several years and can be completed within 10 minutes. Tissue Analysis Right Leg Five sets of three serial cross-sections, lO micra thick, were cut from each muscle using a rotary microtome-cryostat. Each section was identified by muscle, animal number, and date of processing. One section from each set was subjected to one of the following histologic or histo- chemical procedures: a) Harris's alum Hematoxylin and Eosin (H&E) for demonstration of basic morphological characteristics (83), b) Dubowitz and Brooke's method for demonstration of myosin adenosine triphosphatase (ATPase 9.4) activity for differentiation between fast- and slow-twitch fibers (39), and c) as described by Barka and Anderson using NBT, succinic dehydrogenase (SDH) activity was demonstrated for oxidative capacity indication (5). Sections from all animals of a given sacrifice were processed simultaneously to ensure the use of identical techniques between those animals. A representative group of 40 muscle fibers from the midportion of each muscle was selected for further study and only the best of the stained sets was used. The relative staining intensities for both ATPase and SDH for each of the 50 fibers was subjectively evaluated, recorded, and used to verify (see Appendix C) previously reported fiber-type compositions for the muscles examined (3,107). 50 Left Leg Muscle blocks from the left leg were fixed in 10% formal-saline with 0.5% DMSO for 6 hours, washed in distilled water rinses over 16 hours, and then frozen on metal chucks immersed in liquid nitrogen (only the chuck stem, not the tissue, was immersed). A minimum of six serial longitudinal sections, 60 to 70 micra thick, were cut from each muscle and placed in a solution of 10% sugar water. These sections were stained using the combined cholinesterase-Bielchowsky method of Gwyn and Heardman for motor endplate-terminal axon morphology (62). This stain has proven very reliable and has enabled the development of a quantifiable ending classification system (117). In addition it enables evaluation of the majority of endings in a muscle section. Other staining techniques such as the method of Pestronk 33 21, (94) or Cbers' methylene blue (28) have proven unsatisfactory as they either fail to demonstrate the desired ending structures or are unreliable when used with large groups of animals and sections. The morphology of the terminal arborization was evaluated by the primary investigator for each muscle examined. Random spot checks by Dr. Charles Tweedle were used to verify the rating system and to ensure consistency in examining such a large number of slides. A minimum of 100 clearly defined terminal arborizations and end- plate structures were categorized as accessory endings, double endings, branched endings, simple endings, multiple endings, double-innervated endings or sprouts (nodal, preterminal, and ultraterminal) (Figures 3.1 and 3.2). An additional division was created by summing the percentages 51 in the accessory, double, branched, and multiple ending categories and is indicative of the percentage of "complex endings" in the tissue examined. Abruptly ending or 'broken' axons were eliminated from any data collection or analysis. Slides with less than 100 endplates were either recut and re-analyzed or eliminated from analyses (if recuts produced less than the specified number of terminal ramifications). Analyses of Data Comparative statistical analyses were done using the Mann-Whitney U-test (111). A statistical probability (P) of less than 0.05 was con- sidered to indicate significant difference between means. a) b) c) d) e) f) 9) 8—i’i \\.\Tc 8 \\\IAL_3 O \/AL_3 W Figure 3.1. Classifications of motor nerve terminals. LELJULJUv Simple ending. One axon terminating at one endplate innervating a single muscle fiber (see also Figure 3.2d). Accessory ending. An axon with one or more thin branches originating either from the nodes of Ranvier or at the end of the myelin sheath which inserts into one endplate on a single fiber (see also Figure 3 2c Double ending. A bifurcated terminal axon which ends in 2 distinct endplates on one muscle fiber (see also Figure 3. 2d). Branched ending. A branched terminal axon which culminates in 2 end- plates on 2 separate muscle fibers (see also Figure 3.2e). Multiple ending. Three or more branches from a single terminal axon which form more than 2 endplate structures on a single muscle fiber (see also Figure 3. 2f) Double-innervated ending. A single endplate structure on one muscle fiber apparently innervated by two distinct terminal axons (these were very rarely seen). Sprouts. Fine unmyelinated fiber(s) originating either from the nodes of Ranvier, the end of the myelin sheath, or the endplate itself and ending in a growth cone (see also Figure 3.2a and 3.2b) 53 Figure 3.2. Cholinesterase-Bielchowsky stain for morphology of motor nerve endings. a) Nodal sprout b) Ultraterminal sprout c) Accessory ending d) Double and simple endings e) Branched ending f) Multiple ending 54 CHAPTER IV RESULTS AND DISCUSSION Results The results of this investigation will be presented in six main sections corresponding to the terminal nerve ending categories. Each section will be further subdivided to discuss changes in terminal arbori- zations with respect to ageing, muscle fiber type, and exercise. Finally, the discussion attempts to relate the present findings to those of other investigators. Accessory Endings (Figures 4.1 and Tables 4.1 and 4.2) Ageing Effects A significant increase in accessory endings was noted between six weeks and 12 weeks in the soleus (P = 0.001), adductor longus (P = 0.001), gastrocnemius (P = 0.001), and rectus femoris (P = 0.01) muscles of sedentary animals. In addition in the adductor longus muscle between 12 and 16 weeks a further elevation was visible (P = 0.025) after which time no change in mean percentage accessory endings occurred. A similar ageing trend in the soleus was apparent up to 20 weeks (P = 0.01). However, a subsequent decrease then was noted in this muscle between 55 A.moonononnnn unconnnnmnm non axon oomv 56 .mnmn nomnunoxo unm anonnocom no monomze nopuonom nn mmnnnno anommooum omnunoonon .n.¢ onnmnn $3.3 02.2.5: $3.3 wed o. u. . o e mu nu cu m. u. o _ _ _ Pl 0.0 h _ n _ b _ 0.0 on on macaw“. manowm x x \x 06. 8.520855 .11.... \ o.o_ mamfiow oilo xx \x/ on. maozoo 55:84 I \ on... , /. I/ O / 0.8 w 0.8 I no xIIIIIIx\ / 3 . ,x onu $ omu o H. 0.8 A 0.8 3 . m 0.8 0 mm M 9 S mmfimmxw 0. 09. 0.9“ 0.0m 0.00 >mU\bwn Oer-lu-i000 F1 F1 NiV\b-U\fl1r-OD 0.:(01-100b- .: ux .3 Haw—3mm: mph-100F163 ono.41~1~ o1c>c>cuxo N 21.5 2 2 Accessory Double Branched Simple Multiple Sprouts Complex Adductor Longus 57 O\®\OMHU\O\ mNHmFION r-l CDNQJr-i-SO mommaoo MH .3 u'\ \OOv-iwwb-P mNHWOOW (COMP-1.30m Swdgooin .3 Ln PJOPMOO PMHO‘OOO H MOWOHOO FOHHOOO JH .3 \o ONE-Qmomm FMOQOOQ \DQQMOLAN OJOMOOW .3 m HQQOMMb— OHHHOOO r-l HMMOHHQ u§;§.4<£<5<5<5 m U‘\ .3 01:0 0: 0.0 CD h l 1 2 0.0 0. 17. Accessory Double Branched Simple Multiple Sprouts Complex Soleus OLAHU‘OMN \OOHPOOP OawHOr-lN :OOJOOIA M \D m mOmCDOMb- b-HOPOOP PWMMOHW MOOmOO—If m \D m OMPOOOO HOOP-10°F! H H H Hanmooo HOOQOOv—i m \O OU‘V‘OOb-O HDOHCOH H H H CORJHOMW WOOMOOW N l5 N dwmb—Ot-CD \OOH\DOO\O Gar-1000.3 mOHNOOP N P N NHQQOUNQ biplnie-CSCSr- :H:t~0:.ro\ IAHNOOOQ H G) H t’ 'o 0 O o Hoax nonogpo mid Uufl-H 3:4 chagrin->001 8”a5338 <8mm=mo m *1 n O E o In m n 4..) o o 1: ram—3m \DOo-HO (“OWN \Dv-iv-lO N I‘- O\t~0\0 POOQ :WNLfi JOHN N P NCO—3m HCHN H H CLAMP mOo-io-i N P- 000—? 001-10 o-l coxocnm NCO: N (- Accessory Double Branched Simple Gastrocnemius 0.3m 000 000 00d) N 0.0 0.0 or~xo 000 H Chan Multiple Sprouts Complex 16 Weeks S.D. Mean 12 Weeks S.D . Mean Training Duration 8 Weeks S.D . Mean 4 Weeks S.D Mean Category Means and Standard Deviations for Terminal Nerve Endings in Exercised Animals Ending Table h.2. Muscle WNW—3 one-1N H H OMUNO O\O\H\D .3 m P300 [50000 «ions-r4 O\\OCH .: .3 t u o o 030.00 mra 0.4 09:39 0:31:35 0834*: 4 mm m :3 m :5. n 0 +3 0 n '0 '0 <1: 12.2 62.1 9.9 62.2 13.0 50.0 Multiple Sprouts Complex 58 \OQNOOWN JMHQNNQ 00MJO‘PQ HMMPi—ie-(O mv-i J m NUNb-N—S’Oth QNOHCHH H H WOJNNMO mar-100040 m A? UN COP-{\OOPQW 00100000 r-i wmmmcomxo ln\OW1 m m a: QFNJMJH QHHOOOO H H t. '6 o O O Hoax mOSOQvO macaw-1:314 0.059.900. 8323238 <8mm2coo m s 0) F1 0 m NLRCAJOJCD moor—cows H H H Hmmzoa QOHQOO on UN No.9 CDMPQOM POOPOO 7.7 0HOP0 JOH—TC m \0 0.1 35.1 “£2010 001-400 0.0 9.1 Fir-1000 000C130 M \O 0.0 31.0 JUNPMO H0000 H H 11.4 (“"1000 @0000 0.0 0.2 Accessory Complex Double Branched Simple Multiple Sprouts Rectus Femoris 1h.0 7.8 2h.1 7.8 11.5 25.h Accessory Double Gastrocnemius UNMQ 0H0 ma: OHM 6) Ln: Fir-IN r-i me Flt-1H P Branched Simple 000 000) 000 000 01-4!5 own 000 b- N 0001 00¢) 0C) 00 15.9 Multiple Sprouts Complex 59 20 and 28 weeks of age (P = 0.01). After the initial increase in percentage accessory endings in the gastrocnemius and rectus femoris muscles, significant differences occurred in both only between 12 weeks and 28 weeks (gastrocnemius P = 0.05; rectus femoris P = 0.025). Fiber Type Differences Significant differences existed between the primarily fast-twitch and slow-twitch muscles at numerous points. The slow-twitch adductor longus displayed more accessory endings than either the fast-twitch gastrocnemius (P = 0.001) or the fast-twitch rectus femoris (P = 0.001) at all ages except at 6 weeks. At that time there was no notable differ- ence between the adductor longus and the rectus femoris muscle. The slow-twitch soleus muscle had more accessory endings than either the gastrocnemius at all ages (P = 0.001) or the rectus femoris at 12 weeks 0.025), 16 weeks (P = 0.01), 20 weeks (P = 0.01) and 24 weeks (P (P 0.01) of age. No significant difference was apparent between the soleus and the rectus femoris at either 6 weeks or 28 weeks of age (P = 0.05). Differences between slow-twitch muscles occurred only at 6 (P = 0.025), 16 (P = 0.01), and 28 (P = 0.01) weeks of age, while in fast-twitch muscles accessory endings appeared significantly more fre- quently in the rectus femoris at 6 weeks (P = 0.01), 24 weeks (P = 0.01) and 28 weeks (P = 0.025). Exercise Effects No significant differences were seen in the comparison of sedentary animal muscles with those same taken from chronically exercised animals 60 except after eight weeks of training. At that point significant decreases were apparent in the numbers of accessory endings found in the adductor longus (P = 0.002) and soleus (P = 0.05) muscles of the trained animals. A similar decrease, though non-significant (P = 0.10), was present in the gastrocnemius muscle. Double Endings (Figure 4.2 and Tables 4.1 and 4.2) Ageinngffects An ageing trend was seen in the double endings found in slow-twitch muscles. In the adductor longus significant increases in percentage double endings were seen up to 24 weeks of age. These significant differ- ences were noted between 6 and 16 weeks (P = 0.01), 12 and 16 weeks (P = 0.05) and 20-24 weeks (P = 0.02). The soleus muscle was found to contain increasing numbers of double endings up to 20 weeks of age, with major increases occurring between 6 weeks and 12 weeks (P = 0.001) and between 16 weeks and 20 weeks (P = 0.01). No ageing trend was seen in fast-twitch muscles which consistently displayed less than 2.0% double endings. Fiber Type Differences No differences existed at six weeks of age between fast-twitch and slow-twitch muscle terminal arborizations. One percent to 2.2% of the endings seen at this point were doubles. At every point thereafter slow-twitch muscles (adductor longus and soleus) displayed more double endings than fast-twitch muscles (P = 0.001). 61 A.moonononnne unconnnnmnm non axon oomv .mumn nomnunoxo new agapnonom no monomze nonconom an mannuno onnnon ommunoonoa .u.e onnmna “9.30 02.Z_mmmmmaon nonnnaoa on momnosu .m.e onomna mwnomaz 0.1...0wmm Z. mozazw m>mwz 1.42.21m... z. mmoz<10 $0.3. wo< “9.3. wo< 0N ¢N ON 0. N. 0 mm ¢N ON 0. N. 0 L . . . _ . 1 o 1 o 11181111911141ll1oilll 9.1 l 0. l 0. J ON 1 ON 1 on J 0m 1 0¢ .1 0¢ J 00 o\o 1. 00 l 00 l 00 1 on 1 on. XUJQEOU I 85255 .11... x 1 oo 1 oo meDOO ollo E8884 .711. 1 om 1 om UJQZE xlx mamnom 9.0201. m0._.0000< o\o 76 .monsonooaamom ono anoEon moaoon11monomos onnnooom on mmnnono o>ao= noonnnoa on momnogu .m.e onomna mmqomss. 0......ommm z. mozazm w>mmz 442.555... 2. mw02410 $3.3. w0< .23. M04 mN ¢N 0N m. N. 0 mm ¢N ON 9. N. m J 0 I 0. I 0N x11111x11|l1xi I on I 0¢ I On 0\0 1.. on 0\o 1 oo 1 oo X/K\|l/X'l 1] OK. K/l/X 1| ON 1 oo x3228 I x 1 cm x 095255 0116 I 0m unmaoo ollo I om >10mmmoo< xl11x wnagw xlx l 00. l 00. m0..2m200m....m<0 mEOsEu. mahomm 77 compensate for the decreased transmitter release per unit surface area associated with the ageing process. Alternatively the ending/endplate elaboration seen in slow-twitch muscles and with maturation may possibly reflect a system designed to maintain the trophic activity of the motoneuron (116) by providing alternate channels for axoplasmic flow into the expanded endplate area. Exercise Effects on the Neuromuscular Junctions Forced endurance exercise, which presumably produced increased activity levels preferentially in FOG and FG fibers without concomitant muscle fiber hypertrophy (86), produced no consistent pattern of changes in terminal arborization morphology. Similar results have been noted in rats allowed voluntary activity in running wheel cages for 10 weeks (Stephens and Tweedle, unpublished). These results suggest that increased activity levels of an endurance nature which do not alter muscle fiber size or speed (Appendix C) cannot alone produce changes in neuromuscular junction morphology. Tenotomy experiments which result in decreased percentages of double endings (117) as well as causing both atrophy and, in slow-twitch soleus muscle, a shift to a faster contracting muscle (119) further sug- gest the importance of muscle fiber size or speed of contraction in relation to terminal arborization structure. CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of the present investigation was to determine the effects of maturation and a chronic endurance-exercise program on the morphology of the terminal motor ending and endplate in both fast-twitch (gastrocnemius and rectus femoris) and slow—twitch (adductor longus and soleus) muscle from normal, male Sprague-Dawley rats. One hundred animals were brought into the laboratory in two ship- ments and randomly assigned to one of two treatment groups: control (CON) or endurance running (END). CON animals were sacrificed at 6, 12, 16, 20, 24, or 28 weeks of age. END animals were subjected to a progressive, endurance-running program for 4, 8, 12, or 16 weeks and were sacrificed at 16, 20, 24, or 28 weeks of age respectively. Determination of fiber type composition for each muscle taken was made on frozen sections using myosin adenosine triphosphatase (ATPase 9.4) (39), and succinic dehydrogenase (SDH) (5). Muscles taken from the left hindlimb were fixed in 10% formal- saline with 0.5% DMSO for 6 hours, rinsed in distilled water for 16 hours, and then frozen. A minimum of six serial longitudinal sections 60 to 70 micra thick were stained using the combined cholinesterase-Bielchowsky 78 79 method of Gwyn and Heardman (62) for motor endplate/terminal axon morphology. Specific determination of morphological characteristics of the terminal arborizations was made using the categorization system and technique developed by Tweedle and Stephens (117). Both age-related changes and exercise-induced modifications were noted. Comparative statistical analyses were done using the Mann-Whitney U-test (lll). Generalized results of the morphological changes have been summar- ized in Table 5.1. Conclusions The results of this investigation have led to the following con- clusions: 1. Significant differences exist between the neuromuscular axonal terminals of post-pubertal fast-twitch and slow-twitch muscles. Primarily slow-twitch muscles (adductor longus and soleus) exhibit increased percentages of accessory, double, multiple, and complex endings as well as decreased percentages of simple endings when compared with predominantly fast-twitch muscles (gastrocnemius and rectus femoris). 2. Between 6 (pre-pubertal) and 12 (post-pubertal) weeks of age, significant synaptic rearrangement occurs in all muscles as demonstrated by increased ending complexity. After 12 weeks, increased neuromuscular junction complexity appears to be characteristic of slow-twitch muscles. 80 .oononoxo no oxoo: O nouns ooaoosl novnbaisodo ona .osoaoo one na uxooa ON and oswnoa noaonvno one on axoo: mu as unasooaoun Haan: o-nn on nosnuanoo hannonnloo ouoonn nounaa1so~m .N .uuoos Na one o noosaoa nonnluxo conceal ”an nn .300 ono: canonon nnaoon one osonoo on» non: oxoos o as anoouo noun «do an ode-5| sonata 1aoon non» nonnloo one! huanoo nu ounnono on: hunnoanloo consonoon .H onAnonr on: hannounloo eoooononn .H InnnnUa- In: one-s! nuansa1so~m .n xoansoo .omuunl noausaJOOHo nn nano noon ono: uoannaanx .m .nuxooa on on. o a. oaaonnnauno. .vo>noopo cacao. on» non» unaasonno onal ono: nounnaqnl uO.m nnna wood nosono hdnononon cannonoonaooo .N on. AnOnanooxo ona no: nnooa w .onnolon nnaoon as cannon Louosono. noon on: on» ndnu unaanonno no nonnanoo onnnaanx .naoonno o-nonoxo Hosanna .H unnasonno an.~ non» nood nan-so: .A 1non nonwun no: anunOH nonunon< .« nno nanonnm .nuoo: ON and ed nooaaon nno unn-o>o~ ononoa o-nonoou .nonulnn nno: annolon .oononoxo no ouoo: 0 ea nosnnanoo conceal nuanaa1soAm .N cocoon one goons. on» non: nouns hdno and nonoonl noanaa .nnoo: .300: w an anoouo ulna an cannon 1:0uo na nuno noon nno: ownnnno NA and w noonaon conceal and non add an omnnnno onnlan noon odnlno no nounanoonoa voooononn .H nonnnooo hanonualao nu onoonoon < .H voaonannonov sauna. nuannu1no~u .A ounlnm .nxoo: ON on n: osmno~ noaonnoo on» non one .nnoo: mu 0» 95 noon onoa nanoson usaoon nno usulonoonaonu non noooonooo Hoaaann .N .uO.a sodop onoauo one um.~ acaop humononom .oaoonno o-nonoxo uninnnx .a onoa non-unoonoa none-on ado non .u .oapao«> hunnoon non noon» on .H nononnna .nO.N aoHoa suntan no: umnnono cannon ondunoonon 11 noon onoa oaouol noonao1oa¢n an noonano on .u .onoo: ON on noooononn nno~0o one .onoo> o nouns onus: .oxooa am on a: nownanoonon noun Add as wonky nonnn noonaon .oaoonno ouaonoxo Hosannz .u voooonona Ono-on onunOA noaonon< .a noa-nno noonononnav annoanannnw .~ oannoa .nxooa ON one On noonaon mnnnooaoun onenoa ondononn on .nuoon c an ouoonl vohnonno oaonnl nuaniu130uo hano .omn no nuoo: NA nuanaa1aouo coouonouo nan conga-nouns noanoonona .nnooa Na on 9: nova» noann nomoanounon gonna: adanoo no Loans: nu onnonooo o no: ononb .d Inoonon noooononu non nononsl ~H< .4 1anunman can ouoonl noaasa1so~w .« nno-oouo< ooaanon canouoxu vouuuoc and oceanon onus hogan nnowoaoo oanunnnu aoaaoau nose: «can-app an noon-no “can: no noun-so .n.» «nope 81 . Exhaustive endurance exercise appears to have little effect on the modification of motor nerve endings. Apparently normal maturational nerve ending changes are not due to alterations in neuromuscular activity of this type. Recomnendations . A follow-up study should be conducted to assertain the effects of advanced age on terminal motor ending morphology and plasticity. . Exercise programs specifically designed to overload the fast- twitch glycolytic and fast-twitch oxidative glycolytic muscle fibers should be implemented with an identical animal popula- tion. . Cross-innervation and reinnervation investigations should be conducted using the same ending/endplate categorization technique. . Attempts should be made to refine the current cholinesterase- Bielchowsky nerve stain such that identification of individual muscle fibers is more easily made. . Physiological and ultrastructural studies should be carried out in conjunction with future investigations. REFERENCES 10. 11. REFERENCES . Anderson-Cedergren, E. Ultrastructure of motor end-plate and sarco- plasmic components of mouse skeletal muscle fiber. J. Ultrastruct. Res,, Suppl. 1:1-191, 1959. . Anzenbacher, H. and W. Zenker. Uber die GrbBenbeziehung der Muskel- fasern zu ihren motorischen Endplatten und Nerven. Z. Zellforsch. 60:860-871. 1963. . Ariano, M. A., R. 8. Armstrong and V. R. Edgerton. Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21:1:51-55, 1973. . Bagust, J., D. M. Lewis and R. A. Westerman. Polyneuronal innervation of kitten skeletal muscle. J. Physiol. 229:241-255, 1973. . Barka, T. and P. J. Anderson. Histochemistry Theory, Practice, and Bibliography. New York: Harper and Row, 1963. . Barker, 0. and M. C. Ip. Sprouting and degeneration of mammalian motor axons in normal and de—afferentated skeletal muscle. Proc. ,ng. Soc. B. 163:538-554, 1965. . Bennett. M. R. and A. G. Pettigrew. The formation of synapses in reinnervated and cross-innervated striated muscle during development. J. Physiol. (London) 241:547-573, 1974. . Bennett, M. R. and A. G. Pettigrew. The formation of synapses in striated muscle during development. J. Physiol. 241:515-545. 1974. . Benoit, P. and J. Changeux. Consequences of tenotomy on the evolution of multinnervation in developing rat soleus muscle. Brain Research 99:354-358. 1975. Bixby, J. L. and D. C. VanEssen. Regional differences in the timing of synapse elimination in skeletal muscles of the neonatal rabbit. Brain Res. 169:275-286, 1979. Bixby. J. L. Ultrastructural observations on synapse elimination in neonatal rabbit skeletal muscle. J. Neurocyt. 10:81-100. 1981. 82 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 83 Booth, F. W. and M. J. Seider. Effect of disuse by limb immobiliza- tion on different muscle fiber types. In: Plasticity of Muscle, D. Pette (ed.). Walter de Gruyter and 00., Berlin, 1980, pp. 373-383. Brown, M. C., J. K. S. Jansen and 0. VanEssen. A large scale reduc- tion in motoneurone peripheral fields during post-natal development in the rat. Acta Physiol. Scand. 95:3A, 1975. Brown. M. C., J. K. S. Jansen and D. VanEssen. Polyneuronal innerva- tion of skeletal muscle in new-born rats and its elimination during maturation. J. Physiol. (London) 261:387-422. 1976. Brooke, M. H., E. Williamson and K. K. Kaiser. The behavior of four fiber types in developing and reinnervated muscle. Arch. Neurol. 25:360. 1971. Buller, A. J. The motor end-plate: function. In: The Peripheral Nerve, 0. N. Landon (ed.). New York: John Wiley and Sons. Inc., 1976. Buller, A. J., J. C. Eccles and R. M. Eccles. Interactions between motoneurones and muscles in respect of the characteristic speeds of their responses. J. Physiol. 150:417-439, 1960. Buller, A. J. and C. J. C. Kean. Further observations on the force velocity characteristics of cross-innervated cat skeletal muscle. J. Physiol. 233:248, 1973. Burke, R. E. and V. R. Edgerton. Motor unit properties and selection in movement. In: Exercise and Sport Sciences Reviews, J. H. Wilmore (ed.). New York: AEademic Press, 1975, pp. 31-81. Burke, R. E., D. N. Levine, P. Tsairis and F. E. Zajac, III. Physio- logical types and histochemical profiles in motor units of the cat gastrocnemius. J. Physiol. (London) 234:723-748, 1973. Burke, R. E., D. N. Levine, F. E. Zajac, III, P. Tsairis and W. K. Engel. Mammalian motor units: Physiological-histochemical correla- tion in three types in cat gastrocnemius. Science 174:709-712, 1971. Caccia, M. R., J. B. Harris and M. A. Johnson. Morphology and physiology of skeletal muscle in aging rodents. Muscle and Nerve 2:202-212, 1979. Cardasis, C. A. and H. A. Padykula. Ultrastructural evidence indicat- ing reorganization at the neuromuscular junction in normal rat soleus muscle. Anat. Rec. 200:41-59. 1981. Carlson, B. M. The regeneration of skeletal muscle--a review. Am. J. Anat. 137:119-150, 1973. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Carrow. R. E., W. W. Heusner and W. D. Van Huss. Exercise and the incidence of muscle fiber splitting. Brit. Assoc. Sports Med. J. 7:39—41, 1973. Close, R. Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. 173:74-95, 1964. Close, R. Dynamic properties of fast and slow skeletal muscles of the rat after nerve cross-union. J. Physiol. 204:331-346, 1969. C6ers, C. Les variations structurelles normales et pathologiques de la jonction neuromusculaire. Acta Neurol. Belg. 55:741-866. 1955. Cbers, C. Structure and organization of the myoneural junction. Int. Rev. Cytol. 22:239-267. 1967. Cole, W. V. Morphological characteristics of the motor end plate of the rat (mus norvegicus). Anat. Rec. 98:393-400. 1947. Cole, W. V. Structural variations of nerve endings in the striated muscles of the rat. J. Comp. Neurol. 108:445-464, 1957. Cole, W. V. Motor endings in the striated muscle of vertebrates. J. Comp. Neurol. 102:671-716. 1955. Cotman, C. W., M. Nieto-Sampedro and E. W. Harris. Synapse replace- ment in the nervous system of adult vertebrates. Physiol. Rev. 61:3:684-784, 1981. Couteaux, R. Motor endplate structure. In: Structure and Function of Muscle, G. H. Bourne (ed.). New York: Academic Press, 1960. Crockett. J. L.. V. R. Edgerton. S. R. Max and R. J. Barnard. The neuromuscular junction in response to endurance training. Exp. Neurol. 51:207-215. 1976. Dias, P. L. R. Surface area of motor end plates in fast and slow twitch muscles of the rabbit. J. Anat. 117:453-462, 1974. Dias. P. L. R. and J. A. Simpson. Effects of cross-innervation on the motor end-plates of fast- and slow-twitch muscles of the rabbit. Quart. J. Exp, Physiol. 59:213-223. 1974. Orachman, D. B. The role of acetylcholine as a trophic neuro- muscular transmitter. In: Growth of the Nervous System, G. E. Wolstenholme and M. O'Connor (eds.). ChurChill,’Ebnd6n. 1968, pp. 251-273. Dubowitz, V. and M. H. Brooke. Muscle Biopsy: A Modern Approach. Philadelphia: W. B. Saunders Co., 1973. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. SO. 51. 52. 85 Ouchen, L. W. An electron microscopic comparison of motor end- plates of slow and fast skeletal muscle of the mouse. J. Neurol Sci. 14:37-45, 1971. Edds, M. V. Collateral nerve regeneration. anrt. Rev. Biol. 28:260-276. 1953. Ellisman, M. H., J. E. Rash, L. A. Staehelm and K. R. Porter. Studies of excitable membrane. 11. A comparison of specializations at neuromuscular junctions and non-junctional sarcolemmas of mammalian {35; and slow twitch muscle fibers. J. Cell. Biol. 68:3:752-773. 7 . Fagg, G. E., S. W. Scheff and C. W. Cotman. Axonal sprouting at the neuromuscular junction of adult and aged rats. Society for Neurosci. Abstracts 7:473. 1981. Fahim, M. A. and N. Robbins. Ultrastructure of aging at neuromuscular junctions of fast and slow mammalian muscles. Society for Neurosci. Abstracts 7:64, 1981. Fambrough, D. M. and J. E. Rash. Development of acetylcholine sensi- tivity during myogenesis. Devel. Biol. 26:55-68, 1971. Fidzianska, A. Electron microscopic study of the development of human foetal muscle, motor endplate and nerve. Acta Neuropath. 17:234-247. 1971 Fujisawa. K. Some observations on the skeletal musculature of aged rats--III. Abnormalities of terminal axons found in motor end- plates. Exp. Geront. 11:43-47, 1976. Gauthier, G. F. The motor end-plate: structure. In: The Peripheral Nerve, D. N. Landon (ed.). New York: John Wiley and Sons. Inc., Giacobini, G., G. Filogamo, M. Weber, P. Boquet and J-P. Changeux. Effects of a snake a-neurotoxin on the development in innervated skeletal muscles in chick embryo. Proc. Nat. Acad. Sci. 70:1708-1712, 973. Gonyea, W. J. Role of exercise in inducing increases in skeletal muscle fiber number. J. Appl. Physidb. 48:3:421-426, 1980. 6055, R. J. The Physiology of Growth. New York: Academic Press, 1978. Granbacher, N. Uber die GrbBenbeziehungen der Muskelfasern zu ihren motorischen Endplatten und Nerven bei Hypertrophie und Atrophie. Z. Anat. Entwick1.-Gesch. 135:76-87, 1971 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 86 Gruber, H. Die GrUBeneziehung von Muskelfaservolumen und Flache der motorischen Endplatte bei verschiedenen Skeletmuskeln der Ratte. Acta Anat. (Basel) 64:628-633. 1966. Guth, L. Neuromuscular function after regeneration of interrupted nerve fibers into partially denervated muscle. Exp. Neurol. 6:129-141, 1962. Guth, L. 'Trophic' influences of nerve on muscle. Physiol. Rev. 48:4:645-687. 1968. Gutmann, E. and V. Hanzlikova. Age changes in motor end plates in muscle fibers of the rat. Gerontologia 11:2-24, 1965. Gutmann, E. and V. Hanzlikova. Motor unit in old age. Nature (London) 209:921-922, 1966. Gutmann, E. and V. Hanzlikova. Fast and slow motor units in ageing. Gerontology 22:280-300. 1976. Gutmann, E., V. Hanzlikova and B. Jakoubek. Changes in the neuro- muscular system during old age. Exp. Geront. 3:141-146, 1968. Gutmann, E., V. Hanzlikova and F. Vyskocil. Age changes in cross striated muscle of the rat. J. Physiol. 219:331-343, 1971. Gutmann, E. and J. 2. Young. The re-innervation of muscle after various periods of atrophy. J. Anat. (London) 78:15-51, 1944. Gwyn. O. G. and V. A. Heardman. A cholinesterase-Bielchowsky stain- ing method for mammalian motor endplates. Stain Tech. 40:15-18, 1965. Harris, A. J. Trophic effects of nerve on muscle. In: The Ph si- ology of Peripheral Nerve Disease, A. J. Sumner (ed.). Philade1phia: W. B. Saunders Co., 1980, p. 195-220. Harrison, R. G. An experimental study of the relation of the nervous system to the developing musculature in the embryo of the frog. Am. J. Anat. 3:197-220, 1904. Hebb. C. 0., K. Krnjevic and A. Silver. Acetylcholine and choline acetyltransferase in the diaphragm of the rat. J. Physiol. 171:504-513, 1964. Hess. A. Further morphological observation of 'en plaque' and 'en grappe' nerve endings on mammalian extrafusal muscle fibers with the cholinesterase technique. Rev. Canad. Biol. 21:241. 1962. Hinsey, J. C. The innervation of skeletal muscle. Physiol. Rev. 14:514-585. 1934. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 87 Hirano, H. Ultrastructural study on the morphogenesis of the neuro- muscular junction in the rat skeletal muscle of the chick. Z. Zellforsch. 79:198-208. 1967. Ho, K. W., R. R. Roy, C. D. Tweedle, W. W. Heusner, W. D. Van Huss and R. E. Carrow. Skeletal muscle fiber splitting with weight- lifting exercise in rats. Am. J. Anat. 157:433-440, 1980. Hollozy, J. Adaptations of skeletal muscle to endurance exercise. Med. Sci. Sports. 7:155-164, 1975. Ip. M. C. Some morphological features of the myoneural junctions in certain normal muscles of the rat. Anat. Rec. 180:605-616, 1974. Jacobson, M. Developmental Neurobiology. (Second Edition) New York: Plenum Press, 1978. Jansen, J. K.. D. VanEssen and M. C. Brown. Formation and elimina- tion of synapses in skeletal muscle of the rat. Cold SpringHarb. Symp. 40:425-434. 1975. Juntunen, J. and H. Teravainen. Morphogenesis of myoneural junctions induced postnatally in the tibialis anterior muscle of the rat. Acta Physiol. Scand. 79:462-468. 1970. Karpati, G. and W. K. Engel. Histochemical investigation of fiber type ratios with the myofibrillar ATPase reaction in normal and denervated skeletal muscle of guinea pig. Am. J. Anat. 122:145, 1968. Kelly, A. M. and S. I. Zacks. The fine structure of motor endplate morphogenesis. J. Cell. Biol. 42:154-169. 1969. Kerner, J. and A. Sander. The effect of denervation on the metab- olism in rat skeletal muscle. Acta Biochem. Biophysic. 11:214. 1976. Korneliussen, H. Identification of muscle fiber types in "semi-thin" sections stained with p-phenylene-diamine. Histochemie 32:95-98, 1972. Korneliussen, H. and 0. Waerhaug. Three morphological types of motor nerve terminals in the rat diaphragm, and their possible innervation of different muscle fiber types. 2. Anat. Entwickl. Gesch. 140:73-84, 1973. Korneliussen, H. and J. K. S. Jansen. Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibers in newborn rats. J. Neurocytol. 5:591-604, 1976. Kugelberg, E. Adaptive transformation of rat soleus motor units during growth. Histochemistry and contraction speeds. J. Neurol. ‘Sgi. 27:269-289. 1976. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 88 Kugelberg, E. Adaptive fibre and motor unit rransformation in rat soleus during growth. In: Plasticity of Muscle, D. Pette (ed.) Walter de Gruyter and Co., Berlin. 1980, pp. 111-117. Manual of Histologic and Special StainingyTechniques of the Armed Forces Institute of Pathology. New York: McGraw-Hill Book Co., 1960. Mark, R. F. Synaptic repression at neuromuscular junctions. Physiol. Rev. 60:2:355-395, 1980. Maxwell, L. C., J. A. Faulkner and D. A. Lieberman. Histochemical manifestations of age and endurance training in skeletal muscle fibers. Am. J. Physiol. 224:2:356-361, 1973. Mueller, W. Temporal progress of muscle adaptation to endurance training in hind limb muscles of young rats. Cell Tiss. Res. 156:61-87, 1974. Murata, F. and T. Ogata. The ultrastructure of neuromuscular junc- tions of human red, white, and intermediate striated muscle fibers. Tohoku J. Exp. Med. 99:289-301. 1969. Nystrbm. B. Postnatal development of motor nerve terminals in 'slow-red' and 'fast-white' cat muscles. Acta Neurol. Scand. 44:363-383. 1968. O'Brien, R. A. 0., A. J. C. Ostberg and G. Vrbova. Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle. J. Physiol. 282:571-582, 1978. Ogata. T. A histochemical study on the structural differences of motor endplate in the red, white, and intermediate muscle fibers of mouse limb muscle. Acta med. Okayama 19:149-153. 1965. Ogata, T., T. Honda and T. Seito. An electron microscopic study on differences in the fine structures of motor endplate in red. white, and intermediate muscle fibres of rat intercostal muscle. A prelimi- nary study. Acta med. Okayama 21:327-338. 1967. Ogata, T. and F. Murata. Fine structure of motor endplate in red, white and intermediate fibers of mammalian fast muscle. Tohoku J. Exp. Med. 98:107-115. 1969. Padykula, H. A. and G. F. Gauthier. The ultrastructure of the neuromuscular junctions of mammalian red, white, and intermediate muscle fibers. J. Cell Biol. 46:27-41, 1970. Pestronk, A., D. B. Drachman and J. W. Griffin. Effects of aging on nerve sprouting and regeneration. Exp. Neurol. 70:65-82, 1980. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 89 Peter, J. B. Histochemical, biochemical. and physiological studies of skeletal muscle and its adaptation to exercise. In: Contractility of Muscle Cells and Related Processes, R. J. Podolsky (ed.). Englewood Cliffs: Prentice-Hall, Inc., 1970. Peter, J. B., R. J. Barnard, V. R. Edgerton. C. A. Gillespie and K. E. Stempel. Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11:2627-2633, 1972. Peters. A., S. L. Palay and H. deF. Webster. The Fine Structure of the Nervous System: The Neurons and Supporting Cells. Philadelphia: W. B. Saunders Co., 1976. Pulliam, D. L. and E. W. April. Differential ultra-structural changes at end-plate regions of red, white, and intermediate muscle fibers following denervation. Anat. Rec. 184:506, 1976. Redfern, P. A. Neuromuscular transmission in newborn rats. J. Physiol. (London) 209:701-709, 1970. Reed, A. T. Succinic Dehydrogenase and Motor End:plate Choline- sterase in Chronically ExerciSedTRat Skeletal Musc1e. Unpublished Ph.D. Thesis, Department of Health, Physical Education and Recreation, Michigan State University, East Lansing, Michigan, 1972. Reier, P. J. and A. Hughes. Evidence for spontaneous axon degenera- tion in peripheral nerve maturation. Amer. J. Anat. 135:147-152, 1972. Renaud, 0., G. H. LeDouarin and A. Khaskiye. Spinal cord stimulation in chick embryo: effects on development of the posterior latissimus dorsi muscle and neuromuscular junctions. Exp. Neurol. 60:2:189-200. 1978. Riley, 0. A. Multiple axon branches innervating single endplates of kitten soleus myofibers. Brain Res. 110:158-161, 1976. Riley, 0. A. Spontaneous elimination of nerve terminals from the endplates of developing skeletal myofibers. Brain Res. 134:279-285, 1977. Riley, 0. A. Multiple innervation of fiber types in the soleus muscles of postnatal rats. Exp. Neurol. 56:400-409. 1977. Rosenthal, J. L. and P. S. Taraskevich. Reduction of multiaxonal innervation at the neuromuscular junction of the rat during develop- ment. J. Physiol. 270:299-310. 1977. Roy, R. R. Specific Changes in a Histochemical Profile of Rat Hindlimb Musc1e Induced by TWO Exercise Regimens. UnpublishedPh.D. Thesis, Department of Health, Physical Education and Recreation, Michigan State University, East Lansing, Michigan, 1976. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. Sandbank, U. and J. J. Bubis. The Morphology of Motor Endplates: A Review. Los Angeles: Brain Research Institute, University of California, Los Angeles, 1974. Santa, T. and A. G. Engel. Histometric analysis of neuromuscular function ultrastructure in rat red, white and intermediate muscle fibers. In: New Developments in Electromyography and Clinical Neurophysiology. J. E. Desmedt (ed.). Karger, Basel, 1973, V01. 9 ppo '54. Shchitkov, K. G. Changes of the motor nerve endings during active hypertrophy of skeletal muscles. Dokl. Akad. Nauk. SSSR 113:297-300, 1957. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. Srihari, T. and G. Vrbova. The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chick muscles. J. Neurocyt. 7:529-540, 1978. Straus, W. L. and G. Weddell. Nature of the first visible contrac- tions of the forelimb musculature in rat foetuses. J. Neurophysiol. 3:358-369, 1940. Teravfiinen, H. Development of the myoneural junction in the rat. Zeitschrift fur Zellforschupg. 87:249-265. 1968. Tiegs, 0. W. Innervation of voluntary muscle. Physiol. Rev. 33:90-144, 1953. Tuffery, A. R. Growth and degeneration of motor end-plates in normal cat hindlimb muscles. J. Anat. 110:221-247, 1971. Tweedle, C. D. and K. E. Stephens. Development of complexity in motor nerve endings at the rat neuromuscular junction. Neuroscience 6:1657-1662, 1981. Van Harreveld, A. On the mechanism of the 'spontaneous' re-innerva- tion in parietic muscles. Am. J. Physiol. 150:670-676, 1947. Vrbova. G., T. Gordon and R. Jones. Nerve-Muscle Interaction. New York: John Wiley and Sons, Inc., 1978. Waerhaug. O. and H. Korneliussen. Morphological types of motor nerve terminals in rat hindlimb muscles. possibly innervating differ- ent muscle fiber types. Z. Anat. Entwickl.-Gesch. 144:237-247, 1974. Wells. R. L. and W. W. Heusner. A controlled-running wheel for small animals. Lab. Animal Sci. 21:6:904-910, 1971. 91 122. Westerman. R. A., D. M. Lewis, J. Bagust, G. D. Edjtehadi and D. Pallot. Communication between nerves and muscles: post-natal development in kitten hindlimb fast and slow twitch muscle. Symposium on Memory and Transfer of Information, Zippel, H. P. (ed.). New York: Plenum Press, 1972, p. 255-291. 123. Wilkinson, H. J. The innervation of striated muscle. The Med. J. Australia 11:768-793. 1929. 124. Willshaw, D. J. The establishment and the subsequent elimination of polyneuronal innervation of developing muscle: theoretical con- siderations. Proc. R. Soc. Lond. B 212:233-252. 1981. 125. Zacks, S. I. The Motor Endplate. Huntington, New York: Robert E. Krieger Publishing Co., 1973. 126. Zaimis, E. Neuromuscular Junction. New York: Springer-Verlag, Berlin, 1976. APPENDICES APPENDIX A TRAINING PROGRAM 92 APPENDIX A Table A-1. Modified Sixteen-Week Endurance-Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels Total Total Acc- Repe— Time Time Exp. Total eler- Work ti- Bet- Run of Revo- Work Day Day ation Time Rest tions No. ween Speed Prog. lu- Time of of Time (min: Time per of Bouts Shock (m/ (min: tions (sec) Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) min) sec) TER TWT 4=T -2 3.0 40:00 10 1 1 5.0 0.0 27 4o 00 --- --- S=F -1 3.0 40:00 10 l 1 5.0 0.0 27 40:00 -- --- l=M l 3.0 00:10 10 4O 3 5.0 1.2 27 49:30 450 1200 2=T 2 3.0 00:10 10 40 3 5.0 1.2 27 49:30 450 1200 3=W 3 3.0 00:10 10 4O 3 5.0 1.2 27 49:30 450 1200 4=T 4 2.5 00:20 10 30 2 5.0 1.2 27 34-40 450 1200 5:? 5 2.5 00:30 15 2O 2 5.0 1.2 27 34:30 450 1200 1=M 6 2.0 00:40 20 15 2 5.0 1.2 36 34:20 600 1200 2=T 7 2.0 00:50 25 12 2 5.0 1.2 36 34:10 600 1200 3=W 8 1.5 01:00 30 10 2 5.0 1.2 36 34:00 600 1200 4=T 9 1.5 02:30 60 4 2 5.0 1.2 36 31:00 600 1200 5=F 10 1.0 02:30 60 4 2 5.0 1.2 36 31 00 600 1200 1=M 11 1.0 02:30 60 4 2 5.0 1.2 36 31:00 600 1200 2=T 12 1.0 05:00 0 l 5 2.5 1.2 36 35:00 750 1500 3=W 13 1.0 05:00 0 l 5 2.5 1.2 36 35:00 750 1500 4=T 14 1.0 05:00 0 l 5 2.5 1.2 36 35 00 750 1500 S=F 15 1.0 05 00 0 l 5 2.5 1.2 36 35 00 750 1500 1= 16 1.0 05:00 O l 5 2.5 1.2 36 35:00 750 1800 2: 17 1.0 07:30 O 1 4 2.5 1.0 36 37:30 900 1800 3= 18 1.0 07:30 0 1 4 2.5 1.0 36 37:30 900 1800 4=T 19 1.0 07:30 0 l 4 2.5 1.0 36 37:30 900 1800 5=F 20 1.0 07:30 0 1 4 2.5 1.0 36 37:30 900 1800 1=M 21 1.0 07:30 O l 4 2.5 1.0 36 37:30 900 1800 2: 22 1.0 07:30 O 1 5 2.5 1.0 36 47:30 1125 2250 3=w 23 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1125 2250 4=T 24 1.0 07:30 O 1 5 2.5 1.0 36 47:30 1125 2250 5=F 25 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1125 2250 1=M 26 1.0 07:30 0 l S 2.5 1.0 36 47:30 1125 2250 2=T 27 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1200 2400 32W 28 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1200 2400 4=T 29 1.0 10:00 0 1 4- 2.5 1.0 36 47:30 1200 2400 5= 30 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1200 2400 1= 31 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1200 2400 =T 32 1.0 10:00 0 l 5 2.5 1.0 36 60:00 1500 3000 3=W 33 1.0 10:00 O l 5 2.5 1.0 36 60:00 1500 3000 4=T 34 1.0 10:00 O 1 5 2.5 1.0 36 60:00 1500 3000 52? 35 1.0 10:00 O 1 5 2.5 1.0 36 60:00 1500 3000 1=M 36 1.0 10 00 o 1 5 2.5 1.0 36 60:00 1500 3000 =T 37 1.0 12 30 0 1 4 2.5 1.0 36 57 30 1500 3000 32W 38 1.0 12:30 O l 4 2.5 1.0 36 57:30 1500 3000 4=T 39 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1500 3000 5=F 40 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1500 3000 93 Table A-1. (continued) Total Total Acc- Repe— Time Time Exp. Total eler- WOrk ti- Bet- Run of Revo- Work Day Day ation Time Best tions No. ween Speed Prog. lu- Time of of Time (min: Time per of Bouts Shock (m/ (min: tions (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) min) sec) TER TWT 9 1= 41 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1500 3000 =T 42 1.0 12:30 0 1 5 2.5 1.0 36 72:30 1875 3750 3=w 43 1.0 12 30 o 1 5 2.5 1.0 36 72:30 1875 3750 4=T 44 1.0 12 30 0 1 5 2.5 1.0 36 72:30 1875 3750 5a? 45 1.0 12 30 o 1 5 2.5 1.0 36 72 30 1875 3750 10 1=M 46 1.0 12:30 0 1 5 2.5 1.0 36 72 30 1375 3750 2: 47 1.0 16:00 0 1 4 2.5 1.0 36 74 30 1920 3840 3=w 48 1.0 16 00 0 1 4 2.5 1.0 36 74:30 1920 3840 4=T 49 1.0 16 00 0 1 4 2.5 1.0 36 74:30 1920 3840 5=F 50 1.0 16 00 0 1 4 2.5 1.0 36 74:30 1920 3840 11 l=M 51 1.0 16:00 0 1 4 2.5 1.0 36 74:30 1920 3840 2=T 52 1.0 16:00 0 1 5 2.5 1.0 36 90:00 2400 4800 32w 53 1.0 16 00 0 1 5 2.5 1.0 36 90:00 2400 4800 4=T 54 1.0 16:00 0 1 5 2.5 1.0 36 90:00 2400 4800 5=F 55 1.0 16:00 0 1 5 2.5 1.0 36 90:00 2400 4800 12 1= 56 1.0 16 00 0 1 5 2.5 1.0 36 90:00 2400 4800 2=T 57 1.0 21:00 0 1 4 2.5 1.0 36 91:30 2520 5040 3= 58 1.0 21 00 0 1 4 2.5 1.0 36 91:30 2520 5040 4=T 59 1.0 21 00 0 1 4 2.5 1.0 36 91:30 2520 5040 5=F 60 1.0 21 00 0 1 4 2.5 1.0 36 91:30 2520 5040 13 1=M 61 1.0 21 00 0 1 4 2.5 1.0 36 91:30 2520 5040 2=T 62 1.0 20:00 0 1 5 2.5 1.0 36 110 00 3000 6000 3=w 63 1.0 20:00 0 1 5 2.5 1.0 36 110 00 3000 6000 4=T 64 1.0 20 00 0 1 5 2.5 1.0 36 110:00 3000 6000 5:? 65 1.0 20:00 0 1 5 2.5 1.0 36 110 00 3000 6000 14 1=M 66 1.0 20 00 0 1 5 2.5 1.0 36 110 00 3000 6000 2=T 67 1.0 25:00 0 1 4 2.5 1.0 36 107:30 3000 6000 3=w 68 1.0 25:00 0 1 4 2.5 1.0 36 107 30 3000 6000 4=T 69 1.0 25:00 0 1 4 2.5 1.0 36 107.30 3000 6000 5=F 70 1.0 25 00 0 1 4 2.5 1.0 36 107:30 3000 6000 15 1=M 71 1.0 25:00 0 1 4 2.5 1.0 36 107 30 3000 6000 2=T 72 1.0 27:30 0 1 4 2.5 1.0 36 117:30 3300 6600 3=w 73 1.0 27:30 0 1 4 2.5 1.0 36 117 30 3300 6600 4=T 74 1.0 27:30 0 1 4 2.5 1.0 36 117:30 3300 6600 5:? 75 1.0 27 30 o 1 4 2.5 1.0 36 117:30 3300 6600 16 l:M 76 1.0 27 30 0 1 4 2.5 1.0 36 117:30 3300 6600 -T 77 1.0 30:00 0 1 4 2.5 1.0 36 127:30 3600 7200 =w 78 1.0 30 00 o 1 4 2.5 1,0 35 127:30 3600 7200 hzT E9 1 o 0 30: 00 0 l 4 2. S 1 , 0 36 127 : 30 3600 7200 52F 0 1.0 30 00 0 1 4 2.5 1.0 36 127 30 3600 7200 This training program is a modified version of a standard of the Sprague-Dawley strain (100,107). program designed using male rats All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. a double learning situation on the animals and will seriously impair the effectiveness of the training program. Failure to provide this adjustment period will impose APPENDIX B TRAINING, TREATMENT ENVIRONMENT, AND BODY WEIGHT VALUES 0—0 was 94 T! on * a l.— ..EB. .d> o. n. S n. N. .. o. o o a o o o n N . gum; .225 on as oh 8 8 on o... r... 2. on or. 3 on n. o. a >3 .225 o H F F n p h p b h p h p m p b b p n P h b L n n - p b b p h h PIFP - b — P h b b p p p n p h P P - h — h b h h n - - - b h P u n n n b - - p h h n P n b b p L h “.0 AT 4‘ l 9. r .. 8o 1 L 00 I l g. I. W 1 1 a on r . 1 8... H 8. I , : 82 .I 1 ON. m 1 oo- o: u . .. 88 T 1 o! z . . So» 1 83 J L Ogn 95 0:. 23383282233818 fij T I I Q. 5 2:82.. 3.3588 98 £33.51 .52... 63.9!!!» 2.8 8.3 On L..P-...p.............-b.-........p.-.-....L..phr....-..E-...b.......p..b.bt o n u . 8 o. o. a 8.. 93.—kc?!” as HHS-3.: Egg 4 L L a gsgagssesaeaaaz 833.333. >19; 96 onEooom 2492 w. N. 0 ON VN ON — _ _ fic.u.moh mum... wJUmDs. mm vN 0m 0. N . 0 . _ IIIIXIIIIIIK ommammxw I .mm<...023 TIIO 402.200 I @9302: I owmamwxm I .54“. xIIIx Jomhzoo I .54.... xlx 0mm.umwxw I 2.0.6 oIIIo 402.200 I 304m 0'0 mDMJOm 29.3 02.2.42... 3...... wo< 0 0. 0m 0m 0w on o\o 00 0h Om 00 00. 0. N. m .4 mm .VN 0m 0. N. mDOZOI. 20F0300< .33. 02.2.42... .93. weq 0 _ ‘ 0 0. 0m 0m 0c 0m o\o 00 ON 00 Om 00. 98 02.42 020.0253 Iwoz42aozw 024 44.2202 2. mwn.>._. 220....— 240035. 30.3. 02.2.42h w .93 m04 020.0258 I .mm4402: TIIO 402h200 I .mm44023 I 02902200 I .04... xlllx 4022.200 I 2.042 xIIIx owm.02mxm I 3040 OIIIo 402...200 I 3040 To 0.20.22... m:...0m2 40 10. 10N 10m 10¢ 1 On 0\0 100 ION 100 100 l 00. 0. N. 0 v 0N vN ON 0. N. 1744:4472. ’I m3..2w2002...m40 .93. 02.2.42... 0 .93. 204 .II 4 0 0. ON on 04 on .40 00 0h 00 00 00.