THE EFFECTS OF EXERCISE UPON RAT BRAIN CATECHOLAMINES Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY BARRY 8. BROWN 1970 .L I B R A R Y 3.: Michigan State University kAfih . This is to certify that the thesis entitled THE EFFECTS OF EXERCISE UPON RAT BRAIN CATECHOLAMINES presented by Barry S. Brown has been accepted towards fulfillment of the requirements for Ph.D. degree in Physical Education Major professor Date 7/ 29/ 70 0-169 v amnma av Ti" : IIIIMI & SIIII ' V 800K “’L'UERY INC. we”. av “:95 ABSTRACT THE EFFECTS OF EXERCISE UPON RAT BRAIN CATECHOLAMINES BY Barry S. Brown Brain catecholamine concentrations and depletion rates were investigated in male albino rats to observe the alterations accompanying a specific "middle-distance interval-training program" of eight-weeks duration. This program was intended to simulate the type of training used by man for the middle distance running events (i.e., 880- yard or one-mile run). Eighty adult, male, Sprague-Dawley rats were randomly divided into sedentary and exercise treatment groups. An electronically controlled, self-propelled, running wheel was utilized to train the exercise group in a progressive middle-distance interval regimen one hour per day five days per week. Two days following completion of the training program, the sedentary and exercise groups were each randomly divided into three subgroups and subjected to final treatment pro- tocol as follows: Barry S. Brown Final Treatment Subgroups E = Trained rat run through "normal" exercise routine Ewh = Trained rat placed in exercise wheel secured to prevent rotation Esed = Trained rat placed in sedentary cage SCh = Sedentary rat placed in "cheerleader" cage (rat receives same amount of shock as runner, E, but cannot escape) Swh = Sedentary rat placed in exercise wheel secured to prevent rotation Ssed = Sedentary rat placed in sedentary cage One and one-half hours prior to the final treatment (and 2 1/2 hours before sacrifice), each of the subgroups was then randomly divided into two equal parts. One-half of the animals in each group were injected with distilled water and the other half with alpha-methyltyrosine. The latter drug prevents synthesis of catecholamines by com— petitively inhibiting tyrosine hydroxylase. Animals were sacrificed by decapitation and their brains were removed and quick frozen in chilled isopentane. Catecholamines were extracted with perchloric acid, ad- sorbed onto alumina, eluted and analyzed using standard fluorometric procedures. The absolute weights of brain and heart of the seden- tary and exercise rats did not differ significantly. When expressed as per cent body weight these organs were sig- nificantly heavier in the trained rats. Barry S. Brown Motor activity, measured by total revolutions run in the exercise wheel during final treatment, was not signifi— cantly different between runners receiving distilled water and those injected with alpha-methyltyrosine. Higher brain norepinephrine concentrations (p < .05) were found in the trained compared to sedentary rats. Dopamine concentrations were not significantly different. The increased brain norepinephrine concentration found in the trained rats may have been caused by the continuous demand placed upon the sympathetic nervous system by the exercise. Exercise and/or shock among trained rats potentiated alpha-methyltyrosine induced depletion of brain norepi- nephrine. The lack of dopamine depletion among trained rats following exercise and alpha-methyltyrosine suggests con- servation of this amine in the performance of running exercise. THE EFFECTS OF EXERCISE UPON RAT BRAIN CATECHOLAMINES BY ,\ 2‘ Barry Sf Brown A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physical Education College of Education 1970 / on- .- f '3 ., k " -. l'éws :4; (I .~\ .. n g“? (a). " I; j Dedicated to my cherished wife, Gail, daughter, Sherry, forthcoming son, Mom and Dad ii ACKNOWLEDGMENTS A special word of appreciation is extended to those individuals most instrumental in the formation, constructive criticism and completion of this study. Dr. Wayne Van Huss, my major advisor, supplied the initial impetus, needed encouragement and continued dedication that served as the motivational drive toward the accomplishment of the task. The facilities for biochemical analyses of brain tissue were generously supplied by Dr. Kenneth Moore, in spite of the lack of available space. Dr. Moore provided corrective criticism when the need arose. Mrs. Mirdza Gramatins deserves special mention for her patience during the initial phases of the analysis. Voluntary assistance from Messrs. Abe Albenda and George Janes during sacrifice protocol involved many arduous hours, for which I am grateful. Without the assistance of these two gentlemen, results could not have been attained. Training facilities for the rats were provided by Dr. William Heusner, who found it necessary to schedule other projects around my time slot. iii Deepest appreciation is expressed to my wife Gail, whose patience in typing the original manuscript was limit- less, and whose encouragement during times of stress was met with scowls and abusive remarks by her husband. Final sentiment lie among the ashes of the most cooperative subjects any researcher could ever hope to assemble: rattus rattus. iv TABLE OF CONTENTS Page DEDICATION O I O 0 O O O O O 0 O O O 0 ii ACKNOWLEDGMENTS . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . Vii LIST OF FIGURES . . . . . . . . . . . . viii LIST OF ABBREVIATIONS . . . . . . . . . . ix Chapter I. INTRODUCTION . . . . . . . . . . 1 Statement of the Problem . . . . . 2 Rationale . . . . . . . . . . 3 Limitations of the Study . . . . . 3 II. REVIEW OF THE LITERATURE . . . . . . 5 Exercise vs Trained Condition . . . . 5 Effects of Stress Upon the Brain . . . 7 Brain Weight and Stress . . . . . 7 Brain CA Concentration and Stress . . . . . . . . . . 8 Regulation of Brain CA Concen- tration During Stress . . . . . 10 Brain CA Synthesis and Stress . . . 11 Control of CA Synthesis . . . . . 11 "Functional vs Bound" Storage of CA . . 12 Interaction of Alpha-MT with Drugs and Stress . . . . . . . . . . 13 Chronic Effects of Exercise Upon CA . . 16 III. EXPERIMENTAL PROCEDURE . . . . . . . 17 Overview of the Experiment Design . . l7 Receipt and Assignment of Animals . . 19 V IV. V. Routine Animal Care Procedures . . Training Regimen . . . . . Final Treatment Protocol . . . . Catecholamine Analysis . . . . Statistical Analysis . . . . . RESULTS 0 O O I O O O O O 0 Effectiveness of the Exercise Regimen . . Behavioral Response to Alpha- -MT . Catecholamine Response to Alpha-MT Selected Group Comparisons of Brain CA . . . . . . . . . Depletion Comparisons Between Final Treatment Subgroups . . . . . Behavioral Response and Brain CA Concentration . . . . . . Relationship Between Brain CA and Brain Weight . . . . . . . . Discussion . . . . . . . . SUMMARY, CONCLUSIONS, RECOMMENDATIONS Conclusions . . . . . . . . Recommendations . . . . . . . LIST OF REFERENCES . . . . . . . . . APPENDIX vi Page 20 21 24 27 27 29 29 34 35 37 41 43 43 44 47 48 49 51 61 LIST OF TABLES Table Page I. Behavioral Response to Alpha- methyltyrosine Among Trained Rats . . . . 35 II. Factorial Analysis of Mean Brain Dopamine and Norepinephrine Levels (ug/g) : s.e. of Sedentary and Exercised Rats Following Final Treatment . . . . . . 36 III. Selected Group Comparisons of Brain CA- Orthogonal Classification . . . . . . 38 IV. Depletion Comparisons Between Final Treatment Subgroups . . . . . . . . 42 V. Final Treatment Correlations: Behavioral Response and Catecholamine Concentration in the Brain . . . . . . . . . . . 43 VI. Relationship Between Brain CA and Brain weight 0 O I O O O O O O O O O O 44 A-l. Brain Catecholamine Correlations . . . . . 64 A-2. Standard Eight-Week, Medium-Duration, Moderate—Intensity Endurance Training Program for PostPubertal and Adult Male Rats in Controlled-Running Wheels . . . . . . . . . . . . . 65 vii LIST OF FIGURES Figure Page 1. Experimental Design of Treatment Groups During the Eight—Week Training Pro- cedure and for the Final Treatment . . . 18 2. Average Per Cent Expected Revolutions (PER) and Total Revolutions Run (TRR) of Sixteen Rats from the First Week of Training Until Completion of the Program . . . . . . . . . . . . 31 3. Weekly Body Weights of Sedentary and Exercised Rats . . . . . . . . . 32 4. Weekly Weight Gain of Sedentary and Exercised Rats . . . . . . . . . 32 5. Mean Comparison of Absolute Heart and Brain Weight Expressed as Total Gram Weight and Relative Heart and Brain Weight Expressed as Percent Body Weight . . . . . . . . . . . . 33 6. Least Significant Difference Designed Comparison of Per Cent Depletion of Brain NE Levels . . . . . . . . . 39 7. Least Significant Difference Designed Comparison of Per Cent Depletion of Brain DM Levels . . . . . . . . . 4O viii LIST OF ABBREVIATIONS a-MT or alpha-MT alpha-methyltyrosine CA catecholamine(s) CAR conditioned avoidance response CDS cumulative duration of shock DM dopamine DM-B—Hydroxylase dopamine-beta-hydroxylase E epinephrine NE norepinephrine PER per cent expected revolutions TER total expected revolutions TRR total revolutions run ix CHAPTER I INTRODUCTION A large body of literature has accumulated over the years demonstrating changes in the cardiovascular, cardio- respiratory and muscular systems as a result of chronic exercise. Most of the studies, however, can be interpreted only in generalities because the exercise stress utilized has not been clearly defined. Little is known concerning either the differential effects of specific exercise regi- ments or the mechanisms underlying the anatomic and physio- logic changes which are produced by such regimens. An area of investigation of particular interest was the effects of exercise upon brain catecholamine (CA) con- centration. Single bouts of intense swimming had been found to yield brain norepinephrine (NE) levels below control values [67]. An increase in the synthesis rate of brain NE was observed in rats exercised on a treadmill [38]. In discussing the results of these investigations the authors expressed the belief that brain catecholamines are replaced during physical activity and may be involved in the central control of the autonomic nervous system. Arguments have been presented implicating NE and/or dopamine 1 (DM) as transmitters of neuronal networks that modulate: (a) central regulation of the sympathetic nervous system [12, 44], (b) states of alertness and motor activity [16], (c) emotional affective states [87], and (d) temperature regulation [4, 52]. On the basis of these arguments and partial evidence [38, 68] it is reasonable to postulate that brain catecholamines may be involved in the central response to a long-term exercise regimen. A dearth of evidence exists concerning both the differential effects of specific exercise regimens and the chronic effects of such regimens upon neurochemical corre- lates (CA in particular) in the brain. Since a need exists for quantitative evidence of this nature, it was decided to study the effects of a long-term, defined, exercise regimen upon brain CA levels. Statement of the Problem 1. To determine the changes in catecholamine content occurring in the brain of male albino rats following eight weeks of "middle-distance, interval-training," The training program was intended to simulate a middle distance (880-yard or one mile) running regimen. 2. To determine the relationship of various organ weight measures and performance criteria to brain catechol— amine concentrations of sedentary and exercised rats. Rationale Specific physical changes resulting from exercise programs may be influenced by or exert fine control over central nervous system components. Measurable components which might provide further insight into these mechanisms were judged to be the NE and DM levels of the brain. It was reasoned that, following the training program of eight weeks, if half of the animals in each of the groups were injected with a catecholamine synthesis inhibitor, alpha- methyltyrosine (a-MT, 93), and the other half with distilled water, under varying final treatment conditions (including exercise) the degree of catecholamine synthesis attributable to exercise could be estimated. The study was designed around this concept, including appropriate subgroups to provide for controls. Limitations of the Study 1. Whole brain catecholamine analysis may mask sig- nificant changes taking place in smaller areas of the brain (e.g., NE in the hypothalamus and BM in the extrapyramidal tract). 2. Validity of the electronically controlled running wheel in producing a physically trained animal is lacking. Adaptation of rats to shock may be contributing to the changes which are attributed to the chronic effects of exercise. 3. Temperature control was not instituted for the first two shipments of animals due to financial limitations. 4. The inability to maintain shock control animals alongside trained rats for the duration of the regimen pre- vented direct evaluation of the contribution of shock to the training effects produced by the exercise program. CHAPTER II REVIEW OF THE LITERATURE A review of literature encompassing the past twenty years has failed to uncover any studies undertaken to assess the effects of long—term exposure to exercise upon brain catecholamines. The effects of differential physical activity upon possible central neurotransmitter substances remains unexplored. The focus of the following literature review lies in the area of brain CA analyses that have been undertaken within the past twenty years to elucidate the role of central amines under conditions of stress. Exercise vs Trained Condition A popular misuse of the word exercise requires clari- fication, just as the nebulous term drug places burden upon the author to describe with greater precision the treatment employed. Exercise, per se, is neither specific nor infor- mative of the intensity or duration of the physical stress employed. Physical work can be performed in many ways under many conditions for varying lengths of time. A complete description of training procedures is necessary, if one wishes to apply the results of an exercise regimen to similar populations. Effects of swimming exercise may not be applicable to the same species (under identical environ- mental and dietary control) forced to run in a rotating drum. Neither can one generalize results after one bout of severe physical activity to the effects of exercise; rather, attention should be drawn to the acute effects of a spe- cifically defined activity upon previously sedentary, in— dividual (or multiple) housed animals. Swimming procedures have been utilized by a number of authors [4, 9, 30, 31, 40, 68] to demonstrate short-term effects of exercise upon brain CA. All investigators ne- glected to specify in their conclusions the application of their results to acute effects of swimming. Moreover, it has been shown that swimming as an exercise stressor cannot be controlled [21, 61]. Spontaneous motor activity has been determined with devices ranging from a lever-pressing apparatus [39] to actophotometers measuring movement in terms of recorded changes in foot position [63, 98] and to a variety of voluntary running wheels recording number of revolutions run per unit time [13, 23, 65, 78, 79, 82, 83]. The treadmill was used as a training device in four instances by authors investigating CA changes following exercise [10, 22, 42, 91], with only one author [22] giving full specifications of the training program. A more detailed description of the exercise protocol was presented by authors utilizing a motorized drum [2, 5, 42, 96]. How— ever, five sources failed to mention the procedure employed [29, 58, 59, 62, 71]. Treadmill exercise of rats, in general, is a poor technique as only about 50% will run without extensive training [41]. Failure to specify exercise procedures may stem from the authors' use of exercise as a vehicle to stimulate various areas of the central nervous system, especially the central mechanism controlling sympathetic function. It may be argued, chemically speaking, that all forms of stress (physical or otherwise) evoke the same central pattern of response. This viewpoint cannot be accepted as there was a marked disparity in the brain CA response to each of the activities outlined above. These differences are discussed in the sections which follow. Effects of Stress Upon the Brain Brain Weight and Stress The effects of exercise upon rat brain weight have been investigated following three to six months of volun- tary, running activity [25, 26, 43]. A small (4%) but consistent increase in relative brain weight was observed by these authors. The data were not statistically analyzed; therefore, results must be considered speculative. Swim- ming rats to exhaustion (15 to 30 minutes in 15°C water or 4—6 hours in 23°C water) had no effect on brain weight [4]. Reduction of the environmental temperature to 5-7°C for three to five months [66] and isolation or community housing [65] of female rats for 15-17 weeks also failed to alter brain weight significantly. Socially impoverished and environmentally enriched housing conditions had no effect upon total rat brain weight [51]. However, a break- down of the various areas of the brain showed that enriched rats possessed increased cortical and decreased subcortical weight. In addition to increased cortical weight, a series of cortical changes were observed among enriched rats in- cluding: (1) higher cortical ratio of cholinesterase to acetylcholinterase (Che:AChe), (2) larger capillary diameters, and (3) increased number of glial cells [88]. Brain CA Concentration and Stress The application of electric shock to the foot pads of rats has been used as an acute stress to lower brain CA stores [9, 47, 53, 60, 68, 77]. However, Thierry et_al. [95] did not observe any change in rat brain NE levels following three hours of intermittent shock at 0.8 ma; and Iwamoto and Sato [47] obtained an increase in brain NE after four hours of grid shock. The latter authors suggest the rise in brain NE was due to tension and apprehension pro- voked by recurrence of the shock rather than the actual pain. Depletion of brain NE due to immobilization observed by Bliss and Zwanziger [9] was not confirmed under similar restraint stress by other authors [14, 99, 101]. Indeed, Welch and Welch [99, 101] reported increased CA concen- tration in brains of mice made hyperexcitable by eight to twelve weeks of isolation. They suggested the higher levels of brain NE observed in isolated mice may have been due to an increased sensitivity of NE receptors as a result of slower NE release [100]. Sustained swimming for four hours in 23°C water [4, 68] and a survival swim in ice water [40] caused significant decreases in rat brain NE concentration. Both Hamburg [40] and Barchas and Freedman [4] stated that decreased NE levels correlated with behavioral depression following the swim, suggesting that brain NE is important in the behavioral response to stress. Decreased CA levels signify for the most part, increased utilization of CA during stress, such that the stressor applied evokes neural stimulation capable of releasing amine stores more quickly than they can be replenished. Sedentary rats exposed to treadmill exercise of one [38] and three [4] hours duration failed to show any change in brain NE concentration. Maintained levels of brain CA indicate that synthesis of new amine kept pace with the increased CA depletion resulting from the flow of neural impulse. 10 Regulation of Brain CA Concen- tratiOn During Stress The control of endogenous CA levels following stress must be mediated through a feedback mechanism designed to regulate metabolic degradation, physiological inactivation and/or synthesis of the transmitter substance. Intra- neuronal CA is normally metabolically inactivated by deami- nation [34, 48]. Destruction of endogenous CA stores fol- lowing‘stress may be prevented from altering MAO activity, thereby decreasing the normal amount of deaminated metabo- lites formed [33, 54, 60, 101, 102]. Catecholamines would be conserved by this mechanism, maintaining adequate amine stores to be utilized during stress. Active reuptake of physiologically released amine during stress may be necessary to maintain pre-stress levels of endogenous CA [8]. This view is supported by the obser- vations of increased uptake of labelled NE in the rat heart following repetitive stimulation of the cervical sympa- thetic trunk [17] and cold exposure [32]. Brain CA Synthesis and Stress The role that synthesis must play to supply newly formed intraneuronal amine following stress depends upon the degree to which nerve endings have been depleted of its CA stores. Synthesis plays a greater role than uptake in the maintenance of NE following nerve stimulation of the guinea pig hypogastric nerve-vas deferens preparation [1]. 11 Three hours of intermittent shock [11] and one hour of cold [36, 38] increased the turnover rate of labelled NE in the rat brain. Running exercise on a treadmill for one hour caused an increased synthesis rate of NE (0.06 ug NE/g brain/hour) in the rat brain [38]. This evidence supports the concept that the synthesis rate of rat brain CA in- creases during stress. Control of CA Synthesis The formation of CA normally proceeds in the following order: tyrosine————>dopa————9DM-——-9NE. The mechanism underlying control of CA synthesis was elucidated following the discovery of tyrosine hydroxylase [72, 97]. This enzyme was found to be the rate-limiting step in CA forma- tion, catalyzing the conversion of tyrosine to dopa. The presence of free (nonbound) CA is believed to act as its own end-product inhibitor by inactivating a tyrosine hy- droxylase cofactor, affecting tyrosine transport into the cell, or activating other endogenous inhibitors [1, 88]. The end result is to maintain the concentration of functional CA at a near constant level. Release of brain CA following stimulation of appropriate central neurons decreases the content of functional amine. End-product inhibition at tyrosine hydroxylase is, thereby, removed and conversion of tyrosine to dopa proceeds at a faster rate [34, 37]. However, actual levels of tyrosine hydroxylase do not increase following stimulation of the rat submaxillary 12 gland [88] and heart [37]. These authors suggested that NE decreases the rate of synthesis by combining with tyrosine hydroxylase, rather than by altering its levels. A compre— hensive summary of CA metabolism and control of amine synthesis can be found in a recent review [34]. "Functional vs Bound" Storage of CA Incomplete depletion of brain amine stores following total behavioral depression has prompted investigation of the presence of a small functionally active store of brain CA. A number of authors have used tyramine to deplete stores of NE in the rat heart. In heart tissue, there was 'believed to be a tyramine-resistant and a tyramine- releasable pool of NE [35, 49, 50, 80, 81]. This condition created a biphasic decline of non-tracer NE because of in— complete availability of tyramine. Constant infusion of tyramine reduced heart NE stores at a single exponential rate. If infusion is not sustained, tyramine became meta- bolically inactivated. Based upon information gathered from uptake studies of labelled NE [74, 92] and various CA releasing agents [18, 73], a model of intraneuronal NE storage was proposed. Two or more pools of NE were des- cribed; one pool is filled with nonbound "free" NE capable of being bound onto receptor sites at synaptic endings. This represents approximately 10% of the total NE stores. The other pool contains one or more nondiffusable complexes 13 of NE in granules [89, 94], requiring ATP and MG++ [34] for binding. Inhibition of CA synthesis showed that these pools are in ready equilibrium and during such inhibition amine stores decline at a single exponential rate [18, 73, 78]. The existence of a small functionally active store of brain CA has been used to account for the behavioral lag of synthesis inhibitors following prior depletion of CA stores [14, 28, 64]. Interaction of Alpha-MT with Drugs and Stress Alpha-MT has been used to deplete CA stores in various organs and to calculate synthesis rates following exposure to a variety of stressors. The procedure is based upon a single rate of decline of CA disappearance subsequent to drug application [20] and assumes: (l) "a-MT totally blocks tyrosine hydroxylase with a sustained effect, (2) a-MT should not release NE or DM, not affect its metabolism, and (3) no stores of dopa or DM must remain in the neuron for possible conversion to NE following a-MT." The advantage of calculating synthesis rate (based upon a-MT-induced CA decline) compared to measurement of steady-state values is readily evident because endogenous CA levels may not change subsequent to amine release if synthesis is not prevented from replenishing CA stores. Stress may not affect steady-state values but may increase the synthesis rate of brain CA. Measurement of steady-state 14 values only would erroneously indicate that the stress was ineffective in evoking a central adrenergic response. A comparison of techniques for determining CA synthesis rates shows no statistically significant differ- ence between use of a-MT and measurement of Specific activity following administration of labelled amines [45, 46]. The relative ease of the a-MT procedure, and limited budget facing many researchers, have prompted wide-spread adoption of the first method. Alpha-MT administered to rats placed in a cold room for several hours prevented body temperature regulation after a lapse of two hours and depleted brain NE and DM completely [19]. They concluded brain CA is essential to elicit shivering, which, in turn, is necessary to utilize energy substrates mobilized by the peripheral NE still present. Rats exposed to cold (4°C) adapted within seven days and resumed normal synthesis rate of brain NE (DM was not affected) as measured by steady-state kinetics following use of a-MT [90]. It appears that brain NE can be modified subsequent to chronic exposure to environmental stress. Rech §E_31. [85], Dominic and Moore [24], Moore and Rech [70, 71[ and Pirch gt_§l. [79] examined the effects of a-MT upon various forms of behavioral response (shuttle box for CAR; rotarod for muscle tone and coordination; loco- motor activity on a revolving drum to indicate an "uncon- ditioned behavioral response to a non-specific stimulus"). 15 In general, results equate a-MT depletion of brain CA levels with the onset and duration of behavioral depression. Rech et_al. [83] hastened the onset and enhanced the in- tensity of behavioral depression due to a-MT by pretreating animals with reserpine. This procedure exhausted reserve stores of amine and demonstrated that only a small, readily available pool of mediator is necessary for normal function. Measurements of motor activity determined in acto— photometric units by Weissman et_al. [98] indicated that behavioral effects of a-MT are correlated with the in yiyg inhibition of tyrosine hydroxylase, rather than levels of NE. This is in agreement with previous reviews that assume NE is released from a small "functional pool" accounting for the observed behavioral depression. Spector §E_§l. [93] produced mild sedation and im- paired motor activity in cats and guinea pigs by adminis- tration of a-MT. They are generally considered responsible for the discovery of the inhibiting effect of this compound upon tyrosine hydroxylase. The acute effects of running exercise (at l ft/sec) upon rat brain CA concentration were investigated by Gordon §E_gl, [38]. They discovered that one hour of running did not significantly lower NE stores, but enhanced the de- pletion of brain NE (DM was not affected) by administration of a-MT (200 mg/kg) one-half hour prior to exercise. This implies the rate of brain NE synthesis was increased during 16 exercise even though steady-state levels (ug/g) remained unchanged. Chronic Effects of Exercise Upon CA A void exists in the literature concerning the adap- tation of brain catecholamines to chronic exercise regimens. Three days of grid shock at 1 milliampere for one minute per day caused an increase in rat brain NE levels in the frontal pole and caudate nucleus four days following cessation of the treatment [76]. Nielson and Fleming observed, in addition, increased DM in the caudate nucleus after three days of cold stress (daily immersion for ninety seconds in 1-2°C ice water). Thierry gt_al. [95] increased rat brain NE by 22% after three days of intermittent shock at 0.8 milliamperes (followed by one day of rest before sacrifice). These "stress adapted" animals displayed increased NE turnover when subjected to shock immediately prior to sacrifice. In view of the training procedures utilized in the present study, the "chronic" effects of shock stress re- viewed above [76, 95] may have been applicable to the exercise regimen employed. CHAPTER III EXPERIMENTAL PROCEDURE The present study was undertaken to determine the chronic effects of a specific exercise regimen upon rat brain CA concentration. A secondary objective was to de- termine the contributory effects of shock, exercise and a-MT upon per cent CA depletion in the brain. Overview of the Experimental Design The design is illustrated in Figure 1 and first pre- sented briefly for perspective. The animals were randomly divided into sedentary and exercise groups. The exercise group was placed on a middle—distance interval-training program intended to simulate middle distance running (880- yard and one mile run) in man. At the end of the training program the sedentary and exercise groups were each randomly divided into three subgroups for the final treatment: sedentary, wheel secured, and exercise (shock control in the sedentary group). 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Ice 08L tom 32 400 q BODY WEIGHT (ems) O----O Sedentary Rate 380 0-—-0 Exercised Rats L I 1‘ I 1 j I I I I 2 3 4 5 6 7 8 WEEKS Figure 3: Weekly body weights of sedentary and exercised rats. 35- 30! h [:1 Sedentary Rats ,1 Exercised Rate “5’ 25:- 3 z a 20~ o .— I I5- ‘2 ‘4’ 2 l0- < m 2 54 IS? a §—I §—I I I ’ I ' I ’ ' l 2 3 4 5 6 7 8 «X- WEEKS Figure 4: Weekly weight gain of sedentary and exercised rats. Starred comparison is significantly different (p <.05). 33 (OOI X when Kpoqnqbgam uobm) IHSIBM NVSHO BAIIV'TBH .BOVS EooECQm mac mcoBEQEoo motolm .8me of Co .88 Ransom. E3958 8:: _oo_to> .2902 soon 2588 mo powwoaxo £963 :65 can too; 638.9 96 £063 E05 :22 mo oommmaxm Ego; :65 can too; 32030 no 587.3606 522 “m 830E Aha 535/ zme/ _ ES: 2.4mm 1 / _ W / _ + W +/ fl _ / 3.- _ V / _ fl 2: / _ / / _ / 2.... H / _ / _ _ 8L :3. :25.» D H _ 221 3.123 [HIV J. 4 r¢._ In; I@.. IN... I O.N (Stub) 1H9|3M wveao nvaw 34 trained animals is a typical training response. The sig- nificantly lighter body weight among trained animals also has been observed frequently. However, the fact that the difference in weight of the two groups was initiated in the first week of training only is puzzling. This pattern of group differences is different from previous data utilizing endurance swimming and merits further investigation. Behavioral Response to Alpha-MT The ability of a-MT to disrupt CAR and locomotor activity was determined in trained animals subjected to normal exercise routine at final treatment. Cumulative duration of shock (CDS) and TRR were used as criteria to compare exercise rats injected with distilled water (E) to exercise rats injected with a-MT (E ). Table I indicates a-MT that a-MT did not statistically affect TRR or CDS among trained rats. The time course of a-MT administration (1 1/2 hours prior to exercise) was chosen to prevent total sedation of trained animals subjected to the normal exercise routine. This factor coupled with the small sample size may have been instrumental in the lack of a statistically significant difference observed in the behavioral response (TRR and CD8) of physically trained rats to exercise, following injection of distilled water or a-MT (Table I). 35 TABLE I: Behavioral Response to Alpha-methyltyrosine Among Trained Rats. Numbers represent mean values : s.e. Average Cumu- Average Total Final Treatment n lative Duration Revolutions of Shock (seconds) Run (TRR) Exercise 7 489 i 62 648 i 73 Control (H20) Exercise 7 564 i 83 626 i 79 Alpha-MT Calculated t .7274 .2033 value: Catecholamine Response to Alpha-MT Assessment of the ability of a-MT to lower brain catecholamines was performed on each of the final treatment subgroups. A two-way replicated analysis of variance of brain DM and NE was performed using final treatment sub- groups as one variable and the injection media as the other (Table II). The statistical significance of drug treatment observed in Table II demonstrates the ability of a-MT to inhibit synthesis of brain CA stores. The results affirm the purpose for which the drug was employed even though the time course of a-MT activity within each animal was less than that required for maximal synthesis inhibition of brain CA. ommo poCHmqu comm Hoo£3 ooxfim pocflmnuu 33m mmHonoxo pocwmuui m ommo ammucopoml comm Homn3 poxflw mnmucmcomi 23m xoonm humucoooml com ucoEumoHB Assam coHqucoo mcficwmue pcomoqtmsouo #000. we vuno. mo uonum mmom. waoo. m Homo.a ammo. m cofluomuoucH moo.v a Hama.mv Hmmm. H ao.v a moam.¢ ommm. a mama moo.v m onwm.v homo. m ommm. omvo. m ucoEummnB Hagan 6 m>.a>m "m oHumm m mm: mp m>.a>m ”m oflumm m mm: up 3 i i mz cfimum so samum oousom Acmflmoo cmumofiamomv moccaum> mo mHmwamsm hmzrose hug mHG ORG mug mun hflfi mo. + mm. vo. + Hm. mo. + om. vo. + mm. vo. + om. mo. + mm. azimcmad 5mg 0mg “NC Nelda @"C Fug Hz mo. + mv. mo. + me. mo. H mm. mo. H vm. mo. H mm. mo. H Hm. Houmz poaawumflo King 0"“ Qua-H mug OHS F": mo. H «a. no. H om. ma. H mm. mo. H ma. mo. H mm. «a. H mm. Bzimnmad ea. + we. ma. + Nv. NH. + mv. ca. + em. mo. + am. so. + we. Houmz poaawumflo 60mm £3m m @Omm £3m 50m mama H Am\msv ucoEumoHB Hmcflm mcHBOHHom mumm comflouoxm can mumucobom mo .o.m mao>oq ocflunmocfimouoz can ocHEmmoo cflmum cmoz mo mHmwamc< HmHuouomm "HH mamme 37 Selected Group Comparisons of Brain CA Organizing the data into an orthogonal classification3 accomplished two purposes. It gave a composite view of the important comparisons in a single table, and secondly, allowed use of a more powerful statistical tool with which to evaluate the data. The comparisons presented in Table III indicate that the average concentration of brain NE among exercised rats was significantly higher than corre- sponding amine levels of sedentary rats (comparison 01). Prior injection of a-MT did not affect this relationship. The designed comparisons of per cent brain NE and DM depletion over the 2 1/2 hour period subsequent to a-MT injection are graphically presented in Figures 6 and 7, respectively. Ten out of forty-five possible comparisons were chosen for Least Significant Difference analysis in each of the bar graphs. The results give testimony to the CA depleting ability of a-MT upon brain NE concentrations under all final 3Although used with less frequency than undesigned comparisons, such as Duncan's, Tukey's and Scheffe's tests, the Single Degree of Freedom and Least Significant Differ- ence rely upon selection of the meaningful comparisons be- fore observing any results. The Single Degree of Freedom was used in this study to combine raw data of previously selected subgroups, thereby enlarging the sample size of each comparison and increasing the power of detecting a true difference. Least Significant Difference differs from the above mentioned undesigned analyses by eliminating a correction factor, which adjusts for the inflated Type I error incurred if one wishes to calculate all possible comparisons. 38 .emcnanmocs mum wages as» No seen man an mesam> =6. unmonuncmnm n .ocfla HmucoNHHon some numocon mmsoquSm ucwEumoHu Hmcfim mo modam> some ucomoumou Awo nmdounu HOV mGOmHHmmEoom on.a mmmm. mmom.H News. mseo. ”msam> u 3. cm. 3. R. om. mm. mm. am. "Asia use 593 mo.H mama. poem. sums. mess. ”usam> u as. me. me. we. mm. vs. me. am. "can ”so cnmnm mm.a mmhm. ~ms~.H mmam. Hemm.~ "msam> u mm. om. mm. mm. mm. mm. om. mm. “Ezra .m2 593 on.a Show. mamm.a mnba.a geoma.m .msHm> n me. we. as. mm. mm. am. as. am. "cmm .mz cnmnm Ho comm m> nsm pommc3m m> m pomm£3m m> com pomm£3mm m> pommnzmzom some e m I. H Am\msv “Om 0 0 mo 0 moHQMHHm> oswm> MAHOV mGOmHHmmEou accomocuno ocHEmHocomumo .GOHDMOHmemMHU accomocuuormu chum mo mcomwummeou msouw pouooaom "HHH Manda 39 4C)- 6 g 30-s m .J R ul it‘s 2C}— .2 c 2 _\~ a “I In \ ,. 8' c: 3% 3 IO:- 2 2: u: a. I II III IV V VI VII VIII IX X I i i I i I Designed Cormorison of Brain NE Levels (ug/q) Figure 6: Least significant difference dPSIQHQd Comparison 0! nrr cant du- plction 0! brain as luvels. Starred comparisons or. qunlfICdnL to the .05 level. Kean group values Pay hr tuund in fable II. For cent deplvtion of each comparison (llSlOd in Primgry Com- parison Legend below) was dvlutnlnfd by subtracting the mean qroup Va no in column A from the mean group value in Column n, dividing the result by thu mean group value in column B and multiplying by Ian. B-A (—- x 100) A Group Legend Primary Comparison Legend Training Final .. , . Condition Treatment Drug Q E ELELE£2_2£' E -trainud exercise "20 I Ea_MT vs Ewh :cxrrcinc and/or shock upon trained u-MT rats without CA synthesis Ea~MT -traincd °”°ICl'° .‘MT 1! H vs Huh :exvrCIac and/or chock upon trained Ewh -trained fined wheel H20 rats "1‘" CA synthesis III E _MT vs H :a-MT upon trained rats after acute Ewh -trained fixed wheel a-MT “ exercise a‘MT lv Huh vs Ewh :a—MT upon trained rate without Em.d -trainud caqc H20 a-HT exorCise in fixed wheel _ , . _ _ , v r ‘ vs a :a-MT upon trained rate without asedu-MT trained cage a Mr n«_da_M,r eed exercise in cages ~scdcntary shock H20 VI 3 vs s :shnck upon sedentary rats with CA ch ch uh syntheaie Sch -HT -sodcntary 'hOCk a-Hr VII Sch vs swh :shock upon Iedontaty rate without a-HT a-HT CA syntheti- Swh -eeduntary f’xed who°1 "20 VIII Sch vs Sch :a-MT upon sedentary rate after suh —sndvntary fixed wheel a-HT a-M‘ ah"CR a-MT Ix suh ve Sun :a-HT upon sedentary rats in fixed Sfird -svdvntary cage H10 n-H1 wheel x Ss‘d vs sued :a-MT upon Ioduntary ratl without sled "°““"‘dlv cage a-HT L a-HT exercise in cages a- 40 70 Z 9 T L] 60-1 .I it 2 7. O E 40-I :Z 4 «I 0:- a: 30 m \ T 5 3' or u: g 20- 8 a 33 IO-* t I H "I IV V VI VII VHI IX X *- -* Designed Comparison of Brain OM Levels (ug/g) Figure'7: Least significant difference designed comparison of per cent de~ pletion of brain DM levels. Starred comparisons are significant to the .05 level. Mean Group values may be found in Table 11. Per cent depletion of each comparison (listed in Pripary Comparison Legend below) was determined by subtracting the mean group vaIue in co umn A from the mean group value in column B, dividing the result by the mean group value in column B and multiplying by 100. BrA (T X 100) Group Legend Primagy Comparison Leggnd Training Final , V . Condition Treatment Drug 5 9 MEL B -trainod Uercjse H20 I “wt vs Ea-MT :vxvrcise and/or shock upon Itdinvd ‘a-MT rats without CA synthesis La-M'l‘ -trainud cxurvisu a-MT II Ewh vs E :vxvrciae and/or shock upon trained Ewh -truino«l fiXUd wheel “20 rats with LA aYnLhUSl'. III Ea—MT vs u :a~MT upon trained rate after acute Ewh -trninvd fixed wheel a-MT exorcise d-M'I' . _ . IV E vs E :a-MT upon trained rate without Esed trained cage “20 Wha-M’l‘ wh exercise in fixed wheel B -trained cage a-MT V E vs L :a-MT upon trained rate without 8eda-M'I‘ seda-MT sed exercise in cages s -sedcntary shock H20 VI 5 vs S :shock upon sedentary rats with CA ch ch wh . synthe51s scha_MT -aedentary ShOCK a—MT VII Swh vs Sch :shock upon sedentary rats without Swh -aedentary fixed wheel H20 a-MT a-MT CA synthesxs VIII Sch vs sch :a-MT upon sedentary rats after swh -aedentary fiXed wheel a-MT a-MT shock a-MT . . , 1x 8 vs S :a-MT upon sedentary rats in fixed aed sedentary cage H20 "ha-MT wh wheel sed -sedentary cage a-MT x Sscd vs Ssed :a—MT upon sedentary rats without a-MT a-MT exercise in cages 41 treatment conditions and for both experiment groups (com- parisons III, IV, V, VIII, IX and X). Brain DM concentrations experienced significant decline among a-MT treated trained and sedentary rats under sedentary housing conditions (Figure 7, comparisons V and X). A-MT did not significantly lower brain DM among trained rats subjected to exercise (III), placed in a fixed running wheel (IV), or among sedentary rats placed in "cheerleader" cages (VIII) and secured running wheels (IX). Depletion Comparisons Between Final Treatment Subgroups Least Significant Difference was used, in addition, to determine the effect of differences in depletion among the various subgroups listed in Table IV. The results indicate exercise and/or shock potentiates a-MT-induced depletion of brain NE among trained rats (I vs III). DM depletion was not augmented under the same circum— stances. Table IV also demonstrates increased a-MT-induced depletion of brain NE among trained rats subjected to exer- cise compared to shock depleted NE values among sedentary rats (III vs VII). Therefore, exercise and/or shock among trained rats may have evoked greater utilization of brain NE stores than did shock among sedentary animals. No sig- nificant effects were observed for brain DM comparisons. 42 TABLE IV: Depletion comparisons between final treatment subgroups. ‘w - :P-_ ' —-._---...s '-—3' -‘ nu. —£ I ._—._-: ......_... r ' Subgroups ;ean differences (1 - j, uq/g/Z 1/2 hours) i - 3 Brain NE Brain ON I - VII ".03 -.lO IV - IX .02 .04 VI - VII -.04 -.14 IV -' V .02 e08 IX - X -.01 ”.02 III - IV -.01 .11 I - III .07** -.06 III - VII -.10" -.04 VIII - IX .04 -.14 fit p < .05. Group Legend Primary Comparison Leggnd Training Final : . Condition Treatment Drug A E §£££E£2_2£- E -trained exercise H20 I Ea—MT vs Ewh :exercise and/or shock upon trained a-MT rats without CA synthesis Ea-MT -trained exerCise a-MT II E vs Ewh :exercise and/or shock upon trained Ewh —trained fixed wheel H20 rats ”1th CA synthesis LII Ea-MT vs E :a-MT upon trained rats after acute Ewh -trained fixed wheel a-MT exercise a-MT . _ . IV E vs E :a-MT upon trained rats without Esed trained cage "20 Wha-MT Wh exercise in fixed wheel 3 -trained cage a-MT V E vs 8 :a-MT upon trained rats without Beda-MT seda-MT sed exercise in cages sch -sedentary ShOCk “20 VI sch vs swh :shock upon sedentary rats with CA Sch -sedentary shock a-MT synthesis a-MT VII SCh vs Swh :shock upon sedentary rats without swh -sedentary fixed wheel H20 a-MT a-MT CA synthesis 5 —sedentary fixed wheel a-MT VIII Sch vs SCh :a-MT upon sedentary rats after "ha-MT a-MT shock Ssed -sedentary cage H20 IX Swh vs Swh :a-MT upon sedentary rats in fixed a-MT wheel s.eda_MT—sedentary cage a-MT x seed vs Ssed :a-MT upon sedentary rats without a-MT exercise in cages Comparison I vs VII IV vs Ix VI vs VII IV vs V Ix vs x III VB' IV I vs III III vs VII VIII VI IX I Secondary Comparison Legend Meaning :Does a trained rat subjected to exercise and/or shock deplete his CA stores (with- out synthesis taking place) to a greater degree than a sedentary rat subjected to shock? :Does the anxiety of placing a trained rat in the running wheel who expects to run but cannot do so, potentiate a-MT induced depletion of CA compared to a sedentary animal under similar housing conditions? :Does the ability to synthesize CA under shock stress decrease the depletion of CA stores in the sedentary rat, or does behavioral depression (due to a-MT) prevent an anxiety (shock) response in sedentary rats from decreasing its depletion of CA? :Does the anxiety of placing a trained rat in the running wheel who expects to run but cannot do so, potentiate a-MT induced depletion of CA stores compared to trained rats in sedentary cages? :Will sedentary rats respond to a new environment by increasing a—MT induced de- pletion of CA? :Does acute exercise and/or shock among trained rats potentiate a-MT induced CA de- pletion compared to the trained rat placed in a similar environment but prevented from performing his normal (expected) pattern of response? :Does exercise and/or shock potentiate a-MT induced CA depletion among trained rats? - :Comparison of a-HT induced CA depletion after acute exercise among trained rats to shock induced CA depletion among sedentary rats. :Does shock potentiate a-MT induced CA depletion in sedentary rats? J j 43 Behavioral Response and Brain CA Concentration The correlational analysis between brain CA and motor activity presented in Table V may assist in differentiating the relative contribution of DM and NE during exercise. A moderately negative correlation (r = -.62) between brain DM and TRR during final treatment among trained rats subjected to exercise and a-MT may indicate that this amine was utilized at a faster rate than it was synthesized. TABLE V: Final Treatment Correlations: Behavioral Response and Catecholamine Concentration in the Brain. Catecholamine Final Group n TRR CDS E1 7 .33 -.32 Brain NE (ug/g) a-MT 6 -.06 .13 E 7 -.28 .27 Brain DM (ug/g) Ea-MT 6 -.62 .64 1 . . . . E—-trained, exerCISe, H20; E --tra1ned, exerCISe, a—MT a-MT Relationship Between Brain CA and Brain Weight Low correlations were observed between brain CA and brain weight (Table VI). Apparently, the size of the brain does not dictate the concentration of DM or NE contained within. 44 TABLE VI: Relationship Between Brain CA and Brain Weight. Comparison Trained n Sedentary n Brain Weight vs Brain NE .34 39 .39 38 (grams) (ug) Brain Weight vs Brain DM .08 39 .17 37 (grams) (ug) Discussion An evaluation of the training data and body and organ weight comparisons between exercise and sedentary animals shows that the regimen employed did produce a significant training effect. However, too many unanswered questions remain which qualify any conclusions regarding the training program. The accumulation of shock time in the final days of the program may indicate that the exercise group adapted to shock stress. Until this training program is clarified further, the results obtained in this study can be applied only to adult, male, albino rats subjected to a similar training regimen. The implications that can be drawn from the CA analy- sis of brain tissue is speculative at best. The statisti- cally significant results of brain CA comparisons require cautious interpretation until a more specific role is assigned to NE and UN in the functioning of the central nervous system. If one assumes NE modulates the central component of sympathetic tone, then the increased brain NE 45 levels observed among trained rats (compared to sedentary controls) may be indicative of a higher maintained level of sympathetic output. The ability of a-MT to deplete brain NE under all the conditions imposed in the final treatment gives testimony to its function as a powerful synthesis inhibitor, most likely of tyrosine hydroxylase [93]. However, the presence of control brain DM values among trained rats subjected to exercise and a-MT suggests conservation of this amine. Exercise and/or shock among trained and sedentary rats did not significantly lower brain NE or DM concen- trations (Figures 6 and 7, comparison I). Gordon EE.El‘ [38] observed the same result following one hour of tread- mill exercise of previously sedentary rats. However, sig- nificant depletion of rat brain NE following electric shock (5 ma) through a grid floor for one hour [9, 60] was not corroborated in the present investigation. This may have resulted from the lower shock stimulus (1.2 ma) used by this author. Thierry pp pl. [95] obtained results simi- lar to those of the current investigation in rats following intermittent grid shock (0.8 ma) for three hours. Least Significant Difference analysis demonstrated the ability of exercise and/or shock to augment brain NE depletion in trained rats (Table IV, comparison I vs III). This is comparable to the data of Gordon g£_al. [38] indi- cating an increased turnover of brain NE following one hour 46 of running exercise. These authors obtained one-half the depletion following a-MT injection in, roughly, one—half the time period used in this study. Exercise did not increase the rate of brain DM de- pletion in trained rats treated with a-MT. This implies the presence of a mechanism designed to conserve DM under conditions of extreme stress (drug plus exercise). If brain DM were responsible for the maintenance of motor activity as suggested by many authors [7, 15, 75, 86] it would appear that such a mechanism is operating. Stores of brain NE were depleted to a greater extent in trained rats exposed to exercise compared to sedentary rats subjected to shock the final day (Table IV, III vs VIIL The exercise routine appears to have evoked a greater sym- pathetic discharge than shock stress alone. In conclusion, a model is proposed incorporating the theory of Brodie and others [11] placing emphasis upon brain DM stores in the regulation of motor activity under conditions of extreme stress acting somewhat independently of sympathetic stimulation during exercise, modulated by brain NE. CHAPTER V SUMMARY, CONCLUSIONS, RECOMMENDATIONS It was the purpose of this investigation to demon— strate the chronic effects of a specific exercise regimen upon rat brain catecholamine concentrations. Forty male albino rats were exercised in an elec- tronically controlled running wheel five days per week for a total of eight weeks. An equal number of animals served as sedentary controls. Daily records of animal weight and performance were kept during the training period. The animals were divided into six subgroups during the final treatment protocol. Half of each group was in- jected i.p. with distilled water, and the remainder with alpha-methyltyrosine, a potent catecholamine synthesis inhibitor [93]. One and one-half hours following injection, each rat was exposed to his final treatment as follows: E = Trained rat run through "normal" exercise routine Ewh = Trained rat placed in running wheel secured to prevent rotation Esed = Trained rat placed in sedentary cage 47 48 SCh = Sedentary rat placed in "cheerleader” cage (subjecting animal to the identical amount of shock as E) Swh = Sedentary rat placed in running wheel secured to prevent rotation Ssed = Sedentary rat placed in sedentary cage. All animals were decapitated one hour following final treatment. Brain and heart were removed, weighed, quick- frozen and the brain subsequently analyzed for catecholamine content, fluorometrically. Tissue catecholamine content was statistically analyzed using a replicated two-way analysis of variance (factorial design). Variables were further compared using the Single Degree of Freedom and Least Significant Differ- ence designed comparisons. These techniques were employed to gain power in detecting true differences between sub- groups and various combinations of subgroups. On the basis of prior studies investigating the acute effects of a variety of stress situations, it was the author's belief that brain catecholamine levels might be altered in a differential fashion under the experimental conditions imposed. Conclusions Conclusions to be drawn from this study are as follows: 1. Increased steady-state levels of brain norepi- nephrine observed in physically trained rats may have been caused by the continuous demand placed upon the sympathetic 49 nervous system to which this amine has been linked as neurotransmitter. 2. The inability of alpha-methyltyrosine to deplete the brain of dopamine among trained rats exposed to stress may implicate this amine in the control of motor function and may suggest a mechanism which tends to bypass the ef- fects of synthesis inhibition in situations requiring ex- cessive amounts of dopamine. Recommendations 1. It is suggested that training procedures be limited to a maximum of six rats per session (in lieu of twelve per session) continuously modifying the regimen according to the response of each animal. 2. Techniques need to be altered to allow investi- gation of resting heart rate and systolic blood pressure, and their relationship to heart and brain CA concentrations. 3. A time study with larger sample sizes per group is recommended to accurately evaluate turnover rates of brain catecholamines. 4. 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Proc. 27:711, 1968. 60 102. Welch, B. and A. Welch. Stimulus-dependent antagonism of the alpha-methyl-tyrosine-induced lowering of brain catecholamines by amphetamine in intact mice. J. Pharm. Pharmacol. 19:841-843, 1967. APPENDIX APPENDIX A BIOCHEMICAL ANALYSIS OF CATECHOLAMINES Heart and brain tissue was permitted to thaw prior to homogenization in 6 m1 of cold 0.4 N perchloric acid. The toughness of the heart tissue required cold-controlled grinding in a ten broeck pyrex tissue grinder, however, the softer brain substance was quickly ground in a teflon pestle tissue grinder. The homogenates were maintained in an ice bath for 30 minutes and then centrifuged for five minutes at 10,000 x g. The supernatant was obtained, and the pro- cedure repeated, combining final eluates into a single sample adjusted to pH 4.0 with 10 N KOH and 1 N KOH. Cen- trifugation separated the potassium perchlorate precipitate from the supernatant which was then added to 50 ml glass- stoppered centrifuge tubes containing about 400 mg of pre- pared aluminum oxide (Woelm) and 0.5 ml of 0.2 M disodium ethylenediaminetetraacetate. DM (2.0 ug) and NE (0.8 ug) were used as standards and run through the alumina exchange along with samples. The pH of the combined alumina-tissue CO and 0.2 M 2 3 K2C03. The supernatant was removed by aspiration, washed mixture was adjusted to 8.6 to 8.7 with 5M K 61 62 twice with 10 ml H20 and shaken for five minutes subsequent to each washing. The amines were eluted with 8.0 m1 of 0.2 N acetic acid prior to the final shake (of 10 minutes dura- tion) and centrifuged for 10 minutes. The final eluate was divided into two 4 m1 aliquots and transferred to "DM" or "NE" assay tubes for subsequent analysis. No more than eight tubes were shaken concurrently. The pH of the 4 ml "NE" eluate was brought to 6.5 to 6.8 with 5 M K CO 2 3 6.5 phosphate buffer to each tube. Each eluate was then followed by the addition of 0.8 ml of pH divided into two 2.4 ml aliquots, one serving as blank, the other for NE determination. NE Fluorescent Procedure Samples: (Tubes shaken thoroughly after each ad- dition) 1. Start timer and add .05 ml of 0.25% potassium ferricyanide. 2. After 2 minutes add freshly prepared alkaline ascorbate (20 mg ascorbic acid plus 1.0 m1 H20 plus 9.0 m1 5 N NaOH). Blanks: 1. Add 0.25 ml of alkaline ascorbate. Fluorescence was determined within 10 minutes in an Aminco-Bowman spectrophotofluorometer at activation- fluorescent wavelengths of 391 and 510 mu respectively. 63 Mean recovery of heart NE was 70% i 3.4 (S.D.), with a slightly higher average for brain (77% i 7.1). DM was determined adjusting the 4.0 ml acid eluate to pH 6.2 to 6.5 by addition of 2.0 ml of phosphate buffer (pH 8.0). 3.0 ml of the above mixture was removed for assay the remaining 3.0 m1 serving as a blank. DM Fluorescent Procedure Sample: 1. Add 0.2 m1 of 0.5% sodium periodate (mix). 2. Wait 1 minute exactly. 3. Add 1.0 ml of alkaline sulfite (2.65 g of Na SO 2 3 + 10 m1 H20 + 90 m1 of 5 N Na). 4. In rapid succession add: a. 2.8 ml H20 b. 1.0 m1 of 0.5 M Citrate buffer c. 1.7 m1 of 3 M phosphoric acid. Fluorescence was determined ten minutes later in an Aminco-Bowman spectrophotofluorometer at activating- fluorescent wavelengths of 325 and 385 mu, respectively. Recovery of brain DM was 65% i 14.1. Blanks were subtracted from sample fluorescence (at meter multiplier setting .03), divided by the fluorescent value of the standards run through with each analysis and multiplied by the content of CA within the standard cuvettes (0.2 ug for NE and 0.5 ug for DM) to obtain concentrations. TABLE A-l: Brain Catecholamine Correlations. Final Treatment n Brain NE vs Brain DM Groups (ug/g) (ug/g) E 7 .48 Ea-MT 6 ~55 Ewh 6 .28 E 6 .67 wha-MT Esed 7 .62 E 7 .59 seda_MT Sch 7 .66 S 7 .68 cha-MT Swh 6 .77 S 6 .28 Wha—MT Ssed 7 .42 S 5 .83 seda-MT 64 65 TABLE A-2.--Standard eight-week, medium-duration, moderate-intensity endurance training program for postpubertal and adult male rats in controlled-running wheels. 33 s 3 s H ['1 ‘f‘ c c m 5- ()3 i 9 i 3 fi- .- s . := a :5 -: t = a g: s: I 3:: ° :3 i! g3; a- 5:; :E i 3 3 s“ :3 S; i3 “a 3: :3 s: :g :g 33 CI >1 >9 0 Law-4 I 5 5 ‘30 U a) g : a a 28 :5 i: ii I: .3 . a: a: a: as 1 1-M 1 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 2-T 2 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 3-W 3 3.0 00:10 10 30 4 2.5 1.2 2.0 39:45 600 1200 4-T 4 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 SIP 5 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 2 l-M 6 2.0 00:10 10 28 4 5.0 1.0 2.5 51:40 700 1120 2-T 7 1.5 00:10 10 27 4 5.0 1.0 3.0 50:20 810 1080 3-H 8 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 4-T 9 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 SI? 10 1.5 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 3 1-M 11 1.0 00:10 10 27 4 5.0 1.2 3.0 50:20 810 1080 2-T 12 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 3-W 13 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 4-T 14 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 S-P 15 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 4 l-M 16 1.5 00:10 10 26 4 5.0 1.0 3.5 49:00 910 1040 2-T 17 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 3-W 18 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 4-T 19 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 5'? 20 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 5 l-M 21 1.5 00:15 15 19 4 5.0 1.0 3.5 52:00 997 1140 Z-T 22 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 3-W 23 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 4-T 24 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 5'? 25 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 6 l‘M 26 1.5 00:15 15 14 5 5.0 1.0 4.0 53:45 1050 1050 2-T 27 1.5 00:20 20 11 5 5.0 0.8 4.0 55:00 1100 1100 3-W 28 1.5 00:20 20 11 5 5.0 0.8 4.0 55:00 1100 1100 4-T 29 1.5 00:20 20 11 5 5.0 0.8 4.0 55:00 1100 1100 SI? 30 1.5 00:20 20 11 5 5.0 0.8 4.0 55:00 1100 1100 7 l-M 31 1.5 00:20 20 11 5 5.0 0.8 4.0 55:00 1100 1100 2-T 32 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 3'“ 33 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 4-T 34 1.5 00.25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 5'? 35 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 8 1'! 36 1.5 00:25 25 9 5 5.0 0.8 4.0 55:25 1125 1125 2-T 37 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 3-H 38 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 4-T 39 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 SIP 40 1.5 00:30 30 8 5 5.0 0.8 4.0 57:30 1200 1200 This standard program was designed using male rats of the Sprague-Dawley strain. All animals were between 70 and 170 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 PS! 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 impair the effectiveness of the training program. Standard medium-duration, moderate-intensity endurance maintenance program for postpubertal and adult male rats in controlled-running wheels. .: a g 3? ~ 5 “ g; i : 3% '5 '5 5: 5-2;: °. 2: :. :~. §:: 5?: ' 1‘.“ :8 " " b 38' 3 38 :5 .2 a: his: 5: i a: .2: a§ .2: 1.5 00:30 30 8 3 5.0 0.8 4.0 32:30 720 720 "'TITI'ITdfll’flLflflflflfiifliflflflfy’flflflmfl/afllfllfi'“