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"HISTOCHEMICAL AND CONTRACTILE PROPERTIES FOLLOWING NEONATAL DENERVATION IN THE FAST-TWITCH EXTENSOR DIGITORUM LONGUS MUSCLE OF THE MOUSE by Darlene M. Redenbach B.S.R.(PT), The University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Anatomy  We accept t h i s thesis as conforming to Jthe- requiredydta.ri&amp;gf&lt;f  THE UNIVERSITY OF BRITISH COLUMBIA November, 1985 \u00A9DARLENE M. REDENBACH, 1985  \u000CIn  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his  and  scholarly  or  her  thesis  for  of  TO  m  flfUfi  T h e U n i v e r s i t y o f British 1956 M a i n M a l l Vancouver, Canada V6T 1Y3 Date  ^te-fP^/e  &gt;  &lt;/  Columbia  I further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  Department  study.  requirements  It not  be  that  the  Library  permission  granted  is  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  \u000C-  i i  -  ABSTRACT Mature complete and  fast-twitch  differentiation  contractile  development muscle look  is  at  extensor  of  of  At  1  by  on  of  age,  does  (pH  twitch  fatigue  in  histochemical and  of  were  a  not  occur  4.2  and  by  of  role  study  was  effects  these  in  done  of  mouse,  to  properties neonatal  properties  C57/BL6  the  developing  contractile  the  for  histochemical  therefore  This  and  the  for  9.4).  Isometric  tension,  twitch  tension,  velocity  measured, examined  examined  at  at 1  7,  day  at  7,  vitro,  at  14  21  in  the  14  and  and  acid  preincubation,  to  NADH  staining,  post-tetanic  twitch  showed  myosin  tension  and  including  to  one-half  and  twitch  resistance  Denervated age,  were  ATPase  post-tetanic  shortening  that  types  properties,  tension,  of  confirm  fiber  and  twitch  20\u00B0C. days  to  performed.  and normal  to  control muscles  age.  alkaline  muscles  a l l  tetanus  was  Extrafusal  peak  unloaded  of  used  contractile  from  and  was  enzyme  of  of  normal  age,  in  neurectomy  method.  oxidative  time  maximum  sciatic  staining  this  day  days,  of  unilateral  1  according  (EDL)  innervation  dominant  and  examine  development  muscle  a  innervation.  to  tension,  were  also  its  on  fast-twitch  plays  on  twitch  potentiation,  At  longus  its  motorneuron  acetylcholinesterase  time-to-peak  were  pattern  of  characteristics,  denervation,  histochemically  activities  muscles  these  changes  the  day  and  examined  The  depends  age.  reinnervation  peak  the  muscle  maintenance  dependent  digitorum  days  of  more  about  denervation  Silver  many  even  and  properties.  of  some  brought  21  skeletal  fibers  and  typical an  potentiation  stained a l l of  fibers  immature  increase and  for  in  velocity  myosin were  of  uniformly  fibers.  twitch  ATPase  and  unloaded  Over  following oxidative,  the  tetanus  next  21  tension,  shortening,  with  \u000Ca  reduction  and  in  in  time-to-peak  resistance  to  differentiated  into  (anaerobic  the  being  in  type  I.  prolongation with the  controls. maximum  continued  groups  toward  be  The  muscles,  they  muscle.  slowing  the  the  to  studies  neural  In  of  at are  seen  14  suggested in  this  of that  study.  low,  there  of  may  as  14  in  but  was  fiber  evidence  days  only  as  but,  seen  in  These  to  over  differentiation  is and  an  immediate  although  preventing identify  the  of  and  the  digitorum into  significant  denervated normal  the  results  neonatally the  extensor  in  returned  changes  type.  two  fibers  unlike  muscles  the  was  denervated  atypical  IIA  denervated  absent,  there  oxidative,  type  development  age,  by  compared  was  A l l  were  were  muscles,  changes  but 70%  and  significant  and  there  IIA  remainder  a  studied.  these,  The  properties  days  was  fibers  type  the  potentiation  age,  stained  control  pattern  of  ATPase  influence  addition,  the  Of  immature  preceeded  loss  mimick  days  being  with  Histochemically,  oxidative.  of  extrafusal  fibers  remained  ages  relaxation  half-relaxation  twitch  a l l  group  contractile  stalled  changes  of  in  fast-twitch  to  were  properties  results  of  second  properties  removal  myosin  the  there and  shortening  7  one-half  (aerobic),  tension  at  at  for  to  of  H6%  muscle  distinguished.  fibers.  longus  IIB  atrophy.  maturation  staining  that  Further  fatigue,  significant  suggest  becomes  unloaded  dual  physiological  continues  of  to  could  slow  type  denervated  addition,  velocity  of  control  the  with  post-tetanic  showed  immature  k0%  fibers  In  muscles  exhibiting  mouse),  time  Histochemically,  twitch  resistance  fiber  fast  tension,  time-to-peak  marked  some  fatigue.  mature  In  of  twitch  muscle  muscle,  i t  further  maturation.  factors  contributing  \u000C- IV -  TABLE OF CONTENTS ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES GLOSSARY ACKNOWLEDGEMENTS  I  II  INTRODUCTION Neural Influence on Myogenesis D i f f e r e n t i a t i o n Into Fast or Slow-Twitch Muscle Denervation During Myogenesis The Purpose of the Study  v i i Viii ix  1 2 2 4 4  REVIEW OF THE LITERATURE 6 Nerve Dependency i n Early Myogenesis 7 Myogenesis: Independent Stage 8 Coordinated Myogenesis: Dependent Stage 9 (a) Connective Tissue Influences 9 (b) Neural Influence 9 D i f f e r e n t i a t i o n into Fast-Twitch Muscle 11 (a) U l t r a s t r u c t u r a l Changes 12 (b) Sarcoplasmic Reticulum and Calcium Binding Protein Changes . 13 (c) Metabolic Enzyme D i f f e r e n t i a t i o n 13 (d) Myosin D i f f e r e n t i a t i o n 14 (e) Contractile Protein Isozymes 15 (f) Regulatory Protein Isozymes 19 (g) Contractile Properties 19 (h) Summary 21 Models of Neural Regulation 22 Neural Regulation o f Mature Muscle 24 (a) Atrophy 24 (b) U l t r a s t r u c t u r a l Changes 28 (c) Contractile Protein Changes 30 (d) Changes i n Calcium Uptake 33 (e) Alterations i n Contractile Properties 35 Neural Regulation of Immature Muscle 37 (a) Atrophy 38 (b) U l t r a s t r u c t u r a l Changes 38 (c) Alterations i n Contractile Proteins 39 (d) Changes i n Contractile Properties 42 Permanence of Denervation E f f e c t s i n Developing Muscle 43 A Model of Aneural Myogenesis i n Vivo 44 Statement of the Problem 45  \u000C-  IV  IV  V  VI VII  V. -  METHODS Denervation Histochemistry (a) Experimental Proceedures (b) Data Collection Morphometries (a) Experimental Proceedures Contractile Parameters (a) Muscle Dissection (b) Experimental Apparatus (c) Experimental Proceedures (d) Data Collection Data Analysis RESULTS Growth Histochemistry (a) Total Fiber Number (b) Fiber Types Contractile Properties (a) Contraction Time (b) Isometric Twitch and Tetanus Tension (c) Ratio of Twitch to Tetanus Tension (d) Maximum Velocity of Shortening (e) Post-tetanic Twitch Potentiation (f) Resistance to Fatigue  DISCUSSION Growth Morphometries Histochemistry Contractile Properties Summary BIBLIOGRAPHY APPENDICES 1. A Comparison of Sham Operated and Control EDL Histochemical and Contractile Properties at 21 Days of age 2. V e r i f i c a t i o n of Denervation up to 21 days of Age Following Neonatal S c i a t i c Neurectomy . . . . 3 . Preliminary Experiments to Establish pH and Temperature Conditions f o r Developing Normal and Denervated EDL 4. Comparison of Contractile Properties at 20-22\u00B0C and 35-37\u00B0C  46 47 48 48 49 50 50 50 50 51 51 55 55 59 60 63 63 63 76 76 76 82 82 82 88  89 91 93 93 102 112 114 125  126 129 138 141  \u000C- vi LIST OF TABLES I  Myosin isoforms i n fast-twitch muscle development  18  II  Fiber typing of fast and slow-twitch f i b e r s according to myosin ATPase  49  Growth changes i n animal weight, muscle length and muscle weight i n normal mice and those denervated at one day of age. . .  61  Whole muscle cross-sectional area i n normal and denervated EDL at 21 days of age  63  III  IV  V  VI  Twitch tension to tetanus tension r a t i o i n normal and denervated EDL  83  Maximum v e l o c i t y of shortening i n normal and denervated EDL . . .  83  VII Post-tetanic twitch potentiation i n normal and denervated EDL . . 84 VIII Comparison of c o n t r a c t i l e properties of sham operated and control EDL at 21 days of age 127 IX  Comparison of c o n t r a c t i l e properties at 20-22\u00B0C and 35-37\u00B0C i n normal and denervated EDL at 21 days of age  143  \u000C-  LIST 1  Experimental  2  Original day  3  4  5  of  (Vo)  by  and the  determination slack  fiber  21  number  days  of  of  normal  ATPase and  oxidative  EDL  seven  of  12  13  14  15  at  of  unloaded  EDL a t  1  normal  and  denervated  EDL 62  and  pooled age  denervated  from  four 65  EDL a t  1,  7,  14  normal  EDL  at  one  day  enzymes  of  normal  of  age  and  69  denervated  age  71  of  normal  and  denervated  EDL  at 74  type d i s t r i b u t i o n 7 , 14 a n d 21 d a y s  of of  normal age  and  denervated  normal  TTP and  EDL a t  and 1/2RT o f normal 21 d a y s o f a g e  and  EDL 75  O r i g i n a l r e c o r d s o f t w i t c h myograms o f d e n e r v a t e d EDL a t 7 a n d 21 d a y s o f a g e denervated  and 78 1,  7,  14 80  T w i t c h and t e t a n u s t e n s i o n i n a b s o l u t e v a l u e s and those n o r m a l i z e d t o m u s c l e w e i g h t o f n o r m a l a n d d e n e r v a t e d EDL f r o m 1 t o 21 d a y s o f a g e  81  Original record of post-tetanic twitch i n n o r m a l EDL a t 21 d a y s o f a g e  84  Original o f 1 day Fatigue 7,  records of a n d 21 d a y profile  14  and  Oxidative denervated  17  days  Histochemical profile 21 d a y s o f a g e  1, 16  normal  66  Myosin  11  in  age  7  10  velocity of  individual fibers EDL a t 21 d a y s o f  Histochemical profile  Fiber at 1,  of  method  57  6  9  test  age.  Cross-sectional area of normal and 4 denervated Total  FIGURES 53  Whole muscle c r o s s - s e c t i o n o f a t 21 d a y s o f age  and  8  OF  -  apparatus  Vo r e c o r d s  shortening  v i i  Silver  and  denervated  of  21  EDL  the f i r s t three frames of the f a t i g u e n o r m a l a n d 21 d a y d e n e r v a t e d EDL normal  days  enzyme at  of  and  staining of 21  denervated  at  21  muscle  days  of  days  of  regime 86  at  age  87 sham o p e r a t e d ,  control  and  age  acetylcholine esterase EDL  potentiation  age  135 staining of  normal  and 137  \u000CGLOSSARY  ACHE  Acetylcholinesterase: enzyme found i n neuromuscular junction to break down acetylcholine into choline and acetic acid  DEN  denervated extensor digitorum  EDL  extensor digitorum longus muscle: a fast-twitch hindlimb muscle.  myosin ATPase NADH-TR  longus muscle  the adenosine triphosphatase  also referred to as NADH reaction:  on the myosin molecule NADH tetrazolium reductase  oxidative enzyme reaction NORM  normal (or control) extensor digitorum  longus muscle  1/2 RT  one h a l f relaxation time: time from peak tension to one h a l f peak tension  Po  Tetanus tension  Pt  Twitch tension  PTP  post-tetanic twitch potentiation  SR  sarcoplasmic reticulum  VMax  t h e o r e t i c a l value of the maximum v e l o c i t y of shortening at zero load, extrapolated  from measures of shortening v e l o c i t y over  a series of loads Vo  maximum v e l o c i t y of unloaded shortening measured by the slack test method  \u000CACKNOWLEDGEMENTS  The author would l i k e to express appreciation to Dr. B. H. Bressler for  sharing  h i s experience and high  standard  of s c i e n t i f i c  inquiry, as  well as h i s guidance and encouragement throughout t h i s research project. Gratitude W.K.  Ovalle,  assistance assistance.  i s also  expressed  Dr. M.E. Todd  and to Jean McLeod  to the members of the Committee, Dr.  and Dr. W. and Ursula  Vogl Dole  for their  f o r excellent  Special thanks i s due to Dr. W.K Ovalle  the histochemical  advice  and  technical  f o r h i s help  with  study.  F i n a l l y , enormous gratitude i s due to my family support and encouragement.  for their  constant  \u000C1 -  71  I  INTRODUCTION  \u000C- 2 Neural Influence On Myogenesis From neural  an early  influence.  stage  i n myogenesis,  Throughout  muscle  development,  i s highly  dependent on  the motoneuron  continues to  play a dominant r o l e i n d i c t a t i n g the mature c h a r a c t e r i s t i c s of fast and slow  muscle.  physiological and  A  great  changes  the sequence  responsible.  deal  that  occur  of genetic  Strohman  is  known  i n skeletal  expression  and  about  Wolf  the  structural  muscle during  of muscle  (1985)  and  development  proteins  that are  distinguished  between  d i f f e r e n t i a t i o n into muscle, which i s coordinated and determined early i n embryonic l i f e , and maturation, an orderly progression o f the sequence of embryonic to adult muscle protein isoforms. to occur i n v i t r o  D i f f e r e n t i a t i o n can be made  i n the absence of innervation but complete maturation  cannot. Muscle develops from predestined c e l l s of mesoderm which migrate as myoblasts into the developing limb bud and fuse into myotubes. influence  of  the motoneuron,  they  will  begin  m y o f i b r i l l a r proteins and organelles which w i l l  synthesizing  their  properties  morphological, depending  histochemical,  on  the  biochemical  neurotrophic  specific  ultimately determine the  fast or slow c o n t r a c t i l e properties of the mature myofiber. in  Under the  and  influence  These d i f f e r contractile and  impulse  c h a r a c t e r i s t i c s of the motoneuron. D i f f e r e n t i a t i o n Into Fast or Slow-twitch Muscle In response to changing functional to  exhibit  structural  enzyme p r o f i l e , are  reflected  changes  and  biochemical  and c o n t r a c t i l e i n muscle  continue  demands, mature muscle continues plasticity  and regulatory  behavior,  to be controlled  a c t i v i t y pattern of the motoneuron.  i n i t s morphology,  protein  i t s contractile  isozymes. These  properties.  through the trophic  These  influences and  \u000C- 3 At b i r t h , 1978)  with  forming  a l l fibers have polyneuronal  uniform  only  properties of  simple  immature  junctional  innervation  (Obrien  sarcoplasmic  complexes  with  transverse tubules or the sarcolemma (Luff and  a  et a l . ,  reticulum  loose  (SR)  network  Atwood, 1971).  of  They have  uniform moderate oxidative enzyme a c t i v i t y (Reichman and Pette, 1984) high  resistance to  neonatal  form  fatigue.  Myosin  (Whalen et a l . , 1981)  Speed of shortening and twitch and  tetanic  reflecting  an  isozymes and  immature  with  calcium  of  an  embryonic  myosin ATPase a c t i v i t y  contraction times  tension i s low  are  are  slow  a high  binding  and or  is  low.  (Close, 1964).  The  twitch to tetanus  capacity.  Resting  ratio  membrane  potential i s low, and impulse conduction i s slow. F u l l y mature fast-twitch white f i b e r s have an elaborate  sarcoplasmic  r e t i c u l a r network with a c o l l a r at the M l i n e and form frequent t r i a d s at the  Al  junction with  an  abundant  Atwood, 1971). permitting the contraction times. fibers. 1973)i  transverse  rapid  is  corresponds  stable  in  alkaline  and  of  i s high  has  a  relatively  threshold,  and  is  therefore  large  (Luff  calcium  and  for short  labile  (Drachman and in  acid  high g l y c o l y t i c enzyme a c t i v i t y .  motoneuron  system  i s t y p i c a l of fast-twitch white  with rapid rate of muscle shortening.  low oxidative and  frequency  sequestering  Their narrow Z l i n e  Their myosin ATPase a c t i v i t y  tubule  diameter,  recruited  less  Johnston,  conditions  These f i b e r s The  with often,  and  contain  axon from t h e i r  a  high  with  stimulus  short  high  bursts of a c t i v i t y required for rapid high tension performance  of spurt muscles (Jolesz and Sreter, 1981). Slow-twitch readily  red f i b e r s , innervated by small diameter axons which are  excited and  comparitively low  discharge  frequently for long periods of time with  f i r i n g rates, are essential  for the high endurance of  \u000C- 4 postural muscles. and  t h e i r Z l i n e i s broad.  myosin  ATPase  stable  Their oxidative enzyme a c t i v i t y i s high, and  activity  Histochemically, is  They have sparse SR with dyadic and t r i a d i c junctions,  is  slow-twitch  i n acid  and  low  (Drachman  and  Johnston,  1973)\u2022  f i b e r s exhibit myosin ATPase reaction  labile  in alkaline  preincubation  that  conditions.  F i n a l l y , these f i b e r s have a slow contraction time, low maximum tension, a slow speed of shortening, and a high resistance to fatigue. A  third  fiber  type  has properties  intermediate  to these  but i t s  physiological properties are more t y p i c a l of fast f i b e r s . Denervation During Myogenesis Compared denervation with  more  with  denervation  on developing severe  changes  slow muscle  being  metabolic  into  1977).  nerve  enzymes,  fast  muscle,  r e s u l t i n g i n impairment  the  and  regulatory  (Engel slow  denervation  e a r l i e r and  at t h i s  and Karpati,  muscles  proteins,  with  membrane  of  of the development fast  sensitive  1968) and loss of  respect  to Z  properties,  tension, contraction times, physiological maturation of the reticulum  effects  With complete maturation into  dependent,  stage r e s u l t s i n severe atrophy differentiation  mature  muscle have been shown to occur  process (Kumar and Talesara, and  of  and resistance to fatigue (Dhoot and Perry,  band,  maximum  sarcoplasmic  1983; McArdle and  Sansone, 1977; Hanzlikova and Schiaffino, 1973; Lowrie et a l , 1982).  In  fast twitch muscle many of the above parameters return i n the d i r e c t i o n of the slower immature muscle. The Purpose of the Study Mature muscle has been shown to be dependent on i t s innervation for maintenance of i t s fast or slow twitch properties i n studies using reinnervation,  inactivation  by  tenotomy,  casting,  cord  cross  section,  \u000C- 5 denervation,  and  Salmons  Vrbova,  Although  and  little  electrical 1969;  susecptible  innervation purpose  contractile  of  to  during  of  properties (EDL)  deprived  of innervation  muscle.  This  modulated  study  during  was  to  or  1960b;  Bressler, in  1985).  developing  indications are,  a  higher  slow  characterize  developing  and  denervation  with  fast  a l . , 1960a;  i t is  dependency  characteristics. the  histochemical  fast-twitch extensor  on The and  digitorum  compare these properties with those of muscles prior to f i n a l  identifies  development  important information  Webster  procedures  of the  and  1973;  c o n t r a c t i l e properties,  these  study  (Buller et  i s available on  acquisition of  this  longus muscle  Riley,  information  muscle, p a r t i c u l a r l y more  stimulation  in  differentiation  some of the  the  mouse  factors which are  neurally  (C57/BL6J+/+) and  provides  for developmental studies  of the oogenic s t r a i n (C57/BL6Jdy2j/dy2j).  into fast-twitch  of the  dystrophic  mouse  \u000C- 6 -  II  REVIEW OF THE LITERATURE  \u000CNerve Dependency i n Early Myogenesis Muscle  development  begins  when  premyoblast  cells  cease  dividing,  become myoblasts and begin to synthesize c o n t r a c t i l e proteins.  They then  fuse to form myotubes, the precursors to muscle c e l l s and f i n a l l y mature into their adult form. Although myoblasts are capable of synthesizing c o n t r a c t i l e  proteins,  they cannot f u l l y mature into fast and slow muscle f i b e r s i n the absence of  nerves.  culture  In  from  mouse  9.5  day  embryogenesis, somites.  c e l l s cultured from 8.5 conditioned premyogenic from  with  therefore no same  explants  of  nerve muscle were  and  Vivarelli  spinal  cells  occur i n  (1985) found  to produce myosin  cord.  They  able  these  explants  contact. to  Ecob  culture  showed  no  and Whalen  myotubes  only  i n the  (1985) grafted membrane  and  quail  limb  found  accumulate . early gestation,  presence  but  that  forms  only  of  of  spinal  buds  to  grafts  cord. host  myosin  when dissected  light  produce the more mature forms; MLClf and occurs  in  muscle  cell  culture  without  synthesize mature forms of c o n t r a c t i l e  the and  isoforms, Konigsberg  neural  tube  by  16  days  could  they  (MLClf)  neural  and  chorioallantoic  exclude  chains  to include  and  embryo  to  released  striations  Merrifield  dissected  that  (1985), using  with  chick  unless  outgrowth  contractions coincident with the presence of adult fast myosin but  that  concluded  conversion requires presence of factors  Furthermore,  system,  Cossu  positive  day mouse embryos f a i l  to myogenic  nerves.  myosin  tube  MLC3f. Synthesis of myofibrils innervation  proteins.  but  Matsuda  cells  cannot  and Strohmann  (1984; Strohmann and Matsuda, 1985) found that s a t e l l i t e c e l l s from fast and  slow  culture  muscle  that  identical  were  synthesize  culture  able  to  produce  differential  regenerating  forms  of  conditions although they could  myotubes  embryonic  in  myosin  cell under  not mature beyond  the  \u000C- 8 embryonic  stage.  This  suggests  that  there  remain  some  pre-programmed  differentiation capabilities in satellite c e l l s . In-vivo  conditions are  likely  very  different.  Because  i t has  not  been possible to study early aneural development in-vivo, regeneration i n the  absence  of  dedifferentiation al.  innervation of denervation  (1976) autografted  causing  guinea  regeneration.  reinnervating  soleus  They  has  been  with  that of regeneration, Gutmann et  pig  soleus  then  regeneration shows the biphasic decrease soleus  suggesting  dedifferentiation  problem of early results  differ  whereas  nerve  with  in  back  compared  following neonatal  developing  used.  nerve  To  into  depencency  experimental  i t s own  regenerating crush.  as  complete  of developing models  and  site  to with  found  that  They  i t is  in  the  soleus  i n contraction times  reinnervating soleus  i s not  compare  typical  monophasic,  denervation.  muscle  The  i s complex,  conditions  of  under  as  study.  Matsuda and Strohmann (1984) found normal d i f f e r e n t i a t i o n of myosin heavy chain and the  l i g h t chain isoforms occurs  chick,  however  repression  of  following forelimb denervation  immature  forms  of  tropomyosin  of and  troponin does not occur. It can be seen that innervation i s essential  to the early  and continued maturation of muscle during early development. understand  the  changing  characteristics  of  developing  survival  In order to muscles  that  coincide with the onset of innervation, i t i s necessary to review what i s known  about  muscle  development,  beginning  at  the  early  stage  of  embryogenesis but concentrating on the period of maturation into fast and slow-twitch muscle. Myogenesis: Independent Stage Myogenesis can be described i n two independent, the  later  stages. The early stage i s one  of a coordinated,  development.  The  of  independent  \u000C- 9 stage begins with the premyoblast  derived from mesoderm.  In the chick,  somites grafted from any location i n the embryo are capable of providing muscles  for  development  a is  particular  limb  independent.  bud,  indicating  Mesenchymal  muscle, migrate into the limb bud.  that  cells,  this  stage  predetermined  The number of c e l l s  it  unlikely  that  these  cells  can  carry  with  to  be  that migrate i s  small compared with the number of eventual muscles that w i l l making  of  be formed,  them  specific  instructions about the i n d i v i d u a l f i b e r types of muscle they w i l l become (McLachan and and  Wolpert,  1980).  produce a dorsal and  then  stop  myotubes.  dividing  and  The  ventral begin  to  presumptive  muscle c e l l s  block of myoblasts. fuse  with  other  Their nuclei are c e n t r a l l y located, and  f u l l length of the muscle block.  proliferate  These  myoblasts  myoblasts  they w i l l  to  form  extend  the  This can occur i n the absence of nerves.  Coordinated Myogenesis: Dependent Stage (a)  Connective Tissue Influences  A coordinated stage of development now  begins,  and  the  surrounding  connective tissue divides the muscle block providing a template final  muscle  configuration of the  limb.  In the  for the  chick, i f there  is a  disruption of the surrounding connective tissue, for example the altered length of presumptive  bone, the muscle w i l l  accommodate i t s development  to be appropriate to the connective tissue change (McLachlan and 1980).  I f the p o l a r i z i n g region of connective tissue  from  Wolpert,  the t i p s  the d i g i t s i s duplicated, muscles appropriate to that connective  of  tissue  w i l l induce muscle duplication as w e l l . (b)  Neural Influences  In the r a t , pathfinder neurons grow toward  the  earliest  developing  muscle f i b e r s and remain i n apposition with these primary myotubes from  \u000C- 10 the 15th to the 19th day of gestation before forming functional endplates (Rubinstein  and  developing This  Kelly,  cells  cluster,  satellite  (secondary  described by  fibers,  myosatellite c e l l s , myoblast  1981).  Adjacent  myoblasts) Ontell  myotubes  at  to  form  (1977),  the  primary  within  a  consists  different  of  length of the  cluster.  primary  fibers,  development  a l l within the same basal lamina.  full  myotubes,  muscle  of  stages  w i l l become innervated while secondary  have extended  the  Only  the  and  primary  myotubes wait u n t i l  muscle,  separated  away  they  from  the  cluster, and formed their own basal lamina. The  pathfinder  neuromuscular refractory more  are  junction  neurons  of a  the  converge  polyneuronal  i n competition  releases  and  establishes  the  remainder  location  of  the  with  proteolytic  and  innervate  neuromuscular  terminals of  enzymes  the  discouraging  the cell  future becomes  Subsequently, myotube  junction.  other  of  muscle  to innervation (Obrien et a l . , 1978).  motor  formation  neuron  inducing  While  axons the endbulb  two  the  or the  neurons  muscle membrane formation.  The  competing endbulbs are not from the same neuron, but they have been shown to have common conduction properties (Thompson et a l . , 1984).  Retraction  of  axon  unsuccessful  terminals  occurs  and  ultimately  innervate any muscle f i b e r (Riley, 1981). (1978)  used  electrophysiological  ultrastructural  evidence  only  one  In rat soleus, Obrien et a l . ,  (multiple  to show that this  endplate process  potentials)  occurs  between 9 and 13 days, being complete by 18 days of l i f e . innervate a group of myofibers unit. units,  The  innervation.  not  decrease  and  postnatally, One  axon w i l l  forming a functional unit c a l l e d a motor  t o t a l number of functional motorneurons, and  does  will  during  the  elimination  therefore motor of  polyneuronal  Therefore the number of f i b e r s contributing to the tension,  \u000C- 11 and  thus the tension  a l . , 1976) to  developed per  motor unit, must decrease  (Brown et  as each motor neuron reduces i t s number of peripheral synapses  achieve  the  mononeuronal  stage.  This  process  must  decreased a c t i v i t y by tenotomy delayed i t s completion  be  myogenic;  (Riley, 1978)  but  nerve crush with subsequent reinnervation did not (Brown et a l . , 1976). D i f f e r e n t i a t i o n into Fast or Slow-twitch Muscle Mature  whole  muscles  are  heterogeneous  and  their  properties depend on their predominant f i b e r type. theory  that  the  neuron  dictates  fast  or  slow  physiological  In keeping with  the  c h a r a c t e r i s t i c s of  the  muscle, motor units are homogeneous with respect to neural behavior, fast or  slow f i r i n g  1981)  and  Sreter,  pattern  fast or slow-twitch  with  (Engel  the  1964;  and  i s required and  change  1972;  results  1972), enzyme a c t i v i t y  (Nemeth et a l . ,  muscle f i b e r type (Close, 1972;  Jolesz  and  for  slow-twitch  1981)  Innervation muscle  (Close,  Karpati, 1968; from  in differential protein  Shafiq  polyneuronal  Rubinstein  regulatory  maturation  and  to  into  and  et a l . , 1972).  mononeuronal  K e l l y , 1978;  Jolesz  development of organelle synthesis,  fast  This  coincides  innervation  and  Sreter,  (Close,  1981)  and  structure, c o n t r a c t i l e  enzyme production  and  activity,  and  c o n t r a c t i l e properties appropriate to fast or slow innervation. Muscle development i s not Shafiq  et  a l . , 1972).  In  myotube stage at b i r t h and life  the  complete at b i r t h rat  the  majority  (Gutmann et a l . , of  f i b e r s are  1974;  in  the  this number declines u n t i l the second week of  to 10 to 20% at ten days (Engel and  Karpati, 1968).  Type I f i b e r s  are larger at b i r t h , but i n fast muscle the type II f i b e r s soon eclipse the  slow  fiber  in  size.  Growth  satellite  cells  and  incorporation  i s accomplished of daughter c e l l s  by  the  division  into the  of  muscle to  \u000C- 12 are  incorporated  myotube larger  and  (Moss and diameter  synthesize  Leblond  and  proteins  1971).  a rapid  The  to  elongate  neurons  conduction  of  velocity  the  fast  and  developing  fibers  firing  have  a  frequency,  while a neuron to a slow f i b e r i s small and conducts more slowly, (a) Ultrastructure D i f f e r e n t i a t i o n into fast and slow-twitch f i b e r s brings about changes appropriate  to  that  function.  ultrastructural  changes  tubular  in  system  transverse  are  mouse  loosely  EDL  the surface area of the  remaining  triad SR  five-fold  less  than  one  in  EDL  half  in  (SR)  and  soleus.  At  a  described transverse birth  and  same i n fast-twitch  the volume of the SR  two-fold  that of EDL.  in  By  soleus  15  days  junctions per  organized SR network.  sarcomere at  found  no  at  740A  Al  The Z l i n e width was  to 790A but by maturity the EDL remaining  the  into  adult  Z l i n e had form.  difference u l t r a s t r u c t u r a l l y  sarcotubular  system  incomplete  reduced  soleus  age  mature  of  between the  fields  are  subdivided  with  by  a  fast  Schiaffino and  two  highly 740  soleus (1973)  slow twitch  The Z l i n e s are broad and the SR no  triads,  junctional cisterns, coupling with the sarcolemma. myofibrillar  with  to 370A with  and  and  with  similar at b i r t h at about  Hanzlikova  muscle at 18 days gestation i n the r a t . and  junction, and  The  increases  patterns are forming with l o n g i t u d i n a l l y orientated t-tubules forming triad  the  random interconnecting  junctions with the SR.  i s the  muscle, but with maturation  approximately  and  (1971)  They had a longitudinal and transverse  orientation, and form dyads and a few  slow-twitch  Atwood  reticulum  organized  network around the A and I bands.  volume and  and  i n sarcoplasmic  developing  tubules  Luff  the  only  peripheral  With d i f f e r e n t i a t i o n ,  sarcoplasmic  reticulum  and  \u000C- 13 discrete myofibrils form. Soleus  develops  somewhat l a t e r  differences between fast narrow,  the  These extend the entire length of the muscle.  sarcoplasmic  and  than  EDL.  myofibrils,  content being  and  fast-twitch elaborate  In EDL,  fast  i n mitochondrial content  muscle, slow red  fibers  system,  red and  are  sarcotubular system, high  Z bands are  and  mitochondria  greatest diameter,  sarcotubular while  clear  In fast-twitch muscle, fast white  have the  complex  narrow Z l i n e s  higher  weeks of age,  reticulum i s more elaborate  (anaerobic) ultimately  packed  two  slow muscle emerge.  less abundant (Luff and Atwood, 1971). fibers  At  most  low  fibers  densely  mitochondrial  are  similar  have narrower  Z  except  lines.  generally smaller with  mitochondrial content  a  and  In less  thick  Z  lines. (b)  Sarcoplasmic Reticulum and Calcium Binding Protein Changes In  adult  a  comparison  fast  and  of microsomal  slow-twitch rabbit  fraction  from  muscle, the  both developing muscle types strongly resembles muscle.  The  105,000  Mr  Ca  2 +  transporting  protein  during  (Zubrycka-Gaarn binding  and  proteins,  differentiation Sarzala, in  not due  1980).  immature  i d e n t i c a l with those from  into  ATPase  and  slow  fast muscles.  to a l t e r a t i o n i n isoforms.  properties  of  SR,  immature  composition  and  fast  showed muscle,  and from  45,000  that are  Mr.,  activity  twitch these  muscle calcium  immunologically  This indicated the changes are  They concluded  muscle  day)  The amount and  mature  They  (4  that of adult slow twitch  Calsequestrin, are low i n a c t i v i t y and quantity. increases  immature  i s more  closely  that, i n respect to related  to  mature  slow-twitch than fast-twitch muscle. (c)  Metabolic Enzyme D i f f e r e n t i a t i o n Muscles  show uniform  moderate  oxidative enzyme  activity  at  birth  \u000C- 14 (Reichmann and Pette, 1984a).  At about two weeks of age, a wide spectrum  of enzyme levels are seen, which have been categorized loosely fiber  types: fast  slow oxidative nature  oxidative g l y c o l y t i c  (SO) by Peter et a l . (1972).  of muscle probably  rapidly  to  (FOG), fast  i s i t s most  immobilization  (Melichna  The  labile and  to relate (Wirtz  glycolytic aerobic  property  Gutmann,  stimulation and disease (Riley and A l l i n , 1973).  into three (FG) and  or anaerobic  responding  1974),  electrical  Attempts have been made  f i b e r typing by oxidative enzymes with myosin ATPase  et  a l . , 1983),  but  the  relationship  very  i s unreliable  staining (Guth  and  Samaha, 1973; Brooke and Kaiser, 1974), p a r t i c u l a r l y between  f i b e r types  in  a l . , 1982;  different  Reichmann utilize  species  and the  aerobic.  Pette,  (Yellin 1982;  anaerobic  and  Guth,  1984b).  pathway  1970;  Green  In general,  while  the  et  the fast  slow  red  white  fibers  fibers  are  more  Fast-twitch red f i b e r s generally are equipped to u t i l i z e both  energy pathways. (d)  Myosin ATPase d i f f e r e n t i a t i o n Guth  activity, measured  and  Samaha  measured  (1972)  biochemically,  histochemically,  with  surae muscle of the rabbit. muscles,  most  examined  still  50% of adult  toward  alkaline  that  i n both  myofibrillar  i n the fast  stage,  stain  state  of alkaline  exhibit  preincubation.  ATPase  staining,  twitch  triceps  fast-twitch  intensely  f o r myo-  The myosin ATPase a c t i v i t y increases  stability  In addition,  intense  the  ATPase  l e v e l by 10 days of age while the staining  the mature  type  seen  At b i r t h , newly d i f f e r e n t i a t e d  i n the myotube  f i b e r s s t i l l lack reversal. slow  and  development  f i b r i l l a r ATPase at pH 9.4 and 4.2. to  changes  staining  and acid  progresses  lability,  some  there were a few f i b e r s of the after  acid  as  By 21 days of age, a l l f i b e r s  well showed  as  after  reversal  \u000C- I n consistent with the fact that a l l mature f i b e r s contain exclusively fast or slow myosin. was  The routine myosin ATPase reaction used i n early studies  incubated  equivalent dark  at  with  staining  correlate  by  9.4.  dark  It  ATPase  Guth and  Samaha  a c t i v i t y biochemically and  muscle i s low.  fast-twitch muscle  slow  activity (1972).  developing  muscle.  ATPase The  (Barany They  and  activity  contradiction  of  times,  to  Close,  measured  the  shown  1971),  myosin  was  ATPase  of  developing  fast  twitch  be explained by the existance of three d i f f e r e n t  f e t a l , neonatal and  fast adult, found i n  (Whalen et a l . , 1981)  and  muscle, compared  with  concurrs  developing  with the  presence  a l k a l i n e preincubation,  exclusive  staining seen  found  i n mature  Thompson et a l . (1984) found that the lack of reversal i s not  due to mixed innervation of a single f i b e r during the polyneuronal as the neurons at one also  was  found that, i n contradiction to intense myosin  of myosin ATPase staining i n both acid and in  that  contraction  myosin ATPase a c t i v i t y  This may  myosin heavy chains:  thought  f i b e r s with  myosin  ATPase staining, the  was  staining, histochemically.  immature  with  examined  pH  lack  reversal  junction are of the same type. in  development  but  show  stage,  Slow-twitch f i b e r s  reciprocal  staining  to  (actin  and  fast-twitch f i b e r s i n mature slow muscle, (e)  Contractile Protein Isozymes Sequential  myosin)  isoforms  and  synthesized  regulatory at  differentiation  of  the  proteins  embryonic, (Matsuda  major  c o n t r a c t i l e proteins (troponin  neonatal  and  and  Strohmann,  consists of 6 subunits: two heavy chains weight and  two  pairs of l i g h t chains  20,000 (Julian et a l . , 1981). light  chains,  one  dissociated  Adult with  and  tropomyosin)  adult  1984).  stages  The  of  myosin  are cell  molecule  (HC) of 200,000 dalton molecular  (LC) with a molecular weight around fast muscle contains DTNB  called  light  two  classes of  chain  2  fast  \u000C- 16 (LC2f) and two that  can be removed  by a l k a l i  treatment  chains\" referred to here as l i g h t chain one fast three fast (LC3F).  \"alkali  light  (LClf) and l i g h t  chain  Slow muscle has l i g h t chain one slow (LC1S) and l i g h t  chain two slow (LC2s) which migrate to the same position as L C l f and LC2f on the SDS polyacrylamide g e l electrophoresis. to  fast  Kelly,  (anti-fast) and slow  Using antibodies  (anti-slow) adult  1981) and SDS g e l electrophoresis  myosin  raised  (Rubinstein and  for identification  of l i g h t  chains, Rubinstein and Kelly (1978) followed the developmental changes of myosin  isozymes  i n fast-twitch  EDL  muscle  of the r a t . At  gestation, a l l f i b e r s were primary myotubes had positive  15  days  staining with  antibodies against fast myosin and to l i g h t chains LClf, LC2f as well as an embryonic form of l i g h t chain 1 (LClemb). rat,  some secondary myotubes appeared  but neuromuscular  infrequent and primative endplates formed secondary  fibers  stained  By 17 days gestationin the  only  only with antibodies  contacts  were  on primary f i b e r s .  The  against  fast  myosin and  fast l i g h t chains, whereas primary myotubes stain with those against slow myosin as well. and  At 18 days gestation, v e s i c l e s present i n axon endbulbs  the postsynaptic  membrane  functional neuromuscular By  19 days gestation  began  junction.  larger  remain  secondary myotubes had separated from the c l u s t e r  anti-fast  At b i r t h myosin  into  the presumptive antibodies  and  intensely with anti-slow myosin. of age; fast  These authors suggested that primary  as slow-twitch f i b e r s  i n number, d i f f e r e n t i a t e  muscle.  fibers  of a  This coincided with f e t a l movement.  and acquired primative innervation. myotubes  to show the s p e c i a l i z a t i o n  stained  while  secondary  the predominant fast  fibers  fiber  stained  the presumptive  myotubes,  slow  much  type of the  primarily fibers  with  stained  The adult form was achieved by 21 days  exclusively with a n t i - f a s t  and slow  fibers  \u000C- 17 with  anti-slow  purified  myosin.  through  fast-twitch  Using  polypeptide  multiple absorptions,  rat gastrocnemius  with  analysis  Whalen  myosin  et  and  and  antibodies  a l . (1981) examined  found  there  are  three  isozymes of fast myosin heavy chain.  In further studies with polyclonal  antibodies to embryonic, neonatal and  fast myosin, Whalen et a l . (1985)  found coexistance of neonatal and adult myosin i n the same f i b e r s . showed that the t r a n s i t i o n  This  from neonatal to adult myosin i s asynchronous  and occurs within the same f i b e r , not as a r e s u l t of an increased amount of adult myosin i n one f i b e r type.  He referred to these as, myosin heavy  chain embryonic (MHCemb), myosin heavy chain neonatal (MHCneo) and myosin heavy  chain  fast  (MHCf).  findings using antibody  Sartore  to bovine  et  a l . (1982) concurred  with  f e t a l myosin i n rat muscle.  these  Sartore  (1982), using an antibody s p e c i f i c to f e t a l bovine myosin found neonatal and in  f e t a l myosin heavy chains i n fast muscle to be d i f f e r e n t slow muscle, i n spite of i d e n t i c a l  Whalen  et  myosin used  a l . (1981) suggested by Rubinstein and  that  Kelly  from  those  migration on  pyrophosphate  gels.  the  against  antibody  (1978) may  and  Kelly  possibility  (1985) have used  (Rubinstein and  non-denaturing  myosin to follow developmental  Kelly and Rubinstein  Kelly,  1981).  Rubinstein  pyrophosphate gels of native  changes i n whole myosin isoforms i n normal  and denervated rat soleus and EDL.  They found EDL  synthesizes f e t a l and  embryonic form of myosin f l - 4 followed by mature forms FM1-FM3. denervation did not a l t e r t h i s pattern. with  denervation  did.  gastrocnemius,  found  neonatal  myosin  fast  have cross reacted with  neonatal heavy chains i n the developing fast f i b e r . acknowledged t h i s  adult  Butler-Browne  a l l developing and  Neonatal  However, hypothyroidism combined  et fast  a l . (1982), fibers  weakly  working  stained with with  with  rat  anti-fast anti-slow  \u000C- 18 By 14 days of age, 25% of the fast f i b e r s stained with  myosin as w e l l .  antibodies to neonatal myosin, 25% to adult myosin and 50% with both. Whalen et a l . (1979) also found an embryonic form of l i g h t chain one (LClemb),  which  changed  to adult  form  prior  to the heavy  transformation. This indicates that the synthesis o f the embryonic chain  i s not necessarily concurrent  with  that  chain light  o f the heavy chain.  He  showed the developmental pattern reproduced i n Table I.  Table I  Myosin Isoforms i n Fast-twitch Muscle Development:  fetal  7-11 days of age  Adult  MHCemb  MHCneo  MHCf  LClemb-LClf  LClf  LClf  LC2f  LC2f  LC2f  LC3f  LC3f  from the work o f Whalen et a l (1979).  In contrast to the r a t , fast f i b e r s i n the developing chick have been found to contain slow l i g h t al.,  chains  1981; Crow and Stockdale,  (Gauthier  1984).  et a l . , 1978; Stockdale et  However, Gauthier  et al.(1982b)  found an embryonic LC i n both the chicken  and the r a t which they  felt  cross-reacted with adult slow l i g h t chain.  Syrovy and Gutmann (1977) i n  rat EDL and J u l i a n et a l . (1984) i n rabbit psoas, found an increase i n LC3f during development. Very l i t t l e has been reported on the development o f a c t i n isoforms i n developing synthesis  skeletal from  muscle.  Caplan  non-myofibrillar beta  alpha a c t i n during muscle maturation.  (1983)  describes  and gamma a c t i n  a  change i n  to systhesis o f  \u000C- 19 (f)  Regulatory Dhoot and  Protein Isoforms Perry  (1979; 1983b) looked  at isoforms  proteins: tropomyosin and  troponin I, T and  myosin  to  ATPase  stimulation.  responds  for  intracellular  contraction.  Dhoot and  Perry  changes  seen  antibodies raised  against  fast  C.  time  slow  fibers  isoforms.  In  change,  the  switching  chicken,  (beta) which i s repressed  course  troponin,  type I c e l l s stain for both fast and slow forms. the  there  of  the  muscle  fast and  and  During  exclusively i s an  during  At b i r t h , a l l f i b e r s  forms of  way  reticulum, could be  (1980) found that there are  forms of tropomyosin and troponin I,T and with  in  calcium  the a c t i v i t y of  the e f f i c i e n c y of the sarcoplasmic developmental  regulatory  which determine the  These regulatory proteins, along with  myosin ATPase and responsible  increased  C,  of the  to  immature  slow stain  presumptive  differentiation,  synthesis form  of  of  slow  tropomyosin  during development to produce only the mature  form (alpha) (Matsuda and Strohman, 1984). Summarizing to t h i s point, there are d i s t i n c t developmental forms of myosin heavy chains and an embryonic form of the l i g h t chain, which are sequentially fast-twitch begin  by  expressed  in  muscle, the  developing  remaining  fast  muscle.  l i g h t chains  and  synthesis of the fast adult forms with  In  developing  regulatory  proteins  the major change  being  the increase i n synthesis of the proportion of LC3f. (g) The  Contractile properties work of  slow-twitch  Close  (1964) describes  contractile  properties  with  fully  the  changes  development.  i n fast  and  A l l muscles  are  i n i t i a l l y slow to contract and produce only small amounts of tension. development increased  proceeds, tension  with  both  fast  decreased  and  slow-twitch  contraction  time  muscles and  As  generate speed  of  \u000C- 20 shortening,  more so i n muscles subjected  to phasic  than tonic a c t i v i t y .  Fast-twitch muscle i n i t i a l l y has slow membrane properties i n common with slow-twitch transforms  muscle  (McArdle  to a posess  et a l . , 1980),  a higher  but with  resting membrane  differentiation,  potential, a  higher  concentration gradient of sodium, and a higher overshoot and maximum rate of  rise  of the action  potential.  The action  potential i s therefore  propogated more quickly into the increasing network of t Tubules and SR. The release of calcium into the muscle i s then accomplished sooner from a more abundant SR r e s u l t i n g i n the capacity  to produce a greater  l e v e l of a c t i v a t i o n i n mature fast-twitch muscle.  These membrane changes  r e s u l t i n an increased rate of both release and sequestering thereby decreasing  higher  of calcium,  the contraction time of the muscle i n both, i t s time  to peak twitch tension  (TTP) and i t s rate of relaxation, conventionally  measured from the time of peak twitch tension to that of one h a l f peak twitch tension  (1/2RT).  In mature fast-twitch muscle, the m y o f i b r i l l a r  ATPase has a higher a c t i v i t y  (Guth and Samaha, 1972) and, along with the  fast forms of the calcium switch  (Troponin I, T, C, and tropomyosin), the  s i t e s f o r myosin binding on a c t i n are made available more r e a d i l y . rate of a v a i l i b i l i t y of a c t i n binding s i t e s couples release The  and sequestering  of calcium  a c t i n and myosin binding. stimulation  the contraction  time.  of calcium and the a v a i l a b i l i t y and rate of In the case of tension produced by  (tetanic contraction)  the sarcoplasm and the a c t i n  tension i s maximal. by  the more rapid  tension that can be produced i n a muscle depends on the balance of  the release and sequestering  in  to decrease  with  This  the presence of calcium and myosin binding,  repeated  i s maintained  and therefore the  The tension following a single impulse i s influenced  the functioning of the membrane  properties,  the m y o f i b r i l l a r  ATPase  \u000C- 21 and the calcium regulatory processes described. In  contrast  to  slow-twitch  muscle,  with  maturation,  fast-twitch  muscle becomes capable of exhibiting potentiation of the twitch following tetanic  stimulation.  potentation  It  has  been  proposed  (PTP) i s a r e s u l t of the phosphorylation  S t u l l , 1979; 1982; Houston et a l , 1985). i s calcium  that  post-tetanic  twitch  of MLC2 (Manning and  The c a t a l y i s t for t h i s reaction  dependent myosin l i g h t chain kinase and the dephosphorylation  i s c a t y l i z e d by phosphorylase. At b i r t h , fast-twitch muscle, with i t s aerobic metabolism, i s highly resistant to fatigue (Close, 1964).  With d i f f e r e n t i a t i o n the fast white  f i b e r s become more g l y c o l y t i c and lose t h e i r resistance to fatigue, (h)  Summary Before  properties  differentiation, i n common with  the  immature  mature  fast-twitch  slow-twitch  muscle  muscles.  has  These  many  include:  c h a r a c t e r i s t i c s of Z l i n e width, SR and m y o f i b r i l l a r density, content and a c t i v i t y of metabolic,  SR and myosin ATPase enzymes, membrane properties  and c o n t r a c t i l e c h a r a c t e r i s t i c s of contraction times, maximum v e l o c i t y of shortening similarity chains  and resistance to fatigue. of  myosin  i n developing  isozymes, muscle  The myosin heavy chain subject  regulatory  with  isoforms  The puzzling contradiction i s the  those  proteins  of mature  myosin  suggested by Kelly and Rubinstein  light  fast-twitch muscle.  i n early development are s t i l l  to i n t e r p r e t a t i o n but immunologically  explanation  and  appear  to be  (1978) that  somewhat  fast.  fetal  The  muscles  begin fast and are p h y s i o l o g i c a l l y slow because of s e l e c t i v e innervation of  slow primary myotubes, does not give  important  properties  in  developing  adequate weight  muscle  which  do  to the  other  begin  with  \u000C- 22 c h a r a c t e r i s t i c s of slow muscle. Models of Neural Regulation i n Muscle P l a s t i c i t y Much evidence has differentiation  and  been  accumulated  maintainence  properties are nerve dependent directed  to test  this  1981; Kelly, 1984).  to support the theory that  of  muscle  fast  and  slow-twitch  and a large number of studies have been  theory (reviews Gutman, 1976;  To understand how  Jolesz and  Sreter,  these manipulations contribute to  the theory i t i s useful to look at these models of neural input. control of muscle development  the  In vivo  occurs v i a the following mechanisms.  The  f i r s t mechanism i s through a c t i v i t y imposed on the muscle, v i a a c t i v a t i o n at  the neuromuscular  sarcolemma.  The  junction, and  the r e s u l t i n g  depolarization  second i s by a proposed neurotrophic  factor which  been described by Gutman (1976) as long-term maintenance mediated by nerve impulses. to  address  these two  of the has  regulation, not  Many models of neural input have been used  influences  and  assess the  contribution  of each.  There i s some question as to whether muscle a c t i v i t y i s a neurogenic or a myogenic  influence.  Certainly,  i n vivo  activity  i s mediated  by  the  nerve, although the changes themselves may be induced by something within the muscle. regulation activity reduced  The attempts to sort have been  resulting  approached  out the nature and extent of neural  i n eight  from a c t i v i t a t i o n  v i a cordotomy  which  by  i s usually  major  ways.  the central accompanied  Firstly,  nervous by  muscle  system i s  section  of the  dorsal nerve root to eliminate the input from the stretch r e f l e x , a r i s i n g from  the muscle  spindle,  and  render  the muscle  totally  silent.  This  leaves the motor neuron intact to continue i t s neurotrophic influence. second method, denervation, interrupts a l l nerve functions.  A  This can be  accomplished by crushing, or sectioning of the nerve and the r e s u l t s vary  \u000C- 23 dependending (Finol  et  on where the nerve a l . , 1981).  activity  factors.  immobilization activity  eliminates both  decreased  or tenotomy has  been used.  on how  may,  or may  not,  to i t s muscle  neurotrophic  muscle  activity  Neither of  the  (Bray  et  amount al,  of  Changes  factor  in  Nerve  muscles  Colchicine  influence substrates  as  or  Vinblastine  i t i s reasoned  are  is  used  that  the  transported down  the  i n t e r f e r e with conduction of the nerve at the neuromuscular Substances,  at  nerve  properties  are  (Melichna and  Fourthly, some studies have employed the use of chemical  paralysis which comes i n two major forms. by  impulses  released  influenced by the muscle length at which they are produced Gutman, 1974).  eliminate  change neurotrophic influence  neurotrophic  1979).  and  through  these  much that depends on neural a c t i v i t y .  influence  terminals  method  i s made r e l a t i v e  Thirdly,  completely and  depending could  This  section  such  toxin,  a c t i v i t y has been altered by changing  been shown to be  to  control  for  neurotrophic  axon.  These  impulse.  neurotrophic  substance substances  Alternately,  or i t s do  not  paralysis  junction has been used to exclude muscle a c t i v i t y .  as botulinum  muscle by e l e c t r i c a l  Paralysis of axonal transport  stimulation.  have  been used.  Fifthly,  muscle  the pattern of activation  of the  Because the pattern of a c t i v i t y  a more important  factor  than  the amount of  has  activity  (Jones, 1981; Hennig and Lomo, 1985), attempts have been made to mimmick the behavior of either the fast or slow nerves. a c t i v i t y without the neurotrophic influence.  This i s done to assess  The majority of e l e c t r i c a l  stimulation has been applied i n d i r e c t l y v i a electrodes implanted next to the nerve which may  then s t i l l  the nerve i n response to i t s own 1980).  include a trophic  substance  secreted by  f i r i n g pattern (Eldridge and Mommaerts,  Others have used direct muscle stimulation both with or without  \u000C_ 24 denervation.  The assumption  neurotrophic addition  influence  that  electrical  has not been  proven.  of substances to the muscle  neurotrophic  that  stimulation The sixth  are thought  factor produced by the nerve.  excludes the model  i s the  to include the  These substances usually come  from extracts of s c i a t i c nerve, and are injected into the muscle to be studied, or used i n muscle culture medium.  These extracts have not been  characterized yet, and i t i s not known i f they w i l l be found i n both fast and  slow neurons and, i f so, whether they would be s i m i l a r .  A seventh  model involves the cross-reinnervation o f the nerve from a fast muscle to a  slow muscle, and the converse.  These experiments include  periods of  denervation.  Adding an alternate influence, such as fast innervation to  slow muscle,  i s not necessarily  innervation.  To validate influences of dennervation, an eighth method,  the converse  of removing  the i n i t i a l  that of reinnervation, has been used. This i s usually i n association with gentle  crushing  encourage  or  transplanting  reinnervation.  These  differences between denervation  of  the nerve  studies  have  i n developing  under  pointed  conditions up  some  to  major  and mature muscles.  Neural Regulation of Mature Muscle (a)  Atrophy Muscle atrophy i s reflected  content,  total  cross-sectional  area.  With reduction  spinal  cord,  slow-twitch Kelly  Buller  area  of neural  or s p e c i f i c  activity  et a l . (I960)  fiber  following  found  atrophy  cross-sectional  transection to be  of the  greater  in  soleus than fast-twitch EDL of the r a t , as d i d Rubinstein and  (1978).  transection,  i n a decrease i n muscle weight, protein  Atrophy  was  increased  when,  the dorsal roots were severed  in  (cord  addition  isolation),  the muscle s i l i e n t with the motorneuron i s s t i l l i n t a c t .  to  cord  rendering  Deafferentation  \u000C- 25 eliminates  a l l  influence.  In  found  equal  difference work  of  Buller  et  of  a l .  animals  produced  not  slow-twitch  factor  i n  mixed  cordotomy atrophy 1977)  by  fast  (Karpati  than  Engel,  (Gauthier  1980b),  and  1978;  (slow  oxidative  denervation  Davis  and Kiernan, fibers)  on soleus  et a l .  (1972)  soleus.  Herbison  tenotomy,  that  the  et  been  found a l .  weight  glycolytic  Gauthier  atrophied.  preferential  (1977) loss  found  a n d Dunn Reports  Kumar  i n  was g r e a t e r  firing  and  extent  than  of  i n slow  soleus  greater  twitch  muscles  and  Kiernan,  (Niederle  1973)  denervation, i n  muscle  Karpati and  with  and  type  the effects  and Talesara  atrophy  of  but  and Talesara,  1973;  fibers  i n  muscle  produces  Davis  1981;  Eldridge  neurotrophic  lesser  fast  i n  neuron.  (Kumar  i n  and  cordotomized  a  motor  and A l l i n ,  a l . ,  varied.  on  alone  greatest  et  white  1980a;  the least have  i s  of  neural  tenotomy  (Riley  Carraro  the fast  or  fast  effects  twitch  but to a  Denervation  by t h e  However,  depends  The  conversion  maintenance  reduces  atrophy,  atrophy  1982a; i n  fixation  isolation  type  fast  from an i n t a c t  1968).  between  crossover  that  fibers,  by c a s t i n g  Denervation  particularly  Mayr,  Margareth  or cord  fiber  i n  suggests  fiber  illustrated  Denervation  atrophy  transmission  and slow  Hobbs  i n  skeletal  i s  produce  (1968)  gastrocnemius.  isolation.  cats.  fast-twitch  immobilization  1968).  to  This  and Engel,  o r cordotomy  isolation  producing  isolated  muscle.  as impulse  i n  increase  of  neurotrophic  and Engel  cross-reinervation  able  cord  necessarily  fibers  but not i n cord  further  Immobilization produces  where  not  p i g , Karpati  slow  and cord  were  particularly  as well  and  (I960),  (1980)  but  guinea  was e f f e c t i v e  muscle  integrity,  fast  cordotomy,  Mommaerts  i n  i n  adult  cordotomy  soleus  fast-twitch  activity,  cordotomized  between  presence  and  reflex  atrophy  slow-twitch the  the  (1977)  fibers  of and  of rat  casting compared  I  and with  \u000C- 26 fast-twitch gastrocnemius.  The decrease was due equally to fast and slow  f i b e r s , however, i n Plantaris, the white portion had greater atrophy but it  was  severe  i n the  slow-twitch  atrophy  in  fibers.  Webster and  chronically  denervated  post-denervation, with the wet weight of EDL of  controls.  attributed fibers.  The  to  the  variable  results  from  species variation  Other factors may  of  Bressler mouse  (1985) found  by  210  days  38% and soleus 23% of that soleus  the  have  been  percentage  of  partially fast-twitch  also contribute to atrophy; denervated muscle  maintained i n a shortened position shows greater atrophy than when held in  a extended  (1973)  position  suggest  (Melichna and  denervation  changes  Gutman, 1977). are  found  Gauthier and Dunn  preferentially  in  fast  proteins and  not  from  f i b e r s , while tenotomy produces a slow f i b e r l o s s . Atrophy  results  from loss of muscle s p e c i f i c  loss of i n t r a c e l l u l a r f l u i d largely  due  to  decrease  analysis of denervated correlation  between  sectional area. rate  resulting  Others  have  (Metafora et a l . , 1980).  i n myofibillar  r a t , Engel and  the  decrease  proteins.  Stonnington  in  found  a  proportional  greater  In  and  loss  in  with  protein using  protein has been investigated  synthesis i n denervated l4C-L-glycine,  found  denervation i n adult r a t s .  no  muscle. change  cross  fiber  1979).  area. protein  The  loss  i n quantative studies  Pearlstein in  total  myofibrillar  compared with stromal and SR proteins (Herbison et a l . , of m y o f i b r i l l a r  morphometric  decreased at a slower  increase compared  proportional  a  (1974) found a close  myofibrillar  They showed that the area of SR in  The major loss i s  myosin  and  Kohn  of  (1966),  synthesis  after  However, D. Goldspink (1976) found a decrease  i n myosin synthesis for two days post-denervation followed thereafter by an  increase.  In addition  an  increase i n synthetic  activity  could  be  \u000C- 27 accounted  for by  near  sarcolemma  the  (Grampp  et  an  increase i n the number of ribosomes, and  neuromuscular  a l . , 1972).  Use  of  junction,  actinomycin  D,  particularly  following a  denervation  protein  synthesis  i n h i b i t o r , reduced denervation induced membrane changes i n mouse muscle namely:  development  resistant  action  of  extrajunctional  potentials,  fall  in  cholinergic  resting  receptors,  TTX  potential  and  membrane  slowing of the time course of action potential, but only i f used  within  two days of denervation. To further elucidate the source of denervation atrophy, a c t i v i t y been restored to denervated muscle by e l e c t r i c a l stimulation. Allin,  (1973) implanted  electrodes into  caudal nerves  cats and was  able to reverse the p r e f e r e n t i a l white  both  (50Hz) and  phasic  tonic  (lOhz)  stimulation  Riley  of cord  fiber  has and  isolated  atrophy with  patterns.  The  motor  neuron was s t i l l  intact therefore t h i s could be either a neurotrophic or  impulse  influence.  related  Melichna  and  Gutmann  (1974) reversed  the  atrophy i n denervated fast-twitch t i b i a l i s anterior of the rat by d i r e c t stimulation, axonal  suggesting a  transport,  transmission denervated  produced  rat muscle  transmission found  by  has  role  for a c t i v i t y  colchicine, atrophy  without  which  (Ramirez,  only a p a r t i a l  i n atrophy.  was  1983).  interference less This  contribution  equivalent atrophy i n denervated  muscles  than  with that  indicates  to atrophy. with  Blockage  those  of  impulse found  that  in  impulse  Bray treated  (1979) with  TTX to block impulse transmission. However, i t i s possible that blockage of  impulse transmission could i n t e r f e r e with secretion of a neurotrophic  substance.  In an elegant treatment of t h i s question, Davis and Kiernan  (1980a, 1980b) i s o l a t e d , from rat s c i a t i c nerve, a protein extract, shown to  have  neurotrophic  influence  on  denervated  muscle.  Denervated,  \u000C- 28 immobilized  EDL showed  greater atrophy  than  controlateral  controls, primarily i n type IIB fast white g l y c o l y t i c denervated  immobilized  muscles were injected  became equal i n the two groups.  with  immobilized  fibers.  When the  the extract,  atrophy  This showed immobilization accounted f o r  60% while neurotrophic 40% of atrophy i n denervated  fast twitch rat EDL.  Neiderle and Mayr (1978) found exclusive atrophy of the type I I f i b e r s i n denervated  mature EDI of the r a t , up to 42 days post-denervation, and  this was primarily EDL.  There  may  i n the white be  some  fibers  synergistic  which are type effect  IIB i n the  between  activity  rat and  neurotrophic substances but the multitude of studies indicate  that  are  of muscle  both  important  factors  i n regulation  and maintenance  they  properties i n both fast and slow-twitch muscles, (b)  U l t r a s t r u c t u r a l Changes Engel and Stonnington  atrophy  and u l t r a s t r u c t u r a l  gastrocnemius area  (1974) used morphometric techniques to examine  decreased  changes i n denervated  muscles for 84 days post denervation. i n concert by 80% by 84 days with  m y o f i b r i l l a r area.  There was an i n i t i a l  adult  r a t soleus and  Fast and slow parallel  fiber  changes i n  increase i n absolute number and  proportional volume of mitochondria by 8 days although the mitochondria were smaller i n t o t a l s i z e . they  became  muscle.  reorientated  Aggregation of mitochondria was common and longitudinally,  reminiscent  of  developing  There was an increase i n the number of sarcotubular components  (SR and t tubules) with  focal  and SR membrane disruption.  dilation,  irregular  spatial  arrangements  Longitudinal sections showed an increase i n  SR area, however i n crossection, fast f i b e r s showed a marked decrease i n sarcotubular surface area and slow muscles, a f t e r an i n i t i a l 8 days, also showed a decline.  increase to  The t o t a l sarcotubular area declined but  \u000C- 29 not as rapidly  as  f i b e r area, therefore i n spite of some atrophy,  the  sarcotubular elements showed a net increase compared with f i b e r area. A shift loss  i n dominant f i b e r  of one  fiber  type  or  change, i t i s necessary  type  fiber  could type  such  Dunn  a  a  denervation  of  semitendinosis  shift  rat to  be  toward  homogeneous  semitendinosis.  In  a mixed  composed  f i b e r s with narrow Z l i n e s  They  such  muscle,  suggested  altered  longitudinal  in  a  manner  orientation  of  accumulations  of mitochondria  mitchondria.  The  white  fiber  control  follow  this  of  triads,  following  they  that  of the  fast  slow red  of  white fibers.  dedifferentiation;  reduction  remained  found  (FOG), H0%  red  in  subsarcolemmal  but an increase i n i n t e r f i b r i l l a r  fibers  and  i n the number of  red and  suggestive  type  52%  A decrease  fast  Gauthier  muscle,  a deterioration  that the majority were now  were  To  as Z l i n e width.  intermediate (SO) and 8% white (FG) f i b e r s .  fibers  preferential  to use a f i b e r type c h a r a c t e r i s t i c as a marker  to denervation,  found  either  transformation.  which i s r e s i s t a n t (1973)  represent  were  atrophied,  rows of showing  disruption of SR and streaked Z l i n e s . They  also  ribosomes.  found  an  Although  increase  ribosomes  in  the  number  of  subsarcolemmal  accumulate  around  the  neuromuscular  junction, only i n denervation are they found along the entire surface of the  mature  sarcolemma coinciding membrane. inhibitor, membrane inhibitor,  fiber.  Ribosomes  i n newborn with  have  diaphragm  also  been  found  adjacent  to  the  and  Dunn,  1973)  a  time  of  the  (Gauthier  extrajunctional  acetylcholine  Grampp et a l . (1972) used Actinomycin to  investigate the  changes there  following was  a  role  of  denervation.  reduced  protein  sensitivity D,  a protein synthesis  subsarcaolemmal As  a  at  result  synthesis  of  and  ribosomes the  in  protein  reduction  in  \u000C- 30 the  numbers  of  extrajunctional  potential as well as a f a l l  receptors,  the TTX  resistant  action  i n the resting membrane potential  with denervated, non treated muscle.  compared  I t did not a f f e c t the maximum rate  of r i s e and the amplitude of overshoot of the action potential which are slowed by denervation. were  responsible  They concluded that the subsarcolemmal ribosomes  f o r new  protein  synthesis  contributing  to membrane  changes. (c)  Contractile protein changes The high c o r r e l a t i o n between the rate at which myosin hydrolyzes ATP,  the i n t r i n s i c speed of shortening and the pattern of neural control has resulted  i n a primary research focus on myosin ATPase a c t i v i t y  biochemically, myosin ATPase isozymes of myosin  staining measured  separated  histochemically,  by means of denatured myosin  electrophoretograms or non denatured myosin  measured  separated  and the  on SDS g e l  by pyrophosphate  gels.  In spite o f a c l a r i f i c a t i o n by Guth and Samaha (1972) that myosin  ATPase  staining  stability activity. activity ATPase 1972).  at alkaline  pH i s only  of myosin, i t continues Staining  i s i n some  a reflection  to be referred cases  of the a l k a l i n e  to as myosin  correlated  with  myosin  ATPase ATPase  i n adult muscle, but cannot be considered a measure of myosin  activity, In  biochemically,  particularly  this  thesis,  will  be  i n developing only  referred  myosin to as  muscle ATPase  \"activity\"  (Guth  and Samaha,  activity, and  measured  histochemical  determination w i l l be referred to as \"staining\". In  denervation  myosin are a late (Riley and A l l i n ,  studies  on adult  muscle,  histochemical  changes i n  occurance: by 2 months i n caudal muscles of the cat 1973); by 6 months i n the diaphragm and gastrocnemius  of the cat (Carraro et a l . , 1981); 120 days i n EDL of the r a t (Neiderle  \u000C- 31 and  Mayr,  1978)  denervation  and  often  (Metafora  not  seen  et a l . , 1980;  at  a l l in  the  acute  stage  Margareth et a l . , 1972).  of  In  caudal  muscles (mixed f i b e r type) of cord isolated cats, Riley and A l l i n  (1973)  found i n a c t i v i t y did not change myosin c h a r a c t e r i s t i c s , nor did subequent indirect control  tonic or phasic is  by  denervation and  loss  other  stimulation.  than  impulse  This  suggests the  transmission.  In  myosin ATPase  the  same  study,  of the cord isolated cat muscles led to marked deterioration  of  neurotrophic  ATPase  fiber  control.  type  Carraro  differentiation,  suggesting  et a l . (1981), using  of the diaphragm, produced f i b e r s  chronic  a  Gauthier and  of  denervation  that stain at both acid and  preincubation, i n d i c a t i v e of immature f i b e r s .  loss  alkaline  Hobbs (1982),  using antibodies raised against fast and slow myosin and absorbed against Light Chains to ensure reaction with myosin,  found  diaphragm. and  dual  reactivity  As well these  alkaline  the heavy chain component  a l l fibers  of  the  denervated  of rat  f i b e r s stained for myosin ATPase i n both acid  preincubation,  myosins i n dual  of  only  verifying  staining f i b e r s .  a  coexistance  Both acid and  are required to distinguish between the  of  fast  alkaline  and  slow  preincubation  presence of an adult myosin or  dual staining f i b e r s which could represent  an immature or novel myosin.  A  by  more s e n s i t i v e measure has  raised  against  question  fast  because  or  been  sought  slow adult myosin,  crossreactivity  has  staining with  but  been  has  shown  not  antibodies. myosin,  However, i n a  Carraro  et  al.  study  (1981)  using have  gel shown  loss of the slow myosin  and  slow l i g h t  fetal  this and  fast adult myosin  electrophoresis to measure that,  in  gastrocnemius (posssessing mixed f i b e r types), conversion and  clarified  between  neonatal myosins isolated by Whalen et a l . (1981) and  antibodies  chains  denervated  rat  to fast myosin  occurs.  This  lends  \u000C- 32 support to the theory that fast myosin i s synthesized i n the absence of innervation and that only the slow myosin depends on neural input.  There  was also a decrease i n the LC3 component which could be interpreted that the change i s toward an immature myosin. Barany  and  Close  (1971) showed  that  cross  reinnervation  produced  myosin ATPase a c t i v i t y appropriate to the innervating neuron, i n d i c a t i n g a  neural  influence  on  myosin  ATPase  measured myosin ATPase a c t i v i t y  activity.  i n denervated  Syrovy  et a l . (1972)  fast and slow muscles of  the rat and rabbit and found i t decreased r e c i p r o c a l l y with contraction times  i n a l l but the rabbit  contraction  times.  contraction  times  This may  soleus  shows  be  that  related,  which increased while  myosin  findings  may  reciprocally  ATPase  be  activity  with and  confounded due to  marked species differences i n the response to a p a r t i c u l a r manipulation. Although histochemical myosin ATPase staining changes are a l a t e  finding  following denervation, i t has been suggested  a slow  turnover  of myosin.  Gutmann et a l . rat  EDL.  activity  this reflects  changes were seen by  (1972) as early as seven days post denervation i n mature  Using  anterior,  Yet myosin ATPase  that  indirect  comprised  of  low  fast  frequency  white  stimulation  (IIB) and  fast  of  red  rat  tibalis  (IIA) f i b e r s ,  Mabuchi et a l . (1982) found complete conversion from IIB to IIA f i b e r s by histochemical activity.  staining,  Allin  there  was  no  change  of  myosin  ATPase  Either both fast f i b e r types have the same ATPase a c t i v i t y or  the myosin ATPase suggested  yet  by  staining  Guth and  (1973) found  does not measure  Samaha  that  ATPase a c t i v i t y ,  (1972) i n developing  myosin a c t i v i t y  was  muscle.  has been Riley  and  not changed i n i n d i r e c t l y  stimulated caudal muscles of the cat, and suggested that a neurotrophic factor  was  required  to  alter  myosin  ATPase  activity.  The  lack  of  \u000C- 33 c o r r e l a t i o n between histochemical f i b e r typing and ATPase a c t i v i t y i s not an uncommon finding. Light chains of fast twitch muscles  are primarily  of the fast  and remain fast with denervation (Rubinstein and Kelly, 1978).  type  The high  proportion of LC3 to LCI t y p i c a l l y found i n mature fast white f i b e r s was decreased i n single f i b e r analysis of long term  intermittent stimulated  t i b i a l i s anterior muscles of the young rabbit by Mabuchi et a l . (1982) i n denervated r a t t i b a l i s anterior.  They found type IIB f i b e r s of adductor  magnus to be d i f f e r e n t i n isozyme pattern than type IIA or IIB f i b e r s i n the  tibialis  well.  anterior.  Generalizations  The LC1/LC3 from  fiber  ratio  typing  i s significantly  lower  as  may be a reasonable place to  begin, but extreme care must be taken i n assuming that a l l t h e i r myosin properties are necessarily i d e n t i c a l , (d)  Changes i n Calcium Uptake Ultrastructural  disruption of the SR i s minimal  i n denervation and  atrophy occurs at a slower rate i n SR than i n the o v e r a l l and Stonnington, 1974).  fiber  (Engel  Metafora et a l . (1980) found no u l t r a s t r u c t u r a l  changes i n the SR at 8 days post-denervation i n the r a t gastrocnemius. However  the physiological  pronounced calcium  (Heilmann  binding c a p a b i l i t y  soleus of the r a t . total  and  into  Pette,  1979).  of the SR  changes Sreter  are immediate and  (1970)  i n denervated  examined  the  gastrocnemius  and  fragmented  SR v e s i c l e s ,  particularly  rate and i n the  E f f i c i e n c y was impaired with a leakiness of the SR  membrane  and  activity  pattern from  stimulation  biochemical  A marked decrease was found i n the i n i t i a l  calcium uptake  fast white f i b e r s .  and  prolongation of time  to maximum  capacity.  denervation, endurance t r a i n i n g  have the same slowing e f f e c t  Changes i n  or low frequency  on the function of the SR of  \u000C_ 4 3  fast-twitch  muscle.  Denervation  produced  opposite  compared with slow, muscle  (Margareth et a l . , 1972).  was  while  increased  changes  in  soleus,  i n electrophoretic  decreased  pattern.  In  in  long  effects  on  fast,  Calcium transport  EDL,  with  term  concomittant  electro-stimulation  studies using i n d i r e c t , low frequency stimulation, Mabuchi et a l . (1982) showed Early  decreased SR  calcium  changes were  response to endurance 115,000Mr  calcium  uptake  found  tibialis  i n fast  training  binding  in  anterior  r a t EDL  (Green et a l . ,  protein  and  and  in  vastus  the  rabbit.  lateralis  1984), with a decrease i n  increase  in  30,000Mr  protein  content associated with a transformation from type IIB (FG) to IIA and  a  decrease  in  S i m i l a r i l y , Heilmann  cytosolic  calcium  in  binding  protein;  (FOG)  parvalbumin.  and Pette (1979) found changes i n SR function a f t e r  2 days of i n d i r e c t low frequency stimulation of gastrocnemius and EDL i n the  rabbit.  These included: a drop i n calcium dependent  reduced  initial  calcium uptake  Calcium  pumping  protein  affinity  binding  and  total  capacity,  (115,000Mr), calsequestrin  protein  (59,500Mr) on  SDS  gels.  ATPase a c t i v i t y , lower  amounts  (68,000Mr) and Denervation  of  high  changes  could be due to the loss of a neurotrophic substance from fast nerves as reinnervation  of  soleus  with  a  fast  nerve  sarcoplasmic reticulum (Margareth et a l . , 1973).  speeds  function  of  the  For this to be the case  i n d i r e c t stimulation mediated by the fast nerve would have to r e s u l t i n some transformation within is  that  a  the neuron  neurotrophic substance,  itself. common  to  An alternate fast  and  possibility  slow  mediates i t s influence depending on the impulse frequency imposed muscle.  These  effects on SR  contraction time.  function matched  muscles, on the  i n time with changes i n  The correspondence of the changes i n contraction time  with those of SR function lends support to work of Brody (1976), who  used  \u000C- 35 isolated  SR  vessicles  from  i n t a c t crureus  and  soleus  muscles  of the  rabbit, both of which have the same myosin ATPase a c t i v i t y but d i f f e r e n t rates  of calcium  uptake, to demonstrate  that  contraction  time i s more  closely correlated with sarcotubular uptake of calcium than myosin ATPase activity. (e)  Alterations i n Contractile Properties Dynamic properties of fast and slow muscles are l a r g e l y determined by  their s p e c i f i c motor innervation.  Spinal cord section speeds contraction  times i n slow twitch soleus, but EDL i s unchanged Betto and  and Midrio, denervation  1978; or  Eldridge  denervation  and Mommaerts, 1980). alone,  there  contraction  times i n slow and p a r t i c u l a r l y  (Betto  Midrio,  and  1978).  (Buller et a l . , I960;  The  effect  is a  i n fast  of  With  marked muscles  denervation  cordotomy  slowing  of  i n the r a t  i s not  simply  i n a c t i v i t y , as there i s a difference i n contraction time changes between cordotomy and denervation. by cord  isolation,  The fact that a slow muscle, rendered s i l e n t  becomes fast but with subsequent denervation  becomes  slow suggests a maintenance of fast c h a r a c t e r i s t i c s by an i n t a c t ,  albeit  e l e c t r i c a l l y s i l e n t , nerve (Eldridge and Mommaerts, 1980). By alters  reinnervating towards  that  slow muscle of  a  fast  with a muscle.  fast  nerve, contraction  The  converse  cross-reinnervation of a slow muscle (Buller et a l . , 1960b). way,  is  true  time of  In the same  e l e c t r i c a l stimulation has the same r e c i p r o c a l e f f e c t of slowing the  contraction  time of fast  muscles  with  tonic  Verbova, 1969; Heilmann and Pette, 1979).  stimulation  As reported  (Salmons  and  e a r l i e r , the time  sequence and d i r e c t i o n of the changes i n contraction times correspond to functional changes i n SR. Cordotomy p r i o r to cross-reinnervation  of fast  and  slow muscles of  \u000C- 36 the  cat had  no  influence on  t o t a l i s o l a t i o n of the supports  the  theory  the  cord  success of cross-reinnervation, whereas  prevented  that  neurotrophic  determine mechanical properties. interchange shortening  i t (Buller et a l . , 1960b). as  well  Barany and  Close  mechanical properties of soleus and EDL and  contraction  correlated with the  times by  as  activity  cross-reinnervation.  These changes  rate of superprecipitation, a biochemical  correlate  of  and  the  addition  of  calcium,  dissociated a c t i n and myosin i n s o l u t i o n .  to  including v e l o c i t y of  to  by  factors  (1971) were able  of contraction, consisting of increased o p t i c a l density, due actomyosin,  This  magnesium  formation ATP  to  They took t h i s to suggest that  changes i n contraction time are due to a l t e r a t i o n s i n the myosin molecule whose ATPase a c t i v i t y was Many denervation  shown to correlate with these parameters.  studies c i t e prolongation  of contraction time, both  the time to peak tension and relaxation time, with denervation soleus  (Gutmann et a l . , 1972;  1975;  Webster and  1972;  Drachmann and  is  associated  F i n o l et a l . , 1981;  Bressler, 1985).  increase  Prolongation  i n twitch  to  (Lewis,  of contraction  tetanus  tension  and  Johnston,  These are more marked i n EDL  Johnston, 1975).  with an  Drachman and  of EDL  times  ratio,  a  function of the duration of muscle a c t i v a t i o n and related to SR function. The  fact  that  the  contraction  same d i r e c t i o n i n denervation, stimulation  and  as  impaired  properties.  and  i n contrast  cross-reinnervation,  a l t e r a t i o n common to both fast denervation  time of  and  both muscles changes i n to d i f f e r e n t i a l  suggests  that  slow muscles.  the  the  changes with cause  is  an  These occur early i n  coincide with primary changes i n excitable membranes such surface  The  action  potential  and  sarcoplasmic  onset of these changes varies with  the  reticulum  length  of  the  \u000C-  distal  nerve  substance (Finol  stump,  depleted  et a l . ,  changes  suggesting  i n mechanical  paralyzed by blockage  -  they  from the nerve  1981).  37  by a neurotrophic  i n proportion to the remaining  Drachmann properties  are controlled  and Johnston  found  parallel  muscles  whether  (1975)  o f EDL and Soleus  stump  of cholinergic transmission or denervation, but i t  i s not known i f a putative neurotrophic factor would be affected by the former  treatment.  reversed Gutman,  by high  muscles  frequency  influence i n shortened  increased  on  contraction  (1974)  times  or stretched position,  contraction times  changes  stimulation i f started  Melichnia and Gutman  1974).  myogenic  In addition, denervation  respectively.  were  by  can be  early able  (Melichna and to demonstrate  immobilizing  resulting The muscle  partially  denervated  i n decreased or length  used f o r  immobilization also influenced changes i n myosin ATPase a c t i v i t y , and  metabolic  enzyme a c t i v i t y .  These facts  illustrate  atrophy  that the neural  influence on muscle i s an i n t e r - r e l a t i o n s h i p o f neurotrophic and impulse transmission e f f e c t s , and are further influenced by myogenic factors as well. Neural Regulation o f Immature Muscle Neural regulation o f the p l a s t i c i t y o f mature muscle has been studied extensively and although  there i s by no means a clear  manner i n which changes can occur, the  neurotrophic  impulse  twitch muscle myogenic,  there are some consistencies.  influence by a substance  transmission, play a role characteristics.  as indicated  With the developing muscle s t i l l  within  the neuron  i n the maintenance of fast  There are other  by denervation  hormonal (Rubinstein and Kelly,  concensus o f the  1985),  at fixed  factors, different  and  Both the  and slow  some possibly lengths, or  which exert additional influence.  establishing these fast and slow-twitch  \u000C- 38 characteristics,  i t seems  likely  that  the  neural  influence would  be  greater on developing muscle than mature muscle. (a)  Atrophy In  gastrocnemius  Talesara  (1977)  of  found  the  rat,  greater  denervated  atrophy  in  following denervation at 10 days of age, slow muscles. tenotomy.  This d i f f e r e n t i a l  at  fast  maturity,  with  and  muscles  but  equal i n fast  and  than  atrophy was  response  Kumar  age  slow  did not  occur  with  Therefore i t was concluded; there i s a greater dependence on a  neurotrophic  influence making  developing  muscle  more  susceptable  to  denervation. Engel and Karpati (1968) examined denervated gastrocnemius of newborn r a t s .  At 21 days of age  they  found  and soleus  65% of the  f i b e r s were  atrophied and remained i n the myotube stage compared with an absence of myotubes i n 21 day controls. myosin ATPase (pH 9.4) larger  fibers  spindles  were  present.  These f i b e r s were dark staining  t y p i c a l of type  paler  staining  Following  i n routine  II f i b e r s , while the  (type  neonatal  I).  There  denervation  remaining,  were  in  the  soleus, i n which development i s about equal to; a 10 day  no  muscle  guinea  pig  old rat (Engel  and Karpati, 1968), the numbers of p e r s i s t i n g type II f i b e r s was greatest following skeletal  neonatal fixation,  denervation, providing  less  after  additional  cordotomy,  evidence  for  and  least  after  the  role  of  a  and  slow-twitch  neurotrophic factor. (b)  U l t r a s t r u c t u r a l changes Ultrastructural  muscles  in  mature  differences muscle  persist  following  between  denervation  denervation, Shafiq et a l . (1972) found  fast but  ultrastructural  after  neonatal  characteristics  i n Z band thickness and mitochondrial d i s t r i b u t i o n did not d i f f e r between  \u000C- 39 EDL and soleus i n the r a t . in  EDL.  with  However, some maturation of the SR was  A small number of myotubes persisted  myosin  ATPase remained  homogeneous  and  i n both  the  fibers  muscles.  seen  staining  A l l fibers  stained equally with myosin ATPase a f t e r acid and a l k a l i n e preincubation, t y p i c a l of developing f i b e r s . This contrasted with f u l l y mature controls by 3 weeks of age.  Hanzlikova and  of the normal maturation  Schiaffino  i n content and  (1973) also found  failure  orientation of mitochondria,  Z  band structure and t r i a d formation between soleus and EDL which had been denervated i n utero 3 days prior to b i r t h .  Irregular development of the  SR was seen and some f i b e r s showed complete degeneration. Schiaffino and rat gastrocnemius  Settembrini  (1970) found  did not retard  the u l t r a s t r u c t u r a l development of the  sarcoplasmic  reticulum.  c i s t e r n s and  forming an elaborate t system, although  collar  at the M l i n e .  The  neonatal denervation of the  The  SR  hypertrophied,  morphological  developing  implications  lacking should  enlarged the usual predict  a  faster muscle, but i n mature denervated muscle, increased capacity of the sarcoplasmic reticulum has been shown to coincide with a reduced rate of calcium uptake and a leakiness (Sreter, 1970), i n d i c a t i n g an of function i n spite of a s t r u c t u r a l enhancement. with  the  denervated the  limited muscle  myofibrillar  sarcoplasmic (Engel and atrophy,  differential sensitivity  reticular  1974).  which  marked,  was  of muscle c e l l  These changes contrast  atrophy  Stonnington,  impairment  shown  It also and  in  mature  contrasts to  illustrates  a  components to neural influence.  Dedifferentiation i s therefore too simple to explain the complex changes that produce a novel f i b e r (c)  unlike either developing or normal f i b e r s ,  Altered Contractile Proteins Rubinstein and Kelly  (1978) examined the myosin ATPase staining  and  \u000C- 40 myosin  light  chain  composition  neonatal denervation. intensely chains.  i n rat EDL  Soleus was highly atrophied. by  soleus  In both fast and slow muscles,  i n routine ATPase reaction  affected  and  denervation.  However,  (pH  9.4)  and  14  days  the myosin stained posessed  fast  The  light  immature and  chain  adult l i g h t  fast muscle.  without  the  myosin  ATPase  birth,  no  present, as of  type  I  However, i n rats continued  EDL  and  found  longer synthesized at  well as  antibody  a  does  30  small amount of  fibers  which  days.  at  one  staining  to  neonatal  eventually  electrophosesis.  The  this  muscle.  replaced  large  myosin,  Fast  LClslow, with  light  explained prolonged  because  myosin  as  well  Anti-slow myosin was  chains were by  a  myosin  stained  as  anti-fast  found i n larger  with  as  shown  both  with  gel  anti-fast  and  developmental  change.  of rat muscle produced  cells  type  I  As mentioned e a r l i e r , the l i g h t chain complement i s the same i n  neonatal and adult fast muscle making i t ,  cells.  small  denervation.  anti-slow adult myosin, indicating that they were not a t y p i c a l fiber.  at  before denervation at 1 week.  neonatal  fibers  present  (1982) showed  week, Butler-Browne  f i b e r s which could have begun to develop myosin  answer  embryonic  persisted  denervated  myosin i n rat gastrocnemius  Fast  not  immature  (1981) looked at myosin isoforms of neonatal  Ishiura et a l .  rat soleus and  number  information  acid  chains so f a r have been shown to be a l i k e i n  denervated was  light  They concluded that EDL was not as  preincubation reaction, i t i s not known i f these were i n fact fibers.  after  While  alone, an inadequate marker of  Dhoot and Perry (1983b) found neonatal denervation s i g n i f i c a n t atrophy  some type  I  cells  of EDL,  atrophied, a  stained darkly for myosin ATPase with  few  particularly  type II  hypertrophied.  alkaline  All  preincubation but  only the hypertrophied c e l l s stained with acid preincubation too, a sign  \u000C- 41 of immature f i b e r s .  Similarly, at 14 days, a l l f i b e r s responded to fast  troponin I antibodies and fast and  only the hypertrophied  slow antibodies.  exclusively  with  antislow.  By  21  c e l l s responded to both  days, the hypertrophied  c e l l s stained  This shows that synthesis of fast  and  slow  troponin and supression of fast troponin i n slow f i b e r s can occur i n the absence of innervation. T.  C e l l s resistant  denervated  rat  gastrocnemius  The  same r e s u l t s were found with Troponin  to atrophy  EDL  (Engel  have been shown by  and  (Butler-Browne,  Karpati,  1982)  and  1968;  others  i n neonatally  Ishiura,  found  to  stain  1981)  and  with  Ishiura  neonatal  hypertrophied  or  embryonic  increased  with  time.  surprising  finding  was  from reinnervation as In  the  work  the different  are both mixed f i b e r  persisted i n EDL  with atrophy  by  they  Dhoot  results types,  rat  alkaline  Those found by  anti-slow myosin but (1981)  f i b e r s to contain a mixture of fast and  These f i b e r s were not  Although they  myosin.  or  after  preincubation and yet been assumed to be a type I f i b e r . Butler-Browne reacted with adult a n t i - f a s t  C and  found  the  slow l i g h t chains.  decreased and  not  rather than  Perry  (1983b),  i n gastrocnemius from  a population  of large  a  EDL.  fibers  of v i r t u a l l y a l l f i b e r s i n gastrocnemius.  This i s an example of the differences that are found between  apparently  i d e n t i c a l f i b e r s types i n d i f f e r e n t muscles. In a comprehensive study after  denervation  and  of the  synthesis of c o n t r a c t i l e  reinnervation i n the  developing  Matsuda et a l . (1984) found neonatal denervation  and  adult  i n chick did not  synthesis of adult myosin heavy chains and l i g h t chains, but the tropomyosin f a i l e d to be supressed was  evident.  and  proteins chick, prevent neonatal  a discoordinate protein synthesis  This suggests that i n the chick, tropomyosin synthesis i s  under neural control but myosin synthesis may  not be.  This contrasts with  \u000C- 42 the  results  of  influence was  Dhoot  not  and  required  Perry  (1983b),  in  rat  EDL,  to produce mature regulatory  could be a species difference i n rat and  chicken.  where  neural  proteins.  This  Butler-Browne (1982),  in rat muscle and Thibault et a l . (1981) on cultured chick c e l l s , found a similar  lack of neural  cultured  control on  from s a t e l l i t e  cells  of  heavy chain fast  produce mature forms of heavy chain they both synthesised  or  synthesis.  Muscle  slow muscle were not  (Matsuda and  satellite  synthesize  cells  immature forms of heavy chains  immature  in  adult  forms  of  chicken myosin  muscle heavy  able  Strohman, 1984). and  light  but not tropomyosins that were distinguishable from each other. the  cells  are  chains  Yet,  chains, Possibly  preprogrammed and  to  light  to  chains  s p e c i f i c to fast or slow muscle, but not tropomyosin, (d)  Changes i n Contractile Properties In spite of prolonged contraction times i n adult  the d i f f e r e n t i a l between fast and  slow muscles i s retained  Bressler, 1985), but with neonatal denervation to  failure  of  maturation, developing  developmental  in  EDL  (Brown,  denervated muscle,  shortening 1973).  of  (Webster  and  t h i s i s l o s t ; mainly  due  the  After  contraction  stimulation  rabbit muscle, Brown (1973) showed only  the a b i l i t y to reduce contraction time.  Therefore  time,  of  partial  with  denervated recovery  of  a c t i v i t y has a r o l e i n  developmental changes i n contraction time properties. To  assess  developing  the  muscle  influence on  of  contraction  altered a c t i v i t y times,  stimulation i n t i b i l a i s anterior, EDL days of age.  With 10Hz  fast  muscles  were  fast  decreased soleus  unchanged.  of  These  innervated  (1981) applied  and soleus i n developing  stimulation both  while stimulation with 25 Hz  Jones  pattern  and  indirect  rat from 4  slow muscles slowed,  contraction time, but  findings  show  that  the  activity  \u000C- 43 influences  contraction  determinant.  time  with  the  firing  frequency  The histochemical and c o n t r a c t i l e parameters  a  prime  show that  the  e f f e c t s of denervation and a low frequency a c t i v i t y pattern share common features.  A  fast  become f a s t .  firing  pattern may  be  required to  As these were stimulated i n d i r e c t l y  know i f there could be any  make the  muscle  i t would be useful to  retograde influence on the nerve,  such  as,  increased a f t e r hyperpolarization, to account for these changes, or even i f the altered stimulation pattern could a f f e c t release of a neurotrophic protein i n the neuron. Permanence of Denervation Effects i n Developing Muscle To investigate the permanence of any impairment about by denervation, McArdle reinnervation soleus.  following  i n maturation  brought  and Sansone (1977) compared the r e s u l t s of  neonatal and  adult  nerve  crush i n rat EDL  They were examined from 14 to 180 days post denervation.  were able to r e d i f f e r e n t i a t e into  fast and  slow  fibers  i n both  and  Fibers groups,  but the neonatal groups showed persistant signs of denervation such as; atrophy, lack of muscle spindles, presence of myotubes, wide Z l i n e s , and Z l i n e streaming. attributed  to  A reduced  impaired  Ultrastructure  of  the  number of multiple endplate potentials  synaptogenesis  SR  and  sarcolemmal  controls at 6 months following evidenced events  by  remained  neurotrophic under  influences  on  difference  or  activity  calcium uptake,  as  was  Because  parameters  their  not.  properties  mechanical  recovered,  during development must  affected  augmented  patterns  activity.  crush, but  these  be  reflex  as  membrane  may  of  same  yet  Both  lack  properties were the  times  control.  but  axons  neonatal  relaxation  affected  separate  denervation, certain  prolonged  and  was  during  by  concurrently selective  be  during  failure  reinnervation.  the  Such  of a  \u000C-Indifference would include lack of spindle afferents. point  that reinnervation studies  compared activity  cautiously due  with  not  seen  study  which  permitted.  At  nerve  of  2 months,  5-6  soleus  and  that was  only  of controls.  all  fibers  horseradish to  50?  EDL,  remained  both  aberrent  tonic neurons has  unit  crushed  full  and  a  similar  reinnervation  tension  recovery  whereas  fast-twitch muscles,  achieved  tension  There was  oxidative  a reduction  and  fatigue  i n f i b e r number, resistant.  Using  peroxidase, they showed that these differences were not  reduction  of  the  number  between these r e s u l t s and  be  Verbova, 1984); a finding  rats showed  must  been shown to  a l . (1982) did  was  the  motor  Lowrie et  day  anterior  as  (Navarrete and  reinnervation.  tibialis  and  development,  to selective s u r v i v a l by  i n adult  in  following neonatal nerve crush  normal  produce an altered gait pattern  This i l l u s t r a t e s  of  innervating  neurons.  those of McArdle and  The  Sansone  due  difference  (1977) can  be  explained by the fact that neonatal nerve crush coincides with the period of  susceptibility  denervating al.  of  neuron  cell  at f i v e days of age,  and  death  which  occurs  thus avoiding  at  birth.  By  t h i s time, Lowrie et  (1982) were able to show that the persistance of denervation  changes  i n their study were not due to loss of innervating axons. A Model of Aneural Myogenesis i n Vivo Another  approach,  to  d i f f e r e n t i a t i n g muscle, was  studying  the  effect  of  to look at muscle regeneration  described as a r e c a p i t u l a t i o n of development (Matsuda and but with some differences (Gutman, 1976). cross-implanted to the  firing  of their  new  on  which has been Strohman,  1984)  Gordon and Vrbova (1975) found  minced fast muscle grafts w i l l pattern  innervation  innervation  regenerate and  appropriately  become slow.  indicates the program for maturation of fast or slow-twitch  This  properties i s  \u000C- 45 not myogenic.  The increased s u s c e p t i b i l i t y of immature muscles to neural  influence was shown by Riley (1974) who found more complete conversion of cross reinnervated muscles when caused to regenerate  by cold i n j u r y .  He  suggested that a certain amount of d e d i f f e r e n t i a t i o n occurs i n a l l cross innervation studies due to i n e v i t a b l e denervation  and may be responsible  for permitting the reversal of c h a r a c t e r i s t i c s by the new nerve. Statement of the Problem Developing  muscles are more susceptible than  influences of innervation.  information  neonatally  muscle  histochemical p r o f i l e .  Looking  differentiation  opportunity development.  to  into  understand  This  A more comprehensive study i s needed to  about many of the physiological c h a r a c t e r i s t i c s of  denervated  during  to the  Without innervation many fast or slow-twitch  c h a r a c t e r i s t i c s f a i l to develop. provide  mature muscle  study  and  how  at these mature  these  fast-twitch  new  with  the  parameters i n denervated muscle  the i n t e r a c t i v e provides  correlate  muscle,  effect  information  of  provides  an  denervation  on  regarding  the nerve  dependency of the c o n t r a c t i l e and histochemical properties of developing fast-twitch  muscle  i n the C57BL/6J+/+  mouse  and contributes  valuable  information f o r developmental studies of the oogenic s t r a i n of dystrophic mouse.  \u000C- 46 -  II METHODS  \u000C- 47 All  experiments  Digitorum  were  Longus muscle  and raised i n our own  carried  (EDL)  out  on  the  fast-twitch  of the C57/BL6J+/+ s t r a i n  Extensor  of mouse, bred  colony from breeding pairs o r i g i n a l l y obtained from  Jackson Laboratories (Bar Harbor Maine).  For each group, 6 mouse pups of  both  pairs  sexes  neurectomy including twitch  from at  time  32+8  maximum  tension  mated  hrs.  breeding  of  age  (N=42).  isometric twitch  (TTP),  time  to  and  potentiation distribution matched  (PTP),  and  controls at  7,  14  of  right  sciatic  contractile  properties  tension,  time-to-peak  half  peak  twitch tension  shortening (Vo), posttetanic twitch  fatiguability  were measured  The  tetanic  relaxation  (1/2RT), maximum v e l o c i t y of unloaded  underwent  and  histochemical  fiber  i n denervated  muscles and  muscles  and  of  characterize the  21  days  age.  To  from  muscles at a time coinciding with the time of denervation, one six  normal EDL  muscles was  type  examined from unoperated  age  group of  animals at 1 day  of  age. Denervation: Pups were anaesthetized with with t h e i r right leg supported non-sterile  technique,  a  ether and  placed on  in a plasticine splint.  skin  incision  was  made  compartment of the thigh, the underlying f a s c i a was femoris  retracted  exposing  the  sciatic  c a r e f u l l y retracted with fine forceps and greater and  nerve.  peroneal  identification  by  nerves  toe  nail  at  the  clipping  knee. of  left  Using clean but  along  the  lateral  divided, and The  side  nerve  biceps  was  then  excised from the l e v e l of the  trochanter to beyond i t s b i f u r c a t i o n  common  their  into The  the  the  animal  forepaw,  posterior was  tibial  marked  returned  for  to i t s  mother and allowed to recover. In spite of the f l a c c i d paralysis of the right leg, pups were able to develop an e f f i c i e n t gait which consisted of passive extension of the  \u000C- 48 hip  and  knee followed by  denervated  an  abduction-adduction  maneuver to bring the  limb beside the contralateral limb.  therefore  held  ambulation.  i n the  The  same posture  contralateral  limb  as  the  was  The  normal  denervated limb  limb  except  was  during  responsible for supporting the  weight of the hindquarters plus performing a l l the work of ambulation for the  hindlimbs.  Thus,  compensatory  muscle  hypertrophy  was  likely  and  therefore the contralateral limb was not used as a control. All  animals  used  in  the  denervated  group  demonstrated  signs of denervation, including  paralysis,  dragging  of the  right  and complete loss of active movement at the ankle and toes. absence of reinnervation was  positive foot,  In addition,  confirmed by the use of acetylcholinesterase  and s i l v e r staining as described i n appendix 2. Histochemistry: (a)  Experimental The  animal  Procedures was  killed  pinned to a cork board. and  the overlying  with  chloroform, i t s right  The leg was  tibialis  leg removed  and  skinned, the c r u r a l fascia excised  anterior muscle removed, exposing  EDL.  The  proximal and d i s t a l tendons were cut and the muscle placed between layers of  gauze moistened  embedded  in  a  i n 0.9N  block  of  saline. mouse  tragacanth on a cork chuck.  Using  liver  which  in  isopentane  nitrogen, and hour.  was  then  mounted  in  was gum  had  been  The cooled  sample was to  -160  frozen for 30 \u00B0C  placed i n a cryostat cabinet at -20  in \u00B0C  liquid for 1  S e r i a l sections of lOjam thickness were taken from the midbelly of  the muscle and for  which  immediately  forceps, the muscle  The muscle orientated perpendicular to the  chuck i n order to obtain cross-sections. seconds  fine  one  hour  collected and  on glass coverslips.  stained  with  The  Haematoxylin  sections were dried and  Eosin,  NADH  \u000C- 49 Tetrazolium  Reductase  preincubations Dubowitz to  o f p H 4.2,  and Brooke  require  min, and  of  reaction  p H 4.6  of  types were  5 m i n a n d 40  incubation  typing  times  was done  times  myosin  time,  modified  m i n , a n d p H 9.4  according  differences  et al.,  as  found  pH  for  best  o f Brooke  myosin  min and  2.5  ATPase  by  been  min and 5 min  t o the nomenclature  described  Therefore,  p H 4.2,  For the myosin  using  have  and  1983).  follows: 15  reactions,  t o t h e method  temperature  (Gollnick  respectively.  ATPase  according  Age and s p e c i e s  (1973).  fiber  or  o r 9.4  4.6  adjustments  identification ATPase  (NADH-TR)  40  pre-incubation  reaction,  and K a i s e r  fiber  (1970)  a n d B r o o k e e t a l , (1971). (b)  Data  Collection  Using of  Zeiss  one myosin  muscle. assembled was  a  ATPase  section  Counts  o f each  from  the prints  compared  according  photomicroscope,  with  that  t o t h e method  fiber  (preincubation type  (final  at  overlapping  were  mag  p H 4.2  o f Brooke  X800).  to  Typing  According  FIBER  TYPE  4.2)  made  directly  Staining  visualize  were  (mag  made from  of fibers  reversibility  of  of  of Fast  a t pH  t o Myosin  Twitch  ATPase  Fibers  Staining  4.2  4.6  IIA  LIGHT  MEDIUM-LIGHT  DARK  IIB  MEDIUM-LIGHT  MEDIUM  DARK  I  VERY DARK  VERY DARK  IMMATURE  MEDIUM  MEDIUM-DARK  9.4  MEDIUM-LIGHT MEDIUM  each  9.4  staining  II  and Slow  X160)  montages,  e t a l . (1970).  Table Fiber  then  pH  photographs  \u000C- 50 Morphometries: (a)  Experimental Procedures To  examine  the extent of atrophy at  21 days  of age quantative  comparisons were made of t o t a l muscle cross-sectional area and fiber  cross-sectional  muscle p r o f i l e s each  muscle  were traced  attachment,  analyzer. average  onto  calibrated  and  denervated  known  area  projected  The whole muscle  cross-sectional  the d i g i t i z i n g  area was  as: that  onto  muscles.  Whole  of the Zeiss Mop  with a L e i t z  the  digital  cross-sectional area was  Camera Lucida  slide pad  3  and  of  checked  the  image  measured using the  In the same manner the i n d i v i d u a l myofiber  measured  which  board  by means of a micrometer  of three readings.  predefined  normal  A L e i t z microscope, f i t t e d  was  a  in  from the H.and.E. section of the greatest diameter from  Image Analyzer.  against  area  individual  of  a l l cells  runs through  angles to the greatest diameter.  that  the mid-point  fell  on  of and  a at  line right  The section was orientated so that the  l i n e f e l l v e r t i c a l l y , and a l l c e l l s were counted that were touched by the pointer,  as  the  stage  was  moved  vertically.  A l l measurements  were  expressed i n mm . Contractile Parameters: (a)  Muscle Dissection The  secured  right with  l e g was  insect  removed  pins  to  as the  described silgard  previously  bottom  of  and  skinned,  a transparent  dissecting dish and immersed i n oxygenated buffered Krebs solution. crural  f a s c i a was  excised, and  muscles were removed. the to  tibialis  anterior  and  extensor  The  hallicus  The EDL tendons were then c a r e f u l l y freed, cut and  muscle was pinned through i t s tendons, at approximately slack length, the bottom of the dish.  Subsequently, t i e s of 10-0 surgical s i l k were  \u000C- 51 placed at the myotendinous  junctions.  This proceedure was used to reduce  tendon series compliance. (b) Experimental Apparatus The muscle was then transferred, under a bubble of Krebs solution, i n a small polyethylene boat, to the experimental chamber shown i n Figure 1.  With the aid of a dissecting microscope the tendons were t i e d at one  end  to the s t a i n l e s s  steel  extension  o f a force  transducer (resonant  frequency 2.0 KHz) and at the other end to a galvanometer torque motor. The motor was part of a length servosystem which was extremely s t i f f and allowed muscle. aligned was  examination o f the isometric  By means of three way positioners, and the length adjusted.  immersed  NaHCCy  i n Krebs  1. 2mM  MgSO^H^O and  solution  NaH^C^ .H^O;  which  /liter.)  properties  the muscle  Throughout  5.0mM  and 2gm glucose  maintained  appendix  contractile  a Haake  115mM  KC1; .4mM  CaCl ;  gassed  95% 0  3  with  Fe thermostatically  ?  NaCl;  25mM 1.2mM  and 5% CO^  of 21\u00B0C+1\u00B0C (see  controlled  temperature was monitored by means o f a thermistor  the muscle  and  2  at a pH of 7.3, and a temperature  3) using  was c r i t i c a l l y  the experiment  contained,  o f active  pump.  The  (not shown i n  the  figure). (c)  Experimental Proceedures Crow and Kushmerick  at  sarcomere  (1983) found the maximum tetanus tension occurs  lengths of 2.7 microns and the maximum twitch  2.9um i n mouse EDL at 3 to 4 weeks o f age. was  set to the maximum  twitch  tension a t  In t h i s study, muscle length  height,  as  contraction  times and  post-tetanic twitch potentiation were measured from the twitch. was  set at that  which  produced  maximum  twitch  height  Voltage  as well.  For  tetanic contractions, the stimulus frequency and duration were adjusted to  produce  a fused  tetanus tracing.  Stimuli  o f supramaximal  square  \u000C-  Fig.  1  Experimental  Krebs solution arm  52 -  apparatus:  The muscle  i n bath chamber, t i e d at one end to the lever  of the motor and at the other to a s t a i n l e s s  extension of the force transducer. with  i s immersed i n  95% 0,, and 5%C0^.  of a 3-way positioner.  Length  steel  wire  I t i s continuously bubbled  adjustment  i s made by means  Temperature i s monitored by means of a  thermistor and controlled  by c i r c u l a t i o n of water through the  bath chamber (not shown i n diagram).  \u000C\u000C- 54 pulses  of  1.0ms  Stimulator and  duration  were  provided  by  a  displayed on an o s c i l l i s c o p e .  a stimulus pattern of 1 tetanus  Digitimer  DS2  Isolated  Throughout the experiment,  followed by 3 twitches with a 90 second  i n t e r v a l , between each contraction, was  used so that tension decline  to fatigue was minimal, never exceeding 5%.  due  A minimum of 4 tetani and 9  twitches were recorded. Vo was  then measured using the slack test method of Edman (1979).  This consisted of giving the muscle ramp length changes of 100 the plateau of an isometric tetanus zero. The  sufficient  release caused a rapid f a l l  Hz  during  to reduce the tension to  i n tension which remained at  the  baseline while the muscle contracted  to take up the slack.  four d i f f e r e n t amplitudes were used.  The time taken to take up the slack  was  proportional to the amplitude of the  length  change.  Releases of  The  slope  of  this r e l a t i o n s h i p i s therefore the velocity of unloaded shortening. Following the Vo the  muscle  allowed  post-tetanic  twitch  determination, to  rest.  potentiation  the stimulator was  After  a  was  standard  measured.  20 This  pre-twitch, followed i n 90 seconds by a 1 second tetanus l a t e r by a post-twitch ( F i g . 2). have  shown  that,  for  the  mouse  EDL  at  20\u00B0C,  a one  One  the superimposed post-twitch.  a second o s c i l l i s c o p e . second tetanus  muscle length was  and  The  The fatigue p r o f i l e was at the rate of 12  minute  period,  consisted and  of  PTP  is  maximal  data) at  o s c i l l i s c o p e recorded tetanus was  measured using fine c a l i p e r s .  a  20 seconds  recorded  20 the on  studied using a regime of  per minute for 6 minutes.  up to the musculotendinous junctions, placed cup,  o f f and  Bressler and Glotman (unpublished  seconds following the tetanic stimulation. pretwitch and  turned  The muscle was  The  trimmed,  i n a preweighed dessicator  then f i n a l l y weighed on a 6 place balance  i n a temperature  and  \u000C- 55 humidity (d)  controlled room,  Data C o l l e c t i o n The  analogue  signal  from  the tension  d i r e c t l y on an APPLE H E and stored on disc.  transducer  was  recorded  Custom software written f o r  analysis of tension data was used to calculate the twitch parameters of maximum  twitch  tension  ( P t ) , maximum  twitch  tension/muscle  weight  (Pt/mwt), time to peak tension (TTP), time from peak tension to one h a l f amplitude  of peak  tension  (1/2RT) and tetanus  parameters  tetanic tension (Po) and maximum tetanic tension/muscle The  c o n t r a c t i l e responses were also recorded  Pentax camera frame.  with  weight  (Po/mwt).  on 35mm f i l m with  an Asahi  a macrolens and mounted on the o s c i l l i s c o p e  The parameters of Vo, PTP and fatigue were measured d i r e c t l y from  the f i l m . and  fitted  of maximal  The negatives were placed i n a standard  the records  analyzed  from  the projected  photographic  images.  enlarger  For the maximum  v e l o c i t y of shortening, l i n e a r regression analysis was used to calculate the slope of the r e l a t i o n s h i p of the length change to the time as shown i n Figure  2.  (Lo/sec).  Pre- and post-twitch tension measurements were expressed  ratio  A l l Vo values are expressed  of post-twitch  tetanic  over  pre-twitch.  contraction was normalized  with  i n muscle lengths  For the fatigue respect  per second  profile,  to the i n i t i a l  as a each  tetanus  tension of the fatigue regime and plotted against time. Data Analysis Data  f o r groups at 7, 14 and 21 days of age, was subjected to  two-way analysis of variance i n order to assess changes with time (group e f f e c t ) and changes due to denervation changes  that  occurred  from  the point  (group e f f e c t ) . at which  To examine the  denervation  was done,  one-way analysis of variance was applied to data of 1, 7, 14 and 21 days  \u000C- 56 -  Fig  2.  Determination of unloaded  shortening  v e l o c i t y (Vo) by  the slack test i n normal EDL at 1 day of age. Fig.2A,  imposed  length  changes  and  responses for one series of four releases. the  amplitude of the release and  resulting  force  Change i n length i s  change i n time  i s the  time  required to take up the slack. Fig  2B  shows  the  line  representing  the  least-squares  regression of change i n length upon change i n time.  The  slope  i s then divided by the muscle lengths to express Lo i n Lo/sec.  \u000C- 57 -  A  1  1  20  i  24 At  j  28  msec  Slope = 9.36mm/msec-r3.9mm= Fig 2  i  .4Lo/sec  \u000C- 58 of  age.  This  method  of analysis  also  allows  for examination of the  i n t e r a c t i v e effect of denervation and development, that pattern  of  normal  denervation.  Where  development an  has  indeed  been  interactive  effect  was  i s , whether the  interfered found,  with  by  a l l pairwise  camparisons were made using Tukey's t e s t . Total  cross-sectional area of normal and  days were compared using Student T-test.  denervated groups at 21  Results  from measurements of  i n d i v i d u a l f i b e r s form 4 normal and 4 denervated EDL at 21 days of age 2 were collapsed  to groups of 10 ;um  plotted for each group.  and  the frequency d i s t r i b u t i o n  was  \u000C- 59 -  III RESULTS  \u000C- 60 Growth Changes shown  i n growth  i n Table  III.  parameters In  of normal  unoperated  and  operated animals are  animals, there  was  a  significant  increase i n animal weight, muscle length and muscle weight at each period studied.  In operated animals, the increase i n animal weight kept pace  with unoperated controls.  Muscles from both groups continued to grow i n  length at each age studied with a small, but s i g n i f i c a n t , the denervated group when compared continued After  to  increase  i n weight  denervation, there was  with controls.  throughout  a significant  the  The time  difference  reduction i n  normal period  muscles studied.  i n muscle  weight  between normal and denervated EDL by 14 days of age which persisted up to 21 days.  Moreover,  even  increase s i g n i f i c a n a t l y  though  the normal muscle  weight  continued to  from 1 to 21 days, t h i s maturational change  was  arrested i n the denervated muscles by 14 days. A small sample  of whole muscle  cross-sectional  areas, measured i n  normal and denervated muscles at 21 days of age, i s summarized IV.  i n Table  There i s a s i g n i f i c a n t difference between normal and denervated EDL  whole muscle cross-sectional areas was  reduced by 67% i n the denervated  compared  Photomicrographs  with  normal  at  21  days.  of  normal  and  denervated whole muscle cross-sections at 21 days of age i l l u s t r a t e the atrophy produced by denervation ( F i g . 3). fiber  cross-sectional  areas  measured  from  Figure 4 shows the i n d i v i d u a l 4  normal  and  4 denervated  muscles at 21 days of age.  Atrophy was seen to occur i n a l l denervated  muscles by 21 days of age.  The majority of f i b e r s i n denervated muscle  are smaller than the smallest fibers i n 21 day controls.  \u000CTABLE III Growth Changes in Animal Weight, Muscle Length and Muscle Weight (EDL) in Normal Mice and Those Denervated at 1 Day of Age  Days  Animal Weight (gms)  14  Norm  1.57 + 0.05  a  4.37+0.02  7.08+0.14  9.39 + 0.4lt  (6)  (5)  (6)  4.25 + 0.24  7.85 + 0.39  9.25 + 0.29t  (4)  (6)  (6)  3.60 + 0.16  5.77 + 0.11  8.05 + 0.27  9.48 + 0.30t  (6)  (6)  (6)  (5)  4.96 + 0.23*  7.90 + 0.34*  (5)  b  Den  Muscle Length (mm)  Norm  Den  Muscle Weight (mg)  Norm  Den  (5)  (5)  0.284+ 0.044  0.874+ 0.225  1.988+ 0.132  (6)  (6)  (6)  0.516+ 0.212 (5)  a  21  A l l values are means + SE. b ( ) Sample s i z e . * S i g n i f i c a n t difference between normal and denervated muscles, t S i g n i f i c a n t difference between muscles at 7 and 21 days.  8.73 + 0.l8*t (6)  3.598+ 0.4l2t (6)  0.937+ 0.389* 1.102+ 0.294*t (6)  (6)  \u000CFig.3 Whole muscle cross section, Normal and Denervated EDL at 21 d a y s of a g e myosin ATPase pH 4 . 2  DEN  \u000C- 63 Table IV Whole Muscle Cross-sectional Area i n Normal and Denervated EDL at 21 Days of Age Norm #1  0.573  Den #1 Norm #2  0.127 0.579  Den #2 Norm #3  0.108 0.554  Den #3 Norm #4  0.096 0.503  Den #4 Group mean  0.359 0.552  0.173*  SEM t 0.017 \u00B1 0.063 * s i g n i f i c a n t difference between normal and denervated at p ^.005 Histochemistry (a)  Total Fiber Number The t o t a l f i b e r count for muscles at 1, 7, 14 and 21 days of age i s  shown i n f i g . 5.  At a l l age groups studied,  there was no  difference between normal and denervated muscle. 21 days whereas, fibers  there was no change at 21  days  i n total  fiber  the denervated EDL  than i t did at b i r t h .  significant  However, from b i r t h to  number i n the normal EDL  contained  significantly  While there was no s i g n i f i c a n t  less  change i n  the t o t a l f i b e r s i n normal muscles at 7, 14 and 21 days there was a small but s i g n i f i c a n t drop i n t o t a l f i b e r number between the 1 day normal group and 21 days denervated group. (b)  Fiber Types In  the normal EDL, at one day of age, a l l fibers  stained  f o r the  \u000C- 64 -  Fig.  4  Cross-sectional  area of  individual  fibers.  Pooled  fiber  measurements from 4 normal and 4 denervated EDL at 21 days of age.  area  \u000CCross-sectional Fig 4  area  4  x 10\" mm  2  \u000C- 66 -  \u2022 Norm \u2022 Den  12  8  7 14 Time (days) 5 ig.  21  T o t a l f i b e r number o f n o r m a l a n d d e n e r v a t e d EDL d i f f e r e n c e b e t w e e n 1 d a y n o r m a l a n d 21 d a y d e n e r v a t e d  values  \u000C- 67 myosin ATPase reaction a f t e r both acid and alkaline 6).  At myosin ATPase pH 4.2, there  medium-stained fibers  fibers  or presumptive  uniform  were two f i b e r  or presumptive  type  type  A l l fibers  I (Ip).  staining at pH 9.4 preincubation.  preincubation ( F i g . types  discernable:  II ( H p ) and dark-staining showed medium  uniform  In addition, there was moderate and  staining of a l l f i b e r s with oxidative enzymes ( F i g . 6). Typical  staining with myosin ATPase pH 4.2 and NADH, at 7 days of age i n normal and denervated  EDL are shown i n Figure 7.  I t was possible to distinguish  between the two groups of fast f i b e r s , IIA and IIB, however, both the IIA and  IIB f i b e r s  remained oxidative.  again seen at pH 4.2. at  pH 9.4.  exhibited the  Moreover,  profile  (Ip) was  These f i b e r s did not show a reversal of staining as may  uniformly-intense  mature  The darkly staining group  be seen  staining with  of fibers  types  i n Figure  7, a l l the f i b e r s  NADH-TR.  By 14 days of age,  had been  established and remained  unchanged at 21 days. Serial  sections of normal and denervated  muscle stained f o r myosin  ATPase at pH 4.2 and 9.4 and for NADH-TR at 21 days are shown i n Figure 8.  In normal EDL, three f i b e r types could be seen at acid preincubation  (pH 4.2): small dark staining ( I ) , medium to pale staining (IIB) and pale staining  fibers  (IIA).  Using  preincubation of pH 9.4, the small  fibers could be further subdivided reversal  (IIA),  into those which d i d not demonstrate  (Ip), as seen at 1 and 7 days, and those which were pale  I) due to  alkaline i n s t a b i l i t y .  showed reduced  oxidative enzyme reaction with  were more intensely oxidative.  coincide  with  respectively.  named  by  (type  The palest staining f i b e r s at pH. 4.2,  remaining  those  dark  Peter  NADH-TR while the  These f i b e r s  et.al.  (1972)  were labeled to as  FG  and FOG  \u000C- 68 -  Fig. 6  Histochemical  p r o f i l e of normal EDL at one day of age.  S e r i a l sections of EDL at one day of age stained for H and E, NADH-TR, and myosin ATPase at pH 9.4 and 4.2.  Bar = 50 ^um.  spindle (SP) has been marked for orientation.  A muscle  \u000C\u000C- 70 -  Fig. 7.  Myosin ATPase and oxidative enzymes of normal and denervated  EDL at seven days of age.  Photomicrographs of s e r i a l sections: Upper Left:  normal EDL stained with NADH-TR  Lower Left:  denervated EDL stained with myosin ATPase pH 4.2.  Upper Right:  normal EDL stained with NADH-TR  Lower Right:  denervated EDL stained with myosin ATPase pH 4.2  Arrows on NADH-TR micrographs indicate f i b e r s i d e n t i f i e d i n myosin ATPase micrograph bar = 50 /im  \u000C\u000C-  In  the denervated muscles  There  was a s l i g h t l y  minimal  greater  f i b e r s on pH. 4.2 ( F i g . 7). muscles.  are seen at 7 days  changes  difference  i n staining  This varied  i n degree  of age.  of the two fast between denervated  The intensity of oxidative staining was the same i n normal and  denervated result  12 -  muscles  at 7 days  but precipitated  formazan  particles  as a  of the NADH-TR reaction, marking the enzyme location, were more  c e n t r a l l y located i n the denervated muscles.  At 14 and 21 days, i t was  no longer possible to group f i b e r s i n denervated muscle, using the normal criteria  (see f o r example F i g . 8 ) . A dispersed  group  of large  fibers  could be distinguished from the remaining smaller f i b e r s by pale staining with  myosin  ATPase at pH. 4.2.  These  were  equivalent  fibers  yet were moderately oxidative with NADH  fibers  were  heterogeneous  with  respect  variably, at both acid and a l k a l i n e fibers  i n the denervated muscle  (IIAox).  to size  preincubation.  demonstrated  to normal IIA The remaining  and stained,  albeit  Up to 21 days, a l l  uniform moderate  staining  with NADH-TR (see f i g . 8) but differed i n that there was a more central d i s t r i b u t i o n of formazan p a r t i c l e s compared with the oxidative f i b e r s of controls.  A summary of the percent d i s t r i b u t i o n of f i b e r types i n normal  and denervated muscle from 1 to 21 days of age i s shown i n Figure 9\u2022 The proportion  of type one f i b e r s  (type  I and presumptive  remains constant throughout the period studied.  type I f i b e r s )  This proportion declines  gradually up to maturing where there are very few type I f i b e r s i n mature mouse EDL (Parry and Parslow, 1981). IIAox  occurs i n normal  denervated muscle.  development  In can also be seen that the type at 7  days  and i s seen  again i n  I t i s not known i f these two types are a l i k e , or i f  the denervated muscle has d i f f e r e n t properties.  \u000C- 73 -  Fig. 8  Histochemical  at 21 days of age. bar = 50  Fig. 9  p r o f i l e of normal and denervated  EDL  S e r i a l sections stained as indicated,  ^m  Fiber Type d i s t r i b u t i o n of normal and denervated  EDL  \u000C\u000C-75 -  \u2022  Norm  UDen  Fig 9  \u000C- 76 Contractile Properties (a)  Contraction Time  The  difference  muscles  i n contraction  i s illustrated  times between normal  i n the o r i g i n a l  and denervated  records of twitch myograms from  normal and denervated EDL at 7 and 21 days of age  ( F i g . 10).  The  TTP  decreased steadily from day 1 values i n normal EDL up to 21 days of age (Fig.11). during  Following  the f i r s t  denervation, there  week, followed  by a  This parameter remained unchanged  i s an  initial  subsequent  at 21 days.  change of 1/2RT contrast  statistically  decrease at 14  days.  s i g n i f i c a n t l y prolonged  The normal muscle exhibited a s i m i l a r pattern of  with development  to TTP,  i n TTP  In addition, at a l l age  groups studied, TTP of the denervated muscle was compared with controls.  increase  as was  the change i n 1/2RT  significant.  With  seen  with  between 14  TTP.  and  denervation the  However, i n  21  days  1/2RT  was  not  exhibited  an  i n i t i a l increase by 7 days, similar to TTP and subsequently decreased but remained s i g n i f i c a n t l y slowed compared to normal. (b) In  Isometric Twitch and Tetanus Tension the  tetanus  control  tension  denervated  muscles,  both  (Po) increased  EDL,  twitch  absolute  at each age  tension  changes  isometric studied  twitch  (Pt) and  (Fig 12).  were minimal  between  In the 7 and  21  days, although, there was a s i g n i f i c a n t increase when compared with the 1 day  values.  The  tetanus  recorded at 1 day of age. produced  significantly  controls. and  The  was  twitch  significant  normalized with respect  effect  to denervation and  time  never  exceeded  the values  studied, the denervated muscles  and  i n twitch and  tension was due  however  At each age  less  difference  denervated EDL  tension,  tetanus tetanic  at 7, 14 and to muscle  tension  tension  than  their  between  normal  21 days.  weight  (group effects)  When twitch  there was but no  a small  interactive  \u000C- 77 -  F i g . 10.  Original records of twitch myograms of normal  and denervated EDL at 7 and 21 Days of Age. change i n scale)  (note  \u000C7 days  Norm  Den  Fig. 10  21 d a y s  \u000C- 79 -  F i g . 11  TTP and 1/2RT  of normal and denervated  EDL  F i g . 12  Twitch and tetanus tension i n absolute values and  those normalized to muscle weight of normal and denervated  for both figures: * s i g difference between normal and denervated values + s i g difference between 7 and 21 day values  EDL  \u000C\u000C\u000C- 82 e f f e c t of denervation on development.  Normalized tetanus tension showed  a substantial drop by 7 days then remained unchanged i n denervated muscle ( f i g . 12).  S i m i l a r l y , there was no net decline i n Po between 7 and  21  days i n the normal EDL due to a reversal of the trend seen at 14 days, ( f i g 12).  By two-way ANOVA both twitch and tetanus tension showed group  effects for time and denervation but there was no interactive e f f e c t . (c)  Ratio of Twitch to Tetanus Tension The r a t i o of the twitch to tetanus tension i s a r e f l e c t i o n  degree of a c t i v a t i o n i n a muscle. tensions  of the  Table 5 compares the twitch to tetanus  from 1 to 21 days of age  denervated EDL.  The  twitch to tetanus r a t i o decreased from 1 to 21 days i n the normal  EDL.  With denervation, there was reduction  with  significantly  development.  i n normal and  a marked increase at 7 days followed However,  the  denervated EDL  by a  exhibited  higher Pt/Po r a t i o at 7 through 21 days i n t e r f e r i n g  a  with  the normal pattern of maturation. (d)  Maximum Velocity of Shortening Table VI i s a summary of the means + SE of the maximum v e l o c i t y of  unloaded  shortening.  There was  a  significant  difference  at  each  age  studied.  In the normal EDL, Vo increased from 1 to 14 days and remained  unchanged  from 14  to 21  days.  However, there  was  no  parameter i n the denervated muscle at 7, 14 or 21 days.  change  i n this  Moreover, i t i s  noteworthy that the Vo values of the EDL following 21 days of denervation was s i m i l a r to the normal values at one day of age. (e)  Post-tetanic Twitch Potentiation F i g 13 shows an o r i g i n a l record of Post-tetanic Twitch Potentiation  (PTP) i n 21 day normal EDL and the r e s u l t s of a l l PTP measurements are summarized  i n Table VII.  PTP i s minimal i n the normal EDL at day 1 and  \u000C- 83 Table V Twitch Tension to Tetanus Tension Ratio i n Normal and Denervated EDL  AGE  Denervated  N  Normal  1 Day  (6)  0.515  +0.068  7 Days  (6)  0.425  +0.053  (6) 14 Days  (6)  0.370  (6)  0.309  +0.056*  0.708  +0.049*  +0.026  (5) 21 Days  0.847  +0.04lt 0.606  (6)  +0.08l*t  * s i g difference between normal and denervated p i .05 t s i g difference between 7 and 21 day values Table VI Maximum Velocity of Shortening* i n Normal and Denervated EDL  AGE  N  Normal  Denervated  1 Day  (6)  3.080  +0.24  7 Days  (6)  4.392  +0.46  (6) 14 Days  (4)  5.582  (6)  5.552  +0.41*  3.278  +0.05*  3.824  \u00B10.70*  +0.49  (6) 21 Days  3.552  +0.37t  (5) * s i g difference between normal and denervated p^.05 x Lo/sec t s i g difference between 7 and 21 day values  \u000C- 84 TABLE VII Posttetanic Twitch Potentiation i n Normal and Denervated EDL  Days 1(6,-)  Normal b  Denervated a  1.009  + .0l4  7(6,5)  1.035  + .013  1.012 + .007  14(6,5)  1.069  + .018  1.027 + .007  21(6,5)  1.115  + ,013t  1.011  + .007*  a  Values are means + SE, expressed as r a t i o o f posttwitch over pretwitch. k ( , ) Number of normal and denervated muscles tested respectively. * S i g n i f i c a n t difference between normal and denervated muscles, t S i g n i f i c a n t difference between muscles at 1 and 21 days.  F i g . 13  Post tetanic twitch potentiation i n normal EDL at 21 days of. age.  \u000C- 85 -  F i g . 14.  Original records of the f i r s t three frames of the  fatigue regime of 1 day and 21 day normal and 21 day denervated EDL  (note d i f f e r e n t scales)  F i g . 15  Fatigue p r o f i l e of normal and denervated EDL at 1, 7, 14  and 21 days of age  \u000C- 86 -  \u000C- 87  -  1.2  1.0  i  1  270  300  0.8  0.6h  1 Day 7 Days 14 Days 21 Days  0.4  Normal Denervated  \u2022  c o (A  0.2  c  o  O  0  &gt; O  1.2r  30 Fig.  15  60  90  120  150  180  Time  sec  210  240  \u000C- 88 increases  over the next three weeks to reach adult values at 21 days o f  age,  but the denervated muscle shows no post-tetanic twitch potentiation,  (f)  Resistance to Fatigue The o r i g i n a l records using the f i r s t three frames of t y p i c a l fatigue  profiles  are seen  i n Figure  14.  The resistance  to fatigue  of 1 day  normal and 21 day denervated EDL can be compared with the rapid of the 21 day normal muscle.  The fatigue  profile  the means of a l l normalized tetanic contractions in Figure 15.  more  produced by p l o t t i n g  f o r each group i s shown  Normal muscle shows a decrease i n fatigue resistance with  development at each age studied. was  fatigue  fatigue  resistance  than  The denervated EDL from 7 to 21 days controls,  never  returning  equal to those seen at the time of denervation: 1 day of age.  to values  \u000C- 89 -  DISCUSSION  \u000C- 90 Studies of the effects of neonatal denervation on the maturation fast-twitch s k e l e t a l muscle show impairment i n morphological Verbova,  1975;  Shafiq  et  (Engel  and  Kumar  and  a l , 1972;  Schiafino  Karpati,  and  1981;  Butler-Browne  (Matsuda  a l . , 1984;  Strohman  (Brown et a l , 1982;  McArdle  Settembrini,  Karpati  et  properties  al,  1977;  1968;  1972;Ishiura et  Talesara,  and  and  1968;  al,  Matsuda,  McArdle and  (Gordon and  Sansone,  1970),  Engel, et  and  1977;  histochemical Shafiq  1982;),  1985)  of  et a l ,  biochemical  and  contractile  Sansone, 1977;).  Lowrie  et  a l . (1982) reported that neonatal denervation by nerve crush, followed by reinnervation,  permanently  changed  rat  EDL  to  an  oxidative, fatigue  r e s i s t a n t muscle and with a reduced a b i l i t y to produce tension, even with the  number  of  functioning motoneurons unchanged.  McArdle  and  Sansone  (1977) showed a permanent s e n s i t i v i t y to c a f f i n e contractures, suggesting neonatal denervation interferes with the maturation In  this  denervation  work, may  some general  be  findings on  described. From  the  process.  the  effects  of  neonatal  2-way ANOVA analysis of  data,  examined at 7, 14 and 21 days of age, denervation affected a l l parameters measured ability  with of  proceedure series  the  exception  animal  to  of  animal  thrive  was  or the paralysis of one  showed  studied,  the  from  there the  were no  surgical  procedure  be  attributed  to  deprivation  of  i t s innervation. In  not  local  on  any  by  shows the  a l l histochemical  parameters,  of  effects  number, exhibited expected  with  the  muscle properties  1). on  normal  the  the  denervation  The  the  EDL,  results  muscle  may,  due  to  a l l physiological  properties, with the exception of maximum velocity of unloaded and  that  Further, our limited sham  (Appendix  the  This  affected  hindlimb.  effects  therefore,  weight.  exception  of  maturational changes during the  shortening  total 3 week  fiber time  \u000C- 91 period  of  showing a  this  study.  significant  In addition, there all  histochemical  exception values.  of  Denervation  altered  difference between normal and  was  interference with  enzyme p r o f i l e s and  1/2  a l l parameters  RT,  Vo  and  the  studied,  denervated  pattern  groups.  of development i n  a l l physiological properties  normalized  twitch  and  tetanus  with  tension  The muscle showed a small but s i g n i f i c a n t reduction i n growth i n  length and a marked f a i l u r e to increase i n weight, compared with controls. Growth: Changes described  i n growth  i n Table  maturation  III.  i n animal  period studied.  parameters  of  normal  A l l unoperated  weight, muscle  length  Muscle length i s increased  division,  (Moss and that  to  Leblond,  denervated  provide 1971).  synthesis  length  of  t h e i r length was  by  muscle weight division  Moreover, D.  Goldspink  (1976;  denervated  significantly  less  in  Williams  immobilized  and  muscles  in a  for  each  growth  1980)  stretched  showed  postion,  continued  to  increase  with  less than controls by 7 days.  (1976) found  i f their  is  nerve  is  length left  also be  due  age,  Synthesis  i n the absence of change  intact.  therefore possible that the decrease growth i n length i s due reduced neural a c t i v i t y but, i t may  at  to  However, i n t h i s study, although  muscles  Goldspink  due  of myosatellite  proteins  immobilized  are  of the daughter n u c l e i from  of m y o f i b r i l l a r proteins i s therefore able to continue innervation.  growth  necessary  mature muscle,  the  and  showed  animals  of  capable of increased protein synthesis. the  operated  animals  c e l l s and the i n c l u s i o n i n the muscle of one each  and  to  be  It  is  i n part to  to the habitual r e s t i n g  position assumed by the denervated limb. Denervation weight.  interfered with  At 7 days of age,  the  there was  developmental  increase  in  muscle  no s i g n i f i c a n t difference i n muscle  \u000Cweight  between  failed  to gain weight a f t e r  growth.  D.  normal  Goldspink  and  operated  that mainly  (1980) reported  c o n t r a c t i l e proteins decreased that  the  loss  atrophy. days  of  these  denervated  i n denervated  muscle,  i n equal proportion to weight,  protein  atrophy.  found  a  (1972)  i s withdrawn, but  Pearlstein  and  after  Kohn  denervation. in  over  increased  out  that  of degradation  This  which  is  they  myosin  followed them for 16  synthesis of  and  therefore  loss  denervated  muscle was  total  total  myosin  was  synthesis due  to  matched  and  synthesis  protein and  by with  tibialis  Activity  controls  degradation.  of  after  supported  days post operatively.  protein  synthesis  and  further  labelled  time  systhesis  ClM-glycine, prior to denervation i n adult rat gastrocnemius anterior, and  this  innervation occurs  the balance  (1966)  the  points  innervation  major  change  exceeded  proteins i s possible before  degradation  the  cyclicial  degradation  Gauthier  of  indicating  10  and  one  the  D. Goldspink (1980) followed protein synthesis for the f i r s t  to  proteins was  group  to loss of cross-sectional  that  contractile  favors  the  of  However  leading  due  but  components  post-denervation  period.  animals  Atrophy  investigated by Schwartz et a l . (1985) who  of and in  looked  at the source of lysosomes responsible for protein degradation following denervation. disuse,  The fact that degradation i s greater i n denervation than i n  i s explained by  acid phosphotases,  their  findings  that  the  lysosomes,  are released from the severed nerve  containing  terminal as well  as near the neuromuscular junction and from myosatellite c e l l s . and  Scarth  (1981)  examined  autolytic  activity  i n muscle  by  Boegman using  a  chemical axon transport block ( c o l c h i c i n e ) , neural impulse block (TTX), a combination  of the two  or denervation.  They found  enzyme a c t i v i t y occured i n a l l groups but was  increased a u t o l y t i c  highest i n the  combination  \u000C- 93 and  denervation groups.  Therefore, t h i s  neurotrophic and impulse a c t i v i t y . a  role  i n the  denervated  autolytic  muscle,  junction and to the new  the  by  both  This implies that the nerve may  play  activity  presence  of of  influence  denervated  ribosomes  i s mediated  muscle.  near  the  Finally,  in  neuromuscular  immediately adjacent to the sarcolemma has been attributed  synthesis of proteins d i r e c t l y responsible for the increased  TTX r e s i s t a n t channels and ACHE s e n s i t i v i t y of denervated muscle.  These  c h a r a c t e r i s t i c s are also present i n developing muscle. Morphometries: The r e s u l t s of cross-sectional area measurements of whole muscle and individual who  have  fibers, found  that  Stonnington, 1974; which  i n this  denervation r e s u l t s  with the r e s u l t s  i n muscle  atrophy  of others (Engel and  Gauthier and Hobbs, 1973; Webster and Bressler,  i s greater following  Talesara, 1977).  study, coincide  neonatal  Furthermore,  than  adult  denervation  1985),  (Kumar  and  there i s atrophy i n a l l of the denervated  muscles by 21 days, and t h i s atrophy i s due to a decrease i n i n d i v i d u a l f i b e r area. weight  loss  M y o f i b r i l l a r protein loss i s proportional to the whole f i b e r (D.  Goldspink,  1980)  and  myofibrillar  area  decreases  proportion to whole f i b e r area as assessed at the u l t r a s t r u c t u r a l (Engel and Stonnington, 1974)  i n d i c a t i n g the decrease i n f i b e r  represents  decrease  the  extent  of  in  contractile  in  level  diameter  protein.  The  d i s t r i b u t i o n of i n d i v i d u a l f i b e r diameter was random and does not suggest a d i v i s i o n of three f i b e r types according to s i z e . Histochemistry Ontell and Dunn (1978) have shown that there i s no increase i n the t o t a l f i b e r number, a f t e r b i r t h , i n muscles  of the r a t .  They found that  errors are frequent when counting f i b e r s at the l i g h t microscope  level.  \u000C- 94 In  this  study, r e s u l t s  indicate  that  there  f i b e r number i n developing mouse EDL. of Rowe and  Goldspink (1969) and  In the denervated EDL, 21  G.  i s no  change  in  This i s i n agreement with the work Goldspink (1980), also i n the mouse.  t o t a l f i b e r number was  s i g n i f i c a n t l y decreased  days when compared to f i b e r number i n the  could be due  postnatal  1 day  normal group.  to a r e a l f i b e r l o s s , an apparent loss due  by  This  to f i b e r s that  do  not extend the whole muscle length as described i n developing muscle, by Ontell and  Dunn (1978) or d i f f i c u l t y distinguishing the small f i b e r s .  Myosin ATPase a c t i v i t y i n  fast  fibers  a l k a l i n e and  l a b i l e i n acid conditions and  and  in  labile  alkaline  histochemical typing interaction  of  of  the  conditions  f i b e r s by  effects  incubation proceedures, the the  pH of the  typing  alkaline  and  Kaiser,  the  classifications  the  including  using  and  be  stable  Samaha,  of  The  two  1969).  a lack  alkaline  (Green et of  depends on  preincubation  staining,  enzymes and  modified  the and  out  and  of  by  acid  Brooke  preincubation have  a l . , 1982).  correlation  enzyme  The  most commonly used f i b e r  Herman, 1955;  f i x a t i o n at  in  i n acid  myosin ATPase c h a r a c t e r i s t i c s  different  comparing r e s u l t s of oxidative differences  duration  s e n s i t i v i t y (Padykula and  species differences  and  these c h a r a c t e r i s t i c s  been shown to y i e l d d i f f e r i n g r e s u l t s clear  to  i n slow f i b e r s stable  (Guth  preincubation solution.  1970), or  found  temperature at which i t i s carried  methods, according to  and  of  is  There  are  between f i b e r type for  myosin ATPase.  example, In  when  addition,  have been found between f i b e r s of the same type i n d i f f e r e n t  muscles even within  the  same animal  (Muntner et a l . , 1985).  For  these  reasons, many authors warn that f i b e r type terminology should be confined to that  s p e c i f i c a l l y i d e n t i f i e d by  Y e l l i n and Guth, 1970;  the  Brooke and Kaiser,  method used (Green et a l . , 1974).  1982;  \u000C- 95 Brooke and that  are  fibers They  stable  that  inhibited  in  stability  and  staining  pH  in alkaline,  are  further  reversal  Kaiser have subdivided muscle fibers into; type II f i b e r s  is  stable  in  designated acid  labile  acid  the  fully  and  IIA  and  IIB  continue  inhibited.  preincubation  to to  in  alkaline  the  fibers  the  staining  All  of their s t a b i l i t y and  acid  labile  term  conditions  therefore  in  these  a  that  pH  mature  type  I  preincubation. that  fibers  at  and  below  fiber  are  most  show  some  which  types  therefore t h e i r staining  at the  IIA  exhibit alkaline  (see table I I ) . A  stain  fourth type i s occasionally  moderately  at  both  r e v e r s i b i l i t y of staining.  acid  and  seen i n mature f i b e r s alkaline  pH,  and  which  will  therefore,  lack  Lack of r e v e r s i b i l i t y i s t y p i c a l of immature,  regenerating or t r a n s i t i o n a l f i b e r s which stain moderately after acid a l k a l i n e preincubation conditions (Dubowitz and In t h i s study, a l l f i b e r s at 1 day lack of  reversibility  moderate staining was to further lighter  by  characteristic  pH  4.2.  Uniform  i t was  possible  9.4  and 4.2  f i b e r s into those darkly staining  (type I I ) .  Rubinstein and  Kelly  show uniform and  non-reversible staining  referred  to  9.4.  Ishuira  them as  study stained at both pH  IIC  (type I)  (1978) used the  type II for a l l f i b e r s found i n the rat EDL at pH.  and  at  showed t h i s  however, at pH  uniform staining  rat  Brooke,1973)\u2022  of age  staining  seen at pH 9.4,  subdivide the  staining  primitative  ( f i g 6)  II  (Hp).  preincubation,  This fibers  based  could be  term  et a l . (1981) found a l l f i b e r s i n newborn EDL  fibers.  Although  and  the  conditions (immature), i t was  is  and  at b i r t h based on  Soleus of fibers  felt  more accurate to describe them as presumptive type I (Ip) and type  and  on  the  subdivided  findings  into  two  that  in  that i t  to the  this was  presumptive with  acid  groups, those which  \u000C- 96 were very dark and  those which were medium staining.  group i s  consistently  (see  9)  fig  up  to  21  preincubation  does not  declines.  the  In  decreases  to  described by and  is  to  adult the (4.35). of  1%  age,  Kaiser  of  and  total  fiber  while  the  staining  the  number of and  dark  the  intensity,  (Parry  lack  type  Parslow  I  staining  population in  to  the  type  II  acid  of r e v e r s i b i l i t y fibers  1981).  eventually  The  IIC  (1970) i s only moderate staining  correspond  Following the staining seen that the  of  change i n  than  Brooke and  likely  days  adult mouse, the  less  10%  approximately  The  presumptive  fiber  in  named  acid here.  intensity through t h i s three week period i t can  immature f i b e r s progressively s t a i n more l i g h t l y and  d i s t i n c t i o n between IIA  and  IIB  must be  done at  in  a higher  be the pH  This probably represents a converstion to the more mature forms  myosin.  The  oxidative  nature  of  these  fibers  is  shown  by  the  moderate, uniform NADH staining of a l l f i b e r s . The  difference  reversible  staining  between the  dual staining  of mature f i b e r s could correspond  synthesis  of  different  neonatal,  to  fast  myosins  myosin,  during  described  development by  Gauthier et a l (1982) showed that there was slow myosins i n developing muscle and 1978)  Whalen  to  the  from  et  sequential  embryonic  al.  (1979;  crossreactivity  showed further  the  and  1981).  with fast  At  7 days of age,  distinguished  i n t h i s study, a difference  their  stability  fibers.  By  14  i n staining  days, the  remains unchanged at 21 days of  In the mature f i b e r , the to  at  in  fibers.  between type II  p r o f i l e has emerged and  and  (Gauthier et a l . ,  that antibodies to fast myosin correspond to alkaline stable and  slow myosin, to acid stable  due  of immature f i b e r s and  largest  9.4,  could  mature  staining  age.  f i b e r s were c l a s s i f i e d as type  moderate l a b i l i t y  at  be  pH.  4.6  and  IIA  total  \u000C- 97 i n h i b i t i o n at pH. 4.2. In addition, with NADH which indicates glycolytic (1972).  (FG) type  these f i b e r s showed minimal  they are anaerobic f i b e r s probably of the fast  according  to the nomenclature  A second group of alkaline  stable,  acid  of Peter  stain moderate to intensely fast  red  aerobic  oxidative-glycolytic  termed  Ip and with  typical NADH  type  I.  reaction  results  fibers  which  corresponds  These f i b e r s  of Peter  with  et a l .  the remainder  the slow  are at variance with  being  are also oxidative  findings  alkaline oxidative fibers  the  A third  but with 50% remaining alkaline  These f i b e r s  like  al.  with NADH and are therefore oxidative making  (FOG) f i b e r s  f i b e r s are acid stable  et  l a b i l e f i b e r s which did  continue to react at pH. 4.2 are c l a s s i f i e d as IIB f i b e r s .  them  staining  stable  labile  group of which are  are therefore  according  of Peter  of the fast-twitch  fast  to intense  (SO). These fibers  i n the  rat (Melichna and Gutmann, 1974; Niederle and Mayr, 1978) where the large anaerobic f i b e r s are type IIB and the small aerobic fast f i b e r s are IIA. The  r e s u l t s of t h i s study are i n agreement with the findings  and  Pette (1982;1984b) i n fast twitch muscle i n the mouse.  of Reichmann  As the recommendation i s to name the f i b e r s according to the method used  and because the oxidative  with altered  the IIA/FG  some a l t e r a t i o n  was palest  minutes,  conditions.  anterior,  (IIA)  fast  white  fiber.  i s due to a species difference  i n staining  the r a t t i b i a l i s  fiber  particularly  I t must be emphasized however, that  i s the large  whether t h i s difference  in  independently  a c t i v i t y (Green et a l , 1984), the Brooke and Kaiser terms  have been adhered to here. findings,  enzymes vary  at acid  but was the reverse  preincubation, (medium:  I t i s not known i n the mouse or to  I t i s interesting  Samaha et a l .  i n our  to note that,  (1970) found  the largest  i f preincubated  for  30  IIB) i f the preincubation was  \u000C- 98 brief.  This i l l u s t r a t e s the inaccuracies inherent i n myosin ATPase f i b e r  typing. The  myosin  ATPase  terminology  suggests  generalizations  to other  c h a r a c t e r i s t i c s of f i b e r types and the use of IIA f o r a large f i b e r that is  anaerobic could be misleading to those making conclusions from the  rat.  However, there i s r i s k  i n drawing  types even within the same animal. myosin isozymes  and l i g h t  conclusions between l i k e  fiber  Mabuchi et a l . (1982) found d i f f e r e n t  chain patterns i n f i b e r s c l a s s i f i e d  by t h e i r  myosin ATPase staining as IIB, from t i b i a l i s anterior and adductor magnus muscle  of the rabbit.  uptake  of  type  IIA  Similar fibers  inconsistencies  before  and  after  conditions could produce changes i n one parameter f i b e r type by another  were  seen  i n calcium  stimulation.  Altered  while not changing the  parameter.  As has been found by others (Ishiura et a l . , 1981), the typing of the denervated  fibers  found  i n this  study  does  conventional descriptions of adult f i b e r s . f i b e r s , which stain moderately are seen.  not f i t with  A heterogenous  any of the population of  to intensely at both acid and a l k a l i n e pH  They do not posess c h a r a c t e r i s t i c s consistent with any of the  f i b e r types found i n the normal mature fast-twitch muscle as they after  acid  fibers.  as  well  as  alkaline  preincubation, t y p i c a l  immature  I t i s tempting to presume these are again synthesizing neonatal  myosin but there i s no proof that  this  peculiar  to denervated muscle or that  reliable  indicator  population  NADH.  of the type  of f i b e r s ,  shows complete with  of  stain  generally  inhibition  These f i b e r s  after  i s not a novel form  the myosin ATPase staining  of myosin  being  synthesized.  of greater diameter acid  of myosin  preincubatiion  are, by d e f i n i t i o n ,  than  A small  the others,  yet stains  IIA although  is a  they  darkly differ  \u000C- 99 -  from the mature IIA f i b e r s i n controls, i n that there i s no evidence to show that  they are oxidative  they are i n fact  those termed IIA i n the normal EDL.  and  the same fibers  as  To stress that fact, they have been  termed IIA oxidative (IIA ). ox The  f i b e r s that are found  different  from  i n -the f i r s t  the normal group i n that  contrast between staining of the IIA and the one day pattern. In  this  highly  is a  slightly  enhanced from  They remain uniformly oxidative.  thesis,  labile.  there  denervation are  IIB and they have matured  following  neonatal  f a i l e d to convert to an anaerobic muscle. is  week a f t e r  Fast-twitch muscle  manipulations; increased a c t i v i t y  denervation,  fast-twitch  The oxidative nature of muscle  becomes aerobic with  through  EDL  exercise (Green  almost a l l  et a l . 1984),  low frequency stimulation (Pette et a l . , 1973), denervation (Niederle and Mayr,  1978)  and  immobilization  (Melichna  and  Gutman,  1974).  These  changes i n a c t i v i t y of the muscle, be they an increase or decrease, a l l change the muscle i n the same d i r e c t i o n .  What they a l l have i n common i s  the absence of a t y p i c a l fast-twitch motoneuron pattern. spinal  cord  section,  high  frequency  stimulation  of  However, a f t e r  the  disused  fast  muscle resulted i n an increase i n t h e i r g l y c o l y t i c a c t i v i t y . In studies on the effects of neonatal denervation on the maturation of  fast-twitch  alkaline  muscle  using  myosin  preincubation, Engel and  ATAPase  histochemistry  Karpati (1968) i n rat  Karpati and Engel (1968) i n guinea pig gastrocnemius (1983) i n r a t EDL,  found  preferential  atrophy  of  at  gastrocnemius,  and Dhoot and Perry the  type  II  fibers.  Ishiura et a l . , (1981) found a s i m i l a r atrophy of the type II c e l l s . with myosin ATPase at 9.4 light  chains  profiles,  preincubation and  they  found  that  SDS  only  But  gel electrophoresis for  neonatal  denervated  EDL  was  \u000C- 100 still  capable  of  maturation  Unfortunately, they did  did not  in  the  expression  myosin  not present the gel data for the atrophied f i b e r s .  al.  (1972) used both acid and  rat  EDL,  denervated  fibers.  at b i r t h , and  However, Shafiq et  not  differentiated  to the findings presented  able  to express  developmental and delayed.  into mature  i n this t h e s i s .  chains  unchanged  effect  of  mature fast  myosin but  mature forms of myosin they  Rubinstein and  Kelly  i n neonatal  neonatal  of  the  showed dual staining i n the atrophied  (1985) found denervation  denervation  in  the  light  sequence to  chains and  i n rat EDL.  chick  EDL were  using antibodies to  found  the  fibers.  Butler-Browne  a l (1982) and Whalen et a l (1985) found neonatally denervated  eventually  and  This would have  a l k a l i n e preincubation i n a study  f i b e r s , concluding that they had This corresponds  isoforms.  test for reversal i n acid preincubation  provided a d d i t i o n a l information about these  et  of  Looking  fast-twitch  heavy at  muscle  be  the  myosin  isozymes d i r e c t l y , Strohman and Matsuda (1985) found that the s h i f t from immature to adult forms of myosin heavy chains proceed immature isoforms of beta the chick, did not. fast  light  chain  histochemical fast-twitch synthesis  tropomyosin, which accompanies development i n l i g h t chain synthesis showed depression of the  FLC3, high  findings  muscle of  The  but repression of  i n mature  point  when both  contractile  to pH  fast-twitch f i b e r s .  an  inhibition  conditions  proteins  occurs  are in  of  used, which  While  the  maturation  of  a the  discoordinate heavy  expression i s not impaired, the l i g h t chain isoform expression, retarded but the regulatory proteins are s t i l l ,  chain  minimally  or again, of an immature  form. The the  findings of Strohman and  results  reported  here,  Matsuda  assuming  that  (1985) are they  are  i n agreement  atrophied,  with  immature  \u000C- 101 f i b e r s , staining at acid and alkaline pH which may s t i l l have gone on to synthesize mature myosin heavy chain and l i g h t not  regulatory  explained.  proteins.  These  There  fibers  could  are have  denervation and are recapitulating t h e i r development.  Or, the acid  two  chains although  ways  i n which  dedifferentiated  development a f t e r  this  as  a  this  and a l k a l i n e s t a b i l i t y  possibly may  be  result  of  set back to  characteristics  are divorced from the myosin expression as appears to be the case i n the staining of myosin as a predictor  of myosin ATPase a c t i v i t y  (Guth and  Samaha, 1972). The persistence of large c e l l s found i n this study have been shown following neonatal denervation i n guinea pig gastrocnemius by Karpati and Engel  (1968), i n r a t gastrocnemius by Butler-Browne et a l (1982)in r a t  EDL, by Dhoot and Perry (1983b) and i n r a t EDL by Ishiura et a l (1981). I f i t can be assumed these are the same type of f i b e r i n each experiment, they  may  atrophy.  share  the  following  With myosin ATPase  a l k a l i n e by Ishiura et a l . staining  by  alkaline  and  necessarily  Dhoot  and  light an  they were found  (1981) and  Perry  staining  immature  characteristics.  (1983) i n acid  fiber  They  a l l failed  to be l i g h t  to  staining i n  Karpati and Engel (1968) and dual compared in  although  this they  with  dark  study. stained  staining  They with  in  are not acid  and  a l k a l i n e i n the study by Dhoot and Perry and showed a p r o f i l e of l i g h t chains of both fast and slow muscle on single f i b e r analysis.  They have  also been\u2022shown to react to antibodies to mature fast and slow myosin but not embryonic or neonatal myosin (Butler-Browne et a l . , 1982). not  the  prolonged  result  of  denervation  reinnervation (Ishiura  as  they  eventually  et a l , 1981).  Dhoot  They are  atrophied  and  Perry  showed these f i b e r s synthesized both fast and slow troponin.  with (1983)  The f i b e r s  \u000C- 102 found i n t h i s study do not show the same staining c h a r a c t e r i s t i c s but a s i m i l a r process could have resulted i n the sparing of one f i b e r type but be expressed d i f f e r e n t l y i n the mouse. fact  that although  al.,  1981;  Support  these f i b e r s were found  Dhoot and  Perry  1983),  for t h i s comes from the  i n the rat EDL  rat gastrocnemius  (Ishiura et  (Butler-Browne  a l . , 1982) and guinea pig gastrocnemius  (Karpati and Engel, 1968)  not seen  (Dhoot  i n the rat t i b i a l i s  anterior  and  et  i t was  Perry, 1983), a l l of  which possess each of the three mature f i b e r types. Neonatal  denervation of the mouse EDL  results  i n two  fiber  types,  one highly atrophied f i b e r that resembles an immature f i b e r with frequent myotube  formation,  profile  and  form.  It  a second is  denervation,  were  seen  myosin  ATPase  type which  possible rather  dedifferentiation. there was  dual  that  than  i s less  these  a  staining  oxidative  atrophied and  are  result  and  novel  of  may  fibers,  arrested  enzyme  be  a  fast  specific  to  maturation,  or  This i s supported by the fact that a f t e r denervation,  some continued d i f f e r e n t i a t i o n histochemically.  These  before the denervation changes  histochemical  changes  are  delayed,  compared with a l t e r a t i o n s i n the c o n t r a c t i l e properties i n which changes are more immediate. Contractile properties Extensive properties  information of  is  mammalian  available  muscle  Drachman and Johnston, 1973; 1974)  and  following  the  Bressler,  1985;  F i n o l and  Lewis, 1981;  1972;  Melichna  Betto  and  and  with  on  the  changes  development  (Close,  Gutmann and Melichna, 1972;  denervation  of  Midrio, 1978;  mature  Gutmann,  1974  ; Syrovy  and  contractile 1964;  (Webster  Johnston,  Kean et a l , 1974; et  1965;  Gutmann et a l . ,  muscle  Drachman  Gutmann et a l . , 1972;  in  a l . , 1972).  and 1975;  Lewis, However,  \u000C- 103 regarding the denervation e f f e c t s i n developing, muscle the major focus has  been on  little  on  changes i n isoforms of  their  contractile  the  properties.  contractile  proteins but  very  It w i l l  necessary  rely  be  to  heavily on these studies for the interpretation of the mechanical  events  found i n t h i s work. Contraction  time  decreases  with  development  (Close,  1964)  and  increases with denervation of mature muscle (Webster and Bressler, Syrovy  et a l . , 1972;  F i n o l and  Lewis,  1981).  required to develop twitch tension (TTP) may two main events. time  constants  influenced  by  the  the  events  availability  of  a balance  action well  of  energy  cycling  stores and  and  the  the  will  be  activity  of  As well, the duration and maintenance of adequate calcium switch for actomyosin  between the release of calcium i n i t i a t e d  potential  as  time  occur following changes i n  crossbridge  l e v e l s to induce the troponin, tropomyosin is  Prolongation i n the  The rate of development of tension w i l l depend on of  myosin ATPase.  1985;  other  and  by  the membrane  the rate of sequestering of calcium by  proposed  calcium  shunt  mechanisms,  such  binding  as  the  SR  as  parvalbumin  (Heizmann et a l . , 1982). I f a c t i v i t y was with maturation  and  the major regulator of TTP, decrease  with  denervation.  showed that the myosin ATPase a c t i v i t y was  then i t would increase Guth and  Samaha  (1972)  low i n developing muscle and  did not correlate with the intense staining of routine myosin ATAPase (pH 9.4)  at  that stage.  However, Rubinstein and  technical questions about the method used and debate. denervated increase  Syrovy  (1972) found  myosin  ATPase  Kelly  but  not  with  raised  that point i s s t i l l activity  fast twitch muscle of the cat and rabbit i n contraction time  (1985) have  decreased  under in  the  i n concert with  the a l k a l i n e  stability  an  seen  \u000C- 104 histochemically.  I t i s possible, with ATPase a c t i v i t y  not being synonomous, the a c t i v i t y histochemical p r o f i l e . in  rabbit  could  typing  be changing regardless of the  Brody (1976) looked at soleus and crureus muscles  from 7 to 21 days during maturation. These muscles have the  same myosin ATPase a c t i v i t y but d i f f e r i n t h e i r TTP. of  and f i b e r  the SR  activity  changed  with  did not.  the contraction  Due  to contradiction  time  The calcium uptake  but the myosin  ATPase  i n the l i t e r a t u r e ,  i t is  d i f f i c u l t to conclude that the atrophying f i b e r s i n denervated muscle are immature  and therefore l i k e l y  posess a low myosin ATPase a c t i v i t y .  In  any case, the major change i n f i b e r type i s not seen u n t i l 14 days, too l a t e to be a causative factor i n the change i n contraction time, evident by 7 days of age.  In this study, r e s u l t s indicate that the mature state,  i n the normal muscle, has not been achieved u n t i l at least respect  to TTP, which  also  argues against  a major  21 days with  input  from  myosin  ATPase a c t i v i t y because the f i b e r type maturation occurs by 14 days. Other possible contributions to TTP are the rate of conduction o f the action potential, and the maturation of the SR. to  immediately a l t e r  McArdle et a l . , potential, potential  the sarcolemmal properties  1980) including  the rate  of r i s e  and the rate  the reduction  and degree  of conduction  Denervation i s known  (Sellin  et a l . ,  of the resting  of overshoot  1980;  membrane  of the action  of the muscle action  potential.  This change i s complete early a f t e r denervation and could account i n part for the increased TTP. initial  The slowing of the TTP recovers somewhat from the  set back at 7 days and remains p a r a l l e l  to normal development  u n t i l 14 days, therefore some ongoing maturational processes are probably responsible reported  by  for this Close  recovery.  (1964)  In developing  and SR  changes  muscle,  by Luff  the TTP changes  and  Atwood  (1971)  \u000C- 105 follow  a  dilated  s i m i l a r time in  course.  denervated  (Schiaffino and  Although  developing  Settembrini,  the  SR  has  gastrocnemius  been  shown to  muscle  (1970), i t i s poorly  of  developed  There i s an increased maximum calcium uptake due  SR  (1970)  but  the  calcium  transport  activity  rat  (Shafiqu  a l . , 1972). (Sreter  the  to  is  be  et  swollen  impaired.  Gauthier and Hobbs (1982), have described a novel myosin synthesis i n the absence  of  neural  absence of a  the  impaired  contractile  relationship  However,  precisely ordered  coupling could be of  influence.  of  and  proteins  they  suggested  that,  in  the  membrane system, excitation-contraction a slow contraction time occur present.  contraction time  with  Brody the  (1976)  myosin  regardless  compared  the  ATPase a c t i v i t y  and  sarcotubular calcium uptake i n the rat soleus.  He found contraction time  and  The  SR  function to be more c l o s e l y r e l a t e d .  contribution of  each  factor i n t h i s i n t e r a c t i o n cannot be derived from t h i s work, but i t could be speculated  that the development of the SR contributes to the  day reduction i n TTP  and  7 to  the interference with further change may  to the occurance of the denervated f i b e r type. observed i n t h i s study that TTP has  14  relate  F i n a l l y i t has been also  been disrupted by 14 days because of  denervation. The  1/2  RT i n the normal EDL  from 1 day to 14 days and a s i m i l a r f i n d i n g by Close  i n t h i s study decreased  only s l i g h t l y thereafter. (1964) i n the rat EDL.  significantly  This corresponds to With denervation,  the  pattern of change i s the same although the denervated muscles exhibited a significant function,  prolongation which  is  of  their  considered  1/2RT.  responsible  It  could  for  indicate  the  matures by 14 days i n both normal and denervated muscle. RT  are  affected  by  different  events  i n the  SR  that  relaxation  time,  The TTP and  function.  The  SR  TTP  1/2 is  \u000C- 106 influenced by the release of calcium whereas the sequestering of calcium relates  to  the  relaxation phase.  As  mentioned  earlier,  Sreter  showed i n denervated muscle using isolated fragments of SR, of SR  to  take  up  calcium  increased.  increased, the rate of uptake was  However, while  (1970)  the capacity  total  uptake  reduced and further there was  was  a leak of  calcium from the SR prolonging high sarcoplasmic calcium l e v e l s . A single stimulus  produces a  twitch  contraction.  The  unit of force generation i n muscle i s at the crossbridge.  independent  This occurs  means of myosin binding to a c t i n , the s p l i t t i n g of ATP.Pi and energy expended i n force generation (crossbridge) producing work. of a c t i v a t i o n w i l l  by  the  will  fully  tetanus  activate the  tension.  and  therefore  therefore be a result with  s t i m u l i of s u f f i c i e n t  Tension  myofibrillar  of atrophy.  denervation,  cross-sectional area  the  produced  area.  may  isometric  that form i n changes  (Engel and  Stonnington,  1974)  and  whole  the muscle length  decided  to  normalize  This i s v a l i d for adult muscle but  interstitial  could  Because the decrease i n m y o f i b r i l l a r  some error i n this assumption i n developing  area accounted for by  an  fused  frequency  i n mature muscle, i s proportional to the  tensions to muscle wet weight.  and  during  Tension  i s not greatly altered i n denervated muscle, i t was  could be  the degree  However, a  i s d i r e c t l y related to the number of crossbridges  parallel  area  muscle.  , the duration and  resulting  tetanic contraction, produced by repeated  resulting  rotation of the myosin head  In a twitch  influence the  by  there  muscle, where the  tissue i s reduced during  development  the proportion of the contribution of m y o f i b r i l l a r proteins to mass not  be  constant.  It  is  difficult  to  separate  the  factors  contributing to tension development i n whole muscle and interpretations of tension parameters must be made with caution.  Twitch  tension decline  \u000C- 107 can be p a r t i a l l y accounted weight.  However there  During  for by atrophy by normalizing values to muscle  is s t i l l  normal development, the  a reduced fiber  tension due  to denervation.  becomes more densely  packed  myofibrils and the normalized tension decreases as a r e s u l t . that  a greater proportion of m y o f i b r i l l a r  could  account  for  the  difference.  If  area the  per  with  This shows  crossectional  myofibrils  in  area  denervated  developing muscle was more densely packed than controls, i t could explain a  reduced  tension.  cross-sectional  area  It  is  not  i s affected  known  how  the  during neonatal  myofibrillar  denervation.  to  Maximum  tetanus tension i s a measure of the force of f u l l y activated muscle not influenced  by  the  time  course  of  membrane  events.  In  development,  tetanus tension increases as myofibrils are added i n p a r a l l e l . there  is a  small  between 1 and  but  significant  drop  i n normalized  21 days, there i s not between 7 and  Although  tetanus  tension  21 days and the early  drop i s probably due to the greater amount of i n t e r s t i t i a l tissue i n the immature f i b e r denervation,  (compare Figure 6 and Figure 7).  there  i s a profound  tension compared with controls. normalized to muscle weight, of  age.  This  is  also  compared with controls. correspond  level  pattern of  i s less than 50% of what i s was  reflected  in  tetanus  a  high  at one  twitch-to-tetanus  day  ratio  As t h i s change i s evident by 7 days, i t does not type  It would suggest  of the c o n t r a c t i l e mechanism.  changes i n denervated  i n the  I t does not increase with age, and when  with changes i n f i b e r  myosin properties.  alteration  However, a f t e r neonatal  and  i s not  likely  to be  due  to  that the change i s directed at the Effects  of temperature  muscle i s beyond the scope of t h i s  on tension  thesis  but  a  short treatment of this phenomenon i s presented i n appendix 4. The estimated  maximum by  velocity  fitting  of  shortening  a hyperbola  to  of  skeletal  muscle  shortening v e l o c i t i e s  at  has  been  non-zero  \u000C- 108 loads  and estimating  (1979)  compared  the v e l o c i t y  the extrapolated  shortening  at zero  load  shortening  arrived at by the slack  at zero value  load  (Hill,  1938).  of the maximum  (Vmax) to the maximum test  method  Edman  velocity  velocity (Vo). He  of  of unloaded found  both  methods y i e l d the same values i n single frog f i b e r s . Close  (1964)  extrapolated  compared  from isotonic  the  maximum  velocity  contractions at d i f f e r e n t  of loads,  shortening, i n EDL and  soleus and found i t to be faster i n the fast-twitch muscle, but slow i n immature EDL and soleus.  Barany and Close  between Vmax and myosin ATPase a c t i v i t y .  (1971) showed a c o r r e l a t i o n  The r e s u l t s i n this thesis show  that Vo increases between 1 and 14 days i n normal mouse EDL followed by a l e v e l l i n g o f f at 14 days; consistent with the histochemical findings that fiber  types  therefore  are mature at 14 days.  indicate an increase  development activity  as  described  by  The r e s u l t s  i n myosin  Guth  has not been reported  and  ATPase  Samaha  i n neonatal  reported activity  (1972).  here  would  with  normal  Myosin  ATPase  denervated muscle.  The low  l e v e l of Vo, measured i n t h i s study, would suggest that the myosin ATPase a c t i v i t y i n denervated EDL i s low, t y p i c a l of an immature muscle. In denervated mouse EDL, Vo birth. Vmax  Claffin to Vo  shortening  and Faulkner  i n whole  velocity  (1985) have  muscle  of only  does not change from the low values at  and  assessed  concluded  the fastest  that,  fibers.  possible interpretations of the data presented  here.  the r e l a t i o n s h i p o f Vo This  represents results  the  i n two  Either the group of  large IIox f i b e r s seen i n the denervated EDL have a low ATPase a c t i v i t y or the tension they the Vo measure. In  the l a t t e r  produce i s i n s u f f i c i e n t  to contribute noticably to  Only single f i b e r analysis could answer that  case,  the f i b e r s  question.  contributing to the measurement  of Vo  \u000C- 109 would  be the a t y p i c a l  velocity  atrophied  of shortening.  This  fibers would  which, would  be  consistent  a l l have with  a slow  their  other  c o n t r a c t i l e properties. Others have found Vo increased with  development. Reiser and Stokes  (1982), using the slack test method, showed an increase Vo i n developing chick  posterior latissimus d o r s i .  (using  In t h i s  two-way analysis of variance)  study,  statistical  of Vo indicates that  analysis  there  is a  difference between normal and denervated EDL, but that there i s no change with time. effect  In two-way ANOVA, groups are collapsed to look at a general  of time  i n both  groups.  The  high  levels  measurements within each group would make i t d i f f i c u l t effect sources  as ANOVA  i s designed  of variance  o f variance  to see such an  to be s e n s i t i v e to variance.  were sought.  Reiser  and Stokes  in  Possible  (1982) looked at  possible variables influencing the measurement and found that neither the point i n time during the tetanus at which the release was taken, frequency,  or stimulus i n t e n s i t y affected r e s u l t s .  differences was looked  at also by Reiser  stimulus  The e f f e c t of length  and Stokes  (1982), and they  found that stretching prior to measurements changed the r e s u l t s .  Edman  looked at Vo at sarcomere lengths of 1.65 to 2.75um and found that, over this  range, the measurements were stable.  This  center of the plateau of the tension length curve length change, used i n t h i s study, was 9.7%. of EDL was set at the maximum height slightly  shorter  than  i s a change of +24%.  from the  The maximum  A d d i t i o n a l l y , as the length  f o r twitch tension (which i s just  the maximum height  f o r a tetanus)  the releases  brought our muscles closer i n length to the peak f o r tetanus rather than away from i t .  Therefore,  for the variance.  i t i s c l e a r that length effects cannot account  Further muscle length measurements used i n determining  \u000C- 110 Vo  were made on whole  muscle  from  tendon  to tendon.  Errors  due to  differences between the actual f i b e r length which decrease per length of muscle with age (personal communication S. Wylie) would increase than decrease the differences seen i n t h i s work with denervation.  rather It i s  not known what effect this would have i n denervation, but as the muscles grow i t cannot account f o r the t o t a l  lack  of change  i n Vo.  The Vmax  values are proposed to represent the maximum shortening v e l o c i t i e s of a l l f i b e r s i n the muscle rather than just the fastest f i b e r s as i s proposed to occur ( C l a f l i n and Faulkner, 1985) with Vo measurements. (1979)  found  mature cat.  decreased  i n denervated fast-twitch  muscle  of the  Baker and Lewis (1983) found only small decrease i n Vmax i n  denervated adult immature  VMAX  Kean et a l .  r a t muscle.  The mature muscle i s not as affected as  muscle by denervation which could  However, Elumbarak  explain  the smaller  change.  (1985) showed the same increase i n VMAX from 1 to 21  days of age i n both normal and neonatally  denervated r a t EDL.  reported  of shortening  to measure  the maximum  velocity  Vo i s  of only the  fastests f i b e r s which may account f o r the d i f f e r e n t r e s u l t found i n t h i s study.  The conclusion  velocity  of  unloaded  activity,  i s support  of Barany shortening  and Close  (1971), that  i s proportional  f o r the p o s s i b i l i t y  that  to  the maximum  myosin  ATPase  these denervated muscles  have a low myosin ATPase a c t i v i t y t y p i c a l of immature muscles.  However,  the eventual synthesis of mature forms of myosin isozymes i n neonatally denervated  muscle, found by Rubinstein  and Kelly  (1985), prevents the  conclusion that these f i b e r s contain immature myosin isoforms.  As well,  the lack of change i n Vo between 14 and 21 days i n the normal EDL would correspond to the unchanged f i b e r type d i s t r i b u t i o n i n those muscles. Post-tetanic  twitch  potentiation  i s found  i n mature  fast-twitch  \u000C- Illmuscle  but  not  (1979), and the  in  slow  Moore and  phosphorylation  effect  i s not  due  and  that  soleus,  the  time  temperature and  Stull of  and  Stull,  1984).  MLC2f.  Moore and  Stull  of MLC2f but  course they  and  Glotman  and  degree  established  protocol  no PTP  2 weeks post denervation i n mature EDL  shown that therefore  myosin that  i t may  neural c o n t r o l . phosphorylation sustained  LCf2 i s not be  the  that  chain  with  the  maximum  Lewis  PTP  (1972) found the  and K e l l y (1975) have  denervation.  myosin l i g h t  of MLC2f to  be  a  high force contraction.  fiber  varies  It  is  likely  kinase which i s under  Crow and Kushmerick (1982) have suggested a r o l e for the  a fast g l y c o l y t i c f i b e r . of  in  of myosin  of the rat as was  Rubinstein  altered  the  established  obtaining  used here i n t h i s study.  case with denervated and normal soleus.  level  results)  PTP  for  21\u00B0C  that  to  Using the mature mouse  maximum  values at by  which was  a  Stull  i s correlated  to the  (unpublished of  and  (1984) found  the catalyst of the reaction.  Bressler  Manning  (1984) have shown that PTP  to the quantity  l i g h t chain kinase, EDL  (Moore  type and  step  down of  costs  during  a  This would suggest i t would operate i n  Denervation has a  energy  failure  to  resulted i n a marked  produce PTP,  reduction  which supports t h i s  concept. Burke and found The  direct  (1974), i n single motor unit studies  c o r r e l a t i o n between  fatigue  index  and  enzymes and  increase  i n anerobic  f i b e r s by  In t h i s study, the normal immature EDL  of the  oxidative  increasing s u s e c p t i b i l i t y to fatigue corresponds to the  oxidative EDL.  Tsairis  21  cat,  enzymes.  decrease i n  days i n normal  exhibits a high  resistance  to fatigue, which corresponds to the finding of high l e v e l s of oxidative enzymes.  The  persistance  denervated muscle may  of  high  be explained  fatigue  resistance  seen  in  the  by the interference with the change to  \u000C- 112 anaerobic  enzymes that has been demonstrated i n the denervated muscle.  s i m i l a r finding has been reported by Bressler et a l EDL,  where the  increase  resistance to fatigue. been  found  in  A  (1983) i n dystrophic  i n oxidative enzymes coincides with  increased  F i n a l l y , increased resistance to fatigue has also  denervated  muscle  of  the  mature  mouse  (Webster  and  1985).  Bressler, Summary The EDL  r e s u l t s of these  experiments show that the normal fast  begins at b i r t h as a slow muscle with slow TTP  1/2RT, generates  and  low tension, has a slow maximum v e l o c i t y of shortening, and  is  highly  histochemical  resistant  results  to  fatigue.  i n which  a l l fibers  exhibits no  This  correlates  stain  at  preincubation for myosin ATPase and are oxidative.  twitch  acid  PTP  with  and  the  alkaline  With development, the  muscle acquires mature c h a r a c t e r i s t i c s of fast twitch muscle including a greater  anaerobic  metabolism  f i b e r s of type IIA and  IIB.  and The  acid  labile,  alkaline  differentiation  profile. with  The  each  evidence  i s aborted  extent  parameter.  and  ATPase  denervated muscle shows a b r i e f attempt  to d i f f e r e n t i a t e into a fast twitch muscle but the  stable  and  the  i n the absence of nerve  muscle returns  to i t s immature  time frame of d e d i f f e r e n t i a t i o n seems to  This  conclusion  arises  from  the  vary  histochemical  i n which a great number of f i b e r s are i n a myotube form and  dual s t a i n i n g i s more intense. parameters  return  (resistance  to  levels  to fatigue, PTP,  the  Although the majority of the c o n t r a c t i l e less  1/2RT  or  equal  to  those  seen  and  Pt/Po) some achieve  at  birth  more mature  values (Vmax, TTP). It control neonatal  can over  be  confidently concluded the  differentiation  denervation  as  shown  here,  into  that  there  fast-twitch  histochemically  and  is  a  muscle the  loss  of  following  deficits  in  \u000C- 113 contractile  properties  development u n t i l stall  may  the  include  imposed  -  on  the  muscle  d i f f e r e n t i a t i o n becomes s t a l l e d at  membrane changes, the  synthesis  and the disruption of the functioning of the According to the histochemical fibers  types.  immature  Although  muscles,  these  i t is  aborted development.  The  clear  these  fibers.  reticulum  and  possibly  Biochemical studies  fibers i t is  answer  parameters.  properties  not  simply  muscles  in  common  i f these  are  about  with  dedifferentiation  at  or  would  synthesized  the u l t r a s t r u c t u r a l  of changes i n the  to measure d i r e c t l y , the  questions  altered myosin  denaturing gel electrophoresis  these  myosin ATPase oxidative enzymes and would  This  SR.  have  contribution  indicate  mimick  days.  to what isozymes of myosin are  Examination of  l e v e l would help assess the  of an  14  to  p r o f i l e , denervation produces altered  use of non  provide important answers as in  continue  truly  sarcoplasmic  immature  levels  of  calcium uptake of the fractionated  SR  factors  enzyme a c t i v i t y  muscles.  contributing  F i n a l l y , physiological studies  on single f i b e r s or the study of glycerinated  using  the  to  contractile  e f f e c t of  caffine  f i b e r s , could shed l i g h t  the contribution of the SR to changes seen i n t h i s work.  on  \u000C- 114 -  BIBLIOGRAPHY  \u000C- 115 Baker AJ, Lewis DM: The e f f e c t s of denervation on i s o t o n i c shortening v e l o c i t y of rat fast and slow muscle. 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I: a histochemical and morphometrical investigation of normal muscle. J Anat 137:109-126, 1983. Y e l l i n H, Guth L: The histochemical c l a s s i f i c a t i o n of muscle f i b e r s . Exp Neurol 26:424-432, 1970.  \u000C- 125 -  APPENDICES  \u000C- 126 APPENDIX 1:  A Comparison of Sham Operated  and Control EDI Histochemical  and Contractile Properties at 21 Days of Age. Introduction To  investigate  the  denervation could result sham proceedures  possibility  that  from s u r g i c a l proceedures,  were carried out.  Muscles  sham operated mice from the same l i t t e r , for  histochemistry.  effects  attributed  a limited  series  from normal, denervated  were processed  Contractile properties of two  to of and  simulataneously  sham operated  animals  were compared with unoperated controls at 21 days of age. Methods Identical operated sciatic  procedures  mice with nerve  was  the  were  carried  exception  left  ATPase)  and  that a f t e r  undisturbed  muscles were examined i d e n t i c a l l y contractile  histochemical proceedures  out  in  the  on  denervated  making sham  the  and  incision,  operated  group.  for histochemical (NADH-TR and  properties. were from  The  muscles  the same l i t t e r ,  used  but  sham  All  Myosin  for  those  the  the  for the  c o n t r a c t i l e properties were compared with the whole 21 day normal group. Results Oxidative muscles  from  enzyme staining pups of  shown i n figure 16.  the  of normal, sham operated  same l i t t e r  and  and  denervated  stained simultaneously,  are  The normal and sham operated muscles show the same  oxidative f i b e r type d i f f e r e n t i a t i o n , t y p i c a l of findings at 21 days of age,  whereas, the denervated  muscle shows loss  of d i f f e r e n t i a t i o n .  differences could be distinguished between the sham operated and EDL  in  myosin  ATPase  reaction  (not  shown).  Table  VIII  No  control  shows  the  c o n t r a c t i l e properties of two sham operated EDL compared with s i x control EDL at 21 days of age.  The only differences found between the two groups  were i n the tetanus and twitch absolute tension values and muscle weight.  \u000C- 127 -  TABLE VIII Contractile Parameters of Sham Operated and Control Muscle at 21 Days of Age. CONTROL SAMPLE SIZE  SHAM 2  MUSCLE WEIGHT (mg.)  3.598+1.31  MUSCLE LENGTH (mm.)  9.480+0.264  10.000+0.071  13-153+0.890  17.905+0.025  Po/MUSCLE WEIGHT  3.657+0.196  3.740+0.057  TWITCH TENSION (Pt) (gms)  4.007+0.167  5.215+0.145  Pt/MUSCLE WEIGHT  1.117+0.036  1.090+0.014  Pt/Po  0.308+0.015  0.290+0.007  TTP (ms.)  29.587+0.746  30.060+0.785  1/2 RT (ms.)  38.617+2.153  37.935+0.223  1.11483+0.012  1.1415+0.034  TETANUS TENSION (Po) (gms)  PTP  4.792+0.077  \u000C- 128 Discussion All  parameters  were unchanged  with  the  sham  proceedure  exception of twitch and tetanus tension and muscle weight. were higher i n the sham operated group. the  higher weights i n these two  range of normal muscle  which  nerve  crush  studies  of  Lowrie  et  sham  any  contractile  not  indicate  properties as a r e s u l t  possible f a i l u r e to thrive due not  included i n the study.  changes  i n which  The r e s u l t s in  of the operative  to early  the  Tension normalized to  a l . (1982)  from controls.  do  within  This coinicides with the  operated animals were not d i f f e r e n t procedures  Both tensions  were s t i l l  than average.  muscle weight was not d i f f e r e n t between groups. sciatic  the  This could be accounted for by  muscles,  but larger  with  sham  of the  histochemical  or  proceedure or any  postnatal trauma,  hence  i t was  \u000C- 129 APPENDIX 2:  V e r i f i c a t i o n of Denervation up to 21 Days of Age  Following  Neonatal S c i a t i c Neurectomy Introduction: A  variety  of  methods  have  been  used  to  deprive muscle  innervation depending on whether reinnervation i s wanted.  of i t s  For t h i s study  a method of denervation was needed that would prevent reinnervation up to the  longest  range  from  period  studied:  21 days.  electrophysiological  Methods of v e r i f y i n g denervation  evidence  (Dennis and  Harris,  1980)  to  v i s u a l inspection (Dhoot and Perry, 1983b; Ishura et a l . , 1981; Kumar and Talesara.  1977)  or  not  reported  (Brown  Schiaffino, 1973; Shafiq et a l . , 1972). for  reinnervation studies.  et  a l . , 1976;  Gentle nerve crush has been used  Reinnervation can begin as early as 10 days  post nerve crush with normal innervation at 21 days Lowrie  et  a l . , 1982)  and  Hanzlikova and  has  been  shown  to  be  (Ecob et a l . , reinnervated  o r i g i n a l neuron and not by the sprouting of adjacent neurons Harris, 1980). occur  before  by  the  (Dennis and  Reinnervation following s c i a t i c nerve resection does not 28  days  (Ecob  et  a l . 1984;  Following neonatal denervation, Dennis critical  1984;  time was  required  to produce  and  Engle Harris  a viable  Karpati,  (1980) found  1968). that  a  nerve-endplate complex,  a f t e r the regenerating nerve had reached the muscle. preliminary study was  and  The purpose of t h i s  to v e r i f y that the s c i a t i c neurectomy  procedure to  be used for these experiments would produced complete denervation for at least 21 days. Methods: Time mated  females were checked  twice daily  for new  litters  denervation proceedures were performed on the pups 24 hours a f t e r a  and new  l i t t e r was born (24-40 hrs o l d ) . The s c i a t i c nerve was resected from the  \u000C- 130 l e v e l of the greater trochanter to beyond the bifurcation of the s c i a t i c nerve into the posterior t i b i a l  and common peroneal nerves at the knee.  Three methods were explored to v e r i f y the permanence of t h i s proceedure. Clinical  signs recorded  i n animals  v i s u a l inspection of direct  up to 4.5, 7 or 10.0 weeks of age;  stimulation of normal and denervated  muscle  and a histochemical method consisting of dual staining of muscle sections with  Acetylcholine Esterase  nerve i d e n t i f i c a t i o n . thickness  (ACHE)  f o r motor endplate  and s i l v e r for  The staining was done on s e r i a l sections of 16 um  on muscles  sampled  one  i n every  10 sections throughout the  whole muscle, according to the methods of Goshgarian (1977). was  modified  preparation  by to  omitting better  the  oxalic  advantage.  acid  step,  This method  demonstrating  Identification  of  our  innervated  neuromuscular junctions was shown to be r e l i a b l e using either method and this  step  (Goshgarian of  was  not  considered  personal  endplates  and  essential  communication). nerve.  complexes were found  In  to  achieving  Sections were scored  innervated  muscle  nerve  primarily between points one f i f t h  from the proximal end.  valid  f o r presence and  endplate  to three  The presence of both nerve and endplate  i n the same complex was the c r i t e r i a for innervation.  results  fifths  staining  Denervated muscle  from: l , f o u r week; 3.two week; 4, two week; and 2, one week animals were examined with normal controls. Results: The one animal followed up to 4.5 weeks, one to 7 weeks and three to 10 weeks a l l demonstrated c l i n i c a l leg during ambulation innervation.  signs of paralysis;  and absence of a c t i v i t y  dragging  of the  i n the muscles of s c i a t i c  E l e c t r i c a l stimulation was used to check f o r reinnervation  but technical d i f f i c u l t i e s of i n s u l a t i n g other tissue from the electrodes i n small limbs, as well as d i f f i c u l t y finding a nerve stump to stimulate,  \u000C- 131 made this proceedure unreliable. Nerve and endplate staining, on longitudinal sections, resulted i n contraction  of  muscle  fibers  on  the  glass  slide  immediately  after  sectioning of frozen muscle.  This problem has been encountered by others  (Rileyrpersonalcommunication)  Contraction of normal muscle f i b e r s on the  slides  resulted  nonexistant  tearing  and  i n normal  a marked  muscle  increase  even  Nerve  though  in reticular  bundles i n a l l denervated f i b e r s . dispersed nerve tissue remained the  disruption,  i n the denervated muscle.  identifiable There was  in  while  and  it  almost  endplates were  the morphology fiber  was  staining  was  still  damaged.  around  fibers  At 7 days, a fine grained staining of i n some sections but was  different  staining of intact nerve seen i n normal muscle at that age.  from  Silver  staining of nerve was absent i n 14 and 21 week denervated muscle sections and  there was  an absence  of endplate staining.  In innervated  endplates were paler staining at 7 days than at 14 and  muscle,  21 days.  There  were no nerve endplate complexes seen i n any of the denervated muscles up to  21 days.  Figure 16 i s a low  power l i g h t  micrograph  of normal  and  denervated EDL at 21 days of age with a high power view of a part of the section to i d e n t i f y the stained structures. be i d e n t i f i e d connective  i n the normal muscle  tissue  i s seen  Nerve endplate complexes  (arrows).  can  Increased staining of the  i n the denervated muscle  but nerve endplate  complexes are not seen. Discussion: In  reinnervation studies, Lowrie et a l . (1982) found c l i n i c a l  signs of  dragging of the leg and lack of dorsi f l e x i o n persisted up to one year following neonatal s c i a t i c neurectomy. paralysis  do  not  ensure  minimal  The presence of c l i n i c a l signs of  reinnervation  has  not  occured,  but  \u000C- 132 provides a guide that the denervation has been successful. o f motor endplates  are  found  proximal  to the  belly  The majority  of the muscle  and  nerve-motor endplate complexes are found by sampling of normal muscle at and  proximal  staining tissue  to  occurs  the  midbelly  i n the  of  denervated  i n the perimysium.  the  muscle.  muscle  This i s l i k e l y  due  Some to  enhanced  increased  reticular  silver  connective  f i b e r s which  stain  with s i l v e r . In terminals verified  these  preliminary  combined that  with  reinnervation does  neonatal 1968).  (1968), sciatic  who  staining  not  occur  by  21  of  not  find  resection  reinnervation at  days  (Ecob  et a l , 1984;  21  of  the  These r e s u l t s are i n agreement with  did  nerve  silver  Acetylcholinesterase staining  denervation proceedure. Karpati  experiments,  nerve  endplate  using  this  Engel  and  days following  Engle and Karpati,  Ecob et a l (1985), looking at mature mouse muscle, demonstrated  reinnervation  by  21  days following nerve  following denervation did not appear u n t i l i d e n t i f i e d by s i l v e r staining.  crush but  the  earliest  signs  28 days post denervation, as  \u000C- 133 -  Fig.  16  Oxidative  exzyme  staining  of sham  operated,  denervated EDL at 21 days of age. a) 21 day normal EDL stained with NADH. X80 b) 21 day sham operated EDL stained with NADH. X80 c) 21 day denervated EDL stained with NADH. X80  control  and  \u000CFig 16  \u000C- 135 -  Fig.  17  S i l v e r and Acetylcholine Esterase staining of normal  and denervated EDL at 21 days of age. a)  normal  EDL  with  nerve-endplate  complexes  (arrows)  X200  tissue  disruption due to contraction i s frequent. b)  higher  magnification  nerve-endplate  of  above  field  at  large  arrow,  showing  complex. X800  c) denervated EDL X126 d) higher  magnification of above  r e t i c u l a r fibers.X800  field  to i l l u s t r a t e  the staining of  \u000CF,&lt;5  17.  \u000C- 137 APPENDIX  3  Preliminary  experiments  to establish  pH  and  temperature  conditions f o r developing normal and denervated EDL. Introduction  The tension produced by mouse EDL at 1 day of age and i n  denervated EDL at 7, 14 and 21 days of age i s very small. experiments,  these muscles were unstable and tolerated  In preliminary the proceedures  only b r i e f l y as evidenced by t h e i r c o n t r a c t i l e properties. exploratory  experiments  were  done  to  identify  the  A series of  source  of the  instability. Experiment 1 methods:  Twitch  procedure outlined  tension  was  i n methods section,  buffered with 95% 0^ and 5% CO^.  observed  using  the experimental  using Krebs solution  at 35-37\u00B0C  The muscle was given a single  shock  every 90 seconds and the rate of bubbling (airating) was varied . o results: vigorously.(3 (two  In the empty  bath, the pH of Krebs  bubbles/second) was 7.43 to 7.47.  bubbles/second) the pH rose further.  muscle  tension  bubbling.  fell  Variations  from  0.82  to 0.10  at 35 C bubbled  At a  decreased  rate  During the experiment, the grams,  i n the bubbling marginally  inspite  of vigorous  affected  the rate of  decline. conclusion:  The muscle  using t h i s apparatus.  tension  can not be maintained at 35\u00B0C,  The muscle i s sensitive to some condition i n the  bath. Experiment 2 o methods:  The pH of Krebs  was monitored  at 20 C, i n a  closed  10ml beaker and a closed 100 ml beaker, using varying rates o f buffering. results: buffered  The pH  of the Krebs  vigouously, was 7.26.  When buffering  i n the large  enclosed  beaker,  The pH i n the 10 ml beaker was 7.39.  was vigorous f o r 5 minutes  i t reached  7.37  and when  \u000C- 138 7.44  removed for 5 minutes i t rose to conclusion:  In a 10ml meaker, at 20oC the pH could be maintained  7.39.  at pH  Experiment  3  methods: without  The pH of the Krebs i n the bath was  buffering  stimulating bath  -  at  20\u00B0C.  electrodes and  to approximately  10  The  empty  muscle). ml.  bath  The  The  pH  pH  monitored  with and  capacity i s 15ml  (without  electrode would reduce  of the  Krebs  was  measured  the in a  covered and uncovered beaker at 20\u00B0C. The pH of the Krebs, i n the open bath, rose to 8.02  results:  in  15 min unbuffered and subsequently could be reduced only to 7.56  (8  by  i n the  buffering  alone.  The  solution  rapidly  evaporated.  The  covered beaker could be maintained with gently bubbling at 7.3 conclusion: 7.56. of  pH  min)  at 20\u00B0C.  In the open bath the pH could not be maintained below  The control of the pH i s d i f f e r e n t i n the shallow and deep systems  approximately  the  same  volume.  compared with  higher temperatures,  open  As  system.  CO,,  increased surface area. 1)  completely  The and  i s more l a b i l e  pH  i s more  with  a closed  at  higher  stable  at  lower  compared with  temperatures  and  an  with  To overcome the problem:  enclose  the  system  and  work  at  35\u00B0C.  This  was  t e c h n i c a l l y not possible with t h i s bath. 2) t r y an alternate buffer 3)  Work at a lower temperature  buffering  rate  of both  bath  constantly renewing  (for oxygenation)  and  the Krebs with a  the stock  solution,  adding solution at a rate to keep the system i n equilibrium. Experiment  4  methods:  Twitch tension i n 2wk  EDL  using Krebs  pH 7.43  (covered  \u000C- 139 flask) and Hepes buffer pH 7.45  (covered  flask) were compared  at 21\u00B0C  and at 35\u00B0C. results:  At 20\u00B0C, muscle tension  Krebs, i f i t was  renewed  repeatedly.  was  maintained  using  buffered  I t dropped when Hepes was  added,  o At  35 C, the tension dropped and was  buffered  Krebs  improved  the tension  unstable. only  The addition of fresh  briefly  a f t e r which  time i t  declined again. conclusion:  At  35\u00B0C  the muscle  tension  becomes unstable  using  either s o l u t i o n . Experiment 5 methods: The alternate deep muscle bath was tested f o r pH readings at 35\u00B0C both covered and uncovered. results: stablized  at  In  7.60  the with  covered buffered  deep  bath  Krebs.  system  Uncovered  at  34\u00B0C,  the  pH  and  placed  i n the  experimental position, the pH went to 7.76. conclusion:  The alternate bath w i l l not solve the pH problem.  Experiment 6 methods:  Two normal EDL muscles at 2 weeks of age were examined  in Hepes and Krebs solutions at 20 and 3 5 \u00B0 C o results: deterioriated. changing  At 20 C, using The  to buffered  second  Hepes,  muscle  the tension  began  Krebs the tension  to  do  i n the f i r s t the  same  but  muscle after  rose again and s t a b i l i z e d .  At  35\u00B0C the muscle did not remain viable with either buffer. conclusions: 20\u00B0C with constantly stable tension.  Hepes does not keep refreshed  the muscle  maximally buffered  viable.  Krebs at  Krebs can maintain a  \u000C- 140 Appendix 4:  Comparison of Contractile Properties at  21oC and 37oC.  Introduction Mechanical carried  experiments  on  out at both 37\u00B0C and  isolated  20\u00B0C.  skeletal  muscle  have  been  In the experiments reported i n  this study, the muscle bath was very shallow and contained only 10 ml. of  solution.  difficult  to  Due  to  the  maintain a  low  solubility  stable  pH  in  of  CO^  this  at  system.  37^C  it  The  was  immature  muscles were found to be p a r t i c u l a r l y sensitive to pH changes and did not  remain  viable.  As  CO,, i s more soluble  at lower temperatures,  these experiments were carried out at 20-22\u00B0C. Methods In order to compare the r e s u l t s 37\u00B0C,  a. comparison  has  been  from this  made  between  study with those at a  small  number  of  preliminary experiments done from t h i s study, from muscles at 21 days of age, at a bath temperature of 37\u00B0C and the same age group done at o 20-22 C, at  21 days  of age  o converted to estimated values for 37 C  using Q^Q values of muscles tested 1977).  Ranatunga  at both temperatures  (1980) reported some c o n t r a c t i l e properties at both  temperatures i n the mature mouse EDL. of  the r a t , but  (Ranatunga,  the  r a t figure  They were very similar to those  have  been  used  here  because  they  include data from denervated muscle. Results The figures from t h i s study are l i s t e d i n Table IX. for  37\u00B0C  (shown i n brackets) have been  reported by Ranatunga (normal  N=6,  (1977).  denervated  N=2)  estimated using  The values the  Data from the preliminary done  at  37\u00B0C  on  alternate  figures  experiments equipment  designed for larger, whole muscles, are indicated with an a s t e r i s k .  \u000C- 141 Discussion Normal  and  denervated  muscle  both  show  cooling  depression  of  isometric tetanic tension (Po) i n absolute value and when and normalized At 37\u00B0C,  with respect to muscle weight. denervated similar.  the r a t i o  between normal and  EDL i n estimated and preliminary experiment Both  indicate  a decrease  Po values i s very  i n tension with  denervation.  When  normalized to muscle weight the d i r e c t i o n of change i s the same although the  values  tetanic  differ.  This  stimulation,  indicates  the denervated  that  when  fully  muscle generates  activated less  during  tension than  normal muscle and the depression of the tetanus tension, with cooling, i s s l i g h t l y greater i n denervation.  The normal  twitch tension at 37\u00B0C.  similar  and denervated  I f the difference  muscle have  between normal and  denervated muscle twitch tension i s due to a decrease i n the rate of r i s e of the action potential, as suggested  by Ranatunga, then the s i m i l a r i t y  between twitch tension i n normal and denervated muscle could be explained i n the following way. development However,  The safety factor may allow f o r the same tension  i n the presence  i f the i n i t i a l  devervation  effect  of  calcium  of slowing  slightly release  reduced  calcium  i s slowed  on the rate of r i s e  release.  by cooling, the  of the sarcolemmal  action potential i s accentuated and the muscle could f a i l to achieve high enough  sarcoplasmic  potentiation  calcium  levels  of the twitch with  resulting  cooling,  i n reduced  characteristic  tension. of fast  The  twitch  muscle, i s seen i n the predicted value of normal EDL and corresponds to the  preliminary data.  reduced  i n denervated  just given. In denervated  At 37\u00B0C,  the twitch  tension  i s only  compared with normal EDL probably  slightly  f o r the reasons  There i s marked twitch cooling potentiation i n normal EDL. muscle there  i s cooling  depression.  When normalized to  \u000C- 142 weight i t i s found that  the  denervated muscle can  produce more tension  o per gram than normal muscle but  only at 37 C, whereas i t produces l e s s  tension  preliminary  per  gram at  20\u00B0C.  The  r e s u l t s . This could be explained  data  supports the  i f the rate of conduction  potential which i s slowed i n denervation,  was  not  estimated  of the action  slow enough at 35\u00B0C  to a f f e c t the calcium l e v e l s beyond the safety factor but long enough to prolong  the time to peak tension and  generation.  Yet  at  point where calcium things  are  contraction  20\u00B0C  then  time must be  the normalized  conduction  time  could  be  slowed  i s never adequate to produce high tension.  correct  muscle and secondly,  the  thereby give more time for tension  two  criteria  prolonged  must  be  i n denervation  met.  compared with  would not be as great.  differences between normal and  the  I f these  Firstly,  i n a f u l l y activated muscle by repeated  tension  to  the  normal  stimulation,  denervated muscle  Data i n Table IX shows that t h i s i s the  case.  The twitch to tetanus r a t i o of preliminary data and Ranatunga's work are comparable.  With a prolonged contraction time the twitch i s able to  produce more tension, that i s , become more f u l l y activated. of cooling and of denervation, elevated  in  both.  Failure  are the same i n t h i s respect and of  rate of r i s e of AP i s further depressed. i n normal and denervated EDL  35\u00B0C and likewise i n these experiments at 20\u00B0C.  causes  i n which the  This i s borne out by the  i n predicted and  effect  Pt/Po i s  excitation-contraction coupling  f a i l u r e of some f i b e r s to activate, more so with denervation  of TTP  The  ratio  preliminary data at  \u000C- 143 TABLE IX Comparison of Contractile Properties at 20-22\u00B0C and 35-37\u00B0C i n Normal and Denervated EDL at 21 Days of Age NORMAL  Po (gms)  DENERVATED  35oC  20oC  35oC  20oC  (19.1)  13.2  (5.5)  1.6  Po/wt  (5.3)  3.68  (5.0)  1.45  Pt (gms)  (2.8)  4.0  (2.16)  0.95  Pt/wt  (0.78)  1.11  (1.96)  0.87  Pt/Po  (0.16)  0.30  (0.53)  0.60  29.95  (35.60)  72.70  TTP (ms)  (13.0)  Po*  8.4  1.9  Po/wt*  2.6  1.8  Pt*  1.6  1.4  Pt/wt*  0.49  1.3  Pt/Po*  0.18  0.75  TTP*  8.9  24.0  ( ) data estimated from the work of Ranatunga (1981) *  data from preliminary experiments using alternate bath conditions  "@en
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