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A developmental analysis of the reversal response in the nematode Caenorhabditis elegans Chiba, Catherine Meye 1989

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A D E V E L O P M E N T A L A N A L Y S I S OF T H E R E V E R S A L R E S P O N S E I N T H E NEMATODE C A E N O R H A B D I T I S E L E G A N S  by C A T H E R I N E MEYE C H I B A B.Sc,  The U n i v e r s i t y  of B r i t i s h  Columbia,  A T H E S I S SUBMITTED I N P A R T I A L F U L F I L L M E N T T H E R E Q U I R E M E N T S FOR T H E DEGREE MASTER OF  OF  ARTS  in T H E F A C U L T Y OF GRADUATE  STUDIES  Department o f Psychology  We  accept to  this  thesis  the required  as  conforming  standard  T H E U N I V E R S I T Y OF B R I T I S H August  COLUMBIA  1989  © C a t h e r i n e Meye C h i b a ,  1989  1986  OF  In  presenting  degree freely  this  at the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or  her  for  of  B>vg.wot.O^V  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  purposes  representatives.  requirements that  agree  may  be  It  is  financial gain shall not  permission.  Department  I further  the  that  the  Library  an  advanced  shall make it  permission for  granted  by  understood be  for  allowed  the that  without  head  extensive of  my  copying  or  my  written  Abstract The of  nematode C a e n o r h a b d i t i s e l e q a n s  locomotory  responses  b e h a v i o u r s , among t h e m a  which  can  absence of  obvious  mechanical  stimuli.  occur  stimuli,  by  circuit,  whose g e n e t i c s ,  i n the  the  at  least  other  reversal  tap r e v e r s a l In the reversals  one  and  the  and  L 4 ) , and  the  i n response  touch  to  be  to  be  withdrawal  development  Despite  s h o w s no  spontaneous  have  known circuit by  this  apparent  examined  the  reflected  in  reversals  and  two  reported here,  tap reversal stages: adult  under a  for later  stages  light  two the  (young  both  were examined stages adult  a n i m a l s were microscope  spontaneous over  ( L I , L2,  and  4  L3,  day  observed  and  their  behaviour  analysis.  indicated  tap reversal  reflex  four larval  In a l l experiments,  Results  i n the  reflex.  developmental  videotaped  reflex,  number  reversal  behaviour mediated  c h a n g e s may  experiments  individually  a  thought  and  This thesis  responses,  six  olds).  these  of  neuroanatomy of t h i s  touch withdrawal  that  the  well-characterized.  changes over development. possibility  are  neuranatomy,  functional  over development, circuit,  or r e f l e x i v e l y ,  a common n e u r a l c i r c u i t ,  been e x c e p t i o n a l l y  family  spontaneously,  These b e h a v i o u r s  mediated  changes  either  exhibits  that  reflex  elegans.  Young a d u l t  frequency  of spontaneous  both  spontaneous  show d e v e l o p m e n t a l  animals  showed an  reversals i i  reversals  changes  in  and C.  increase i n the  relative  to the  other  developmental of  stages; this  increased a c t i v i t y  Larval  animals  different stages,  w i t h L2  response  half  a l l stages  animals  Although  to touch,  previously  taps,  undescribed  larval  animals  The  tap reversal  showing a s i g n i f i c a n t  magnitude  also  larval  taps  animals  one,  the  larval  pattern  responding  adult pattern.  o f known n e u r o n a l  patterns  accelerations.  two  to  single  approximately  t o be  of  graded, with  accelerations  fewer  stages,  reversals  and  animals. phases  L4  i n the  from  development  newly hatched the  other,  changes  from  i n the  transition be  neural from  the  the  result  of  LI  when  the  larval  with  circuit. larval  of  the  t o young a d u l t ,  periods correlated  might  a  Increasing stimulus  stages;  to taps  change  of responding  found  but  increase i n magnitude  showed  stage  These  that the  on  in  While  i n response  probability  adult  larval  from  reversed  only reversals,  also  transition  to subsequent  hypothesized  still  t o be  transition  the  a l l stages not  to adult  pronounced  f o r a l l developmental  than  appeared  reflex:  of  was  increased the  to single  There  animal  response,  s t i m u l u s magnitude.  more a c c e l e r a t i o n s  the  of  trend to  function  to taps r e l a t i v e the most  elicited  reflex  a  significantly  showed a c c e l e r a t i o n s  responses.  increasing  showed  showed r e v e r s a l s  the  though  also  animals  neither  i n sensory threshold.  showing  taps  consistently  relative  change  patterns of responding  differences.  adults  of  or  i n c r e a s e was  It  to  periods was  to adult the  addition  of  a specific  sensory  neuron,  connect ions.  iv  AVM,  and  i t s associated  Table  of  Contents  Abstract Table  ii  of Contents  List  of Tables  List  of Figures  v v i i viii  Acknowledgement  i x  INTRODUCTION  1  BACKGROUND  6  The  Life  Cycle  The  Behaviour  6 9  The T o u c h C i r c u i t Post-Embryonic General  10  Changes  i n The Touch C i r c u i t  Methods  17  Animals  17  Establishment Verification  of Synchronous  Apparatus  C o l o n i e s and  of Stages  Characteristics and General  17  of Developmental  Stages  Technique  Scoring  18 20 23  Statistical Experiment  14  Analyses  27  1: S p o n t a n e o u s R e v e r s a l s  29  Introduction  29  Method  31  Results  31  Discussion  32  Experiment  2: R e f l e x R e v e r s a l s t o T o u c h e s  Introduction  and Taps....37 37  v  Method  3  Results  39  Discussion  44  Experiment  3: G r a d e d R e s p o n s e  8  45  Introduction  45  Methods  46  Results  4  7  Discussion  6  5  GENERAL D I S C U S S I O N  68  Conclusion REFERENCES  vi  8  0  8  1  List 1. M e a n A c c e l e r a t i o n Tap  Stimuli  Tap  Stimuli  Magnitudes  o f Three  2. M e a n R e v e r s a l  of Tables  Different  Magnitudes  of Three  to Intensities  63  Intensities:  64  to  Different  vi i  List 1. T h e L i f e  Cycle  Apparatus in  f o r Studies  of  of Varying  Stimuli  of Varying  and 41  to  Intensity  51 to  Intensity  53  Between Response Type and  Intensity  on Response M a g n i t u d e  Response Magnitudes Varying  35  of Animals Reversing  Interactions  of  Period  of Animals Reversing  8. P r o p o r t i o n s  10.  Active  t o Touch and Tap S t i m u l i  Stimuli  Stimulus  33  of Animals Reversing  7. P r o p o r t i o n s  9.  Time  Reversals  Period  10 m i n O b s e r v a t i o n  Accelerating  Tap  Behaviour 21  10 m i n O b s e r v a t i o n  6. P r o p o r t i o n s  Tap  12  C. e l e q a n s  5. M e a n P e r c e n t in  7  o f C. e l e q a n s  4. M e a n N u m b e r S p o n t a n e o u s in  Figures  o f C. e l e q a n s  2. T h e T o u c h C i r c u i t 3.  of  Intensity:  56  f o r Stimuli Larvae  viii  and A d u l t s  61  Acknowledgements I  didn't  do t h i s  alone.  Thanks t o Ann Rose a n d h e r s t u d e n t s f o r a l l t h e i r help: t h e i r a s s i s t a n c e , advice, and e s s e n t i a l s u p p l i e s f o r t h e c a r e a n d f e e d i n g o f C. e l e g a n s ( a n d t h e i r p r o v i s i o n o f C. e l e g a n s i t s e l f ) made t h i s w o r k p o s s i b l e . Thanks e s p e c i a l l y , A n n , f o r i n t r o d u c i n g o u r l a b t o C. e l e g a n s i n the f i r s t p l a c e : i t ' s a wonderful animal. T h a n k s a l s o t o R o d Wong, who w a s t h e r e a t t h e beginning, and introduced a romantic t o the study of animal b e h a v i o u r . Thank y o u f o r y o u r c o n s i d e r a t i o n , good w i l l , and encouragement. I have c h e r i s h e d y o u r p o i n t o f view. F i n a l l y , I w o u l d l i k e t o t h a n k C a t h a r i n e R a n k i n , who h u n g o n t o t h e e n d , a n d i n d o i n g s o t a u g h t me a l e s s o n i n f r i e n d s h i p , craftsmanship, and t e n a c i t y that I never thought I had t o l e a r n . And I t h o u g h t I was s t u b b o r n . T h i s t h e s i s i s d e d i c a t e d t o J o h n T.M. Kam: I n t h e w o r s t o f i t y o u w e r e my s o l a c e , my s a n c t u a r y , my s a n i t y . T h a n k y o u . Y o u made t h e d i f f e r e n c e .  ix  A the  fundamental  q u e s t i o n o f how  general in  agreement  i s that  lead  t o changes  is  that  changes  not always One  systems  way and  the  nervous  One  clear  and  nervous  system:  this  the r e l a t i o n s h i p s nervous  system  entire  comes  levels,  necessarily  no  and  levels  conclusions  guarantee  and  level  of  circuits  about  start and  with  the us  system  relationships  behaviour are  that  ranging  neurons;  to the  changes  from  level  i n changes  precludes our  a t one  at another  from the  of of  level  these are  level.  interactions  at  any  anatomical, or  ability  the r e l a t i o n s h i p  not  and  t a k e p l a c e a t any  i t molecular, cellular, often  nervous  can g i v e  interactions  the sheer complexity of  level—be  behavioural,  neural  nervous  a  it,  of organization,  Changes can  reflected  Furthermore, one  systems  of molecules to the  with  systems  between  i n determining the  nervous  at several  organisms.  from  function.  are products of  of neurons  role  however,  between nervous  identical.  level  follows  relationship,  assembly  as  level  is  a key  of nervous  i t assembles  between t h e two,  the  that  1 is  cut.  difficulty  activities  plays  A d e v e l o p i n g animal does not  Both  biology  There  b e h a v i o u r i s t o s t u d y b e h a v i o u r as  into  The  assumption  i n the structure  of behaviour during  structure  system  to analyze the r e l a t i o n s h i p  functioning  insights  i s produced.  i n b e h a v i o u r . The  system develops.  nature  i n b o t h p s y c h o l o g y and  behaviour  producing behaviour.  this  a  issue  to  between  draw interactions  2 across  levels.  Yet out to  of  ultimately,  the workings  integrate  understand reflected  to understand of a nervous  information across  how  c h a n g e s a t one  i n another;  interact.  No  behaviour;  a l l appear  is  how  to  single  integrate  how  system,  level  i s the  t o be  the  we  levels.  how  sole  m u s t be  Our  goal  these  able  i s to  levels  producer  interdependent.  i n p u t s and  arises  o f o r g a n i z a t i o n may  to understand level  behaviour  be  may  of  The  question  outputs of various  levels. One animal  approach  such  as  t o the  the  m a n y r e s p e c t s , C. an  integrative  promoted 70's  (Brenner,  problem our  this  of  eleqans  potential  is ideally  simple  who  s p e c i e s as  a model  organism  i n the  1974;  see  also  a major  the  He  saw  study had  1988)  hindrance  processes  function.  organism  Brenner  and  early  recognized  the  to progress  eleqans  of nervous  such  selected  of development  i n C.  In  for just  Brenner,  t o become t o t h e  model  suited  Sidney  development what b a c t e r i a powerful  a very  approach.  c o m p l e x i t y as  system  i s to study  nematode C a e n o r h a b d i t i s e l e q a n s .  understanding of  nervous  problem  in  and  the  systems  and  been t o m o l e c u l a r b i o l o g y :  from which  to extract  a  first  principles . With  o n l y 302  highly  determinate  highly  conducive  neurons,  consistent,  development,  and  precise,  and  a genome a n d  to genetic dissection,  C.  eleqans  lifestyle is  one  3 of  the most  intensively  multicellular than  of  organisms  a millimeter  transparent,  developmental  Such choice In  of  of  putative wiring  adult  diagrams  One  C.  elegans  exhibits  of  which  itself  on  a  map  see  1988).  tempting  of  &  system. the  Karn, lineages for  hermaphrodite  Schierenberg, White,  system,  neuroanatomical  including  behaviours  &. map  locations  and  of  anatomical  (White,  Southgate,  1986). in this  animal  studied—behaviours related a number o f response  or r e f l e x i v e l y f o r w a r d on  locomotory that  can  pass  to  down t h e  substrate,  l e n g t h of  been  locomotion.  behaviours,  ( C r o l l , 1975).  a solid  has  occur  i t s s i d e w i t h u n d u l a t o r y waves of that  (e.g.,  developmental  e l e c t r i c a l synapses  is a reversal  contraction  system  as  a l s o Wood,  Brenner,  a complete  of behaviours  well  swims  as  aging  elegans  adult  less  tiny,  diverse  physical  Sulston,  nervous  Brenner,  spontaneously  i n the  1977;  and  especially  normally  type  this  i n s e a r c h of a model  completed  for several  family  1980b;  made C.  complete  as w e l l  chemical  Thomson, &  s t u d y has  exist  every  1983),  the e n t i r e  1980a;  Measuring  i s the model  and  (Coulson, Sulston,  (Sulston & Horvitz, Thomson,  adult,  toxicology,  to a nearly  t h e r e now cell  today.  nematode  neurobiologist  genome  extensively described  o f p r o b l e m s as  genetics,  f o r the  wild-type  every  study  a young soil  i n Zuckerman,  addition  1986),  l o n g as  intensive  and  i n biology  free-living  choice f o r the  references  studied  either  The  animal  propelling muscular  i t s body  from  one  4 head  to t a i l .  When t h e a n i m a l  waves r e v e r s e s d i r e c t i o n , tail  t o head r e s u l t i n g  This  behaviour  stimulation,  response,  results  i n the animal's  1981).  Other  the t a i l  stimuli,  s u b s t r a t e beneath  latter  reversals  response  such  the tap reversal  &  1988; R a n k i n ,  The t a p r e v e r s a l  of and  reflex  We  i s also  1988; R a n k i n ,  capable  to  the t a i l ,  s u g g e s t i n g an i n t e r a c t i o n  studied  (Rankin, unpublished  Sulston, 1984;  by M a r t i n C h a l f i e 1981; C h a l f i e ,  Chalfie,  dish  (Rankin  i n press).  eleqans  of long-term  i s inhibited  memory  i n press).  by g e n t l e  between  touch  competing  data).  reflex  has been  extensively  and h i s c o l l e a g u e s  Thomson,  S u l s t o n , White,  i s capable  dishabituation  Beck, & Chiba,  the tap reversal  withdrawal  this  has been t h e s u b j e c t of  addition,  The t o u c h  called  a s i t c a n be  of the animal's  In  responses  through  elicit have  non-associative learning—habituation,  (Rankin & Chiba,  A  & Sulston,  passing  reflex,  of behavioural p l a s t i c i t y .  sensitization—and  mechanical  reflex,  can a l s o  Beck, & Chiba,  motion.  (Chalfie  a vibration  i n adult animals.  from  and i s termed the  withdrawal  the animal,  these  & S u l s t o n , 1981).  forward motion  by t a p p i n g t h e s i d e  studies  to direct  (Chalfie  touch  elicited Chiba,  backward  t o t h e head,  reflex  similar  reflexive  i n response  as touch  head touch w i t h d r a w a l  the  the contractions passing  i n the animal's  can occur  such  reverses, the flow of  (Chalfie  & S u l s t o n , 1983;  Southgate,  Thomson,  &  Chalfie, &  5 Brenner, 1989). this  1985; W a l t h a l l The  functional  & Chalfie,  anatomy o f t h e n e u r a l c i r c u i t f o r  a combination of genetic lesions  microsurgery Both reflex  t o determine  the roles  are thought  t o be m e d i a t e d  by C h a l f i e  Southgate,  Thomson, & B r e n n e r ,  critical  elements  addition,  many f e a t u r e s  is the  by t h e n e u r a l Sulston,  1985)  Animals  insensitive also  Chalfie  present throughout occurence  1981; C h a l f i e ,  1984; W a l t h a l l  The r e v e r s a l  systems  thus  suitable  of t h i s  Thomson,  to touch  i n the  &  1988)  i s that i t despite  circuit's  1981).  form  a family  of  f o r the study of the  between developmental  interesting given that  changes  i n nervous  They a r e  at least  one  the touch withdrawal r e f l e x , appears  substantial  detailed  & Chalfie,  and t h e b e h a v i o u r they produce.  behaviour,  responses  development,  changes  & Sulston,  responses  especially  particularly  reflect  post-embryonic  (Chalfie  in  and  and h i s c o l l e a g u e s have  of substantial  anatomy  relationships  defective  1989).  & Sulston,  1983; C h a l f i e ,  responses  White,  show a b n o r m a l  i n t r i g u i n g aspect of t h i s response  neural  cells.  circuit  o f t h e neuroanatomical development  (Chalfie  Sulston,  of i n d i v i d u a l  et a l (Chalfie,  tap (Rankin & C h a l f i e ,  circuit  laser  of the touch withdrawal c i r c u i t  known t o be t o u c h  In  and  t h e t o u c h w i t h d r a w a l r e f l e x and t h e t a p r e v e r s a l  elucidated  One  & Au,  b e h a v i o u r has been worked out i n c o n s i d e r a b l e d e t a i l  using  to  1988; C h a l f i e  neuroanatomical  not to  changes as they  occur  6 over  development.  both  The  following  experiments  the  touch  the  development. threefold:  and The  (1)  reflex  and  the  possibility neural (3)  and  to  these the  of  experiments  behaviours—the  tap reversal  reflex;  the s u b s t a n t i a l  may  be  reflected  (2)  changes  f o r an  of the  of  throughout were  between  touch  two  withdrawal  to examine  the  i n the u n d e r l y i n g  i n tap reversal  reflex;  examination  behavioural plasticity  characterization  examination  response  relationship  l a y the groundwork  development detailed  aims of  similar  that  circuit  tap reversal  to examine  topographically  r e p r e s e n t an  i n C.  of  elegans  tap reversal  the by  reflex  a  over  development. Before describe  the  development  outlining  the experiments  I will  first  relevant  characteristics  of the  animal's  and  neural  anatomy.  BACKGROUND The C.  elegans  reproducing at  20°  three  molt  adult  after  through form.  and  Cycle  rapidly,  proceeding  from  i n a p p r o x i m a t e l y t h r e e and  (see F i g u r e 1 ) .  hours  passes adult  C  develops  Life  Eggs a r e  fertilization,  four  larval  molts  Egg-laying begins  c o n t i n u e s f o r about  laid  and  egg  a half  to  days  approximately  the developing  animal  before reaching i t s final  several  hours  f o u r days,  after  after  the  which  last  the  7 Figure  1.  presented  The  life  of  i n B y e r l y e t a 1,  typical  life  20°  Eggs are  C.  cycle  cycle  of  laid  laid.  spend  eggs a t of  approximately  development. eggs h a t c h  3 to  Times are  L4;  approximate lines  indicate  adulthood  3.5  given  thick  11  end  of  of  p e r i o d s of  testing  at  eggs  in larval begin  view  after  to  lay  indicate  of  time  On  early  average,  being  laid.  l a r v a l development lines  Boxes w i t h  begins  are  egg-laying.  i n hours.  p e r i o d s of  animal  i n days  the  Arrows  v e r t i c a l black  approximate  the  after  days and  days.  hours  of molting.  e n d s when t h e  Approximate asterisks.  the  times  2.5  data shows  at which  shows a more d e t a i l e d  approximately  through  time  effective  Dark r e c t a n g l e s mark s t a g e s LI  1(a)  3 hours  embryogenesis),  peak e g g - l a y i n g and 1(b)  the  approximately  (including  Figure  Figure  approximately  0 represents  development  1975.  From  Caenorhabditis elegans  fertilization; Animals  e1egans.  indicate  diagonal  lethargus. to  are marked  from  lay  eggs.  with  Young  T H E LIFE C Y C L E  (a)  (b)  egg laying maximal egg laying ends  _l_  t to approx. 21 days larva  egg  (b)  egg  young adult  reproducing adult  old adult  9  animal about  may 21  As  continue to live  t o a n e s t i m a t e d maximum o f  days. shown  i n F i g u r e 1, e a c h  of the four larval  lasts  approximately ten to fifteen  stage  being  larval  continuously cell is The  cuticle,  between  about  The a n i m a l  under  (see S u l s t o n ,  ends a g i v e n  larval  two h o u r s  the longest  and the p a t t e r n  individuals  consistent  animal  1, o r L I .  between m o l t s ,  division  highly  stage  hours,  after  stages  grows  and t i m i n g of  similar  conditions  1988, f o r summary).  s t a g e when entering  i t sheds i t s  a period of  inactivity. The Immediately locomote, such  or through  with sinusoidal  o f i t s body.  i s able to  semi-solid substrates  waves t h a t  I t responds  the head and t h e t a i l and,  hatching, the animal  swimming o v e r  as agar  length  after  Behaviour  propagate  t o gentle touch  b y s w i m m i n g away  from  which  body waves.  (See C r o l l ,  locomotory The  appear  behaviour  animal  also  independent  reversal  reflex,  stimulus  i s t r a n s m i t t e d t o the animal  substrate. reflex  In studies  i s elicited  containing  head  analysis of  elegans.)  shows a s i m i l a r  which  both  of the sinusoidal  1975, f o r a d e t a i l e d  i n C.  on  the stimulus,  when swimming u n d i s t u r b e d , shows c o n t i n o u s  oscillations  down t h e  i s elicited  reflex,  when a  the tap  vibrational  v i a the underlying  of non-associative learning,  by t a p p i n g t h e s i d e  t h e worm on a g a r o s e  gel.  of a  Both  this  plate  resting  and  10 forward-travelling backwards the  animals  f o r some d i s t a n c e .  primary  focus  of  the  The The  tap  head  control,  1985;  are  Thomson, &  6 touch  circuit.  elements  of  the  microtubule  The  cells,  three  cells, cells  respectively:  see  the  1988;  of  the  1983;  touch  receptors  of  groups based left  and  (abbreviated White et  as  a b b r e v i a t i o n s ) , are  either  of  the  a n t e r i o r h a l f of  processes  anteriorly.  right  posterior lateral  Two  Au,  circuit  the  i n C.  of  eleqans  and  69  as the  adult  animal  can  position.  anterior  lateral  ALMR,  f o r a more d e t a i l e d positioned the  other  microtubule  1984;  1989).  a l s o known  ALML and  a l , 1986,  genetic  Brenner,  upon t h e i r  right  of  their  to  Chalfie,  elements  the  Sulston,  interneurons,  sensory  by  been  Thomson, &  receptors,  the  has  Chalfie &  explanation side  mediated  (Chalfie &  Southgate,  touch are  t o be  circuit  5 p a i r s of  s i x touch  into  this  Sulston,  Chalfie,  receptors,  The  divided  White,  is  here.  f u n c t i o n a l neuroanatomy,  colleagues  basic  reversal reflex  r e v e r s a l response  C h a l f i e and  microtubule  Two  appears  by  Sulston,  undulating  Circuit  of  motor neurons.  be  The  reverse,  tap  development  Walthall & The  Touch  u n d e r l i e s the  touch.  Chalfie,  Chalfie,  The  and  described 1981;  that  and  experiments reported  reversal reflex  same c i r c u i t gentle  stop  laterally  animal, cells,  cells  the  on  extending left  (PLML and  and  PLMR,  11 respectively) of  are positioned  the animal's  anteriorly, other  These  microtubule  the anterior cells  extending  PVM  i n the posterior  half  anteriorly  into  animal.  Of t h e s e  cells,  directly  to the withdrawal  anterior  touch.  k n o w n , b u t may  The  involve  i n the anterior anteriorly;  and  extending i t s  portion  of the  a r e known t o c o n t r i b u t e  responses  seen  of the sixth,  to posterior PVM,  and  i s not y e t  the mediation  of other  touch  circuit  i n C. e l e g a n s  c a n be d i v i d e d  interconnected units:  anterior  touch  posterior  (head  touch-  touch  (tail  and  touch).  L) c e l l s  a n d t h e AVM  for  anterior  touch.  cells  These  response  (2) t h e n e u r a l  are the sensory are coupled  electrically  When s t i m u l a t e d , t h e s e  t o gentle touch  circuit for  (See F i g u r e 2 ) .  cells  gap j u n c t i o n s and a r e thus as a u n i t .  into  (1) t h e n e u r a l c i r c u i t f o r  touch)  and  working  AVM  behaviours.  The  by  two  respectively) are  the anterior  function  The  ventral  i t s process  five  side  processes  midline.  of the animal,  process  on e i t h e r their  and p o s t e r i o r  m e d i a l l y and v e n t r a l l y :  of the animal,  two  extend  longitudinal  (AVM a n d PVM,  half  related  also  to the animal's  cells,  positioned  tail.  posteriorly,  The ALM receptors  t o each  other  coupled, cells  cause  v i a gap j u n c t i o n s t o a p a i r  of  t h e AVDs, w h i c h  synapses  the motor neurons r e s p o n s i b l e f o r backward  movement,  t h e A a n d AS c e l l s .  connections  AVD  excitatory  the  interneurons, onto  form  (R  also  makes  chemical  indirect  t o these motor neurons v i a a chemical  synapse  12 Figure  2.  The  touch  hermaphrodite). al.,  1985,  and  Catharine  the  touch  touch  cell  represented  receptors  hatching 1977).  elegans  personal  (adult in Chalfie  communication  are  LUA.  indicated  (|—|).  et  to  the  form A  solid  *)  t o AVB  arise  interneurons  and  Both  shown.  are  are  circles. (-•=>) a n d  gap  excitatory  lines  indicate  excitatory.  synapses w i t h of the  the  40  Sulston &  identical  the  anterior  latter  approximately  & S u l s t o n , 1981;  not  as  postembryonically; the  j u n c t i o n s between are  lines  o n l y AVM  i s completed  (Chalfie  dotted  chemical  cells;  r e c t a n g l e s , as  w i t h arrows  Inhibitory  s y n a p s e s w i t h AVB.  Gap  as  Interneurons  also depicted:  cells  not  represented  diamonds; motor neurons  connections,  (marked w i t h AVM  are  connectors,  are  AVB  but  Chalfie,  w i t h bars  inhibitory The  C.  information presented  cells  as  connections  from  from  synapses  junctions  cells  From  of  Rankin.  The  Chemical  circuit  motor  AS touch  connections connection hours  after  Horvitz, neurons  and  FORWARD MOTOR NEURONS  •YAVEO  INTERNEURONS  SENSORY NEURONS HEAD TOUCH  TAIL TOUCH  14 to  AVA, a  the  large interneuron coupled  A a n d AS m o t o r  also  thought  posterior  circuit,  The  inhibitory  circuit  analogous  t o AVA  t o AVD  touch  chemical  the  touch  t o AVB, t h e i n t e r n e u r o n circuit.  circuit  i s structured  synapses onto  t h e PLMs a c t i v a t e  f o r w a r d movement.  AVD, m a k e s a s e c o n d  are  similarly.  electrically  (like  which  t o a c t as e x t e n s i o n s  t o make  anterior  chemical AVBs o n t o  circuit  synapses, t h e AVA  thought  connections  AVA)  of  to the B  forms gap are also  o f i n t e r n e u r o n s , t h e LUAs, o f t h e PLMs, a l l o w i n g  t h e AVA  (Chalfie  along with  a n d AVD  cells  e t a l , 1985).  These  from t h e  i n t e r n e u r o n s a n d t h e AS m o t o r  neurons,  t o be i n h i b i t o r y  (see Figure 2 ) .  Changes  of the elements  post-embryonically.  chemical  of  synapses  Post-Embryonic Many  to a pair  contact with  touch  half  i t s counterpart  The PLM c e l l s  coupled  them both  like  set of excitatory  the B c e l l s .  neurons.  t h e B m o t o r n e u r o n s v i a PVC,  electrically appear  the B motor  t o the posterior  PVC,  n e u r o n s v i a AVB, w h i c h  junctions with  are  are  2 ) : one t o PVC, t h e  r e c e p t o r s , t h e PLM c e l l s ,  animal,  eliciting  the  onto  i n the posterior  When s t i m u l a t e d b y g e n t l e t o u c h  motor  cells  v i a gap j u n c t i o n s t o PVC, an i n t e r n e u r o n m a k i n g  excitatory  the  connections  (see F i g u r e  i n the touch  posterior  sensory  coupled  T h e A L M a n d AVM  a n d o n e t o v i a AVM  analogous  The  t o make  touch  interneuron  neurons.  by gap j u n c t i o n s t o  i n the Touch  Circuit  of the adult touch  The n e w l y  hatched  circuit  LI animal,  arise  although  15 capable  of locomoting  (Chalfie  &. S u l s t o n ,  substantially The which  different  the four  AVM a n d PVM a r i s e AVD,  to gentle  1981), has a nervous  newly hatched  only  pairs  and responding  from  that  LI starts  lateral  head  touch  circuit  in  system  of the adult. with  touch  a touch  receptors  post-embryonica1ly.  are present;  The  AVA, P V C , a n d AVB a r e p r e s e n t ,  interneuron but the  connections  b e t w e e n AVBs a n d t h e ALMs a r e m i s s i n g  the  o f AVM.  absence  well, for  so t h a t  a n t e r i o r touch The  ventral  cord motor  B  types  neurons),  from  of motor neurons,  of these  have  2 2 o f t h e 76  as an a d u l t  larva  Changes b e g i n hours a f t e r reinitiated.  (White,  Within  comprise  A (DA), the adult  ( t h e VA, V B , a n d VD  neurons,  motor  In addition,  t h e DD  motor  i t s pattern of connectivity A l b e r t s o n , & Anness,  i n the larval  hatching.  hatching,  including the ventral  a n d t h e AS n e u r o n s .  reverses  to adult  the dorsal  types,  c l a s s e s of motor  completely  At  (DD), a r e p r e s e n t ;  neurons  the C neurons,  of these  that w i l l  has only  i twill  consists of eight  counterparts  neurons,  b e t w e e n AVB a n d t h e c i r c u i t  L I animal  neurons  as  missing.  (DB), and d o r s a l D  complement  one  i s also  neurons a r e absent  1976; S u l s t o n & H o r v i t z , 1977).  three  dorsal  connection  newly hatched  (Sulston, only  this  T h e AS m o t o r  due t o  At t h i s the next  nervous system  time  cell  10 h o u r s ,  the adult nervous  1978). three  division i s many o f t h e c e l l s  system  are  generated.  The do  touch  r e c e p t o r s AVM  the v e n t r a l  neurons. they  not  not  extend  by  B,  arise  ventral  new  cells  processes  D,  functional.  the  as  as  during this  are  of  onset  late  the  AVM  to their  as  AS  motor  born  at  this  Some  of the  may  (see W a l t h a l l  &  The  occurrence  of such  changes  seem  correspondingly is  large  a p p a r e n t l y not  touch withdrawal (Chalfie  &  The that  To  as  case:  neither  of  spontaneous  need  to  there should  at  molt; migrate before  neural  be However,  the head and  are observed  may  1988).  i n the  i n behaviour.  both  the  this  tail  a l l larval  stages  1981). reported here  changes  i n the  reversals these  address  touch  to test  reversals  and  the  the  possibility  circuit  might  by  circuit,  the  tap reversal  b e h a v i o u r s have been  r e s p e c t to development. designed  larval  Chalfie,  i n other behaviours mediated  spontaneous  therefore in  Sulston,  that  changes  reflex  connectivity  date  with  the  experiments  reflected such  indicate  be  larval  appropriate targets  functional  would  may  second  fourth  neuron,  large  time,  However, o t h e r s  the  16 as  structural  neurons)  becoming  circuit  time,  and  t h e DD  et a l , 1978).  until  such  PVM  rewiring  White  complete  some n e u r o n s , and  the  complete  (eg., see be  these  necessarily  (such as  essentially molt  ventral  Although  are  changes  A,  and  and  i n the  reflex.  examined  These experiments f o r developmental  be  were  differences  tap r e v e r s a l  reflex.  17 General  Methods  Animals C. e l e q a n s room  temperature  seeded  NGM  All each  agar  (as described  Juvenile  when m o s t  egg-laying.  animals were animals  hours  after  hatching  thus  tested  immediately  animals have reached  (Sulston,  five  as c e l l  hours  of  i n the adult  t h e "young a d u l t "  of hatching,  Establishment  ages  Op50)~  1974).  i n the f i r s t  The L I a n i m a l s were  three  (strain  by B r e n n e r ,  s t a g e , and a t v a r i o u s  ( a t 55-60 h o u r s ;  days,  N2) w e r e m a i n t a i n e d a t  ( 2 0 ± 2° C) o n E. c o l i  L3, and L4; a d u l t  molt  (strain  a n i m a l s were t e s t e d  larval  animal. L2,  Bristol  i n early L I , after  the final  stage),  and a t 4  their  peak r a t e  tested within division  the  of first  resumes 3  hours  1988).  o f Synchronous  Colonies  and V e r i f i c a t i o n  of  Stages In  order  t o ensure  same a g e , s y n c h r o n o u s allowing seeded  plates  animals for  several  The  adults  invisible  t o l a y e g g s o n E.  just  prior  hours  the development  given  i n thick  established  tested  were e s t a b l i s h e d  f o r 3 0 t o 90 m i n u t e s .  are almost  temperatures  the animals  colonies  gravid  L I a n i m a l s were  bacteria  that  As t h e  by coli-  youngest  bacteria,  on p l a t e s  were t h e  dotted  colonies with  to the addition of adults. i n Figure  of the animals.  under which  these  1 served  as rough  However,  given  a n i m a l s were kept  guides to that the were  subject verify  to small stages.  hatching  or  pharyngeal colony)  These  lethargus pumping  prior  particular  checked using one  approximately microscope, variation  are  the time  head  light  with  Slightly  no from  older L i s  body  and t a k e s  coloured,  body  with  from the  L3 a n i m a l s  along  their  the future  The gonad  one-third  tail.  (no d a r k body lengths.  The  length.  L3 a n i m a l s  ( B y e r l y e t a l , 1976) .  are clear  long  up  approximately  A f t e r the second molt,  4 8 0 \xm l o n g  mottled  oval  length  3 7 0 urn  ( B y e r l y e t a l , 1976).  i s transparent,  as a narrow white  approximately  are approximately  molt  i s darkly  L3 a n i m a l s .  faintly  are  c a n be d i s t i n g u i s h e d  one-third of the animal's  the animal's  molted  also  inserted  the d i s s e c t i n g  t o r p e d o - l i k e shape.  of the f i r s t  o f t h e body  appearing of  LI animals  transparent,  c o l o u r i n g , and  L2 a n i m a l s  of the animal  approximately rest  of  was  device  as  cylindrical.  L2 a n i m a l s . at  as w e l l  a  Stages  Under  are completely  i n body  consistency  within  eyepieces).  2 0 0 jim l o n g .  L i s by t h e i r  more  Size  Newly hatched  they  of m o l t i n g ,  for  lack of  characteristic  of Developmental  animals.  and  18 to  used  of the animals  (a measurement  of the microscope  LI  time  were  the animals  inactivity  for features  a gradicule  Characteristics  older  ( d e f i n e d by  and t i m e s .  methods  checking  i n the majority  checks  stages  other  included  to the expected  making v i s u a l  into  fluctuations,  are  Newly-  colouring)  As L 3 a n i m a l s  and become  19  older,  the  body  posterior  darkly  coloured,  although  light-coloured. elongated the  animals. lose  again.  After  (Byerly  et  pharyngeal  head remains  is thin,  a short  shedding  their  animal's  flat,  white  future round  head  body  with  bulb  becomes  clear and  line  and  slightly  on  one  side  of  with  the  take the  the  L3  transparent  the  640  jim  soon a f t e r . up  about  as to  a  The  bulb  is  white  l o c a t i o n of  appears  body;  time  o n e - f i f t h of  large  the  gonad  long  for a short  pharyngeal  a grayish  animals  as  a  in older  the  tight,  L4s,  f l a t t e n e d quarter-moon  head  one-fifth  of  (YAP).  animals  a l , 1976).  the  still  e a r l y L4s,  adult  including  outside;  cuticles,  apposed  a w i d e and  adult  young et  to  molt,  become  transparent  and  end  third  the  against  body.  Young  (Byerly  and  gonad appears  semicircle against  blackish  the  colouring  appears  The  L3  are  length,  In  gonad resembles  molt,  their  body  its flat  vulva.  p r i o r to  colouring  L4s  dark  visible.  crescent  dark  a l , 1976)  regain  clearly  Shortly  their  light-coloured  a  the  gonad  i n appearance,  animals  the  The  the  animal. L4  but  to  pharyngeal body.  however,  transparent.  no  the  the bulb)  The  850  adult  taking has  visible  up  nm  long  and  approximately  opened and  larval  proportions,  body a n t e r i o r t o  vulva  eggs are  final  approximately  They have normal  ( p o r t i o n of  the  are  After  the  to  the  animal  is  20 Adults.  Four  distinguished and  large  visible under  from young  size  (1 mm  throughout  adults  by t h e i r  darker  colouring  R i p e n i n g eggs a r e  uterus; these  microscope.  the rest  (4D) c a n be  and l a r g e r ) .  i n the animal's  the light  appear  day o l d a d u l t s  eggs a r e g r a y i s h  A d u l t s c o n t i n u e t o grow  of the l i f e  span,  t o become m o t t l e d w i t h d a r k ,  and t h e i r  blackish  intestines  pigment as  they age. Males.  Males  approximately Horvitz, only  once every  & Brenner,  found  removed.  1000 a n i m a l s  were used  This  by t h e i r  at L3 o r e a r l i e r ,  original firmly  test  plates  tail  As  t h e L3 stage, Adult males a r e  tails.  Animals  however, were m a i n t a i n e d  and General  a l l experiments,  until  their  on  their  sex could  Technique  o b s e r v a t i o n s w e r e made t h r o u g h  (see Figure 3 ) .  under b r i g h t  for  1979).  established.  videotaping  To  (Hodgkin,  experiments, any  shape.  W i l d M3Zoom s t e r e o m i c r o s c o p e e q u i p p e d  Panasonic  a t 20° C  after  spade-shaped  with bacteria  Apparatus For  i n these  c a n be done v i s u a l l y  t o the male's d i s t i n c t i v e  tested  i n populations  i n t h e s t o c k and s u b j e c t p o p u l a t i o n s were  distinguishable  be  spontaneously  1979; Rose and B a i l l i e ,  hermaphrodites  males  due  arise  field  D5000  facilitate individual  low-light,  scoring,  w i t h a phototube f o r  A l l o b s e r v a t i o n s were  illumination  made  and v i d e o t a p e d u s i n g a  high-resolution  the date,  a  time,  video  and time  a n i m a l s were superimposed  camera. elapsed  on t h e v i d e o t a p e d  21 Figure C.  3.  Apparatus used  elegans.  equipment plate  and  holder  description.  Top  f o r s t u d i e s of  depicts microscope  stimulus generator; and  tapper.  See  behaviour  with video  in  recording  b o t t o m shows d e t a i l  General  Methods  for  of  detailed  22  23 record  using a Panasonic  Animals were fine  hair  Petrie  removed  (thicker  transferred  hairs  from were  plates used  to a bacteria-free  plate)  were then  WJ810 t i m e - d a t e g e n e r a t o r .  containing  placed within  10 mL  for larger  plate  ( a 5 cm  o f NGM  agar.  a h o l d e r mounted  MM33 m i c r o m a n i p u l a t o r o n a m a g n e t i c allowed still  the dish  allowing  videotaping. using  to vibrate  In Experiments  micromanipulator. controlled  freely  smooth t r a c k i n g  a mechanical  with  a Grass  a and  diameter plates  Marzhauser  The  holder  when t a p p e d ,  while  during  2 a n d 3, t a p s w e r e  number S88  animals)  in a  base.  with  These  of the animal  t a p p e r mounted The  for testing  delivered  i n a separate  and r a t e  of taps  was  stimulator.  Scoring In Experiment total  number  active. for of  1 v i d e o t a p e d r e c o r d s were s c o r e d f o r  of spontaneous  In Experiments  described  animal (head body.  The methods  of Spontaneous  of spontaneous was  response  t y p e s and of s c o r i n g  said  to t a i l )  Reversals.  reversals  t o be r e v e r s i n g waves were seen  This normally resulted  scored magnitudes  are  were  occasionally  When c o u n t i n g  i n Experiment  1, t h e  i f backward-propagating p a s s i n g down t h e a n i m a l ' s  i n the animal's  m o v e m e n t ; h o w e v e r , some o f t h e s m a l l e r particular)  time  below.  Numbers numbers  observed.  and p e r c e n t of  2 and 3 t h e r e c o r d s were  frequencies of s p e c i f i c responses  reversals  animals  backward (Lis  u n s u c c e s s f u l i n moving  in  24  backward  (sometimes  appeared  to  continuous a s one  "bunch bout  the a n t e r i o r up"  i n front  the in  of  of the  animal  the t a i l ) .  Each  o f b a c k w a r d - p r o p a g a t i n g w a v e s was  spent a c t i v e  which  In Experiment  i n the o b s e r v a t i o n  nearest second  using  backward-  or  1,  t h e amount  period  a stopwatch. Only  scored to  those  periods  f o r w a r d - p r o p a g a t i n g waves  down t h e a n i m a l ' s b o d y w e r e  c o u n t e d as  periods  (momentary pauses  transitions  caused  backward  t o f o r w a r d movement w e r e  in  an  which  animal remained  were s c o r e d as head  portion were  occasionally: of the body  thus  involving active  inactive  ignored  Response Experiments  increases of  periods,  even  movements  Responses  3 fell  into  (for definition,  a b o v e ) , and  the animal  2 seconds  responses occurring  o f two  prior  within  seconds total  touch i n major  categories: Spontaneous  defined  as  to the average  to the stimulus.  1 second  contraction  the  see Numbers o f  relative  pharynx  period.  (2) a c c e l e r a t i o n s ,  i n swimming speed  moved  that  to the  number o f  t o t a p and one  periods  undulation  only  by waves o f  i n the o b s e r v a t i o n  from  i f the animal  involving  The  "active"  without  immediately anterior  o f t h e body.  Type.  2 and  reversals  Reversals,  stationary  were  Those  then converted to a percentage of  number o f s e c o n d s  (1)  ignored).  i f unaccompanied  the r e s t  was  by  of  was  passing  its  counted  reversal.  P e r c e n t Time A c t i v e . time  part  speed  Only  of s t i m u l u s completion  25 were  counted.  In cases  occurred within classified that  fell  a single  as w h a t e v e r into  but were  classified  as  detectable  change  animals video  different  of  each  measurements  b o d y was  expressed  Thus  distance  travelled  body  covered  / body  length),  the  a d u l t body  travelled  allowing  the t a i l  magnitude  size  by  filmed image  body  immediately  resuming  of  a distance length).  the path  travelled  following  the  t h e end of either  forward  s c o r e d by t r a c i n g  of the  distance  e n d e d when t h e a n i m a l  of the animal  was  the effects  the actual  lengths travels  body  (distance  rather than  i t s own  own  an e x a m i n a t i o n  s c o r e d by t r a c i n g  A c c e l e r a t i o n s were  2  stop-frame  of the animal's  t o body  or changed d i r e c t i o n ,  by  i n Experiments  t r a c e d , and a l l  of response  t o 8 t o 10 t i m e s  stimulus. A reversal  travelled  the paths  traversing  the head of the animal  stopped  no  of v a r i o u s s t a g e s were  on r e s p o n d i n g  R e v e r s a l s were by  i n which  were  occurred.  also  relative  (an L I animal  by two  equivalent  acceleration  to the stimulus  responses  i n terms  the measure  of stage  size  or  Responses  m a g n i f i c a t i o n s , the length of the video  length.  effects  was  first.  of responses  As a n i m a l s  responses  the response  responses  i n behaviour  of  acetate sheets w i t h the a i d of  animal's  travelled  clearly  Magnitudes  analysis.  at  types  of the r e v e r s a l  o b t a i n e d by t r a c i n g  onto  both  occurred  " o t h e r " , as were  Magnitude. 3 were  second,  type  neither  categories  and  i n which  motion. path  immediately  following  26 the  stimulus.  obtained tail  by  tracing  2 seconds  averaging Both to  of  prior  to the onset  the d i s t a n c e  determine  changes  distance  defined  judged  as  the  and  seconds. half  second  t o have o c c u r r e d i f the  i n t e r v a l i n the  the stimulus  t r a v e l l e d per The  the s t i m u l u s  i n i n t e r v a l s o f one  was  following  stimulus.  animal's  t r a v e l l e d f o r t h e two  travelled in either  immediately  of  the  was  i n speed.  acceleration  distance  the p r e - s t i m u l u s speed  t h e p a t h t r a v e l l e d by  r e c o r d s were marked  An  the  A measure  exceeded  i n t e r v a l i n the  beginning of  second the  average  2 seconds  prior  the a c c e l e r a t i o n  i n t e r v a l i n which  this  increase  to  was  in  speed  occurred. The  end  interval  intervals  the of  the animal  or decreased t o a speed  speed  stimulus  i t was or  also  between  of  accelerations tracings which  on  the  whichever  a digitizing the  the  stopped,  changed  of  to either 2 seconds  the  first  occurred f i r s t .  b e g i n n i n g and  paths  as  f o r two c o n s e c u t i v e  the speed  end  of  the  (a) prior  the to  interval The  time  acceleration  acceleration. t r a v e l l e d by  or reversals  transferred  either  than or equal  r e c o r d e d f o r each  Lengths  defined  t r a v e l l i n g i n the  (b) h a l f  the  was  i t s speed  less  the a c c e l e r a t i o n ,  elapsed was  the a c c e l e r a t i o n  a f t e r which  direction,  average  of  were  pad  image  animals  o b t a i n e d by  in either  retracing  (Summagraphics B i t Pad into  a Macintosh  SE  the  Plus),  27 microcomputer. computed  The l e n g t h s o f t h e images were  u s i n g MacMeasure,  measuring  a program s u i t a b l e f o r  the lengths of irregular Statistical  Two b a s i c t y p e s experiments: of  Ratio changes  were  ratio  types  observed  analyses of analyses of  whether  between g r o u p s a r e l a r g e enough  being  attributable  occurs  within  (e.g.,  stage)  being  see Bailey,  to the kind of variation  pp. 3 2 4 - 3 5 9 ) .  1981, pp. 99-114;  The t e s t  statistic,  present  error.  ANOVAs c a n b e m o d i f i e d  design,  the primary  change  Glass  to suit  being  &  that (for Hopkins,  F, i s a r a t i o o f  attributable  t e s t e d t o t h e amount o f v a r i a t i o n  t o be  tested,  a l l groups r e g a r d l e s s of treatment.  amount o f v a r i a t i o n  error  frequencies  f o rage-related  used f o rt h e i n i t i a l  rather  being  Numbers  a c t i v e , and  to test  o f measurements,  to the factor  the  of time  i n the  measurements.  In order  attributable  1984,  obtained  measurements.  T h e ANOVA a l l o w s o n e t o d e c i d e  differences  discussion,  were  measurements;  categorical  (ANOVAs) w e r e  data.  percents  Measurements.  i n these  variance the  type  Analyses  and c a t e g o r i c a l  m a g n i t u d e s were  response  curves.  o f measurements  spontaneous r e v e r s a l s ,  response of  ratio  then  to the factor  attributable to the experimental  i n composition  of the  term. A s t h e ANOVA t e s t s  significant  value  a l l groups simultaneously, a  for F indicates  b e t w e e n g r o u p means e x i s t s ,  only  n o t where  that a  difference  a difference  occurs.  28 For most  analyses,  probability with  an  of  test  response This  i n those  underlying in  reaching  I e r r o r of of  method,  magnitude. used  test  the  .05  or  less  magitudes  was  i n which  of  variable,  and  determining  whether the  variation  present  trend across  levels  (For d i s c u s s i o n of  Keuls,  and  multiple  comparisons,  368-392,  and  see  in particular  Categorical  Data.  categorical  data,  analysis  variance.  of  they  As  pp.  could  not  Instead,  f o r the  association  ( G l a s s &. H o p k i n s ,  p r o p o r t i o n s of  with to  respect  the  the  that  stages  there  p r o p o r t i o n of  attribute.  No  1984,  exist  pp.  396-391). types using  285-289)  test  i t tests  an  tests  values  f o r the  of  applied  observed is  similar  the  d i f f e r e n c e s between exhibiting  were  t a b l e s were  chi-squared  This  in that  choosing  1984,  accelerations  individuals  theoretical  for  analyzed  pp.  tested.  no  Newman-  response  and  be  the  the  contingency  r e v e r s a l s and  the  be  analyzed  a n a l y s i s of v a r i a n c e  hypothesis in  to  data  of  is  interested  can  Hopkins,  374-376,  counts  constructed  to  and  is  of  basic rationale Glass  Experiment  continuum  one  independent v a r i a b l e . trend analysis,  f o r making  in  is a  independent  to s p e c i f i c  followed  F-statistics,  there  a  graded  the  attributed  with  An  used  to stimuli  which generates  situations  was  between means.  trend analysis,  test,  significance  Newman-Keuls method  pairwise comparisons  alternative to  type  application  multiple  3  any  a  groups  given  proportions  29 expected  are  assumed; on  rather, expected  calculated  based  works w e l l  e v e n when t h e  response  in a cell  Hopkins,  1984,  As  with  statistic  (x )  in  a  can  Significant  (JD <  (see  with  actually  be  .05)  Glass  compare t h e  contrast with  the  ratio  having  freedom equal data  set.  that  no  of  i n Glass  that  the &  a chi-squared to  one  less  pp.  variance  were  the  (pp.  observed which  thus  followed  proportions  391-392).  of  with  degrees groups  proportions  interval  280-281.  shows a t  least  tables  Glass  &  Reversals  p r e v i o u s l y , i s the occurs  1:  two  i n response  types reflex  to  of  in  contrast,  number o f  f o r the  These  tested  the  distribution  confidence  proportions  to  proportions  intervals  using  as  between the  1984,  than  test  1984).  discussed  reversal  of  different.  values  the  expected  Confidence  elegans  test  statistically  considered  Spontaneous  One,  notes  indication  Experiment  C.  frequency  between p r o p o r t i o n s  & Hopkins,  the  a  comparisons  variance  for binomial  Hopkins,  (see  This  ANOVA, a s i g n i f i c a n t  chi-square  were a l s o c o n s t r u c t e d , given  expected 2  f o r the  table,  the  the  as  obtained.  are  288).  with multiple pairwise  of  low  difference exists  proportions  tests  average  F-test  contingency  observed  actually  indicates only  2  significant  data  i s as  p. the  the  frequencies  reversal.  reversal:  mechanical  a  in  30 stimulation.  Both  the  head  the  tap r e v e r s a l  reflex  seen  frequently  in adult  touch withdrawal  are  reflex  animals,  reversals. i s the  uninduced  reversal.  direction  w h i l e swimming u n d i s t u r b e d on  surface, factors by  and  Adult animals  a l t h o u g h t h e y may  i n the  agar  observers, the  or to minute  response  exogenous mechanical Evidence that  from  frequency Stopfer, Aplysia  of  &  frequency  of  the  1987)  newly  circuit  t h e r e may  a general principle upon b e h a v i o u r , in  the  then  occurrence  of  by  to  any  be  i n those  found of  unseen  obvious  suggests  i n the et a 1  that  (Rankin,  juvenile  spontaneous gill  that  and  siphon.  decreased  as  new  as  elements.  elements  development  similar  spontaneous  If this and  i n the  in a of  reflects i t s effect  increases w i l l  activity  The  the  a concurrent high rate  of neuronal perhaps  species  Rankin  contractions  This suggests  activity  agar  not d e t e c t a b l e  reflected  developed  animals matured.  spontaneous  be  frequency  of spontaneous  develop,  caused  or  reverse  a bare  vibrations  reversals.  Carew,  showed a h i g h  contractions  frequently  invertebrate  c h a n g e s may  spontaneous  Marcus,  other,  spontaneous  responding  i s not  The  and  stimulation.  another  developmental  be  response  occur  touch  c ircuit. The frequency  first of  development.  experiment  spontaneous  was  thus  reversals  designed as  they  t o examine occurred  the  over  31  Method Subjects Ten  animals  were used  (60  f o r each  animals  of  in  the  s i x developmental  stages  total).  Protoco1 Single prior  to  filming.  microscope animals  animals  stage  were  bright-field Animals  at  allowing  then  minutes  a l l experiments  stage  best  was  was  left  were  on  after  under and  plate the  which  the  filmed of  to equate  3  min  lighted these  same  a l l stages,  held at a constant  combination  In order  to a test  3 minutes,  f o r 10  illumination  the  required  plate  f o r these  For  each  tracking.  The  filmed  illumination.  were t r a n s f e r r e d  the level.  at m a g n i f i c a t i o n s resolution  the  and  amount o f  ease  of  tracking  between stages, m a g n i f i c a t i o n s g i v i n g  approximately  equivalent-sized  video  screen  images  were  chosen. Scoring  and  Statistical  Videotapes reversals  used the  Analyses  were s c o r e d  ( i n ten minutes)  for total and  One-factor  between groups  to analyse  the  between groups  data.  number o f  percent analyses  Developmental  of  time  spontaneous spent  active.  of v a r i a n c e were s t a g e was  set  as  factor. Results  Developmental mean number o f  stage  had  a  significant  spontaneous r e v e r s a l s  10-minute o b s e r v a t i o n p e r i o d ; F ( 5 ,  effect  observed  54)  = 4.84,  on  the  during  the  p_ <  .005.  32  Newman-Keuls  posthoc m u l t i p l e comparisons  the young a d u l t stage showed s i g n i f i c a n t l y of r e v e r s a l s than any other age group 4) .The number of spontaneous all  revealed  that  l a r g e r numbers  (p_ < .05; see F i g u r e  r e v e r s a l s was s i m i l a r across  other stages t e s t e d . Animals of a l l stages were a c t i v e f o r most of the ten  minute o b s e r v a t i o n p e r i o d .  The percentage of time an  animal spent a c t i v e showed a s i g n i f i c a n t e f f e c t of developmental comparisons  stage; F(5, 54) = 3.106, p_ < .02.  showed that the LI animals spent  Multiple  significantly  l e s s time a c t i v e than any other stage (p_ < .05; see F i g u r e 5) .  Other developmental  different  stages were not s i g n i f i c a n t l y  from each other. Discussion  Spontaneous r e v e r s a l s occur at s i m i l a r r a t e s across most developmental  stages t e s t e d .  immediately f o l l o w i n g the l a s t  However, i n the hours  l a r v a l molt, young a d u l t  animals show a g r e a t l y e l e v a t e d tendency to engage i n spontaneous  reversals.  T h i s increase was not the r e s u l t  of a increase i n locomotor a c t i v i t y  i n g e n e r a l , as a l l  stages with the e x c e p t i o n of LI were a c t i v e f o r approximately the same percentage of time. it  i s at approximately t h i s stage  Interestingly,  (about 40 hours p o s t -  hatch) that C h a l f i e and S u l s t o n (1981) r e p o r t that the AVM-ALM connections become  functional.  33  Figure  4.  minutes,  Total  number o f s p o n t a n e o u s  a l l developmental  stages.  reversals in ten  Black  bars  indicate  mean number o f s p o n t a n e o u s r e v e r s a l s s e e n d u r i n g minute  observation period  young a d u l t stage other  stages (which  square  root  Glass of  tested.  Only the  different  from the  Means a r e p r e s e n t e d  i n t h e case  o f t h e ANOVA,  o f {the a v e r a g e d  & Hopkins,  standard  (*) w a s s i g n i f i c a n t l y  tested.  errors  f o r each stage  standard  a 10  with  standard  i s * V M S / n' p  the  d e v i a t i o n / rty ; s e e  1984, pp. 217, 233, 436, f o r d i s c u s s i o n  errors).  Mean Number  L1  L2  L3  Spontaneous Reversals  L4  developmental  YAD stage  4D  35 Figure  5.  observation spend  Mean p e r c e n t period,  significantly  developmental  stages  time  active  i n 10  ± standard errors. l e s s time  LI animals  a c t i v e than  tested, with  minute  the  (*)  other  the e x c e p t i o n of  L2.  36  Mean  Percent  Time  developmental  Active  stage  37 It  is  reversal  possible  frequency  manifesting to  connectivity reversals, change this  and  of  the  However,  change  of  examine  stimulus  spontaneous  a n e u r o a n a t o m i c a 1 change  is  another  threshold.  to  in  o c c u r i n g i n the  result  in sensory  change  due t o  observed  changes  or the  the  in behaviour.  the  problem i s  source  is  itself  say whether  that  the  it  circuit factor,  is  result  of  mediating s u c h as  a  addressing  behaviours  for reversal  difficult  direct  One method o f  reflex  is  i n which  clearly  the  identified  quantifiable.  E x p e r i m e n t 2: Reflex In t h e reversal stimuli is  Reversals  adult,  reflex.  A l t h o u g h we h y p o t h e s i z e  that  the  circuit  in s l i g h t l y  sending  vibrations  stimulating of  the  receptors Thus,  different through the  ways.  i n the  head  it the  tap operates  by  may a c t u a l l y  be  i n b o t h the  head and the  tail  experiment  across  stages.  the  touch  the  responses  those e l i c i t e d  This  i n d e t e r m i n i n g whether younger all,  circuit,  it  compared t o  tap at  both  alone.  second  developmental  the  i n t e r a c t i n g with As t h e  agar,  by head t o u c h e s were  both  are  The head t o u c h s t i m u l a t e s  i n the  that  touch withdrawal  two s t i m u l i  touch receptors  animal.  T o u c h e s and T a p s  b o t h t a p and h e a d t o u c h e l i c i t  a r e a c t i n g upon t h e  possible  to  experiment animals  and i n d e t e r m i n i n g w h e t h e r  elicited  by  was  taps useful  respond to  responses  the  differed  38  as a f u n c t i o n of developmental  stage.  Method The general methods and apparatus  for this  are d e s c r i b e d under "General Methods". the r e l e v a n t f e a t u r e s of Experiment  experiment  D e t a i l e d below are  2.  Subjects Twenty animals were used developmental animals  stages  (total:  f o r each of the s i x 120 a n i m a l s ) . H a l f the  i n each stage were randomly assigned to a "touch  first"  group,  first"  group.  while the other h a l f were assigned to a "tap T h i s was  done to c o n t r o l  f o r order e f f e c t s .  Protocol All  animals were g i v e n two s t i m u l i , 7-15  minutes  apart, one a g e n t l e touch to the head of the animal, d e l i v e r e d u s i n g a f i n e h a i r glued to the needle of a 1 cc s y r i n g e ; the other a s i n g l e tap, d e l i v e r e d to the s i d e of the d i s h with a mechanical S c o r i n g and S t a t i s t i c a l  tapper.  Analyses  Responses to touches and taps were scored f o r both response  type and response magnitude.  Frequencies of  animals g i v i n g s p e c i f i c response types were analyzed u s i n g chi-squared t e s t s of a s s o c i a t i o n to t e s t developmental  stage.  Reversal and  acceleration  frequencies were analyzed s e p a r a t e l y . responses  ( r e g a r d l e s s of response  f o r e f f e c t s of  Magnitudes of  type) were  first  analysed with a 2 - f a c t o r repeated measures ANOVA with  39 developmental  stage  stimulus  as  stage  type  (as each  tested  on  as  the  between groups  the within-groups  animal  factor  factor,  nested  i n a given developmental  i t s responses  to both  the  touch  and within  stage  and  the  was  tap).  ResuIts In experiments reversals. reversed  In t h i s  to the  undescribed  response  than  stationary had  prior  of  the  animal  of  the  speed  not  reversals  I.  travel  i f they  of  had  faster stage, on  forward been  i f they the  speed  average  43.3%  acceleration.  of developmental reversals  the  and  case  from  accelerations  to  and  head  animals  of  touches  (see F i g u r e elicited  ( s e e F i g u r e s 6a  acceleration  &  a s s o c i a t i o n were  6a).  both 6b). applied  frequencies exhibited  to  in  taps.  Frequency  exhibiting  stage,  f o r taps, which  chi-squared tests  reversal  response  previously  they  s t i m u l u s was  during  sometimes  Frequencies  T h i s was  the  forward  Regardless  to the  elicit  acceleration.  s t i m u l u s , moving  forward.  always  animals  sometimes showed a  moving  t o the  attained  elicited  Separate  almost  accelerate to a tap,  prior  Regardless always  taps larval  t o t a p , an  backward,  been moving  Response  experiment,  t a p , and  When a n i m a l s rather  with adults,  of  reversals  The  i n response  to tap  significantly  as  .0001.  p r o p o r t i o n s and  These  a  Reversals.  function  of  p r o p o r t i o n of differed  stage; x (5) 2  their  animals  = 41.6,  p_ <  associated confidence  40 intervals  are p l o t t e d  comparisons old  adults  reversals adults not  of the proportions showed  were  than  indicated higher  observed  Frequencies  with 6b.  proportion indicated  t h e i r associated A chi-squared  of Young  proportions  were  Multiple between  on t h e s e  proportions  significantly (p_ <  that  although  on t h e  of  between  stage  stages;  pairwise  comparisons  a l l larval  more a c c e l e r a t i o n s  .05 o r s m a l l e r ) ,  to tap are  of a s s o c i a t i o n  Multiple  of  intervals in  as a f u n c t i o n  indicated  proportions  i n response  a significant difference .0001.  The  confidence  test  of a c c e l e r a t i o n s  ( 5 ) = 4 2 . 4 4 , _p_ <  stage  both  of Acceleration.  plotted  showed  stages.  no s i g n i f i c a n t d i f f e r e n c e  exhibiting accelerations  2  t h e 4 day  proportions  f o r young a d u l t s .  animals  X  that  stages.  II.  Figure  pairwise  d i f f e r e n t from L2s and L3s. but  L i s or L4s, although  comparisons revealed larval  Multiple  than any of t h e l a r v a l  significantly  that  6a.  significantly  to taps  from e i t h e r  lower  i n Figure  than  stages  either  the hypothesis  L4 a n i m a l s were d i f f e r e n t f r o m y o u n g a d u l t s  was  adult that  not  rejected. III.  Other  showed r e s p o n s e s accelerations observable of  stages  Responses. that  t e n o u t o f 120  were not c l e a r l y d e f i n e d  or reversals.  response  Only  Of  these,  t o t a p (1 a n i m a l  L I , L 2 , a n d YAD;  2 animals  five  animals as  showed  o u t o f t h e 20 o u t o f 20  either no i n each  i n L4),  41  Figure  6.  Proportions of animals  accelerating represents with  both  data  6(b)  of animals  bars)  stimuli.  f o r 20 a n i m a l s ;  tap and touch.  proportions (black  t o t a p and t o u c h  each  touch  hatched  bars,  animal  to touch).  accelerated.  P r o p o r t i o n s of response  (*) than  more  and fewer  Figure  types  ata l l  none to tap are  Both  adult  stages  higher proportions of reversals  reversals  L I , L 2 , a n d L 3 s t a g e s ; 4D  reversals  touch;  intervals.  proportions of accelerations  significantly  bars).  A l lanimals  r e v e r s e d t o head  depicted w i t h 95% confidence  to tap  to tap; non-existent  stages  lower  tested  that accelerated to the  developmental  showed s i g n i f i c a n t l y  was  a t each stage  (black bars, responses responses  Each bar  (hatched  shows p r o p o r t i o n o f a n i m a l s  same s t i m u l i  and  F i g u r e 6 ( a ) shows t h e  reversing  and t o head  reversing  than  and fewer  (**)  accelerations  larval  stages  (YAD  accelerations  significantly  than  and  a l l larval  more stages.)  (a)  Reversals to Tap and T o u c h  1.0 n en •5J  0.8 H  > £  0.6-  •  c .2  0.4 H  o Q. o 0.2 H 0.0 •+• developmental  (b)  tap touch  stage  Accelerations to Tap and  Touch  4  paused o r ceased  the  t a p (1 a n i m a l  slight  movement just  in  As v e r y  "other" evenly were  beyond  the 1 second  cut-off  responses  and as they  between groups,  appeared  that  point  fell  a  (1 a n i m a l  into the  t o be  distributed  the frequencies of these  responses  not analyzed.  All relative  Magnitudes magnitudes were e x p r e s s e d to the animals'  significant  effect  the magnitudes 9 8 . 8 9 7 . p_ <  of the responses  larger  f o r touches  0.13 b o d y  stimulus  age  response  relative  magnitudes  than  were d e l i v e r e d difference  also  .0008) a n d age b y  effects  .001)  were  As t h e a c t u a l  controlled  nor quantifiable  effects  manifested  magnitude of (touches  by hand w i t h a n o n - c a l i b r a t e d h a i r ) , Additional  of v a r i a n c e performed  accelerations  There were  t o touch.  was n o t p u r s u e d .  to  2 . 3 3 ± 0 . 1 3 a n d 1.04 ±  = 4 . 5 6 1 ; p_ <  but these  responses  means a n d s t a n d a r d  ( F ( l , 1 1 4 ) = 4 . 4 1 7 ; p_ <  magnitude,  t o u c h was n e i t h e r  analyses  (overall  on  F ( l , 114) = showed  (F(5,114)  i n responses  touch)  t o touches  and taps were  interaction  T h e r e was a  (tapversus  observed;  lengths respectively).  significant  lengths.  travelled  Responses  taps regardless of stage errors  own b o d y  as d i s t a n c e  of stimulus type  .0001.  significantly  only  43 following  1 second  f o l l o w e d by a r e v e r s a l  few of these  category,  Response  on  f o r more t h a n  i n L4; 2 i n 4D), and 1 e x h i b i t e d  forward  occurred L2).  moving  this  one-factor  on t h e r e v e r s a l s  t o t a p showed no s i g n i f i c a n t  and  effects  of  44 stage  upon m a g n i t u d e s  observed; =  F(5,54)  1 . 2 9 9 , p_ >  detectable  =  . 25  of  0 . 5 5 6 , p_ >  0 . 2 8 3 ; p. >  f o r any  .85  .7  or  i n v e l o c i t y or  of  the  stages  f o r v e l o c i t y ; F(4,  accelerations  for reversals;  f o r a c c e l e r a t i o n s ) , nor  differences  acceleration  either reversals  were  there  duration  tested 45)  =  F(4,  45) any  of  (F(4,  0.698,  45)  =  >  .5  for  larval  and  p  durat ion). Discuss ion As adult This  Chalfie  animals was  Larval  not and  tendency adults  animals,  elegans,  to  tap.  the  only  as  pauses,  It  least  is also  are in  a  only  do  they  elicit  day  old  larval reverse.  as  and  animal  data,  c l a s s of  in  responses  once the  from t h i s  In  learning  infrequently,  occurred  significant  their  response.  been r e c o r d e d  is clear  4  as  non-associative  only  tap.  in  whereas  unexpected  tested.  to  and  accelerate  appeared  however,  responses  to  animals.  from the  produce d i f f e r e n t response  adults  to  an  of  larval  clear  responses  taps,  typically It  a l l ages  to  r e v e r s a l s had  accelerations at  young  likely  in studies  been h a b i t u a t e d .  tap,  taps:  Other responses  some, s u c h  that  as  touch at  both  differed significantly  always reversed almost  found,  however, w i t h  a c c e l e r a t i o n s were  C.  had  to  (1981)  t o head  animals  reverse  a n i m a l s were  adult  case,  adult  to  Sulston  reversed the  nearly  The  and  data  patterns  that i n C.  d i f f e r e n t response  touches  and  elegans.  types  across  taps Not  45 developmental magnitudes Responses  stages, they also  of response  of response  developmental In  effect  difficult  The  of developmental  significantly  response  magnitude  of developmental  the magnitude  t a p , h o w e v e r , was  of  about  the  not  stages.  The  final  the e f f e c t s  different  of  to tap  i t is to  controlled. Animals  gave  across  experiment  tap s t i m u l i  magnitudes  an  magnitudes  of uniform magnitude.  developmental  to test  However,  response  t o u c h was  equivalent responses  quantifiably  larger  t o t o u c h showed  stage.  approximately  designed  stage.  than d i d taps across a l l  t o make s t a t e m e n t s  as  different  stages.  addition,  apparent  touch  regardless  to touch e l i c i t e d  magnitudes  elicit  across  was  thus  of  developmental  stages.  Experiment  while  magnitude.  multiple  personal  response Large, order  Single  taps e l i c i t  communication).  examine the  effects  magnitude  Response  to determine  small  whether  development.  of  to taps  short  longer reversals Experiment  i n each  response  taps e l i c i t  of d i f f e r i n g  i n t e r m e d i a t e , and  throughout  Graded  e 1 e q a n s show a g r a d e d  Adult increasing  3:  3 was  reversals,  (Rankin, designed  s t i m u l u s magnitudes  the developmental magnitude  the response  of  to on  stages.  t a p s were used i s graded  in  46 Method Subjects Sixty  animals  were used  (total:  of each  of the s i xdevelopmental  stages  360 a n i m a l s ) .  Protoco1 Each stage,  animal  animals  (a) weak taps)  received only a single  were  (single  tested  for their  (train  of s i x taps)  S88 s t i m u l u s g e n e r a t o r was u s e d  60 v o l t ,  tapper:  tapper;  msec s i g n a l trains  single  25 m s e c s i g n a l  mechanical  o f 8.5  (train  to  t o generate  In  either  A  Grass  the taps v i a a by a  single  t o an e l e c t r o m a g n e t i c r e l a y  trains  of three o f 8.5  b y a 60 V,  pulses per  taps  each  of three  stimuli.  t a p s were g e n e r a t e d  with a frequency  of s i x taps  frequency  response  t a p ) , (b) i n t e r m e d i a t e  o r (c) s t r o n g  mechanical  stimulus.  b y a 60 V,  i n the 300  pulses a second;  600 msec s i g n a l  and  with a  second.  Scoring Responses were v i d e o t a p e d and  magnitude  Statistical  and s c o r e d  as d e s c r i b e d i n the General  of Response.  Separate  t a b l e s were  constructed f o r reversals  Chi-squared  tests  frequencies  of response  second  effects  Methods.  Analyses  Frequency  and  f o r frequency  sets of and  o f a s s o c i a t i o n were used  as a f u n c t i o n  type,  first  contingency  accelerations. t o examine  as a f u n c t i o n  of stimulus type.  o f age and s t i m u l u s t y p e were  o f age  As b o t h t h e  significant.  47 additional to  chi-squared  responses  differences results  a t each stimulus across  on t h e s e  pairwise  t e s t s were  Magnitude.  group  with  2-factor  were  variance  (where  multiple  previously.  o f r e s p o n s e s were  analyses  with  magnitude.  separate  appropriate)  tested  of variance,  and s t i m u l u s  followed with  Significant  followed with  Magnitudes  applied  to test f o r  stages.  as d e s c r i b e d  f a c t o r s of stage  results  magnitude  developmental  comparisons,  initially  t e s t s o f a s s o c i a t i o n were  one-factor  and trends  between  Significant analyses  of  analyses.  Resu1ts Response I.  Freguencies  animals and  Freguencies  exhibiting  stimulus  stage;  of stimulus  under  stage  .0001 f o r  .0001 f o r m a g n i t u d e .  magnitude,  o l d adults reversed.  adults  Larval  tended t o  a n d 4 9 o u t o f 60  animals  t h e same c o n d i t i o n s , o n l y  tended  four  not to  19 o u t 6 0 L i s , 4  o f 60 L 2 s , 13 o u t o f 60 L 3 s , a n d 16 o u t o f 6 0 L 4 s  reversed. three  single  However,  different  respect  out  2  4 0 o u t o f 60 y o u n g a d u l t s  reverse: out  m a g n i t u d e ; x ( 5 ) = 1 0 4 . 1 9 1 , p_ <  2  reverse:  The p r o p o r t i o n o f  r e v e r s a l s was a f f e c t e d b y b o t h  x ( 2 ) = 2 1 . 0 5 5 , p- <  Regardless  day  of Reversals.  when  stimulus  to developmental taps  produced  of a possible  elicited  frequencies  of r e v e r s a l s to the  magnitudes were examined stage,  the data  the greatest  indicated  that  number o f r e v e r s a l s (67  120 r e v e r s a l s ) , w h i l e  fewer r e v e r s a l s  without  multiple  taps  ( 3 8 o u t o f 120 f o r 3 t a p s ,  36 o u t  48 of  120  for 6 taps).  determine  Data were  then analyzed  e f f e c t s of developmental  stage  separately  at each  to  stimulus  magni tude. Single taps p_ <  taps.  differed  L4) were  less  frequencies  given  taps.  showed only  stage  _Fewer animals taps.  tended  stages taps.  effect  stages  trend  7c).  significant  (see Figure The  train  Developmental effect  and L4, a l lshowed  (see  significance. when stage  2  ( L I , L2, L3, and  of 3 taps  than  L4)  adults,  different  from the  also produced  trend  was  continued  .0001.  significantly  a  t o those  not uniform  upon t h e p r o p o r t i o n s  2  stages  7b).  stage  o b s e r v e d ; x ( 5 ) = 3 9 . 5 2 4 ; p. <  lowest  x ( 5 ) = 3 4 . 0 4 4 ; p_ <  of s i x taps  though t h i s  the  upon t h e  f o r fewer r e v e r s a l s r e l a t i v e  single taps,  Figure  showed  adults  developmental  t h e L i s and L2s were s i g n i f i c a n t l y  general  ( L I , L2, L3, and  to reverse  However,  of r e v e r s a l s observed; a l l larval  of the  to s i n g l e tap than  fewer r e v e r s a l s t o t r a i n s  Six  L3,  animals  d i f f e r e n c e d i d not reach  of three  Although  two a d u l t  2  comparisons  L2 animals  t o have a s i g n i f i c a n t  proportions .0001.  larval  to single  x ( 5 ) = 52.075;  of r e v e r s a l s to tap of a l l larval  a train  continued  stages;  Pairwise  to reverse  Larval  7a); this  Three  for  likely  of responses  across  7a).  indicated that  e i t h e r age.  Figure  proportion  significantly  .0001; s e e F i g u r e  proportions  of  The  given (see  to exert  a  of r e v e r s a l s  Larval  stages  fewer r e v e r s a l s  L2, than  49  did  4 day o l d a d u l t s ;  fewer r e v e r s a l s differ  than young  significantly  II.  Frequencies  accelerations reversals.  function  of stage;  and m u l t i p l e  taps  than d i d s i n g l e  2  stage,  taps.  the fewest  .0001).  significantly  accelerations  frequently  84 o u t o f a p o s s i b l e  as a  Similarly,  the proportion  120 a c c e l e r a t i o n s ) ; m u l t i p l e more  of  significantly  of  as a  function  .0001.  2  produced  more  produced  the proportion  to taps d i f f e r e d  differed  seen f o r  accelerated  m a g n i t u d e ; x ( 2 ) = 2 6 . 1 3 1 , p_ <  accelerations and  animals  magnitude,  surprisingly,  the trends  x ( 5 ) = 9 6 . 5 1 8 , p_ <  accelerating  possible  larval  of developmental  stimulus  taps  Not  of stimulus  regardless animals  of Acceleration.  overall  accelerating  significantly  LI animals d i d not  t o the taps mirrored  more a c c e l e r a t i o n s  animals  adults.  show  stage.  than d i d adults,  Regardless  also  from e i t h e r adult  In general,  frequently  of  L3 a n i m a l s  Single  (48 o u t o f a taps  produced  (79 o u t o f a p o s s i b l e  120 f o r 3 a n d 6  120  taps,  respect ively) . Single single  taps  46.25  ; p <  taps.  differed .0001;  indicated  larval  accelerate  than  except adults.  The p r o p o r t i o n  L I were  of accelerations  significantly  see Figure  across  8a).  Pairwise  a n i m a l s were s i g n i f i c a n t l y 4 day o l d animals;  also  The a d u l t  from each other.  stages:  significantly  stages d i d not d i f f e r Again,  L2 a n i m a l s  x (5) 2  =  comparisons more  a l l larval different  to  likely  stages  from  young  significantly  showed t h e  highest  to  50 frequencies  of  stages,  were s i g n i f i c a n t l y  but  acceleration  to single  taps  different  of  a l l larval  only  from  Lis  and  adults. Three animals  taps.  given a  proportion  of  Regardless train  animals  given  a single  8b).  Developmental  effect X (5)  tap  upon the = 31.751;  2  of  stage  p <  significantly Six  was  4 day  The  not  Developmental effect X (5)  than  uniform stage  upon the  d i d not  the  differ  significantly  the  gave  adult to  and  animals.  produce this  8c).  significant  accelerating;  stages the  more  also  tap; however,  animals  from  showed  tended  to exert a  different  observed;  to  L2s  (compare F i g u r e 8a  Larval  significant  likely  than young  .0001.  Figure  did adults;  more  The  single  continued  animals  a  stages  s i x taps  p r o p o r t i o n s of  were s i g n i f i c a n t l y animals  of  to  to have  than  old adults.  train  = 3 9 . 5 2 4 ; p_ <  2  3 taps  higher  compare w i t h  were s i g n i f i c a n t l y  more a c c e l e r a t i o n s effect  of  stage, a  accelerations  more a c c e l e r a t i o n s  taps.  relative  .0001. A l l l a r v a l  L3s  than  produced  continued  p r o p o r t i o n s of  Lis,  accelerate  three taps  ( s e e F i g u r e 8b;  to trains  and  developmental  accelerating  accelerations L2s,  of  L2,  4 day from  L3,  and  olds.  LI  either  L4  adult  stage. III. only  Other  8 out  occurred  of  Responses.  the  total  i n response  360  Other  responses  responses.  to single  tap,  Of  three  contributed these,  5  i n response  to  Figure of  7.  varying  Proportions  of animals  magnitude.  Twenty  combination animals in  Figure  animals Figure with  animals  o f age and s t i m u l u s  accelerating 8.  Figure  7(a) represents  7 ( c ) , to 6 taps.  were  magnitude;  i n each of these  reversing to single  95% confidence  reversing  f o r each  proportions are  proportions  A l lproportions  intervals.  tested  groups  tap; Figure  51 stimuli  to tap  depicted of  7(b), to 3 are  of  taps;  presented  52  REVERSALS (a)  1 Tap 1.0-1  (b) 3 Taps 1.0n O)  £  0.6 H  O  0.2-  L4  YAD  4D  L4  YAD  4D  (c) 6 Taps 1.0-1 O) <0 I—  a>  £  0.6-  O  0.2-  developmental  stage  53 Figure  8.  stimuli for  each  Proportions  of v a r y i n g magnitude. combination  proportions are  proportions  are  i n Figure of animals  to 3 taps;  presented  Figure  with  accelerating  Twenty animals  o f age and s t i m u l u s  of animals  depicted  8(b),  of animals  95%  reversing 7.  Figure  i n each of these 8(a)  to 6 taps.  confidence  were  tested  magnitude; groups  represents  reversing to single 8(c),  to tap  tap; Figure A l l proportions  intervals.  ACCELERATIONS  L4  YAD  (b) 3 Taps 1.0 -| Bui CO  •  0.8-  accel  0.6-  c o  0.4-  propoi  a>  0.2-  0.0  (c) 1.0-1  L1  L2  L3  L4  developmental  YAD  stage  a  train  of  Response  3 taps,  and  none  stage  and  i n response  significantly  a f f e c t e d the  accelerations,  the  next  and  regardless  of  developmental  effects  stimulus  of  two-factor  stimulus  type  stage.  and  analysis  revealed  stimulus  magnitude  as  was  and  response  f o r stimulus magnitude;  p_ <  .0001  f o r response  Both  increase  (F(2,  variables.  as  stimulus  of  The that,  (see  regardless  response  of  w e l l as  of of  larval  predominant  assess  response,  factor. of  This  =  15.12,  68.271,  significant  3 . 8 1 2 , p. <  .05)  between  is plotted in  r e v e r s a l s show a increase;  of  r e v e r s a l s at  a  both  =  a  the  using  Figure  tendency  to  however,  In a d d i t i o n ,  a c c e l e r a t i o n s were u n i f o r m l y  Figure  analyses  on  F ( l , 346)  interaction  magnitudes  average magnitudes  magnitudes  =  to  (F(2,346)  i n c r e a s e more m a r k e d l y .  average magnitudes  the  This  as  346)  a c c e l e r a t i o n s and  accelerations  than  type)  and  whether  performed  type  .0001  effect  type  main e f f e c t s  p_ <  interaction  In order  a between s u b j e c t s  significant  reversals  i n magnitude  response  a n a l y s i s of v a r i a n c e  of  to determine  reversals differed  response magnitude  9.  55  taps.  magnitude  frequencies  s t e p was  accelerations  two  6  Magnitude.  Because both  the  to  greater  a l l stimulus  9).  frequencies  of  response  stimulus magnitude, animals  response  of  to  taps  adult  was  the  types  showed  predominant  an a c c e l e r a t i o n ,  animals  a  reversal.  56  Figure  9.  Interactions  magnitude  on r e s p o n s e m a g n i t u d e s ,  developmental (relative are  stages.  t o body  accelerations  steeply  reversals.  Distances  length) rather  as a f u n c t i o n  when t h e r e s p o n s e s  standard  between response  travelled  are accelerations travelled  and  stimulus  across by  animals  when t h e r e s p o n s e s  reversals,  of increasing  Mean d i s t a n c e s  errors.  collapsed  are greater than  type  and i n c r e a s e  stimulus rather  more  magnitude than  are presented  with  57  Response Magnitudes : Acceleration vs. Reversals  58 Because  the interaction  response  type  significantly  responses  seen,  magnitude  differed  addressed  with  and w i t h  both  developmental  stage  effects  Trend  p <  positive  linear  accounted Regardless  .0001 f o r s t i m u l u s  increased  ( F ( l , 169) = 17.07,  tended  from 1  When  examined,  stage,  stage;  magnitude.  of a c c e l e r a t i o nf o r showed  p_ <  f o r most o f t h e v a r i a b i l i t y  accelerations  were  .005 f o r d e v e l o p m e n t a l  stimulus magnitudes  of larval  animals.  of acceleration;  a n a l y s i s o f t h e mean m a g n i t u d e s  each o f t h e three  animals  and s t i m u l u s magnitude had  upon m a g n i t u d e s  169) = 5.016, p <  was  animals.  ( L I , L2, L3, and L4) animals  = 11.744,  and  response  to accelerations i n larval  of accelerations i n larval  larval  F(2,169)  as t o whether  to reversals i n adult  only  F(3,  magnitude  as a f u n c t i o n of s t i m u l u s magnitude  respect  Magnitude  stimulus  a f f e c t e d the magnitudes of  the question  respect  significant  between  that  a  .001) t r e n d  i n the data.  mean m a g n i t u d e s o f  to increase  as s t i m u l u s  to 3 to 6 taps.  However,  the difference  between  means a t 3 a n d 6 t a p s  was  than  the difference  between  means a t 1 a n d 3 t a p s  (mean o v e r a l l  magnitudes [ i n  body  lengths]  regardless and  and standard  of larval  3.43 ± 0.23  stage  less  magnitude  e r r o r s f o r 1, 3, a n d 6 were  taps  1.84 ± 0 . 2 7 , 2 . 8 5 ± 0 . 2 3 ,  respectively).  As  there  were  between  stage  and s t i m u l u s magnitude  p_ > . 3 0 ) , s e p a r a t e  no s i g n i f i c a n t  interaction  effects  ( F ( 6 , 169) =  o n e f a c t o r ANOVAs w e r e u s e d  1.141;  t o examine  59  the  effects of stimulus  within all  each  showed  larval  stage.  L4).  = 6 . 3 9 6 , p. <  <  stages,  3 5 ) = 5 . 2 2 8 , p. <  trends  showed  from 3 t o 6 taps  increased,  =  but tended 1 t o 3 taps  L2 a n i m a l s  also  seemed  trend,  an a n a l y s i s  of variance  .002 f o r o f t h e mean trends  11.379,  tended  from  Although  .05 f o r L I ;  .001 f o r L 4 )  than  10a).  to  to  increase  increase  (see Figure  t o show an upward  showed t h a t  b e t w e e n means was n o t s i g n i f i c a n t ( F ( 2 , p  magnitude on  F(l,43)  of acceleration  magnitude  size  L 3 , and L4  significant linear  .05 f o r L I ;  mean m a g n i t u d e s  stimulus  less  LI,  analyses  .01 f o r L 3 ; F ( l , 4 1 ) = 1 4 . 4 9 7 , p <  Again, as  stages  .005 f o r L 3 ; F ( 2 , 4 0 ) , p_ <  a c c e l e r a t i o n magnitudes  p  Larval  ( F ( 2 , 3 5 ) = 3 . 3 2 8 , p_ <  In each o f these  (F(l,  on a c c e l e r a t i o n  s i g n i f i c a n t effects of stimulus  a c c e l e r a t i o n magnitude F(2,43)  magnitude  the differences  51) = 0.468,  > .5) . Acceleration  analyzed to  due s m a l l  12 f o r a g i v e n  magnitude). in  Table  magnitudes and h i g h l y combination  unequal  s t a g e s were not n's ( r a n g i n g  of stage  and  These r e s u l t s a r e summarized  from  0  stimulus f o r comparison  1.  Magnitude significant  of reversals  e f f e c t of both  p  <  .0001) a n d s t i m u l u s  p  <  .001).  reversals  f o radult  Trend  i n adult stage  ( F ( l , 83) =  magnitude  analysis  animals.  (F(2.  T h e r e was a 24.025,  83) = 8.008,  o f t h e mean m a g n i t u d e s o f  t o s t i m u l i of increasing  magnitude  regardless  of  adult p_ <  stage  .001)  trend.  animals, stages  showed a  tended  overall  1.01 for  there  p  >  examine  stage.  1.573  no  the  Reversal  =  6.594, p  significant accounted  <  linear  Although young  of  trends  ( F ( l , 46)  adult  the  =  (F(2,  also  i n c r e a s i n g magnitudes  both adult  stages  are  .10).  not  plotted  analyzed  n's  (ranging  from 0  to  9  and  stimulus  magnitude).  i n each  animals  magnitude  <  .01)  i n the  appeared  of  of  (F(2. a  data.  to  show  reversal  stimuli  adult  (see  a in Figure  significant  Mean r e v e r s a l m a g n i t u d e s  Mean r e v e r s a l m a g n i t u d e s but  performed  adult  d i f f e r e n c e s b e t w e e n means were not >  =  trend  variability  animals  old  83)  9.555, p  to  recorded,  day  increasing magnitudes  2.176; p  in  0.14  that  response  =  ±  a n a l y s i s showed  give  (F(2,37)  but  (mean  1.72  magnitude  stimulus  to  the  and  magnitude  four  tendency  10b),  adult  interaction effects  stimulus  e f f e c t of  f o r much o f  3 taps  f a c t o r ANOVAs w e r e  magnitudes  .003);  combined  6 taps, r e s p e c t i v e l y ) .  one  e f f e c t s of  larval  for reversals  ± 0.13,  stimulus  separate  60 16.072,  =  magnitude,  1 to  errors  significant  and  showed a s i g n i f i c a n t 46)  and  f o r the  from  ± 0.11, 3,  for  stimulus  standard  stage  .5),  with  increasing  1,  (F(l,83)  accelerations  and  were  between a d u l t  to  with  increase  magnitudes  lengths As  to  most when  a d u l t s were  0.63,  As  linear  mean r e v e r s a l m a g n i t u d e s  increased  body  significant  due  in Figure  10b.  for  stages  to  larval small  for a given  and  highly  combination  These r e s u l t s are  were  of  for  also  unequal stage  summarized  in  61 Figure  10.  magnitude;  Response larvae  and a d u l t s .  response magnitudes tap;  open  bars,  number o f a n i m a l s  10(a) r e p r e s e n t s Figure aduIts.  (relative  to three  Means and s t a n d a r d 2  magnitudes  taps;  forstimuli Hatched bars  t o body black  of varying represent  length) bars,  e r r o r s are presented;  to single  to s i x taps.  see Tables  c o n t r i b u t i n g t o e a c h mean.  mean a c c e l e r a t i o n m a g n i t u d e s  10(b) represents  mean  1 and  Figure f o r larvae:  mean r e v e r s a l m a g n i t u d e s f o r  62  (b) Reversal  Magnitudes:  Adults  1 TAP  •  •  YAD developmental  stage  3 TAPS 6 TAPS  Table  63  1  Mean A c c e l e r a t i o n Different  Magnitudes  Intensities:  To Tap  A l l Developmental  Stimulus Stage  Stimuli  1 Tap  of  Three  Stages  Intensity-  3 Taps  6  Taps  Larvae LI  0.56  ± 0.62  2.41  (9) L2  2.91  1.82  ± 0.44  3.25  1.07  ± 0.43  ± 0.59  2.73  ± 0.47  2.00  2.92  (10)  ± 0.48  0.52  ±  0.34  (17) 4.19  (16)  ± 0.59  ±  (13)  (19)  (10) L4  2.43  (16)  (18) L3  ± 0.47  ±  0.42  (20) 3.11  (15)  ±  0.44  (18)  Adults YAD*  0.13;  no S E  2.61  (1) 4D*  ± 0.41  3.88  (7)  —  2.75  (0)  ± 0.90 (6)  ±  0.50  (8) 4.80  ±  1.52  (4)  Note. Means shown w i t h s t a n d a r d e r r o r s when a p p l i c a b l e ; number o f a c c e l e r a t i o n s c o n t r i b u t i n g t o mean i n b r a c k e t s (n). T o t a l p o s s i b l e a c c e l e r a t i o n s i n e a c h c e l l = 20. A l l magnitudes i n body l e n g t h s . *n's f o r t h e s e g r o u p s were t o o s m a l l and t o o u n e q u a l f o r a p p l i c a t i o n o f t h e ANOVA. In these cases, standard errors (SE) w e r e c a l c u l a t e d a s Vs / rT. O t h e r w i s e , S E = V M S /n*. 2  e  64 Table 2 Mean R e v e r s a l  M a g n i t u d e s To Tap  Stimuli  Intensities:  A l l Developmental  Stages  Stimulus Stage  1 Tap  of Three  Different  Intensity  3 Taps  6 Taps  Larvae LI*  1.12  ± 0.19  1.14  (9) L2*  1.01;  0.68  no S E  —  ± 0.17  0.95  0.81  ±  2.00  0.58  (9)  0.24  ±  0.34  (3)  0.24  (4)  ± 0.19  ±  (7)  (0)  (9) L4*  0.82  (3)  (1) L3*  ± 0.43  ± 0.27  (0) 1.00  (5)  ±  0.70  (2)  Adults YAD  0.74  ± 0.15  1.14  (19) 4D  1.26  ± 0.19  1.19  (13)  ± 0.15  2.00  (20)  ± 0.19 (13)  ±  0.24  (8) 1.98  ±  0.17  (16)  Note. Means shown w i t h s t a n d a r d e r r o r s when a p p l i c a b l e ; number o f r e v e r s a l s c o n t r i b u t i n g t o mean i n b r a c k e t s ( n ) . T o t a l p o s s i b l e r e v e r s a l s i n e a c h c e l l = 20. A l l m a g n i t u d e s i n body lengths. *n's f o r t h e s e g r o u p s w e r e t o o s m a l l a n d t o o u n e q u a l f o r a p p l i c a t i o n o f t h e ANOVA. In these cases, standard errors ( S E ) were c a l c u l a t e d as V s / n\ Otherwise, S E = V M S /n\ 2  b  Table  65  2.  Other Features Not for  of  Accelerations  surprisingly,  a l l developmental  effects  of stimulus  accelerations, increased  stages,  there  magnitude  with  duration  (F(5,194)  acceleration  when a c c e l e r a t i o n s w e r e  79),  a n d 7.2  found  .02).  ± 0.46  s f o r 6 taps  f o r stage;  stage  s f o r 3 taps  ±  (n =  No  or stimulus  F ( 5 , 194) =  of  w e r e 4.1  (n = 8 4 ) .  e f f e c t s of e i t h e r stage  for acceleration velocity;  p_ > .80  Mean d u r a t i o n s  of developmental  s f o r 1 t a p ( n = 4 8 ) , 5.7  significant  of  i n c r e a s i n g as magnitude  0.59  s ± 0.45  significant  on t h e d u r a t i o n  = 4 . 1 1 , p_ <  regardless  were  examined  type  were  0.418,  F ( 2 , 1 9 4 ) = 0 . 4 9 3 , p, > .60  f o r stimulus  magnitude.  Discussion The showed more  a n a l y s i s of graded  three  frequently  magnitudes; of  major trends: to taps  developmental  stage;  to e l i c i t  addition,  adults  of  experiment  animals  accelerated  at a l l stimulus  elicited  greater  regardless  (3) l a r g e r s t i m u l u s  responses  magnitudes  than magnitudes changes  than  single taps, and  in this  (1) l a r v a l  (2) m u l t i p l e t a p s  a c c e l e r a t i o n s than  tended  response  frequencies of  magnitudes  l a r g e r magnitudes.  of a c c e l e r a t i o n s tended  t o be  In greater  o f r e v e r s a l s , and were more s e n s i t i v e t o  i n stimulus  magnitude.  As t h e p u r p o s e o f t h i s  experiment  was  to determine  66 w h e t h e r r e s p o n s e m a g n i t u d e was stimulus  magnitude,  differed  significantly  reverse  or  accelerate  stimulus  magnitude,  response  types  Overall,  both  reversals of  than These  in  L2  LI,  adults; response  less  and  the  from  function It  L3,  3 two  and  to  as  6  two  stimulus  therefore in  of  with  but  tap  larval  response magnitudes  did  (see at  animals tap  and  accelerations  The  increased  the  increasessmaller  Only show a  differentiate larval  least  their  group  as  1 and  a  2).  with  reversals  in  increase  largest  stage  significant  increase  Tables  day  responses  although to  larval  four  larger to  taps.  in  i n the  seemed  stimuli  function  multiple  gave  not  a  than were  and  trend  that,  i n magnitude.  and as  although  response, the  to  predominant  stimuli  taps  of  analyzed.  both  stimuli.  magnitude  appears  the  for accelerations  animals  stage  either  regardless  of  for reversals  largest  adult  consistent  as  multiple  and  to  animals  of  of  animals  i n magnitude  taps,  larger stimuli,  young  increase  larval  between s i n g l e  L4,  taps  groups were  in  to  function  adult  magnitudes  increase  both groups of  accelerations  taps  1 to  occurring  to  magnitudes  to  a  tendencies  were g r a d e d  towards gradedness  means were  the  these  tended  between the  trend  by  t r e n d s were m a i n t a i n e d  stages old  the  as  and  i n response  between the  those  in their  magnitude:  i n magnitude  larval  accelerations  reversals  occurring  as  only  given  in adults  stimulus  and  and  graded  adults, as  increases  the in  67 response magnitude  occur  as  single  taps;  however,  to m u l t i p l e  differentiate trend  also  l e s s between  appears  animals  giving  stimuli  of  not but  less)  to  multiple It  tap  by  magnitudes  in  before  the  Experiment  Experiment  that  that  3  As  more taps  to  single  to  of  tap  stage  The  r e l a t i v e to  a  note  than L2  taps  the  stimulus  time  that  2,  and  the  to  distance  (and  results  single for  larval  either  animals  of  of  sole  fewer r e v e r s a l s  reversals  single  reverse  in velocity-  r e s u l t s obtained  give  (and  gradedness  for responses  of  single  ended.  in Experiment  larval  proportion  accelerations)  3  accelerate  differing total  responses  pattern  to  to  l i e i n the  the  tended  interestingly,  the  accelerated.  i n t e r e s t i n g to  2.  accelerations  lowest  noted  i n Experiment  replicated  to  reflected in differences  seemed t o  is also  6  for  taps.  between responses  passed  obtained  be  3  of  of  of  r e l a t i v e to  consisting of  This  proportions  tended  accelerate  tap  taps.  regardless  more a n i m a l s  to  accelerations  necessarily  animals  differences  r e f l e c t e d i n the or  from  tend  multiple  taps  also  not  animals  two  magnitude.-  consisting  should  attained  It  not  increase  to m u l t i p l e  a multiple  r e s p o n s e was  that  stage,  did  the  stimuli  either reversals  reverse)  taps,  be  increasing  developmental (and  to  the  highest  single  animals and  adult again  tap tap in  more  stage. showed  More the  proportion  a l l developmental  of  stages.  68 GENERAL Both reflex  spontaneous r e v e r s a l s  show  Experiment the  DISCUSSION  developmental  c h a n g e s i n C.  1, y o u n g a d u l t  frequency  and the t a p r e v e r s a l  animals  the other developmental  3,  larval  animals  stages.  of a l l stages  patterns  of responding  the  stages.  Although  in but  response  to touch,  a previously  Larval  animals  responses  Increasing  true  showed  showed  accelerations  than  magnitude  also  relative  reversals not  merely  over  elevated  (except  L i s ) showed  failed  t o show  elevated  e l e v a t i o n was  also  Young  the  taps.  the  though  taps;  this  larval  and more  adults  showed  to exhibit  observation  of increased  stages  The  to single  to single  stages,  tendency  a ten-minute  a function  half  animals.  Spontaneous Reversa1s. significantly  reversed  reversals,  increased  fewer r e v e r s a l s  of  and 4 day o l d a d u l t s  i n response  adult  either  accelerations.  reversals  f o r a l l developmental  animals s t i l l  than  on a p p r o x i m a t e l y  young a d u l t  of accelerations  2 and  significantly  not only  response,  in  relative  of a l l stages  elicited  reversals  while  stimulus  probability was  showed  increase  occurring  to taps  animals  undescribed  to taps,  consistently  taps  an  In  In Experiments  showed  different adult  showed  of spontaneous r e v e r s a l s  to  eleqans.  spontaneous  period.  activity,  similar activity  rates  a  of spontaneous  as  This  was  other  rates  and  reversing.  u n l i k e l y t o be t h e r e s u l t o f a  difference differ of  in sensory  significantly  response  to  Although frequency related  of  young  other  studies  spontaneous  see  seemed t o  indicate a general  age  (data  ages of  Adult  the  adult  adult  as  an  event  is  change,  i s that  a r e s u l t of  touch  the  circuit.  Bekoff,  1981,  developing  showed, the  unit  age  the  a  redidivus, a  related  towards as  a  that  not  of the  specified. reversals  phenomenon.  differ  from  is a  occurs  new  that  however,  only  elements  Carew,  1987;  spontaneous  larval  function  i n spontaneous  i n c o r p o r a t i o n of  data  function  spontaneous  not  the  increasing  1980);  were  Rankin,  &  at  long-term adjustment.  see  and  Pollock  increase  increase  the  specifically  of  f o r a d i s c u s s i o n of  motor  1975;  time  do  i f this  As  touch withdrawal  throughout  Chalfie & reflex  development:  Sulston was  One reversals into see  (1981)  a l l animals  in  at a l l  the  also  activity  present  of  briefly,  systems). Taps.  69  not  magnitude  elegans  transient  i t i s one  than  (e.g.,  Touches and  elegans  4 days of  rather  possibility  trend  a  at  (1976) o b t a i n e d  studied  be  did  i n the  looked  i n C.  Dusenberry,  to  i n d i c a t i n g that  some n e u r o n a l  also  in frequencies  appears at  per  animals  increase  animals  stages,  reversals  stage  Croll,  Samoiloff  r e c o m p u t e d by  The i n young  and  adults  taps.  In P a n a g r e l l u s  Pollock  of  or  looked  nematode.  frequencies  young  other  have  none have  stage.  as  reversals  (e.g.,  1976),  adult  f r o m any  e i t h e r touches  nematodes  Samoiloff,  threshold  C.  in  70 developmental It  was  not  occurred  c l e a r whether  neither  question could  stimuli Frey  were  that  be  t o head  differences  over development,  experiments This  stages responded  as  the  addressed,  nor  i n these  quantifiable.  however,  by  o f known magnitude  tap r e v e r s a l  reflex  also  using  (e.g.,  present  However,  developmental  stages reversed i n response  animals  a l l animals  sometimes a c c e l e r a t e d ,  stationary, response likely  not  was  development.  moving  to single  faster tap,  to accelerate  animals  between  nearly  larval  pronounced  and  at  touch von  3 showed t h e  tap  relative  The  pattern  Experiment  at a l l  a n i m a l s were  r e v e r s e ; young  adult  reversed.  2 was  significantly  of responses  or d u r a t i o n s of  the s i n g l e  developmental  the  tap d i d not  i n both  stages.  The  day  Experiments  stages tap  to  s t a g e s , nor  2  single  tested. in 3.  tap d i d not  accelerations,  single  as  four  of r e v e r s i n g  to single  differ  least  and  i n Experiment  between developmental  In  difference  obtained for single  thus reproduced  initially  particularly  which  lowest p r o b a b i l i t y  larval  i f  at  adult  This  s t a g e s was  stage L2,  of r e s u l t s  to tap:  forward  to a l l other developmental  Magnitudes  velocity  larval  as  moving  throughout  i f already i n motion.  always  larval  and  that  magnitude  hairs). The  old  reversing.  i n response  touches  controlled  apply force  t o u c h by  did  differ the  suggesting  i n magnitude  across  tap d i d , however,  differ  in  apparent  intensity  greater magnitudes Graded occurred stimuli tap  had  tap  response  touches  than  two  greater  effects:  of  (a)  response  proportions  of  the  (trains  a  and  changes  when a s s e s s e d  Increasing  with  tap  magnitude  of  tendency (b)  71 elicit  to  taps.  S i m i l a r developmental  reversal reflex  general  tended  single  from s i n g l e to m u l t i p l e  greater magnitudes elicit  touch:  v a r y i n g magnitude.  stimuli  taps  of  Response.  i n the of  from  3 or  to  of  6)  elicit  a tendency  to  accelerations relative  to  reversa1s. The of  tendency  response  f o r animals  to  tap  stimuli  indicated  that  the  tap  graded  as  a  elicit  larger responses.  tended  t o show s i g n i f i c a n t l y  animals  as  magnitude  were  only  the  pronounced  enough t o  Interestingly, significant  tap  magnitude  i n those  larval  increased,  Young a d u l t the  trends  statistical  stages  where  differences occurred,  relatively responses  increased little  to  three  and  were  stimuli  animals  i t appeared  d i f f e r e n c e appeared s i x taps.  while  adult  reversals L2 not  to This  that  occurred  exist  the as  the  taps; between  finding  as  animals  statistically  from s i n g l e to m u l t i p l e  versus  is  significance.  d i f f e r e n c e i n response magnitude  stimuli  larger  l a r g e r magnitude  in which reach  elegans  l a r g e r magnitude  increased.  stages  i n C.  magnitude:  In g e n e r a l ,  showed s i g n i f i c a n t l y  magnitudes  i n c r e a s i n g magnitude  stimulus  stimulus  stimulus  largest  of  greater  reversal reflex  f u n c t i o n of  accelerations  to give  may  72 indicate stimuli  that  greater  indicate  that  magnitude  to  tests help  there  using to  than  3  and  be  in  magnitude  response  so  low  responses were not (see  that  inconsistent  would have trends  to  could  The  of  occurred  not  of  and  were  with  2);  would  in adults were  reversals Second,  at  in  and  to  larger  give  in  a  single  proportion  some s t i m u l u s  i n each of  two  combine  adult  not  for  stimulus  to  the  to  animals magnitudes  possible. these  of  The  categories  the  trends  for graded  response  however,  larger  numbers of  responses  before  a  clear picture  of  these  established. of  increasing  response  r e s u l t s obtained  proportions  Additional  restricted  i t unwise  a n a l y s e s were  obtained  be  effect  proportions the  be  in  magnitude  increases  making  were o b t a i n e d  1 and  to  also  close  animal.  varying  exhibiting reversals  meaningful  Tables  the  in general  magnitude.  accelerations  that  i t may  been too  reversals  accelerations  animals  responses  3 taps;  r e s p o n s e was  and  reversals,  measure of  exhibiting  graded  accelerations  of  larval  by  for  issue.  of  magnitudes  of  have  more s e n s i t i v e t o  than  effect  more w i d e l y  larvae  First,  m a g n i t u d e and  of  this  analysis  reasons.  a magnitude 6 t a p s may  stimuli  accelerations  ceiling  distinguishable  clarify  The  is a  types  as  added  i n Experiment  accelerations  only  stimulus  a  another 2.  relative  function  of  magnitudes  The to  on  dimension shift  to  in  reversals  developmental  stage.  but  also  as  Experiment a  a 3  function  of  indicated  s t i m u l u s magnitude.  t h a t as  s t i m u l u s i n c r e a s e d from  proportion  of  animals  the  single  number o f  adult stages  as  adult  still  t o r e v e r s e more  taps  than  adult  larval  animal  tended  stages  well  a t any  responding  as  to multiple a  single  tap.  greatest difference  occur  between the  relative  to  responses roughly  the  animal  patterns elicited  patterns e l i c i t e d  to three taps  similar  and  overall  in  the  Although  frequently to  t a p s was  t o a c c e l e r a t e as the  taps,  larval.  likely  Again,  given  stimulus magnitude,  larval  73 of  i n a l l stages  increased—in animals  taps  to multiple  accelerating  Results  by  by  almost  responding  single  both  to  taps taps;  to s i x taps  f r e q u e n c i e s of  as to  seemed  multiple  responses  an  showed  response  types. The stimuli  dual  observed  observed suggest the  effects  a number o f  relative  of  the  to touch  accelerations, area  varying  increasing  i n Experiment  between touches  nature  major  of  areas  stimuli make  of  increases  seen  proportion  of  the in  the  used:  what  i s the  nature  stimuli  are  (a) r e s p o n s e  accelerations.  tap  i n Experiment  exploration. f e a t u r e s of  likely  in larval  of  differences  observed  for further  s t i m u l u s m a g n i t u d e s on  what a s p e c t s  2 and  taps  i t more  especially  to explore  and  magnitude  to  the  responses  tap  Another  effects  to tap,  responsible for magnitude  the  elicit  animals? of  One  and  (b)  of  namely, the  2 is  74 The r e l a t i o n s h i p was  an i s s u e  only  always  stages,  of  raised  produced  they  eliciting  first  also  larger  developmental  between  appeared  magnitudes of  stage.  Was  of response  the  issue, forces  able  one n e e d s applied  to find  animal  both  magnitude. about with  magnitude  by b o t h  the touches  the s i n g l e  produce  the physical  address  magnitudes  so t h a t  a n d be  controllable  provide  information  t o u c h and t a p s t i m u l i underlying  of  one c a n g i v e an  and t a p s o f known and  the neural c i r c u i t s  taps  i f both are  In order to  would  used  larger  t o t a p s , even  the stimuli  i n which  regardless  t h e t a p and t h e t o u c h ,  Such an a n a l y s i s  t h e ways  stimuli,  of response  than  always  t o know  Touches not  intense  because  magnitudes?  calibrate touches  this  relative  equivalent physical  this  t o be more  magnitudes  stimuli  2.  stimuli  at a l l developmental  overall  Or do t o u c h  and t a p s as  i n Experiment  reversals  were o f g r e a t e r p h y s i c a l used?  touches  interact  the responses  observed. Another the  nature  In the introduction  speculation  put f o r t h  from both  whereas the head head.  bears  of the sensory signals  represent.  receptors  question that  This  acceleration  was  that  the head  response  appears  that  exploration i s  touches  t o Experiment t h e t a p s may  and t h e t a i l  touch stimulates  i s an a t t r a c t i v e  further  and  3, be  taps  one stimulating  of the animal,  receptors only  i n the  hypothesis given that t o be s i m i l a r  to the  the tail  75 touch withdrawal r e s p o n s e — t h a t forward—and neural  circuit.  stimulates while  might  be m e d i a t e d  In addition,  the anterior  tap stimulates  responses,  i tmight  are  larger  always  that  the  be t h a t  t o be  either  of s t i m u l i  Mutants  throughout reversals missing AVM)  less  addition,  stimulates  mediated  tail  touch receptors respond  (perhaps  and t a i l a pair  raised  stage.  with  Mutants  show stage.  of the animal  of hairs)  by E x p e r i m e n t s  the increases i n response were  to taps  of developmental  by t a p .  accelerations  (PLMs)  t o u c h r e c e p t o r s (ALMs a n d  elicited  of  lack  touch r e c e p t o r s (but not  of r e s p o n d i n g over development  whether  that  a n i m a l s , t h e use of a s t i m u l u s that  t h e head  issue  inhibited  i n mutants  patterns  Another  or  should always  to tap, regardless  simultaneously  to tap, given  by t h e p o r t i o n o f  of developmental  development  both  t o touch  response.  should always  i n normal  touch  h y p o t h e s i s i s t o examine the  a l l of the anterior  throughout  that  magnitudes  inhibited  missing the t a i l  regardless  of the  posterior  magnitudes  or posterior  development  accelerations In  and  over development  the a n t e r i o r  both).  this  portion  receptors directly,  mediating the opposing  One w a y t o t e s t effects  touch  response  responses  moves  i f i t i s supposed  than responses  by c o m p e t i n g  circuit  by t h i s  both a n t e r i o r  they are l i k e l y  later  head  i s , the animal  produced  should similar  elicit t o those  2 and 3 i s  magnitude  b y t h e same  and frequency features of  76 stimulus stimuli  magnitude. used  varied  Examination  taps)  the  magnitude  over development  . while  way t o r e s o l v e  i n magnitude  taps  of varying  oscillations. exhibiting  in  shift nature are  force  this  issue  only  one p a r a m e t e r .  could content  tap stimulus,  i n proportions  magnitudes.  i s a function  than  the s h i f t  force  of the stimuli  striking  perhaps  i n proportions  the animal only  showing  Similarly,  a  i f the  of the qualitative  i s , the fact that  varying  a  o c c u r when  single, or i n rapid  singular—then  cause  of animals  i s solely  i tshould  a r e used,  of the s t i m u l u s — t h a t  single  corrections  i n proportions  then  s i n g l e taps  Thus,  that  i n t h e a g a r ) made t o damp o u t  of the force  effect at highest  than  i s compare s t i m u l i  be u s e d , w i t h  I f the shift  multiple rather  rather  causes:  t o s i n g l e t a p d i d n o t change  a c c e l e r a t i o n s and r e v e r s a l s  magnitude  ceiling  independent  that  t h e number o f a c c e l e r a t i o n s  of the magnitude  a given  high  along  as lower water  function  2 suggests  did.  vary  (such  of the  number o f t a p s .  c h a n g e s may h a v e  of the response  3  amount o f f o r c e  ( t h e sum o f t h e f o r c e s  of the r e s u l t s of Experiment  two b e h a v i o u r a l  One  i n Experiment  (1) t h e t o t a l  a n d (2) t h e t o t a l  the  e1icited  i s r a i s e d because the  response  two p a r a m e t e r s :  the animal  individual  issue  t o t e s t graded  along  striking  This  these  seen,  were e q u i v a l e n t .  even  the taps succession  parameters  should  i fthe total  These  experiments  should  also  stimuli  help  are  to  determine  responsible  what a s p e c t s  f o r the  increases  of  the  seen  77  tap  in  response  magnitude. The  r e s u l t s of  number of  issues  taps reversal first be  of  animals  simple  to  measure  response  occur  magnitude to  tap,  learning.  strictly  i t may  be  of  response  or  not) to  instance.  Studying  the of  to  magnitude:  i f i t i s the  to  t h e n we  large might  reversals of  the  the  during  stimulus  an  classifying and  habituation  and  that  habituation, to  of  to  i n the  due  responses—into  to  tendency  for touch to  stimulus  smaller  tend  to  stimuli,  p r o b a b i l i t y of  when t h e become  a  a  responses,  the  did  occur  use  accelerations to  a  response  may  responses  whether  reversals  increase  i s thought  of  function  case  no  use  of  that be  in  yes/no,  a measure  changes  pauses,  is a sole  stimuli see  of  assessing  accelerate  occur  tap  than  to  larval  to  (i.e.,  The  appeared  as  informative  the  animal.  behaviours  occurrence  c a t e g o r i c a l method  a n o t h e r way  of  and  a l t e r n a t i v e would  accelerations,  be  more  rather  reversals,  may  in this  a  in  accelerations  types  track  Another  as  raise  plasticity  than r e v e r s a l s ,  show b o t h  i n order  of  3 also  over development  i n magnitude to  2 and  analysis  is practical:  tended  response  f o r the  reflex  these  greater  Experiments  perceived  intensity  progressively  sma11er. The are  two  r e s u l t s of key  these  transition  experiments  points  i n the  indicate that development  of  there the  78 tap r e v e r s a l from t h e  reflex.  newly  These  hatched  are  larval  the  stage LI to  and the  transition  adult.  Newly h a t c h e d L i s were t h e  developmental lowest  frequency  accelerations Experiments  from l a r v a l  stages, of  single  2 and  3.  frequencies  any o t h e r  stage,  started  responding,  to  Both of  these  with  periods  periods  juvenile  (embryonic)  time, the  nervous  system.  all  1976,  C h a l f i e et  addition, the  the  connections  all  showed of  taps  in  relative  stage  pattern  rather  the  showed  reversals  behavioural  is  arise  the  at  to  which  of  than  existing  lose  dorsal  appear  within  the  the  are born d u r i n g touch  ventral time  circuit.  to this  AVM,  A and v e n t r a l B (Sulston  White et  al,  D motorneurons,  embryonic p a t t e r n s  completely  to  the  switch  &. H o r v i t z ,  1978).  rewired  In  responsible  c o n t r a c t i o n between m u s c l e  their  and become  the  1985;  change  period in which  system begins  during this al,  change  New c e l l s  and t h e  c o o r d i n a t i o n of animal,  of  three  only  first  neuronal  nervous  AS motor n e u r o n s ,  i n the  of  of  L2 s t a g e  motoneurons  for  the  i n c l u d i n g many members o f  1977;  young  tap.  The L I t o  adult  spontaneous  s t a g e L2  frequency  of  not  show an a d u l t - l i k e  animal.  the  Young a d u l t s  but were a l s o to  the  active  and h i g h e s t  t a p s and t r a i n s  of  larval  L4 t o  least  predominantly reversing  accelerating  correlate  stage  represented  L2s c o n s i s t e n t l y  reversals  increased  animals  and the  to  transition  groups  of  (White  et  al,  79 1978).  The y o u n g  transition: coupling  adult stage  at approximately  between  t h e AVM  represents 40 h o u r s  a  second  after  a n d ALM n e u r o n s  hatching, the  becomes  funct iona1. Figure circuit Martin  2 represents relationships  f o r touch Chalfie  Chalfie,  sensitivity  circuit  depicted  appears  tenable:  i s correct, Adult  than  connection  between  addition If  routes  larval  there  PVC, w h i c h  goes  from  exciting  from  t h e AVM  interneurons connections the  anterior  inhibit  going  from  (tail  prevent  because of  connections  will  inhibited, i t  f r e q u e n t l y to taps there  a r e two  the anterior touch)  One  goes  forward  t o AVB.  i n the posterior  (head  circuit; from  movement  circuit.  i n larval  t h e ALMs t o by  circuit  whose  preventing The  other  inhibitory coming  They a r e a l s o t h e o n l y  neurons  touch)  The AVBs a r e t h e o n l y  a r e not m i r r o r e d by c o n n e c t i o n s  the motor  inhibitory  t o t h e B m o t o n e u r o n s a n d AVB. cell  more  AVM.  In the adult,  i s o n l y one.  should  and p o s s i b l y  a d u l t s r e v e r s e more  to the posterior  animals,  o f a new  that a response  animals.  hypothesis  to the taps  number o f i n h i b i t o r y  of i n h i b i t i o n  circuit  PVC  why  I f the  the following  because  neuron,  f a r by  e t a l , 1985:  reverse  AVB v i a AVM,  the probability  do  then  animals  out thus  t o C. R a n k i n ) .  animals  of asensory  becomes c l e a r e r than  larval  the r e l a t i v e  affects  (Chalfie  communication  frequently  the  as worked  and c o l l e a g u e s  personal  between t h e n e u r a l  producing  from  neurons  the competing  that  response  80 directly. The c r i t i c a l a n i m a l s may t h u s consequent Because  be the  a d d i t i o n of  AVB makes  anterior reduces touch  difference  touch the  two  workings routes  of  of  are  the  the  simultaneously  that  show t h e  the  Thus,  two  touch  or g e n e t i c  circuit,  a d d i t i o n of AVM t o lesion  p a t t e r n of  the  neuron  by the  tail  the  while  anterior  t o AVB.  upon  inhibiting  to  larval  larval  this  c i r c u i t has more r o u t e s  b l o c k e d by t h e  ablation  i n h i b i t i o n of  backward movement  (or more p a r t i c u l a r l y , the laser  AVM, and  i n h i b i t o r y connections  f o r w a r d movement.  touch  and  an i n h i b i t o r y c o n n e c t i o n  i n h i b i t i o n of  probability posterior  a d d i t i o n of  circuit,  c i r c u i t while  between a d u l t  the  inhibit  AVM.  of  the  these  Removal o f AVM  AVB c o n n e c t i o n ) should r e s u l t  via  in  adults  responding.  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