sucmmc DEHYDROGENASE AND v. . MOTOR END-PLATE CHOLINESTERASE m CHRONICALLY EXERCISED ' RAT SKELLTAL MUSCLE ' - Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ALFRED T. REED ‘ 1972 11.1111"11ll 1 __..___4— 111111111 L1 1D 4x . r . 1 Michigan State “ University This is to certify that the thesis entitled Succinic Dehydrogenase and Motor End-Plate Cholinesterase in Chronically Exercised Rat Skeletal Muscle presented by Alfred T. Reed has been accepted towards fulfillment of the requirements for Ph.D. Jesreein Physical Education 111/14 Major professor Date June 28, 1972 0-7639 rumours In“ ‘ HUME & SDNS 1' 800K BMDEIRYD INC 51'1“; i IIIIIIIIIIIIIIIIIII ABSTRACT SUCCINIC DEHYDROGENASE AND MOTOR END-PLATE CHOLINESTERASE IN CHRONICALLY EXERCISED RAT SKELETAL MUSCLE By Alfred T. Reed The purpose of this study was to determine the effects of seven chronic physical activity programs upon succinic dehydrogenase (SDH) and motor end-plate cholinesterase (MEP-ChE) in the left soleus and tibialis anterior muscle of adult male albino rats. One hundred seventy-six, 72-day-old, male, albino rats (Sprague- Dawley strain) were brought into the laboratory and were randomly assigned to seven treatment groups. The treatment groups were seden- tary control (CON); voluntary running (VOL); short-duration, high- intensity running (SHT); medium—duration, moderate-intensity running (MED); long-duration, low-intensity running (LON); electric stimulus control (ESC); and endurance swimming (SWM). Animals were provided with food and water ad Zibitum. Treatments were administered Monday through Friday under controlled environmental conditions. The healthiest and best trained animals were selected for sacri- fice. Animals were sacrificed at the onset and 4, 8, and 12 weeks after the start of treatments. The final sample consisted of 102 animals. Alfred T. Reed During sacrifice, animals were anesthetized by intraperitoneal injection of sodium pentobarbital. The left soleus and tibialis anterior muscles were excised and immediately frozen in an iSOpentane-liquid nitrogen system. Histochemical processing involved a 24-hour softening period in room temperature Ringer's solution, during which tissue samples were teased apart. Teased tissues were double-stained, first for SDH by the nitro blue tetrazolium (NBT) method and then for MEP-ChE with the acetylthiocholine (ATC) method. The SDH staining intensity was measured photometrically. The MEP-ChE staining pattern was traced from micro- projector images of 200x magnification. Surface areas of MEP-ChE tracings were measured by polar planimetry. The SDH photometer readings and MEP-ChE surface areas were analyzed with a two~way, fixed effects ANOVA model. The Scheffé method of multiple contrasts was used for post-hoc 'analysis. Of the two muscles investigated in this study, the soleus displayed more specific patterns of adaptation than did the tibialis anterior. In the soleus, the VOL, SHT, MED, and LON programs produced increases (p<.10) in MEP-ChE surface area. The SHT program appeared to accelerate this effect. The same programs increased (p<.10) soleus SDH staining intensity with the programs of relatively high-intensity, VOL and SHT, producing earlier increases than those produced by the lower-intensity programs, MED and LON. In the tibialis anterior, a general non-significant trend toward decreased MEP-ChE surface area was noted. A significant increase (p<.10) in SDH staining intensity for all groups from 0 to 4 weeks of treatment appeared to be an age effect. A nearly significant (p=.ll) training effect on tibialis anterior SDH appeared to warrant further investigation. SUCCINIC DEHYDROGENASE AND MOTOR END-PLATE CHOLINESTERASE IN CHRONICALLY EXERCISED RAT SKELETAL MUSCLE By ( 11* m Alfred r!” Reed A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR.OF PHILOSOPHY Department of Health, Physical Education and Recreation 1972 Dedication To my wife, Lynn 11 ACKNOWLEDGEMENTS Appreciation is extended to both Dr. W. D. VanHuss and Dr. W. W. Heusner for their guidance during my graduate program and throughout the course of this study. Gratitude is extended to Dr. E. Eisenstein and Dr. R. Carrow for their service on the guidance committee. Special thanks are due Dr. Carrow for suggestions leading to the study and for the use of the facilities of the Neuromuscular Research Laboratory, Department of Anatomy. A thank you is extended to Barbara Wheaton for technical help in the processing of tissues, to KwokAWai Ho for diligent care of the animals, and to Dr. J. F. Taylor and T. Gilliam for thought-provoking discussion. This study was supported by National Institutes of Health Grant HD 03918. 111 Chapter I II III IV TABLE OF CONTENTS THE PROBLEM. . . . . . . . . . . . . . . . . REVIEW Rationale . . . . . . . . . . . . . . Statement of the Problem. . . . . . . Significance of the Problem . . . . . Limitations of the Study. . . . . . . OF RELATED LITERATURE . . . . . . . . Skeletal Muscle Metabolism. . . . . . Succinic Dehydrogenase. . . . . . . . Neural Influences upon Skeletal Muscle Metabolism The Alpha Mbtor Neuron. . . . . . . . Motor End-Plate Cholinesterase. . . . RESEARCH mTHODS O O O O O O O O O O O O O 0 Sample. . . . . . . . . . . . . . . . Treatment Groups. . . . . . . . . . . CON. . . . . . . . . . . . . . VOL. . . . -.- . . . . . . . . Controlled Runni g Groups. . . ESC. . . . . . . . . . . . . . SWM. . . . . . . . . . . . . . Duration Groups . . . . . . . . . . . Treatment Procedures. . . . . . . . . Animal Care . . . . . . . . . . . . . Sacrifice Procedures. . . . . . . . . Histochemical Procedures. . . . . . . Tissue Analysis Procedures. . . . . Statistical Procedures. . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . Treatment Results . . . . . . . . . . Treatment Environment and Body Results. . . . . .'. . . . . . Activity Level Results . . . . Histochemical Results . . . . . . . . Moror End-Plate Cholinesterase Succinic Dehydrogenase Sin'1 Per Cent Light Absorption . . . . . . . Discussion. . . . . . . . . . . . . . iv Daub-IN H coco-b b 10 13 15 15 16 16 17 17 18 18 19 19 20 21 22 23 27 30 30 31 32 32 33 39 46 Chapter Page V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. . . . . . . . 48 sumary . O O C C O O C O O O O 0 O O O . O C I O O 48 Conclusions . . . . . . . . . . . . . . . . . . . 49 Recommendations . . . . . . . . . . . . . . . . . 50 LIST OF REFERENCES 0 o o o o o o o o o o o o o o o o o o o o o o '0 o 51 APPENDICES A TRAINING PROGRAMS. . . . . . . . . . . . . . . . . . . . 60 B TRAINING, TREATMENT ENVIRONMENT, AND BODY WEIGHT VALUES. 65 C ADJUSTED CELL MEANS OF HISTOCHEMICAL MEASURES. . . . . . 72 Table 10 11 12 13 14 LIST OF TABLES Some differences among mammalian red, white, and inter- mediate skeletal muscle fibers related to their metabOI 18m 0 O O O O O O O O O O O O O O I I O O O O O O O 0 Final cell frequencies. . . . . . . . . . . . . . . . . . . Cage assignments and activity programs for each treat- ment group O O O O O O O O O D O O O O O O O O O O O O O O 0 Muscle fiber sampling reliabilities . . . . . . . . . . . . Two-way ANOVA table for soleus MEP-ChE surface area . . . . Summary of complex Scheffe contrasts within durations across treatments for soleus MEP-ChE surface area . . . . . Summary of complex Scheffe contrasts within treatments across durations for soleus MEP-ChE surface area. . . . . . Summary of standard Scheffé contrasts between duration cell means within the VOL and SHT treatment groups for soleus MEP-ChE surface area . . . . . . . . . . . . . . . . Summary of standard Scheffé contrasts between treatment cell means within the eight-week duration group for soleus mp-CIIE surface area 0 O O O O O O O O O O O O O I O O I I O Twodway ANOVA table for tibialis anterior MEP-ChE surface area 0 O O O O O O O O O O I O O O O O O O O O O O O O O O 0 Summary of complex Scheffe contrasts within treatments across durations for tibialis anterior MEP-ChE surface area 0 O O O O O O O O O O O O O O O O O I O O O O O O C O 0 Summary of standard Scheffe contrasts between duration cell means within the VOL and E80 treatment groups for tibialis anterior MEP-ChE surface area. . . . . . . . . . . Two-way ANOVA table for soleus Sin-1 SDH per cent light absorption. 0 O O I O O I O O O O O O I I O O O O O I 1 O O 0 Summary of complex Scheffé contrasts within treatments across durations for soleus Sin-1 SDH per cent light absorption. 0' O O O O O O O O O O O I O O O O O O O O O O 0 vi Page 15 16 27 33 34 34 35 35 37 38 38 42 42 Table Page 15 Summary of standard Scheffé contrasts between duration cell means within the VOL, SHT, MED, and LON treatment groups for soleus Sin‘1 SDH per cent light absorption . . . 43 16 Two-way ANOVA table for Sin.1 tibialis anterior SDH per cent light absorption 0 o o o o o o o o o o o o 'o o o o o o 44 17 Summary of complex Scheffé contrasts within treatments across durations for tibialis anterior Sin'l SDH per cent light absorption 0 O O O C O O I O O O I C O O O . O O 45 18 Summary of standard Scheffé contrasts between duration cell means within all treatment groups for tibialis anterior Sin"1 SDH per cent light absorption. . . . . . . . 45 A-l Standard eight-week, short-duration, high-speed endurance training program for postpubertal and adult male rats in contra lled running Wheels 0 O O O O O O O O O O O O O ‘ O O 61 A-2 Standard eight—week, medium-duration, moderate-speed endurance training program for postpubertal and adult male rats in controlled running wheels. . . . . . . . . . . 62 A—3 Standard eight-week, long-duration, low—speed endurance training program for postpubertal and adult male rats in controlled running wheels. . . . . . . . . . . . . . . . 63 A-4 Standard eightdweek, endurance, swimming training program for postpubertal and adult male rats. . . . . . . . . . . . 64 B-l Treatment environmental and body weight values for SHT, MD, and LON O O O O O O O O O O O O O O I O O O O O O I O 0 7o B-2 Treatment environment and body weight values for SWM. . . . 71 C-1 Adjusted cell means for soleus MEP-ChE surface area (sq. micra) 0 O O I O O O O O O I I O O O I I O O O O I I O 73 C-2 Adjusted cell means for tibialis anterior MEP-ChE surface area (sq O micra) O O O I O O O O O O O O O O O O h. C C O ’0 O 73 C-3 Adjusted cell means for soleus Sin-1 SDH per cent absorption 0 O O I O O O O O O O O O O O O O O O O O O O O O 74 C-4 Adjusted cell means for tibialis anterior Sin-1 SDH per cent absorption . . . . . . . . . . . . . . . . . . . . . . 74 vii Figure 3-4 LIST OF FIGURES Teased muscle fibers from the soleus of a CON animal Stained for SDH and MEP-ChE. o o o o o o o o o o o o o Teased muscle fibers from the tibialis anterior of a CON animal stained for SDH and MEP-ChE . . . . . . . . Adjusted cell means (least squares means) for soleus MEP-ChE surface area 0 o o o o o o o o o o o o o o o 0 Adjusted cell means (least squares means) for tibialis anterior MEP-ChE surface area. . . . . . . . . . . . . Adjusted cell means (least squares means) for Sin-1 soleus SDH per cent absorption . . . . . . . . . . . . Adjusted cell means (least squares means) for Sin-1 tibialis anterior SDH per cent absorption. . . . . . . Mean daily per cent shock free time (PSF) and per cent expected revolutions (PER) for CRW SHORT . . . . . . . Mean daily per cent shock free time (PSF) and per cent expected revolutions (PER) for CRW MEDIUM. . . . . . . Mean daily per cent shock free time (PSF) and per cent expected revolutions (PER) for CRW LONG. . . . . . . . Mean daily total revolutions run (TRR) for VOLUNTARY and CRW SHORT, MEDIUM, and LONG. . . . . . . . . . . . viii Page 25 25 40 40 41 41 66 67 68 69 AChE ATC ATP BuChE CDS CON CRW ESC EST HCP LON MEP-ChE NBT PER PET PSF SDH SHT LIST OF ABBREVIATIONS Acetylcholinesterase Acetylthiocholine Adenosine triphosphate Butyrylcholinesterase. Cumulative duration of shock (sec.) received by experimental (SHT, MED, LON) and the control (ESC) animals during all work periods of all bouts of a given training period. Cholinesterase Sedentary control Controlled running wheel Electric stimulus control (paired with SHT) Expected swim time (min.) Histochemical photometer Long-duration, low-intensity running exercise (long CRW program) ' Medium-duration, moderate-intensity running exercise (medium.CRW program) Motor end-plate cholinesterase Nitro blue tetrazolium Per cent expected revolutions PER - 100 TRR/TER Per cent expected swim time PET - 100 STC/EST Per cent shock free time PSF - 100-(100 CDS/TWT) Succinic dehydrogenase Short-duration, high-intensity running exercise (short CRW program) ix STC SWM TER TRR TWT VOL Swim time completed (min.) Swimming exercise (in individual tanks) Total expected revolutions that the animal would run, during all work periods of all bouts of a given training program, if he would run at the prescribed speed. Total revolutions actually run by the experimental animal, during all work periods of all bouts of a given training period. Total work time (sec.) during all work periods of all bouts of a given training period. VOluntary running exercise CHAPTER I THE PROBLEM Recent evidence (96, 59, 70, 41) suggests that one of the bases of the effects of exercise upon skeletal muscle fibers lies in altered metabolic states which are specific to the exercise regimens employed. Histochemical methods have contributed to the emergence of this concept by permitting identification and subsequent classification of indi- vidual muscle fibers through demonstration of relative enzyme and substrate levels which reflect the metabolic state of the fiber. Succinic dehydrogenase, a mitochondrial enzyme of the tricarboxylic acid cycle, often is used for this purpose (101, 89, 23, 7, 118). One outcome of histochemical investigations, suggested by earlier physiological studies (19, 9, 10), is the finding in a variety of mammalian skeletal muscles of heterogenous fiber populations (101, 89, 23, 7, 118). Although the classification of muscle fibers as red, white or intermediate has received some acceptance (80), the hetero- geneity of muscle fiber populations precludes application of a comparable classification scheme to whole muscles. Therefore, the physiological groupings of slow and fast, based on whole muscle measurements of relative contractile properties, often are employed as a muscle classi- fication scheme. For example, the soleus muscle is physiologically classified as slow and histochemically reveals a red and intermediate fiber population, while the tibialis anterior is physiologically fast with a mixture of red, white and intermediate fibers. 1 The specific mechanisms responsible for the observed metabolic changes in exercised muscle fibers are not known. Surgical alterations of the innervation to skeletal muscles affect their histochemical, biochemical, and physiological prOperties, yet the relationship between these surgically-induced neural changes and those resulting from chronic exercise is not clear. It has been shown that the soma and axon of the motor neuron respond to exercise (40, 31, 65, 60, 22, 105); however, the terminal aspect of the motor neuron, the motor end-plate, has received little attention. At the motor end-plates of mammalian skeletal muscle, cholinesterase enzymes display a fiber-specific distribution pattern (78, 62, 53, 46, 98, 20). Functionally, cholinesterases participate in the termination of neuromuscular transmitter action (61, 93) and serve as a model for the "trophic" influence upon muscle fiber metabolism (47). Thus, a study was undertaken to determine the effects, in a fast- slow antagonistic muscle pair, of several levels of chronic physical activity upon two enzymes. One of these, succinic dehydrogenase, is associated with oxidative muscle metabolism; the other, cholinesterase, is fundamental to motor end-plate function. W The level of succinic dehydrogenase activity and the pattern of motor end—plate cholinesterase localization vary among different types of muscle fibers. If one hypothesizes that the effects of various exercise regimens are specific for different types of muscle fibers, then the above fiber-specific parameters should respond differently in the soleus and tibialis anterior--two muscles known to have markedly different fiber pOpulations. 3 Statement of the Problem The purpose of this study was to determine the effects of seven chronic physical activity programs upon succinic dehydrogenase and motor end-plate cholinesterase in the left soleus and tibialis anterior muscles of adult male albino rats. Significance of the Problem The effects of chronic physical activity upon the motor end-plate are not known. The particular aspect of motor end-plate structure studied in this problem, motor end-plate cholinesterase localization, differs among muscle fiber types as does the level of succinic dehydrogenase activity. Therefore, the problem represents a test of the hypothesis that the adaptation of skeletal muscle to different levels of chronic physical activity manifests itself by cellular changes. Limitations of the Study l. The results may be specific to the papulation of adult male albino rats that favorably respond to the training methods employed. 2. Data on succinic dehydrogenase activity reflect.relative stain- ing intensities, not quantitative enzyme concentrations in individual muscle fibers. 3. The results may be specific to the particular muscles studied and not reflect the total range of response of skeletal muscle. 4. Characterization of the responses of the muscles studied are limited by selection of a sample of muscle fibers to represent the total pOpulation of fibers. 5. The total sample size limited the power of the statistical analysis. CHAPTER II REVIEW OF RELATED LITERATURE The purpose of this study was to determine the effects of seven chronic physical activity programs on SDH and MEP-ChE in the left soleus and tibialis anterior muscles of adult male albino rats. The following review of research related to the problem has been organized into five sections. The first deals with the general relationship between exercise and skeletal muscle metabolism. It is followed by a discussion of the research specifically related to exercise and SDH, the latter being one of the enzymes involved in this study. The influence of the nervous system upon skeletal muscle metabolism is the next area covered before focusing on the literature relating exercise and a specific part of the nervous system, the alpha motor neuron. The final section focuses on an enzyme associated with the alpha motor neuron, MEP-ChE, the other enzyme involved in this investigation. Skeletal Muscle Metabolism Mammalian skeletal muscle fiber heterogeneity was reported by early microscopists; however, the implications of these first histological observations of skeletal muscle metabolism were not realized until the advent of the more striking techniques of enzyme histochemistry (80). Numerous examples of mosaic responses of skeletal muscle fibers to histochemical staining techniques now exist (7, 118, 108, 101, 89, 23). 5 As an outgrowth of the demonstration of skeletal muscle fiber heterogeneity, there have been numerous attempts to arbitrarily classify muscle fibers according to histochemical (89, 118, 101, 7, 30, 69) and morphological (80, 67, 39) criteria. More recently, classification schemes have been reported for ultrastructural differences in muscle fibers (79, 80). Although clarification of the relationship between fiber typing schemes and muscle function is needed, the existence of at least two, and probably three, types of muscle fibers with distinctly different metabolic profiles has emerged from fiber typing studies. The use of the terms red, white, and intermediate to describe these fiber types has received some degree of acceptance (80) and will be used in this discussion. Some of the differences related to metabolism among mammalian red, white, and intermediate skeletal muscle are summarized in Table 1. Table 1. Some differences among mammalian red, white, and intermediate skeletal muscle fibers related to their metabolisml Fiber Type Measure White Intermediate Red Glycolytic enzymes High Intermediate Low Mitochondrial High to inter- oxidative enzymes Low' mediate High Lipolytic enzymes Low Intermediate High Intracellular glycogen High Intermediate High Capillary to fiber High to inter- ratio Low mediate High Number of mitochondria Low High to inter- High mediate 1For a more complete listing see Engel (35). 6 If the possible range of metabolic demands placed upon muscle fibers represents a continuum from aerobic to anaerobic limits, Table 1 reveals that red fibers are best adapted for metabolism under aerobic conditions, white for anaerobic conditions, and intermediate for limited levels of both conditions. This implies that the metabolic organization of skeletal muscles is at the fiber, or cellular, level. The above concept offers a potential framework to discuss the mechanisms under- lying responses to conditions affecting general metabolic state, such as chronic exercise. Elucidation of cellular adaptations in chronically exercised skeletal muscle has centered around morphological, biochemical, and histochemical studies. Steinhaus' (102) review of strength implicated hypertrophy, rather than hyperplasia, of cellular material as the means by which muscle size increases with overload training. Although several recent studies support this position, as indicated in the review of Jeffress and Peter (59), reports of fiber "splitting" in surgically-induced hypertrophy (106, 87) and in certain exercise regimens (12, 28) of the type used in this investigation indicate that this question is not yet resolved.' Other reported cellular morphological changes resulting from exercise include increased mitochondrial content (42, 56), capillary to fiber ratio (11, 12), and packing density of myosin and actin filaments (84). Biochemical alterations of both aerobic and anaerobic pathways were initially reported by Russian investigators (83). Although later attempts to confirm these findings were not successful (49, 50, 51, 45), the source of disagreement was possibly in the exercise intensities and experimental techniques employed by the investigators (42). That iii 7 exercise intensity can markedly affect the outcome of biochemical investigations was demonstrated by Holloszy (56). He confirmed an earlier report (49) of a lack of effect of moderate exercise upon mitochondrial oxidative enzymes, but also demonstrated marked effects with a program of greater intensity. Several reports exist indicating the ability of chronic exercise, especially the more demanding regimens, to increase aerobic metabolism (56, 42, 27, 4). However, a similar stimulation of anaerobic metabolism has not been clearly identified. While a few investigators have demon- strated an initial increase in anaerobic (glycolytic) enzymes, these effects have not persisted (5, 41). Such results may reflect a shift, or adaptation, from anaerobic metabolism to other energy sources as training progresses due to a failure of the exercise regimen employed to present a chronic anaerobic stress. Histochemical techniques, although generally not quantitative, have provided insight into qualitative adjustments to exercise stresses by permitting observation of intact muscle fiber populations. Tracking of fiber populations of several mammalian skeletal muscles has revealed a.more dynamic potential of muscle fibers to respond to chronic exercise than was previously recognized. Consistent with biochemical findings previously mentioned, demonstrations of increased numbers of red fibers with Chronic exercise have outnumbered the reverse effect (103, 27, 29, 4, 59, 13). However, this finding is subject to limitations similar to those expressed above, e.g., that the exercise programs employed may have been unable to present a chronic anaerobic exercise stress. The combination of increased mitochondrial content, mitochondrial oxidative enzyme activity, and number of mitochondrial-rich red muscle 8 fibers all indicate a resultant increased cellular aerobic metabolism with chronic exercise. Succinic Dehydrogenase Padykula and Gauthier, in a recent review of muscle fiber typing, have reported mitochondrial content to be a reliable morphological fiber typing criterion (80). Activity of mitochondrial oxidative enzymes, particularly SDH, is an accepted histochemical fiber typing criterion (101, 89, 23, 7, 118). High levels of SDH activity are found in red fibers, relatively low levels in white fibers, and high to intermediate levels in intermediate fibers. SDH, an enzyme of the tricarboxylic acid cycle, catalyzes the oxidation of succinic acid to fumaric acid. One result of this reaction is the release of two electrons which are subsequently delivered, via the electron transport chain, to intracellular oxygen for subsequent oxygen reduction and ATP formation. The entire process occurs within the mitochondria (113). Thus, SDH plays a role in the aerobic produc- tion of ATP, a molecular storage form of cellular energy. Chronic exercise generally has increased levels of SDH activity (56, 100, 13, 107, 29, 66). Reports of no exercise effect upon SDH activity (49, 45) may be specific for moderate exercise regimens (41, 56). Decreases were reported in SDH activity levels with anaerobic type exercise such as "weight-lifting" (100) and high-intensiy, short- duration wheel running (14). Neural Influences upon Skeletal Muscle Metabolism Surgical alterations have been used to demonstrate a neural influence upon skeletal muscle metabolism. These surgically-induced neural effects were shown with denervation, reinnervation, and cross-innervation experiments. 9 Denervation of skeletal muscle causes a decrease in the activity of enzymes of energy metabolism (34, 74, 90, 55). Although all fiber types are affected, the rate of enzymatic activity loss is directly related to its normal level. Thus, white fibers display the most rapid reduction of glycolytic activity while red fibers show a similar effect for oxidative and lipolytic activities (90, 55). Consequently, the net’ effect of denervation is a loss of skeletal fiber heterogeneity, which is not explained solely by the concomitant muscular atroPhy with nerve section (37). This effect appears to be related to the neuromuscular transmission process since neuromuscular transmission blockade by various pharmacologic agents produces effects similar to those of surgical denervation (24, 25). Reinnervation of a muscle by its original motor nerve reverses the effects of denervation and generally restores its metabolic profile (91, 92). One exception to this reversal is an alteration of the motor unit spatial pattern. Normally, motor units display a."scattered" spatial pattern. Although the muscle fibers of motor units are thought to be of a single type (8, 33, 32, 68, 91, 92, 117), they usually are intermingled, or scattered, among other types of fibers from other motor units. After reinnervation, these motor units diaplay a more condensed, or "clustered", pattern despite the observation that the overall population of fibers is quantitatively similar to unoperated controls (33, 91, 92, 117). Denervation appears to be a necessary pre- requisite for successful reinnervation (8) and multiple reinnervation, often by nerves originally innervating muscles of different properties (8). The effect of multiple reinnervation is comparable to that of cross-innervation. 10 Cross-innervation of characteristically slow and fast skeletal muscles reverses their physiological, biochemical, and histochemical properties although the reversal is rarely complete (21, 91, 92, 17, 117, 88). Mutation of prOperties from white to red muscle is more complete than the reverse effect following cross-innervation experiments (9, 10). The implications relevant to the present investigation to be drawn from the above neuron-surgery experiments are that a neural influence exists upon both quantitative, redness vs. whiteness, and qualitative, heterogeneous vs. homogeneous, aspects of skeletal muscle fiber metabolism. This dependency of skeletal muscle metabolism upon neural innervation led to the designation of the neural influence as a "trophic" factor (47). Since both an axoplasmic and a synaptic flow of materials has been identified (47), three probable mechanisms have been hypothesized to explain the neuron's trOphic effect on skeletal muscle: nerve-to- mmscle flow of either a trophic substance, normal impulse traffic, or a combination of the two (47). In any case, the specific anatomical "carrier" of the skeletal muscle trophic effect is believed to be the alpha motor neuron, not sensory or sympathetic neural elements (120). The Alpha Motor Neuron Striking similarities exist between certain anatomical and physio- logical properties of alpha motor neurons and the skeletal muscle fibers they innervate. Generally, these findings support the view that the motor unit is more structurally and functionally integrated than previously thought. Alpha motor neurons innervating white skeletal muscles have larger somata and motor end-plates as well as higher levels of discharge ll frequency and conduction velocity than motor neurons to red muscles (8, 47, 46, 78, 72, 116, 52). Literature regarding the effects of exercise upon the alpha motor neuron is scanty. Present knowledge relates to exercise effects upon the motor neuron's axonal structure, somal structure, and somal metabolism. The evidence as to the effects upon the number and size of peripheral nerve fibers to exercised muscles is inconclusive (1, 3, 105). A similar situation exists for those studies where functional overload was surgically produced (26, 2, 110, 36). The most recent and comprehensive study in this area is that of Tomonek and Tipton (105), who investigated the effects of tenectomy, acute exercise, and chronic exercise upon the number and size of fibers in the medial gastrocnemius nerve of rats. Only tenectomy had a significant effect, reducing the number and size of nerve fibers. . Acute exercise was shown either to have no effect (63) or to reduce slightly (63, 54) alpha motor neuron somal size. Histochemical results have revealed a metabolically challenged cell, i.e., decreased meta- bolic substrates (57, 65) with lower enzyme (65, 109) and nissl sub- stance (65, 31) levels. Increases in alkaline phosphatase and thiamine pyrophosphatase activities (60, 65), although not conclusive, may reflect an increased enzyme synthesis (65). Investigations of the effects of chronic exercise upon the alpha motor neuron are few (31, 40). Of particular interest are two studies, using the same rats as subjects, which employed three chronic activity levels; sedentary, sedentary plus daily 30-minute swims with a tail weight equal to 3 per cent of body weight attached, and voluntary activity plus two daily 12 30-minute swims with a 4 per cent tail weight attached. Gerchman (40) investigated the lumbar alpha motor neuron somata while Edgerton (27) studied specific lower limb skeletal muscles. The reported significant increases of red fibers in the plantaris and decreases, though not statistically significant, in alpha motor neuron size of the voluntary- plus-forced exercise rats would support a hypothesis of a nerve-muscle, white-to-red "mutation." However, such support is limited since no specific functional relationships between neurons and muscles were established. Another finding of Gerchman's was that the sedentary- plus-forced exercise group, which had difficulty coping with the swimming regimen, displayed alpha motor neuron somata of reduced metabolic activity. Edgerton reported no comparable muscular changes. The voluntary-plus-forced exercise group, although subjected to higher intensity exercise than sedentary-plus-forced animals, performed better and showed indications of increased metabolic activity of alpha motor neurons. Although acute and chronic exercise have affected some aspects of the alpha motor neuron, too little evidence is available for extraction of a clear pattern of response. Limited as the preceding information is, even less is known of the exercise effects upon the neuromuscular junction. The neuromuscular junction of mammalian skeletal muscle is the site of a chemically-conducting synapse.at which acetylcholine is the transmitter. In mammalian skeletal muscle, a single neuromuscular junction per muscle fiber is the predominant mode of innervation. Contraction of skeletal muscle occurs when neural impulses release, frombpresynaptic synthesis or storage sites, sufficient ACh to bind to post-synaptic receptor sites and initiate local, non-propagated 13 depolarizations of the post-synaptic membrane (114, 61). If the local depolarization is large enough, a propagated muscle action potential is triggered. Although the specific mechanism.linking neural excitation and muscular contraction is not known, ACh apparently can effect release of calcium ions, essential for contraction, previously bound to sarco- plasmic reticulum (18). The termination of ACh action is accomplished, at least in part, by extracellularly located cholinesterase enzymes. These enzymes catalyze the rapid hydrolysis of ACh to acetate and choline (114). Since cholinesterase enzymes are found in several places in the body, those localized at extracellular sites of neuromuscular junctions are designated as motor end—plate cholinesterases. Motongnd-Plate Cholinesterase Acetyl(true)-butyry1-, and pseudo-cholinesterases have been demon- strated at mammalian motor end-pretes. Early investigations attempting to pinpoint the location of MEP-ChE enzymes led to the identification of several sites, both pre- and postsynaptic and intra- and extracellular (58, 20, 85, 6). The identification of such a multitude of MEP—ChE sites was not consistent with neuromuscular transmission theory (114, 61, 93). IMore recent studies have identified methodological, rather than theoretical, problems which led to the earlier discrepancy between theory and research upon MEP-ChE (104, 77). Recent histochemical and biochemical studies have identified AChE as the primary ChE enzyme at mammalian motor end-plates (104, 77, 76, 75, 38, 44, 119, 20). In human intercostal muscle, 90% of MEP-ChE activity is due to AChE (76). Its location is extracellular and pre- dominantly post-synaptic (76, 114). Although selective localization of AChE at end-plates of fast muscles and BuChE at those of slow muscles 14 has been reported (48), the results were not confirmed in subsequent studies (68, 98). However, the above research did stimulate attempts to relate MEP-ChE to muscle fiber type. Larger, structurally more "complicated" end-plates, as reflected by MEP-ChE activity, were observed in white than in red muscle fibers where SDH activity was the fiber typing criterion (78). More recent electron microscopic studies have revealed details of motor end-plate structure. The greater "complexity of end-plates on white fibers than red fibers was identified as relatively more_branched and anatomosed junctional folds (of post-synaptic membrane). It also appears that both white and intermediate fibers have larger axon terminals and greater areas of potential nerve-muscle interaction, in terms of apposed axoplasmic and sarcoplasmic membranes, than do red fibers (73, 82). Although more detail is needed of motor end-plate structure, these findings lend support to the notion of fiber-specific motor end-plate structures. This can be demonstrated by MEP-ChE histochemical tech— niques because the end-products of such stains are localized primarily in post-synaptic membranes where marked differences exist among fiber types . CHAPTER III RESEARCH METHODS Sample An initial sample of 176 normal, 72-day-old, male, albino rats (Sprague-Dawley strain)1 were brought into the laboratory in four shipments of 48, 48, 40, and 40 animals, respectively. The animals were randomly assigned to seven treatment groups and allowed 12 days to adjust to laboratory conditions before treatments began. Applica— tion of selection criteria, to be discussed later, resulted in the final sample consisting of 102 animals (Table 2). Table 2. Final cell frequencies Duration , Treatment Odwk 4-wk 8-wk 12~wk CON 2 4 4 4 VOL 2 4 4 5 ESC 2 4 4 5 SET 2 4 4 . 4 MED 2 4 4 5 LON 2 4 z. 5 SWM 2 4 4 4 1Obtained from Hormone Assay Laboratory, Chicago, Illinois. 15 16 Treatment Groups Cage assignments during the adjustment and treatment periods and activity programs for the seven treatment groups used in this study are presented in Table 3. Table 3. Cage assignments and activity programs for each treatment group- W Adjustment Treatment Period Experimental Experimental Period Group Housing Period Housing Activity CON sedentary sedentary no special treatment VOL. voluntary voluntary exercise at will SHT voluntary sedentary short-duration high- speed endurance running MED voluntary, sedentary medium-duration moderateespeed endurance running LON voluntary sedentary long-duration low- speed endurance running ESC voluntary sedentary shock control for SHT program SWM voluntary sedentary long-duration low- intensity endurance swimming CON These animals received no special treatment and were housed in standard, individual, sedentary cages (24 cm, long x 18 cm, wide x 18 cm, high) during the adjustment and treatment periods. l7 XQL_ These animals received no special treatment during either the adjustment period or the treatment period but were housed in standard, individual, voluntary-activity cages which allowed access to freely revolving activity wheels (13 cm. wide x 35 cm. diameter). Individual records of total revolutions run were recorded from attached revolution counters . Controlled Running Groups These animals were housed in individual voluntary-activity cages during the adjustment period and in individual sedentary cages during the treatment period. Utilizing small-animal controlled running wheels (CRW), each of these animals was subjected to one of three programs of interval training. The following descriptions of the three running programs used include only the final exercise requirements which were first imposed on the 37th day of training (for complete programs see Appendix A). SET, This group was subjected to a short-duration, high-speed endurance program and was expected to complete eight bouts of exercise with 2.5 minutes of inactivity between bouts. Each bout consisted of 6 repetitions of 10 seconds of work alternated with 40 seconds of rest. This group ran at a speed of 5.5 ft./sec. during the work intervals. ‘MEQ, These animals were subjected to a medium-duration, moderate- speed endurance program and were expected to complete five bouts of exercise with.5 minutes of inactivity between bouts. Each bout consisted of 8 repetitions of 30 seconds of work alternated with 30 seconds of rest. This group ran at a speed of 4.0 ft./sec. during the work intervals. 18 Egg, This group was subjected to a long-duration, low-speed endurance program of continuous running and was expected to complete four bouts of exercise with 2.5 minutes of inactivity between bouts. Each bout consisted of l repetition of 12.5 minutes of continuous work. This group ran at a speed of 2.0 ft./sec. during the work intervals. fig These animals were housed in individual voluntary-activity cages during the adjustment period and in individual sedentary cages during the treatment period. Each ESC animal was permanently paired with a SHT animal. During the treatment period for the SHT animals, the ESC animals~ were placed in stimulus control cages attached to the CRW (21.5 cm. long x 14 cm. wide x 10.5 cm. high). The ESC animals received electrical shock through a grid floor comparable to that of the CRW. Each ESC animal was exposed to the same total light stimulus and electrical shock. as its paired mate in the SHT group. §E§_ These animals were housed.in individual voluntary-activity cages during the adjustment period and in individual sedentary cages during the treatment period. Each animal swam in an individual cylindrical tank (76 cm. high x 28 cm. in diameter) in 70 cm. of water (ZS-32°C.). 0n the last four days of the eighth week of this program, each of these animals was expected to swim one 60-minute bout with an attached tail weight equal to three per cent of his body weight. 19 Duration Groups To provide chronological perspective of the various treatment effects, animals were sacrificed at 0, 4, 8, and 12 weeks after initia- tion of treatments. CRW, ESC, and SWM 12~week groups followed their respective 37th day routines on each of the last twenty-three training days (see Appendix A). Treatment Procedures The exercise treatments were begun when the animals were 85 days of age. Animals designated as O-wk received no special treatment and were sacrificed at the end of the 12-day adjustment period. The SHT, MED, LON, SWM, and ESC experimental treatments were con- ducted once a day five days per week, between 12:30 p.m. and 5:30 p.m. Monday through Friday, in the Human Energy Research Laboratory, Michigan State University, East Lansing, Michigan. Body weights for SWM. MED, LON, and ESC animals were recorded before and after each treatment period while only pre-treatment weights were taken for SWM animals. For each animal in the VOL group, total revolutions run during the previous 24 hours were recorded,.Tuesday through Friday, between 10:00 a.m. and_11:00 a.m. The SHT, MED, LON, and ESC groups.received treatment in the CRW which is described as: "...a unique animalepowered wheel which is capable of inducing small laboratory animals to participate in highly specific programs of controlled reproduce ible exercise." (111) During the first 40-min. learning period in the CRW, the animals run in response to electrical shock. By the end of the third learning period, most animals are conditioned to run in response to a stimulus of light which precedes the shock stimulus. 20 Initially, animals were placed in individually braked running wheels. For each running period, a light above the wheel signaled the start of a work interval and remained on for a predetermined time, the acceleration period. Loss of the light stimulus and application of the shock stimulus occurred for animals not obtaining prescribed wheel speeds during the acceleration period. Animals running slower than the specified speed had the light-shock sequence repeated., Maintenance of the light stimulus without shock occurred for animals obtaining or exceeding prescribed wheel speeds. Typically, running programs con- sisted of alternate periods of work and rest. TRR and CD8 values for the SHT, MED, and LON animals were recorded from a result unit attached to each CRW after_each treatment period. The ESC animals used SHT values. These values, along with TER and TWT (Tables A—l through A-3, Appendix A), were used to calculate PER and PSF. For the SWM group, STC was recorded after each treatment period and-used, with EST (Table A-4, Appendix A) to calculate PET. Animal Care Since rats are normally more active at night than during daylight hours, the light sequence in the animal quarters was automatically timed to reverse the rats' active period by having lights off from 1:00 p.m.. to 1:00 a.m. and on from 1:00 a.m. to 1:00 p.m. Thus, animals were trained during the active phase of their diurnal cycle and at a conven- ient time for the laboratory staff. Standard procedures were observed to-maintain a relatively constant environment for the animals. Daily handling, temperature and humidity control, and regular cage cleaning were included in those procedures. 21 Throughout the experiment, all animals had access to water and a com- mercial animal dietl ad Zibitum. Sacrifice Procedures Fourteen biweekly sacrifices of seven animals were conducted from November 11, 1970, to May 24, 1971. All animals in each sacrifice were of the same duration group. Each sacrifice involved one CON animal plus pairs of animals from each of the VOL, MED, and LON groups or from each of the SHT, ESC, and SWM groups. This sacrifice schedule, while not balanced, was judged to be necessary to be compatible with treat- ment schedules. Animals were selected after the last treatment on Friday for sacrifice on the following Monday on the bases of their health and performance histories. Only animals subjectively determined to be in good general health were selected. A criterion PER value of 75 was set as the minimal acceptable standard for execution of the CRW programs. Only SHT, MED, and LON animals whose mean PER values were above this criterion were selected for sacrifice. Proximity to a mean PET value of 100 was used as a sacrifice selection criterion for SWM animals, since most animals in this group were able to execute SWM program requirements. Animals were weighed and then sacrificed under anesthesia by an. intraperitoneal injection, 4 mg./100 g. body weight, of 6.48% Halatal2 (sodium.pentobarbital). Laparotomy, gentle rotation of abdominal viscera, and partial removal of the parietal peritoneum.were performed to permit withdrawal of 1-2 ml. of venous blood from the inferior vena cava with lWayne Laboratory Blox, Allied Mills, Inc.,-Chicago, Illinois. 2FromJensen-Salsberg Laboratories, Division of Richardson- Merrell, Inc., Kansas City, Missouri. 22 a 20-gauge hypodermic needle. Following barrel exchange, each animal received an injection of 4 ml. of physiologicink.l After three mdnutes of in viva ink circulation, the heart was removed. The right hind limb then was skinned to permit removal, after reflection of overlying tissue, of the right triceps surae and plantaris as a unit. Similar procedures were followed for the left hind limb except the plantaris, gastrocnemius, and soleus were separated. The tibialis anterior and a distal segment of the nerve to the soleus also were removed from the left hind limb. Only the left soleus and tibialis anterior were used in the present study. The remaining tissues which were removed at sacrifice were analyzed by other investigators. Once removed, the left soleus and tibialis anterior muscles were immediately frozen in an isopentane-liquid nitrogen system {-140 to -185°C.). Frozen musculature was stored at -20°C. until histochemical processing. Histochemical Procedures The histochemical procedure employed in this study was previously reported (86). Briefly, the technique involves 24 hours of tissue "softening" in room temperature Ringer's solution during which tissue samples were "teased" apart, i.e., separated into small, dispersed seg- ments. Teasing disoriented muscle fibers from their normal spatial relationships. Teased tissues were double-stained, first with the NET method for SDH (97) and then with the ATC method for MEPACha(&4, 43). Randomly selected, stained tissues then were mounted, in a glycerine jelly 1Pelikan Ink. Obtained from John Henschel and Co., Farmington, Long Isle, New York. 23 medium, on clean glass microscOpe slides. Mounted tissues were immediately surveyed under a light microscope for the presence of MEP-ChE staining. Samples of stained tissues were mounted and surveyed until sufficient appropriate tissues for analysis were obtained. Pictures of stained muscle fibers from the soleus and tibialis anterior of CON animals are presented in Figures 1 and 2, respectively. All histochemical procedures were conducted without knowledge of tissue origin by the use of code number identifications. Tissue Analysis Procedures Analysis of histochemical sections was performed on the Histochemical Photometer or HCP (112). This device has been described as consisting of a "...Prado microprojector, a photocell with associated circuits to measure light intensity, and a digital voltmeter readout" (112). The HCP was calibrated for each slide such that zero per cent readouts equaled zero light transmission to the photocell and 100 per cent read- outs equaled transmission of that amount of light passing through the cover slip, glycerine jelly, and microscope slide, but no tissue. Thus, SDH staining intensity was recorded as per cent light transmission and later changed to per cent light absorption by subtracting the obtained value from 100. Per cent absorption is directly related to staining intensity. SDH readings were taken with a HCP setting such that the projected image had a X200 linear magnification. Magnification was verified by a calibrated microscOpe slide. This magnification permitted tracing of the perimeter of MEP-ChE reactive end product localization. Earlier work had indicated that, due to the often irregular shape of the MEP-ChE reactive end product localization, perimeter measurements were more reliable than diameter measurements (78). 24 Figure l. Teased muscle fibers from the soleus of a CON animal stained for SDH and MEP-ChE. (SOOX) Figure 2. Teased muscle fibers from the tibialis anterior of a CON animal stained for SDH and MEP-ChE. (500x) 25 26 Since teasing did not permit control of orientation of mounted tissue and produced some uneven staining and destruction of muscle fiber integrity, the following criteria were used to select muscle fibers for analysis: 1. The maximum permitted range of SDH in per cent light trans— mission, within a muscle fiber, was 10. 2. The MEP-ChE reactive end product localization was entirely visible. 3. The MEP-ChE reactive end product was sufficiently localized to permit accurate perimeter tracing. The first of these criteria listed above defined evenly stained muscle fibers. Fluctuations of more than 10 per cent SDH light trans- mission within a muscle fiber were generally visually verifiable. The second and third criteria defined acceptable fiber orientation and MEP-ChE reactive end product localization. A typical muscle fiber analysis consisted of insertion of a slide in the microprojector stage, calibration, survey to find an acceptable fiber, MEP-ChE tracing, and notation of SDH per cent transmission values at four different points on the longitudinal axis of the muscle fiber, two on each side of the motor end-plate. Four SDH readings were neces- sary and sufficient to characterize staining intensity of muscle fibers selected in this study (test-retest Rho-+0.951). Data were collected on 10 fibers from each of the soleus and tibialis anterior muscles for each animal. Table 4 shows the test— retest rank—order correlation coefficients (Rho) of 10 fiber sampling reliabilities at each treatment duration. Surface areas of tracings of MEP-ChE reactive end product locali- zation were measured in square centimeters by polar planimetry. The 27 Table 4. Muscle fiber sampling reliabilities Duration Tibialis (wks.) Soleus~ Anterior O .933 .900 4 .940 .904 8 .906 .877 12 .901 .857 mean of 5 such planimeter measures gave a reliable (Rho - .989) measure. These means were converted to square microns by multiplication by the calibration factor 2500, which adjusted for magnification and measure- ment units. Mean SDH per cent light absorptions and MEP-ChE surface areas were calculated for the soleus and tibialis anterior of each animal. Statistical Procedures Treatment data were analyzed for appropriate groups and days by computer calculation of means, standard deviations, and simple correla- tion coefficients for training performance, environmental conditions, and pre- and post-treatment body weights. Since the diameters of the wheels attached to voluntary-activity cages are less than CRW diameters, daily TRR values of VOL animals were multiplied by a calibration factor of 0.9163 to equate the TRR values of the VOL animals to those of SHT, MED, and LON animals. The Bartlett and F-max tests were applied to ascertain if the histo- chemical data met the variance homogeneity assumption of analysis of variance (ANOVA). Variances among cells were not significantly different 28 at P-.05 for soleus and tibialis anterior MEP surface areas. However, SDH per cent light absorption values were found to have nonhomogeneous variances, a situation often encountered with per cent data where values do not fall between the values of 25 and 75 (99). Application of the angular transformation and subsequent retesting with the Bartlett and F-max tests indicated that the transformed values could be analyzed by ANOVA methods. A two-way, fixed effects ANOVA model, with treatment and duration of treatment as independent variables and animal body weight at sacri— fice as a covariate, was employed to analyze the histochemical data. Complex Scheffé contrasts were used to detect significant categories within each independent variable. These complex Scheffé contrasts consisted of all possible contrasts of pairs of cell means within each category of the independent variable shown to be significant by the overall ANOVA. Within significant categories, standard Scheffé con- trasts were used to identify the particular cell means responsible for the observed significance. For the ANOVA analyses, an alpha level of .10 was selected as being a realistic criterion in view of the small number of observa- tions per cell. For the Scheffé test an alpha level of .20 was selected. Scheffé (95) has suggested a .10 "nominal" level for contrasts run post-hoc to .05 F tests. Thus, a .20 alpha level for the Scheffé tests appeared to be appropriate, especially in light of the possibility of rejecting potentially fruitful research areas simply due to limi- tations of sample size in this study. Since there was no particular interest in or control of the body weight of the animals used in this investigation, body weight at sacri- fice was treated statistically as a covariate. In a strict sense, body 29 weight is not a legitimate covariate due to its dependency upon both independent variables. Its treatment as a covariate represented a compromise between statistical protocol and the desire to remove the body weight effect, if any. No interpretation of the covariate was intended. CHAPTER IV RESULTS AND DISCUSSION The results of this study will be presented in two main sections. The first set of data describes the experimental treatment, environment, and activity levels of the animals while the second set of data was derived from the histochemical analysis. The discussion will focus on the histochemical results since they represent the effects of the independent variables under investigation. Treatment Results On the basis of the programmed values of TER for the various CRW treatments (see Appendix A), animals of the LON group should have had a large mean daily TRR increase over the training period, the MED animals a moderate_increase, and the SHT animals a slight increase followed by a gradual decrease. From Observation of the mean daily TRR values, it can be seen that the SET, MED, and LON animals met their respective program requirements (Figure B—4, Appendix B). The mean_daily TRR values of the VOL animals did not display a con- sistent position relative to those of the CRW groups for the first 5 weeks of training, although a definite trend toward a position between the SHT and MED groups is evident from 6 to 12 weeks. This observation is limited in that the speed and duration of wheel revolutions run by VOL animals cannot be equated to those by SHT, MED, and LON animals. The SHT, MED, and LON animals generally maintained PER values above 80, 30 31 thus exceeding the criterion PER level of 75, which was set as the minimum acceptable standard for execution of the CRW programs (Figures B-l, B-2, B-3, Appendix B). This level of performance compares favorably with other groups of animals subjected to similar training programs (94). The animals generally responded to the light, rather than the electrical shock, stimuli. Comparisons across treatment durations of PSF values for the SHT, MED, and LON groups show that the SHT animals and their ESC counterparts received the greatest relative amount of electrical shock (Figures B-l, B-2, B-3, Appendix B). Almost without exception, PET values for SWM.animals were 100 (Table B-2, Appendix B). Therefore PET was not plotted across duration of SWM treatment. Treatment Environment and Body Weight Results The SHT, MED, and LON animals were exercised under conditions of relatively constant air temperature, barometric pressure, and humidity (Table B-1, Appendix B). That these values did not affect PER and PSF is reflected in the low correlations among the parameters. However, animals with relatively high pre-treatment body weights tended to display low PER and PSF values. Animals showing relatively large weight losses tended to display high PER and PSF values. The high correlation coef- ficient between PER and PSF confirms the nearly parallel plots of these values (Figures B-l, B-2, and B-3, Appendix B). The SWM animals exercised under controlled conditions, including water temperature, which were comparable to those of the SHT, MED, and LON animals (Table B-2, Appendix B). None of the environmental or pre- treatment body weight values were highly correlated with PET. 32 Activity Level Results Before discussing the histochemical results, it seems necessary to establish if the treatments employed in this investigation did, in fact, represent seven different levels of chronic physical activity. That the CON animals represented a low activity level is assumed due to the physical restrictions of their cages. Coupling the observation that controlled running and swimming program expectations were markedly different (Appendix A) with the high levels at which the animals met these expectations (Figures B-l to B-3, Tables B-1 and B-2, Appendix B), it appears that the SHT, MED, LON, and SWM groups do represent four different levels of physical activity. A divergence of activity levels, on the basis of mean daily TRR, of SHT, VOL, MED, and LON groups.was observed (Figure B-4, Appendix B). The decision to pair ESC and SHT animals in anticipation of the SHT animals receiving the largest relative amount of electrical shock stimuli was correct (Figures B-l to B-3, Appendix B). Although the chronic activity levels of ESC animals is not known, they are not equatable to CON animals due_to their physical responses to the noxious stimuli. Based upon the above treatment results and conditions, the seven treatment groups of animals appear to represent distinctly different levels of chronic physical activity. Histochemical Results Histochemical results will be discussed separately for each depend- ent variable: soleus MEP-ChE surface area, tibialis anterior MEP-ChE surface area, sin-Isoleus SDH per cent light absorption, and sin-1— tibialis anterior SDH per cent light absorption. 33 Motor End-Plate Cholinesterase Soleus. Adjusted cell means for soleus MEP—ChE surface area are' presented in Table C-l, Appendix C. The cell means are graphically presented in Figure 3, page 40. A general trend toward an increase'ina soleus MEP-ChE surface area is apparent in all groups except the CON group. These means were generated from the raw data as were the overall ANOVA results given in Table 5. Table 5 shows that both treatment and duration significantly affected soleus MEP-ChE as well as significantly interacting with each other. The covariate, body weight at sacrifice, was also significant: an expected result. Table 5. Twoeway ANOVA table for soleus MEP-ChE surface area Summary of Analysis of variance Source SS df MS F P Treatment 2972.54 6 495.42 2.11 0.062 Duration 2557.90 3 852.63 3.63 0.017 Training x Duration 7093.42 18 394.08 1.68 0.064 Covariatel 2370.44 1 2870.44 12.22 0.001 Error 17149.87 73 234.93 Total, 32644.17_ 101 4847.50 1Body weight at sacrifice. Subsequent complex Scheffé contrasts identified the VOL and SHT treatment categories and the 8-week category of duration as the sources of the treatment and duration significance observed in the overall ANOVA (Tables 6 and 7). 34 Table 6. Summary of complex Scheffé contrasts within durations across treatments for soleus MEP-ChE surface area Dependent variable O-wku 4dwk. 8-wk. 12-wk. Soleus MEP-ChE surface area N N S N N-not significant. S-significant at the .20 level. Table 7. Summary of complex Scheffé contrasts within treatments across durations for soleus MEP-ChE surface area Dependent Variable CON VOL SHT MED LON ESC SWM Soleus MEP-ChE surface area N S S N N N N N-not significant. s-significant at the .20 level. The significant treatment-duration interaction appears, upon inspec- tion of the means in Figure 1, page 40, to have been caused by the increase in MEP-ChE size for the three forced running groups, SHT, MED, and LON, at 12dwks. duration. Standard Scheffé contrasts between pairs of cell means within the VOL and SHT programs and the 8-wk. duration are presented in Tables 8 and 9, respectively. The results summarized in Table 8 indicate that significantly larger-soleus MEP-ChE surface areas had emerged in the VOL animals after 12 weeks of treatment. Only 8 weeks of the SHT treatment were necessary 35 Table 8. Summary of standard Scheffé contrasts between duration cell means within the VOL and SHT treatment groups for soleus MEP-ChE surface area Dependent Duration Means Contrasted ~ Variable Treat. 0-4 0-8 0-12 4-8 4-12 8-12 Soleus MEP—ChE VOL N N + N N N surface area SHT N + N + N - N-not significant. -=significant decrease with increasing duration of .20 level. +-significant increase with increasing duration at .20 level. Table 9. Summary of standard Scheffé contrasts between treatment cell means within the eightdweek duration group for soleus MEP-ChE surface area f f 8~wk. Treatment Soleus MEP-ChE Means Contrasted Surface Area CON vs. VOL CON vs. SHT CON vs. MED CON vs. LON CON vs. ESC CON vs. SWM VOL vs. SHT VOL vs. MED VOL vs. LON VOL vs. ESC VOL vs. SWM SHT vs. MED SHT vs. LON SHT vs. ESC SHT vs. SWM MED vs. LON MED vs. ESC MED vs. SWM LON vs. ESC LON vs. SWM ESC vs. SWM l2222+2222+2 222222! N-not significant. +-treatment mean to the right is significantly greater than that to the left. -treatment mean to the right is significantly less than that to the left. 36 to produce a similar significant increase, although this increase was not maintained through 12 weeks. Table 9 shows that the MEP-ChE surface areas of the SHT animals were significantly larger than those of any other group at 8-wks. duration. The marked interaction between the SHT training program and 8~wks. duration of this program warrants further discussion. Inspection of the progression of exercise intensity, i.e., running speed, in this program (Table A-1, Appendix A) shows that the SHT animals had reached the point of maximal running speed during the eighth week of training just prior to sacrifice. The succeeding program requirements for the SHT group, in effect from the 38th to-the final day of training, actually represent a plateau in the progression of running speed at 5.5 ft./sec. The fact that the significantly increased MEP-ChE surface area in the soleus of the 8~wk. animals could not be maintained may be related to the fact that the progression in running speed was not maintained. Taking the net increases in MEP-ChE surface area for the three forced running groups with the significant increase of the same measure from 8-12 weeks of VOL treatment, it appears that running exercise, particularly the high-intensity type, augments soleus MEP-ChE surface area. Recalling the fact that the soleus of the albino rat normally has a red-intermediate fiber papulation, these results would indicate a possible shift of MEP-ChE surface area toward a more completely inter- mediate or white-intermediate pattern, since larger end-plates have been associated with intermediate and white muscle fibers (73, 78, 82). Tibialis anterior. Adjusted cell means for tibialis anterior MEP-ChE surface area also are presented in both tabular (Table C-2,~ 37 Appendix C) and graphic (Figure 4, page 40) form. These data reflect a general trend toward decreased MEP-ChE surface area for all groups. The results of the overall ANOVA are presented in Table 10. Table 10. Twoeway ANOVA table for tibialis anterior MEP-ChE surface area Summary of Analysis of Variance Source SS df MS F P Treatment 1335.29 6 222.55 1.15 0.344 Duration 1211.68 3 403.89 2.08 0.110 Training x Duration 1984.34 18 110.24 0.57 0.911 Covariatel 280.59 1 280.59 1.45 0.233 Error 14163.15 73 194.02 Total 18975.05 101 1211.29 1Body weight at sacrifice. None of the effects met the criterion alpha level of .10 although the duration effect was nearly significant (P-.ll). Subsequent evalua- tion of the power of this test yielded a low value (Power=.43) indicating that any final judgment of the effect of treatment duration upon tibialis anterior MEP-ChE surface area should be reserved. Although further analysis of the nearly significant duration effect is not consistent with strict statistical protocol, the post-hoe Scheffé tests were performed in order to extract all potentially valuable results. The pooling of O-wk. animals and the utilization of a .20 criterion alpha level for the complex Scheffé contrasts increased the power of the test sufficiently to identify the VOL and ESC groups as those causing the nearly significant duration effect in the overall ANOVA analysis. 38 These complex Scheffé contrasts are summarized in Table 11. Table 11. Summary of complex Scheffé contrasts within treatments across durations for tibialis anterior MEP—ChE surface area Dependent Variable CON VOL SHT MED LON ESC SWM Tibialis anterior MEP-ChE surface area N S N N N S N N-not significant. S=significant at the .20 level. Standard Scheffé contrasts identified a potentially important decrease in tibialis anterior MEP-ChE surface area in the VOL animals at 8 weeks which persisted until 12 weeks. The ESC group also showed a decrease but not until lZ—wks. duration. These standard Scheffé con- trasts are presented in Table 12. Table 12. Summary of standard Scheffé contrasts between duration cell means within the VOL and ESC treatment groups for tibialis anterior MEP-ChE surface area Dependent Duration Means Contrasted Variable Treat. 0-4 0-8 0-12 4-8 4-12 8-12 Tibialis anterior VOL ' N - - N N N MEP-ChE surface area ESC N N - N N N N-not significant. --significant decrease with increasing duration at .20 level. +-significant increase with increasing duration at .20 level. The decrease in tibialis anterior MEP-ChE at 12 weeks is exactly opposite to the significant increase in the same measure, in the same 39 animals, observed in the soleus. In general, inspection of the means (Figure 2, page 40) reveals a similar, though nonsignificant, trend for all groups. MEP-ChE surface area generally increased in soleus but decreased in tibialis anterior.‘ The most likely explanation for this divergence of effect lies'in‘ the markedly different fiber papulations of the two muscles, the BOIEUS“ displaying a red-intermediate profile while the tibialis anterior con- tains all three fiber types. Further study, on an individual muscle fiber level, now appears necessary to specifically test the above explanation. Another explanation could lie in the difference in functional roles between the soleus, an ankle plantar flexor, and the tibialis anterior, an ankle dorsiflexor. Possibly both explanations are involved in some integrated manner. Succinic Dehydrogenase Sin-1 Per Cent Light Absorption Soleus. Adjusted cell means are presented in Table C-3, Appendix C, and the cell means are shown ianigure 5, page 41. These data. reflect a general increase in all groups in this measure of SDH staining intensity. Overall ANOVA results are summarized in Table 13. Table 13 shows that duration of treatment and the covariate, body weight at sacrifice, significantly affected the soleus SDH measure. Complex Scheffé contrasts within treatments across durations are sum- marized in Table 14. The four running groups, VOL, SHT, MED, and LON, showed the duration effects indicated by the-overall ANOVA. Follow-up standard Scheffé analysis to identify the specific sig- nificant duration contrasts within the four running groups are presented in-Table 15. 40 3.4 88.5w mcoldwz 8:024 265$ .8 $50.2 8.4 32.8 wco ems. 320m toe 8:82 $.83 $8.: 8022 :00 3.334. ”v or. «083m 33.: 38.2 :8 02864 .m 9.1 s 3 3 o A m 3 o m A o m 1.. m m m m m m M m a M m w W N H W N I 3 1 W S 1 1N. 3 .I W 1 Suzhdmmb a 1.. m m m w m 9 a m m m w m m I.— nA m I. 1A , , , 1 oo - - - , 1 0.. m 2.. 3.. 5.. .2 8.. 1 cm N. 8.. 3.. 3... 1 co. m_ m 8.»: 3 2...: V L 00. 88: l Om. % V , w 1 1 IS'vol J cm W 1% 1 In A J Oh We: gaze. _ . — 3.: W 3. .1 om % 1 00. w m. 23 m 38. co. m. Nona P q 00. .32 L 9: x J om 1 1 1 Oh W § I _ _ v l .3. .8 W 006» 00.x. the. I. 00 q zno I. CO- V sau- \MI 1 oo. 1 on. .( 1 J 00 J on 1 om o .5. 1 oo. o 00.0. 1 oo. 1 on. 41 53384 22.. E8 to 8:834 22.. 10m 50:25... 305:. 1.5 no» A9602 :80 com 18 maflom TEm cow Ago—2 mocoaom .805 882 :00 “.2364 up .2... 8.30m 38.: 83.2 =8 BEAR Na. .9“. S O O S A m m m m m m m m m m m I 3 1 a I EQEDE a m. m n m m m m 1 m 1 Jon Ow 11111111111111: 3% wmoau m §® laoHs M80 g- mum-.0. l - - 10w — —1ow N. b 00.: .d 3 H O 3 w. 1 2. .2 1 oh 1 m , , , - , 1 on - . 1 mm m m 1 0w t. 4 mm m w 3.: an». .3 8.3 3. 3.3 on... W 1 Ch 3.: 8 L Oh N nH - 1 on - - 1 mm A O m V 1 cm 1 mm ¢ W N 006. 1 on. .2 z... 1 9. 42 Table 13. Two-way ANOVA table for soleus sm‘l SDH per cent light absorption Summary of Analysis of Variance Source SS df MS F P Treatment 9.03 6 1.50 0.96 0.456 Duration 22.75 3 7.58 4.86 0.004 Training x Duration 13.75 18 0.76 0.49 0.955 Covariatel 7.25 1 7.25 4.65 0.034 ‘ Error 113.95 73 1.56 Total 166.73 101 18.65 1Body weight at sacrifice. Table 14. Summary of complex Scheffé contrasts within treatments across durations for soleus Sin'1 SDH per cent light absorption Dependent Variables CON VOL SHT MED LON ESC SWM Soleus Sin_l SDH per cent light N S S S S N N absorption N=not significant . s-significant at the .20 level. Significant increases in soleus SDH were observed.for.the VOL and SHT groups at 8 weeks, for the MED group at 12 weeks, and for the LON group at—4 and 12 weeks. Given the empirical observation that the VOL animals-tend to do their wheel running in short, quick bursts, the two groups with.the relatively high-intensity, low-duration activity (VOL and SHT) displayed increased SDH measures earlier than_the two groups 43 Table 15. Summary of standard Scheffé contrasts between duration cell means within the VOL, SHT, MED, and LON treatment groups for soleus Sin'“1 SDH per cent light absorption Dependent Duration Means Contrasted Variable Treat. 0-4 0-8 0-12 4-8 4-12 8-12 VOL N + N N N N Sin-1 soleus SDH per cent SHT N + N N N N light absorption MED N N + N N N LON + N + N N N N-not significant. -=significant decrease with increasing duration at .20 level. +-significant increase with increasing duration at .20 level. with relatively lower intensity and longer duration activity (MED and LON). The significant 0-4 wk. LON result is an exception to the above pattern. However, considerably more "over-running", i.e., PER values in excess of 100, occurred in these animals than in their SHT and MED counterparts during the first four weeks of training (Figures B-l through B-3, Appendix B). Taylor (103) analyzed SDH measures of the right soleus of 98 of the 102 animals of the present study. Using subjective ratings of NET stained sections, he also found a significant increase in staining intensity of 12~wk. LON animals but not in the other running groups. Assuming these observed differences in soleus SDH to be a general adaptation to physical activity in that muscle, the findings would indicate that the relatively short-duration, high-intensity activity precipitates a more rapid adaptation than activity of longer duration and lower intensity. 44 Tibialis anterior. Means for Sin.1 tibialis anterior SDH per cent absorption are presented in Table C-4, Appendix C, and the cell means are shown in Figure 6, page 41. The means of all treatment groups are increased from 0 to 12 weeks. Table 16 contains the results of the overall ANOVA. Table 16. Two-way ANOVA table for Sin-1 light absorption tibialis anterior SDH per cent Summary of Analysis of Variance Source 88 df MS F P Treatment 36.40 6 6.07 1.79 0.112 Duration 205.96 3 68.65 20.31 0.005 Training x Duration 62.11 18 3.45 1.02 0.448 Covariatel 2.41 1 2.41 0.71 0.401 Error 246.76 73 3.38 Total 553.64 101 83.96 1 Body weight at sacrifice. A significant duration effect was observed in the tibialis anterior SDH measure. The nearly significant (P-.11) treatment effect upon the tibialis anterior SDH measure would appear to merit further scrutiny, especially in light.of the power value (Power=.23). subsequent complex Scheffé contrasts did not identify any signifi- cant treatment categories to explain the nearly significant treatment 1 tibialis effect. However, a relatively large increase in the mean Sin- anterior SDH measure for the LON group was observed (Figure 6, page 41). Complex Scheffe contrasts within treatments across durations are sumr marized in Table 17. 45 Table 17. Summary of complex Scheffé contrasts within treatments across durations for tibialis anterior Sin’1 SDH per cent light absorption Dependent Variable CON VOL SHT MED LON ESC SWM Tibialis anterior Sin-1 SDH per cent 8 s s s s s 8 light absorption S=significant at the .20 level. Table 17 shows that duration effects were present in all treatment groups. Standard Scheffé contrasts on the treatment groups are presented in Table 18, which shows that the duration effects had occurred in all groups by four weeks and persisted until twelve weeks. Table 18. Summary of standard Scheffé contrasts between duration cell means within all treatment groups for tibialis anterior Sin"1 SDH per cent light absorption Dependent. Duration Means Contrasted Variable, Treat. 0-4 0-8 0-12 4-8 4-12 8-12 CON + + + N N N Sin’l tibialis. VOL + + + N N N anterior SDH per cent light SHT + + + N N N ab3°rpt1°n MED + + + N N N LON + + + N N N ESC + + + N N i. N SWM + + + N N N N-not significant. -significant decrease with increasing duration at .20 level. +-significant increase with increasing duration at .20 level. 46 The-lack of significant differences within duration across treat- ments and the lack of any significant interaction indicate that the above duratiOn effects were uniform for all treatments and treatment- duration combinations. This most likely represents an age effect which was not anticipated. The reader is reminded that the overall ANOVA‘ showed a nearly significant treatment effect on tibialis anterior SDH with relatively.low power. Discussion Of the two muscles investigated in this study, the soleus displayed more specific patterns of adaptation to the seven chronic physical activity levels than did the tibialis anterior.. The three forced exercise treatments (SHT, MED, and LON) and volun- tary activity (VOL) activity produced the most marked effects upon 1 soleus MEP—ChE and SDH. It appears that running exercise causes an increase in MEP-ChE surface area and that high-intensity running, of the type in the SHT program, accelerates the rate at which this occurs. The exact physiological significance of this finding needs further study. The possibility that the larger endrplate structure is signaling a shift in the soleus from its normal red-intermediate profile to.one with more intermediate fibers or even a white-intermediate profile, appears to be a potentially fruitful area for further study. The failure of the SHT group to maintain an enlarged MEP-ChE measure when the pro- gram intensity was plateaued appears to warrant further study of MEP- ChE in high-intensity programs of sustained progression and the tracing of MEP-ChE during detraining. Soleus SDH was also increased by running exercise and this-increase was also accelerated by high-intensity running. The increase in SDH 47, with chronic exercise is in agreement with most previous studies (13, 29, 56, 66, 100, 107). This increase in SDH is compatible with the explanation given earlier which implied a shift in the soleus from.its usual red-intermediate fiber population to one with increased inter— mediate fibers. The mechanism by which high intensity exercise speeds this adaptation process remains to be elucidated. Perhaps the most striking finding in the tibialis anterior is that its response was dissimilar to that of soleus. In contrast to the soleus, a trend toward decreasing MEP-ChE size was observed in the tibialis anterior of all groups. Of particular interest was the def- inite decrease in the MEP-ChE surface area in the tibialis anterior of the VOL animals while the soleus MEP-ChE of the same animals was significantly increased at 12 weeks. This implies that the inherent fiber population differences between the soleus and tibialis anterior are related to the divergence of exercise effects. Indeed, the shifts in end—plate structure implied for soleus would-not appear to be as etiologically feasible in the tibialis anterior, a muscle which normally has a large population of intermediate and white fibers. Increases in tibialis anterior SDH were present in all groups and this effect appears to be primarily related to age. However, the presence of nearly significant training results despite relatively low power appears to warrant further study on animals in age ranges other than those used in the present study. CHAPTER V SUMMARY,_CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of this study was to determine the effects of seven different chronic activity levels upon SDH and MEP-ChE measures in the left soleus and tibialis anterior of adult male albino rats. One hundred two animals were randomly assigned into CON, VOL, SHT, MED, LON, ESC, and SWM groups. Within each group sacrifices occurred before the onset of training and 4, 8 and 12 weeks later. Tissues for histochemical analyses were obtained at 14 biweekly sacrifices. Left soleus and tibialis anterior muscles were surgically removed from sodium pentobarbital anesthetized animals. Upon removal, muscles were immediately frozen until histochemical processing. Central portions of the muscles were softened in Ringer's solu- tion for 24 hours during which the tissue was teased apart. After softening, teased tissues were double-stained with the NET method for SDH and the ATC method of MEP-ChE. SDH staining intensity was photo- metrically measured as per cent light absorption. MEP-ChE surface areas were determined by tracings of micrOprojector images and measured by polar planimetry. MEP-ChE surface area significantly increased in the soleus of SHT and VOL animals, but showed a marked decrease in tibialis anterior of VOL and ESC animals. A significant interaction between the running 91W- 48 49 exercise programs and duration of treatment was observed for MEP-ChE surface area in the soleus. All significant changes in SDH Sinfl per cent light absorption were increases, regardless of the muscle. In the soleus, significance was found only in the running groups: VOL, SHT, MED, and LON. The increases generally appeared earlier in those animals doing relatively higher intensity activity than in those animals running at lower intensi- ties. In tibialis anterior, no such pattern emerged. The observed increases in SDH in all groups at 4 weeks of treatment appeared to be an age effect. The relatively large increase in tibialis anterior SDH in the lZdweek LON group, although not significant, appeared to warrant further investigation. Conclusions The results of this study have led to the following conclusions: 1. At least one aspect of the motor neuron's terminal muscle connection, MEP-ChE surface area, is affected by chronic physical activity. 2. Changes in the MEP-ChE surface area of a skeletal muscle appear to be related to the fiber pepulation of the muscle and the particular type of activity to which that muscle is subjected. In_ the present study, the most striking changes appeared in a red- intermediate muscle subjected to relatively high-intensity activity. 3. 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The histochemical localization of acetylcholinesterase in the fine structure of neuromuscular junctions of mouse and human intercostal muscle. J. Histochem. Cytochem. 9:317, 1961. Zalewski, A. A. Effects of reinnervation on denervated skeletal muscle by axons of motor, sensory, and sympathetic neurons. Amer. J. Physiol. 219:1675, 1970. APPENDI CES APPENDIX A TRAINING PROGRAMS 60 61 Table A-1. Standard eight-week, short-duration, high-intensity endurance training program for postpubertal and adult male rats in controlled-running wheels. Total Total Acc- Repe- TIms Time Exp. Total eler- Work tI- Bet- Run of Revo- Work Day Day atIon Time Rest tIons No. ween Speed Prog. Iu- Time of of TIme (mIn: TIme per of Bouts Shock (ft/ (mIn: tIons (sec) Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TNT =T -2 3.0 40:00 IO I I 5.0 0.0 I.5 40:00 --- --- 5=F -I 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- --- I=M I 3.0 00:IO IO 40 3 5.0 I.2 I.5 49:30 450 I200 2=T 2 3.0 00:I0 IO 40 3 5.0 I.2 I.5 49:30 450 I200 38W 3 3.0 OO:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200 4-T 4 2.5 OO:I0 IO 40 3 5.0 I.2 2.0 49:30 600 I200 53F 5 2.0 00:I0 I0 40 3 5.0 I.2 2.0 49:30 600 I200 Ian 6 I.5 00:IO I0 28 4 5.0 I.2 2.5 5|:40 700 II20 2=T 7 I.5 00:IO I5 27 4 5.0 I.2 3.0 59:00 8I0 I080 38H 8 I.5 00:I0 I5 27 4 5.0 I.2 3.0 59:00 8I0 I080 4=T 9 I.5 00:I0 IS 27 4 5.0 I.2 3.0 59:00 BIO I080 5=F I0 I.5 00:I0 I5 27 4 5.0 I.2 3.0 59:00 8I0 I080 I=M II I.5 OO:I0 I5 27 4 5.0 I.2 3.0 59:00 BIO I080 2=T I2 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920 3th I3 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920 4-T I4 I.5 OO:I0 20 23 4 5.0 I.2 3.5 59:40 805 920 5=F l5 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920 I-M I6 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920 2=T I7 I.5 00:I0 25 20 4 5.0 I.0 4.0 60:00 800 800 38W I8 I.5 00:I0 25 20 4 5.0 I.0 4.0 60:00 800 800 4=T I9 I.5 00:IO 25 20 4 5.0 I.0 4.0 60:00 800 800 5=F 20 I.5 OO:I0 25 20 4 5.0 I.0 4.0 60:00 800 800 I-M 2| I.5 OO:I0 25 20 4 5.0 I.0 4.0 60:00 800 800 2=T 22 I.5 00:IO 30 I6 4 5.0 I.0 4.5 55:40 720 640 3-H 23 I.5 OO:I0 30 I6 4 5.0 I.0 4.5 55:40 720 640 43T 24 I.5 00:I0 30 I6 4 5.0 I.0 4.5 55:40 720 640 5=F 25 I.5 00:I0 30 I6 4 5.0 I.0 4.5 55:40 720 640 I=M 26 I.5 00:I0 30 I6 4 5.0 I.0 4.5 55:40 720 640 2=T 27 2.0 00:I0 35 IO 5 5.0 I.0 5.0 54:35 625 500 38W 28 2.0 00:I0 35 I0 5 5.0 I.0 5.0 54:35 625 500 4-T 29 2.0 OO:I0 35 IO 5 5.0 I.0 5.0 54:34 625 500 52F 30 2.0 00:IO 35 I0 5 5.0 I.O 5.0 54:35 625 500 Is" 3I 2.0 00:I0 35 IO 5 5.0 I.0 5.0 54:35 625 500 2=T 32 2.0 00:I0 35 7 8 2.5 I.0 5.0 54:50 700 560 33H 33 2.0 00:I0 35 7 8 2.5 I.O 5.0 54:50 700 560 4-T 34 2.0 00:I0 35 7 8 2.5 I.0 5.0 54:50 700 560 5-F 35 2.0 00:I0 35 7 8 2.5 I.0 5.0 54:50 700 560 II" 36 2.0 00:I0 35 7 8 2.5 I.O 5.0 54:50 700 560 2=T 37 2.0 00:I0 40 6 8 2.5 I.O 5.5 52.IO 660 480 3=N 38 2.0 OO:I0 4O 6 8 2.5 I.0 5.5 52 I0 660 480 4=T 39 2.0 00:IO 40 6 8 2.5 I.O 5.5 52 I0 660 480 58F 40 2.0 00:I0 40 6 8 2.5 I.0 5.5 52 I0 660 480 This standard program was deslgned uslng male rats of the Sprague-Dawlev Strain. AII anImaIs were between 70 and I70 days-of-age at the begInnIng of the program. The duration and IntensIty of the program were established so that 75 per cent of all such anImaIs should have PSF and PER scores of 75 or hlgher durIng the fInaI two weeks. AIteratIons In the rest time, repetItIons per bout, number of bouts, or time between bouts can be used to affect changes In these values. Other straIns or ages of anImaIs could be expected to respond differentIy to the program. All animals should be exposed to a mInImum of one week of voluntary running In a wheel prIor to the start of the trainIng program. Failure to provIde thIs adjust- ment perIod wIII Impose a double IearnIng situatIon on the animals and wIII serlouslv ImpaIr the effectiveness of the traInIng program. 62 Table A-2. Standard eightdweek, medium-duration, moderate intensity endurance training program for postpubertal and adult male rate in controlled-running wheels. Total Total Acc- Repe- Time Time Exp. Total eIer- Work ti- Bet- Run of Revo- Work Day Day atIon Time Rest tions No. ween Speed Prog. Iu- Time of of Time (min: Time per of Bouts Shock (ft/ (min: tions (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER THT 0 4=T -2 3.0 40:00 I0 I I 5.0 0.0 i.5 40:00 --- --- 58F -l 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- --- I IHM I 3.0 00:i0 i0 40 3 5.0 i.2 i.5 49:30 450 I200 2=T 3.0 OO:IO l0 4O 3 5.0 i.2 i.5 49:30 450 l200 3=W 3 3.0 OO:IO IO 40 3 5.0 i.2 i.5 49:30 450 I200 4=T 4 2.5 OO:I5 i5 I9 4 5.0 I.2 2.0 52:00 570 il40 58F 5 2.5 00:i5 I5 I9 4 5.0 i.2 2.0 52:00 570 iI4O 2 I-M 6 2.0 OO:I5 I5 i9 4 5.0 I.2 2.0 52:00 570 iI40 2=T 7 2.0 00:i5 i5 I9 4 5.0 I.2 2.5 52:00 7I2 II40 38W 8 I.5 00:i5 I5 I9 4 5.0 i.2 2.5 52:00 7l2 II40 4-T 9 I.5 OO:I5 I5 I9 4 5.0 l.2 2.5 52:00 7i2 iI4O 5-F l0 i.5 OO:IS l5 i9 4 5.0 i.2 2.5 52:00 7I2 II40 3 II" ii I.5 00:i5 I5 I9 4 5.0 I.2 2.5 52:00 7l2 ii40 2=T I2 I.5 OO:I5 I5 I8 4 5.0 I.2 3.0 50:00 8|O I080 33W l3 i.5 00:i5 I5 I8 4 5.0 i.2 3.0 50:00 8I0 I080 4-T I4 I.5 00:l5 i5 I8 4 5.0 I.2 3.0 50:00 8I0 I080 58F I5 i.5 OO:I5 I5 l8 4 5.0 I.2 3.0 50:00 8l0 I080 4 i-M l6 i.5 OO:I5 I5 I8 4 5.0 I.2 3.0 50:00 8I0 l080 2=T I7 I.5 OO:I5 i5 I8 4 5.0 i.0 3.5 50:00 945 I080 38W l8 I.5 00:i5 I5 I8 4 5.0 I.0 3.5 50:00 945 I080 4-T l9 I.5 OO:I5 i5 I8 4 5.0' l.O 3.5 50:00 945 i080 5-F 20 i.5 OO:I5 I5 i8 4 5.0 i.0 3.5 50:00 945 I080 5 III 2i i.5 OO:i5 i5 I8 4 5.0 i.0 3.5 50:00 945 i080 2=T 22 i.5 OO:I5 I5 i4 5 5.0 l.0 4.0 53:45 I050 i050 38W 23 I.5 OO:I5 I5 I4 5 5.0 I.0 4.0 53:45 i050 i050 4=T 24 i.5 OO:I5 I5 I4 5 5.0 l.0 4.0 53:45 l050 I050 58F 25 i.5 00:i5 I5 i4 5 5.0 l.0 4.0 53:45 l050 i050 6 ll" 26 I.5 OO:I5 I5 I4 5 5.0 i.0 4.0 53:45 I050 i050 2=T 27 I.5 00:20 20 II 5 5.0 i.0 4.0 55:00 IIOO iiOO 3-W 28 i.5 00:20 20 II 5 5.0 I.O 4.0 55:00 IIOO iIOO 4-T 29 i.5 00:20 20 Ii 5 5.0 l.0 4.0 55:00 IIOO iIOO 5-F 30 i.5 00:20 20 II 5 5.0 l.0 4.0 55:00 IIOO IIOO 7 II" 3i I.5 00:20 20 II 5 5.0 l.0 4.0 55:00 IIOO iiOO 2=T 32 I.5 00:25 25 9 5 5.0 l.0 4.0 55:25 II25 Ii25 38W 33 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 II25 il25 4-T 34 i.5 00:25 25 9 5 5.0 i.0 4.0 55:25 il25 ll25 5-F 35 I.5 00:25 25 9 5 5.0 l.0 4.0 55:25 ii25 ii25 8 l8“ 36 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 II25 II25 2=T 37 i.5 00:30 30 8 5 5.0 i.0 4.0 57:30 i200 i200 33W 38 I.5 00:30 30 8 5 5.0 i.0 4.0 57:30 i200 i200 4-T 39 i.5 00:30 30 8 5 5.0 l.0 4.0 57:30 I200 I200 5-F 4O i.5 00:30 30 8 5 5.0 i.0 4.0 57:30 l200 I200 This standard program was designed using male rats of the Sprague-Dawiey stra All animals were between 70 and I70 days-of-age at the beginning of the program. The duration and Intensity of the program were established so that 75 per cent of all such animals should have PSF and PER scores of 75 or higher during the final two weeks. Alterations In the work time, rest time, repetitions per bout, number of bouts, or time between bouts can be used to affect changes In these values. Other strains or ages of animals could be expected to respond differently to the program. All animals should be exposed to a minimum of one week of voluntary running In a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation on the animals and will seriously Impalr the effectiveness of the training program. in. 63 Table A-3. Standard eight-week, long-duration, low intensity endurance training program for poatpubertal and adult male rate 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. Iu- Time of of Time (min: Time per of Bouts Shock (ft/ (min: tions (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TVT 0 4=T -2 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- --- 58F -I 3.0 40:00 lO I I 5.0 0.0 I.5 40:00 --- --- I I-M i 3.0 OO:IO IO 40 3 5.0 I.2 I.5 49:30 450 I200 2=T 2 3.0 OO:IO IO 40 3 5.0 l.2 I.5 49:30 450 I200 r 33W 3 3.0 OO:IO IO 40 3 5.0 I.2 I.5 49:30 450 I200 i 4-T 4 2.5 00:20 IO 30 2 5.0 I.2 I.5 34:40 450 i200 5=F 5 2.5 00:30 I5 20 2 5.0 I.2 I.5 34:30 450 i200 2 II" 6 2.0 00:40 20 I5 2 5.0 I.2 2.0 34:20 600 I200 2=T 7 2.0 00:50 25 i2 2 5.0 I.2 2.0 34:IO 600 i200 3=W 8 I.5 OI:OO 30 IO 2 5.0 I.2 2.0 34:00 600 i200 4=T 9 I.5 02:30 60 4 2 5.0 I.2 2.0 3I:OO 600 I200 . 58F l0 I.O 02:30 60 4 2 5.0 I.2 2.0 3I:OO 600 I200 ' 3 i-M ll I.0 02:30 60 4 2 5.0 I.2 2.0 3I:OO 600 I200 2=T I2 I.O 05:00 0 I 5 2.5 I.2 2.0 35:00 750 i500 3=W i3 I.O 05:00 O I 5 2.5 l.2 2.0 35:00 750 I500 4=T I4 l.O 05:00 0 I 5 2.5 l.2 2.0 35:00 750 I500 51F l5 I.O 05:00 O I 5 2.5 I.2 2.0 35:00 750 I500 4 l-M I6 i.0 05:00 O I 5 2.5 I.2 2.0 35:00 750 I500 2=T I7 I.O 07:30 0 I 4 2.5 I.0 2.0 37:30 900 I800 3-W l8 I.O 07:30 O I 4 2.5 I.O 2.0 37:30 900 I800 4-T I9 I.O 07:30 O I 4 2.5 I.O 2.0 37:30 900 i800 58F 20 l.0 07:30 0 I 4 2.5 I.O 2.0 37:30 900 I800 5 It" 2i l.O 07:30 0 i 4 2.5 I.O 2.0 37:30 900 I800 2=T 22 i.O 07:30 O I 5 2.5 I.O 2.0 47:30 II25 2250 36W 23 l.O 07:30 0 I 5 2.5 I.0 2.0 47:30 ll25 2250 4-T 24 i.0 07:30 0 I 5 2.5 I.O 2.0 47:30 II25 2250 5=F 25 I.0 07:30 O I 5 2.5 I.O 2.0 47:30 ll25 2250 6 lsM 26 I.O 07:30 0 I 5 2.5 I.0 2.0 47:30 Il25 2250 2=T 27 i.O IO:OO 0 I 4 2.5 l.0 2.0 47:30 l200 2400 3=W 28 I.O IO:OO O I 4 2.5 I.O 2.0 47:30 I200 2400 4=T 29 I.O IO:00 O I 4 2.5 I.0 2.0 47:30 I200 2400 5=F 3O i.0 l0:00 O I 4 2.5 I.O 2.0 47:30 I200 2400 7 I=M 3i I.O IO:OO O I 4 2.5 I.O 2.0 47:30 i200 2400 2=T 32 l.0 IO:OO O I 5 2.5 I.0 2.0 60:00 i500 3000 38W 33 I.O I0:OO O I 5 2.5 I.O 2.0 60:00 I500 3000 4=T 34 I.O I0:OO 0 I 5 2.5 I.O 2.0 60:00 l500 3000 52F 35 I.O l0:00 O I 5 2.5 I.0 2.0 60:00 I500 3000 8 I-M 36 i.O IO:OO O I 5 2.5 I.O 2.0 60:00 i500 3000 2=T 37 I.O I2:3O O I 4 2.5 i.0 2.0 57:30 I500 3000 38W 38 I.O I2:3O 0 l 4 2.5 I.0 2.0 57:30 l500 3000 4=T 39 l.0 l2:30 O I 4 2.5 I.0 2.0 57:30 l500 3000 5=F 40 I.O I2:3O O I 4 2.5 l.0 2.0 57:30 I500 3000 This standard program was designed using male rats of the Sprague-Oewiey strain. All animals were between 70 and l7O days-of—age at the beginning of the program. The duration and intensity of the program were established so that 75 per cent of all such animals should have PSF and PER scores of 75 or higher during the final two weeks. Alterations in the work time, number of bouts, or time between bouts can be used to affect changes In these values. Other strains or ages of animals could be expected to respond differently to the program. All animals should be exposed to a minimum of one week of voluntary running In a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation on the animals and will seriously impair the effectiveness of the training programs. 64 TABLE A-4.-- Standard eight-week, endurance, swimming training program for postpubertal and adult male rats. Expected Per Swim Day Day Cent Time of of Tail (min) Wk. Wk Tr. Weight EST i |=M I O 30 2=T 2 O 40 3=W 3 C' 50 4=T 4 C 60 5=F 5 C 60 2 I=M 6 2 40 2=T 7 2 40 3=W 8 2 4O 4=T 9 2 45 5=F l0 2 50 3 I=M II 3 3O 2=T i2 3 3O 3=W I3 3 3O 4=T I4 3 35 5=F I5 3 35 4 I=H l6 3 35 2=T I7 3 4O 3=W I8 3 40 4=T I9 3 4O 5=F 20 3 4O 5 I=M 2| 3 40 2=T 22 3 45 3=W 23 3 45 4=T 24 3 45 5=F 25 3 45 6 I=M 26 3 45 2=T 27 3 50 3=W 28 3 50 4=T 29 3 50 5=" 30 3 50 7 I=M 3| 3 5O 2=T 32 3 55 3=W 33 3 55 4=T 34 3 55 5=F 35 3 55 8 I=M 36 3 55 2=T 37 3 6O 3=W 38 3 60 4=T 39 3 6O 5=F 4O 3 6O 'C = clothes pin only. This standard program was designed using male rats of the Sprague-Oawley strain. All animals were between 70 and 90 days-of-aqe at the beginning of the program. The duration and intensity of the program were established so that 75 per cent of all such animals should have PET scores of 75 or higher during the final two weeks. Alterations in the per cent tail weight or expected swim time can be used to affect changes in these values. 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Adjusted cell means1 for soleus MEP-ChE surface area (sq. micra) Duration Treatment O-wk. 4~wk. 8-wk. lZ-wk. CON 101.420 93.884 85.944 97.660 VOL 83.150 84.430 85.044 97.660 SHT 78.115 86.272 130.674 113.595 MED 101.436 102.483 100.461 125.686 LON ~89.857 107.030 105.200 119.762 ESC 80.043 87.619 96.292 97.598 SWM 94.621 95.479 99.624 101.294 1 Least squares means. Table C-Z. Adjusted cell means for tibialis anterior MEP-ChE surface area (sq. micra) Duration , Treatment Odwk. 4dwk. 8dwk. lZ-wk. CON 89.398 82.967 74.520 81.203 VOL 97.684 74.904 64.987 68.054 SHT 90.273 77.766 84.087 75.472 MED 87.830 74.497 72.199 79.786 LON 90.823 74.091 80.992 77.543 ESC 98.265 87.345 93.621 78.658 SWM 96.288 77.155 75.423 87.020 1 Least squares means. 74 Table C-3.‘ Adjusted cell means1 for soleus Sin-1 SDH per cent absorption Duration Treatment O-wk. 4de. 8de. 12-wk. CON 68.224 69.017 68.172 68.469 VOL 68.120 70.136 69.991 69.432 SHT 67.194 68.751 69.248 69.263 MED 67.051 70.236 69.685 70.240 LON 67.693 70.005 68.629 69.614 ESC 68.724 69.790 69.273 69.361 SWM 67.476 69.682 69.110 69.195 ““7 1Least squares means. Table C-4. Adjusted cell means1 for tibialis anterior Sin-1 SDH per cent absorption Duration Treatment O-wk. 4dwk. 8-wk. 12~wk. CON 58.420 65.489 65.434 65.088 VOL 59.975 65.091 65.582 64.440 SHT 58.000 63.676 64.563 64.069 MED 62.325 65.450 64.320 65.093 LON 56.836 63.827 63.847 66.116 ESC 58.045 65.551 64.904 64.428 SWM 56.901~ 63.756 64.728 64.784 1Least squares means. "liiiiiii'iil’liiii