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THE FUNCTIONS OF THE FISH PINEAL ORGAN  by  JAMES CLARKE FENWICK  B.Sc,  U n i v e r s i t y o f Manitoba,  1962  M.Sc,  U n i v e r s i t y o f Manitoba,  1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA January,  1969  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  I further for  shall  the  his  of  this  agree that  written  of  be  available  g r a n t e d by  gain  QQ  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  (  j  Ok  f  for  for extensive  permission.  "Z.  British  the  It i s understood  for financial  Department of  Date  University  permission  representatives. thesis  f u l f i l m e n t of  make i t f r e e l y  s c h o l a r l y p u r p o s e s may  by  in p a r t i a l  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and c o p y i n g of  that  not  the  that  Study.  this  thesis  Department  c o p y i n g or  for  or  publication  allowed without  my  i  ABSTRACT  The r o l e of the f i s h p i n e a l organ has been s t u d i e d u s i n g the g o l d f i s h C a r a s s i u s auratus and the P a c i f i c chus tshawytscha.  salmon Oncorhyn-  To t h i s end, the e f f e c t s of pinealectomy i n  g o l d f i s h on v a r i o u s b e h a v i o u r a l responses, endocrine and the r e p r o d u c t i v e system were s t u d i e d .  systems,  The p i n e a l  organs  and the r e t i n a l t i s s u e from mature and immature salmon were examined by t h i n - l a y e r chromatography and fluorometry to d e t e r mine i f melatonin, a mammalian hormone, i s present i n the fishes.  G o l d f i s h were i n j e c t e d with melatonin to see i f the  e f f e c t of exogenous melatonin was  opposite to that of p i n e a l e c -  tomy. Pinealectomized g o l d f i s h l o s t the photo-negative response seen i n normal g o l d f i s h .  B l i n d i n g had the same e f f e c t  on  p h o t o t a x i s as pinealectomy and a combination of the two had the same e f f e c t as b l i n d i n g or pinealectomy alone.  I t was  concluded  that the normal p h o t o t a c t i c response depended upon both the p i n e a l organ and the eyes.  Pinealectomy, b l i n d i n g , or both  followed by a marked i n c r e a s e i n swimming a c t i v i t y . t h i s i n c r e a s e was  Although  c o r r e l a t e d with a decrease i n the whole b r a i n  s e r o t o n i n i l e v e l , a c a u s a l r e l a t i o n s h i p was not e s t a b l i s h e d tween the two.  was  F u r t h e r , pinealectomy alone produced  no  be-  signifi-  cant changes i n whole b r a i n s e r o t o n i n l e v e l . Melatonin was l o c a l i z e d w i t h i n the p i n e a l organ o f salmon and i t s c o n c e n t r a t i o n i n t h i s t i s s u e was analyzed.  The  melatonin s t o r e v a r i e d d u r i n g the r e p r o d u c t i v e c y c l e and  pineal was  ii  found i n lower c o n c e n t r a t i o n s i n the p i n e a l organs of mature salmon.  Stored melatonin could not be found i n the r e t i n a l  tis-  sue d e s p i t e evidence f o r an a c t i v e tryptophane metabolism i n this tissue. I n j e c t i o n of melatonin i n t o g o l d f i s h i n h i b i t e d the i n c r e a s e i n gonad s i z e under l o n g photoperiod; t h i s was accompanied by l a r g e r gonadotrophs  i n the melatonin i n j e c t e d f i s h .  Removal of  the p i n e a l organ from g o l d f i s h held under short photoperiod caused an i n c r e a s e i n gonad s i z e s i m i l a r to that seen i n unt r e a t e d g o l d f i s h exposed  to l o n g photoperiod.  The e f f e c t of  pinealectomy on the gonads was l i m i t e d to t h a t season d u r i n g which the gonads could be s t i m u l a t e d by i n c r e a s i n g day l e n g t h . At  other times of the year, n e i t h e r photoperiod nor pinealectomy  produced was  any s i g n i f i c a n t e f f e c t on the gonad s i z e .  From t h i s i t  concluded that the p i n e a l gland of the g o l d f i s h i s r e l a t e d  to the r e p r o d u c t i v e c y c l e and that i t s f u n c t i o n depends upon photoperiod and the p r o d u c t i o n of melatonin. Pinealectomy had no e f f e c t on the i n t e r r e n a l t i s s u e , t i s s u e , plasma osmotic c o n c e n t r a t i o n , or plasma l e v e l s of —  Na , +  ++  CI , or Ca are  thyroid  specific  , i n d i c a t i n g that the e f f e c t s of t h i s o p e r a t i o n f o r the r e p r o d u c t i v e system.  The d a t a obtained from these s t u d i e s support the hypothesis that the p i n e a l organ o f f i s h e s serves a s e c r e t o r y as w e l l as a sensory f u n c t i o n .  F u r t h e r , the f u n c t i o n a l a s p e c t s of the mam-  malian and f i s h p i n e a l organs are d i s c u s s e d and i t i s concluded that the r o l e of the p i n e a l organ i s s i m i l a r i n the two  groups;  that i s , the p i n e a l organ of mammals and f i s h i s i n v o l v e d i n the  iii  t i m i n g of r e p r o d u c t i v e  events.  iv  TABLE OF CONTENTS Page ABSTRACT  i  LIST OF TABLES  vii  LIST OF FIGURES  ix  ACKNOWLEDGMENTS  x  PREFACE  1  GENERAL INTRODUCTION  2  SECTION I  The Sensory R o l e o f the P i n e a l Organ i n the G o l d f i s h ,  Carassius auratus L .  INTRODUCTION  5  MATERIALS AND METHODS  9  O p e r a t i v e Procedures  .  9  Apparatus and E x p e r i m e n t a l Procedures  11  S e r o t o n i n L e v e l s i n Whole B r a i n s  17  RESULTS  22  P i n e a l e c t o m y , B l i n d i n g , and P h o t o t a x i s  22  P i n e a l e c t o m y , B l i n d i n g , and C o n d i t i o n i n g to L i g h t and Sound. . . . . . . ,. ••>-> . . . . . . .  . . . 22  P i n e a l e c t o m y and Response to E l e c t r i c a l Stimulation P i n e a l e c t o m y , B l i n d i n g , and Swimming A c t i v i t y  26 28  E f f e c t of P i n e a l e c t o m y , B l i n d i n g , or b o t h on Brain Serotonin L e v e l s . . . . . . . . DISCUSSION  .28 3k  V  Page SECTION I I  Demonstration in  and E f f e c t of M e l a t o n i n  Fish  INTRODUCTION  41  MATERIALS AND METHODS  44  Melatonin Injection  44  Studies  Q u a l i t a t i v e and Q u a n t i t a t i v e  E s t i m a t i o n of  • M e l a t o n i n and S e r o t o n i n  49  RESULTS  57  Demonstration  of S e r o t o n i n and M e l a t o n i n i n f i s h . . .  57  S e r o t o n i n and M e l a t o n i n L e v e l s i n Immature and Mature Salmon  58  E f f e c t s of M e l a t o n i n I n j e c t i o n  60 6 6  DISCUSSION SECTION I I I  E f f e c t o f P i n e a l e c t o m y on the  Repro-  d u c t i v e and E n d o c r i n e Systems i n G o l d f i s h INTRODUCTION  72  MATERIALS AND METHODS  75  E s t i m a t i o n of R e p r o d u c t i v e C y c l e  75  Effect  of P h o t o p e r i o d  Different  on the Gonad S i z e at  Times of the Year  E f f e c t s of P i n e a l e c t o m y on the GSI S t a t i s t i c a l Methods RESULTS  75 7 6 . . . 8l 82  Seasonal  V a r i a t i o n i n Gonad S i z e  Seasonal  V a r i a t i o n of the E f f e c t of P h o t o -  p e r i o d on the Gonad S i z e  82  82  vi  Page Effect  o f Pinealectomy on the Gonad S i z e  Effect  o f P i n e a l e c t o m y on the H i s t o l o g y of  R e p r o d u c t i v e and E n d o c r i n e T i s s u e E f f e c t o f P i n e a l e c t o m y on the System DISCUSSION GENERAL DISCUSSION  85  88  Electrolyte 88 89 94  SUMMARIES  100  LITERATURE CITED  103  vii  LIST OF TABLES Table I  Page Reproducibility, extraction efficiency, and s p e c i f i c i t y of s e r o t o n i n d e t e r m i n a t i o n s  II  E f f e c t of pinealectomy, b l i n d i n g ,  . . . .20  or b o t h  on the c o n d i t i o n i n g o f g o l d f i s h to l i g h t 25  and sound III  E f f e c t of p i n e a l e c t o m y on the response o f 27  g o l d f i s h to e l e c t r i c a l s t i m u l a t i o n IV  Swimming a c t i v i t y o f g o l d f i s h showing d i u r n a l rhythm and e f f e c t  of pinealectomy,  blinding, 30  or b o t h V  E f f e c t of p i n e a l e c t o m y , b l i n d i n g , or b o t h on the s e r o t o n i n c o n t e n t i n g o l d f i s h b r a i n s .  VI  . . . .  S e r o t o n i n and m e l a t o n i n content o f mature and immature salmon p i n e a l s and r e t i n a t i s s u e  VII  exposure 6l  to l o n g p h o t o p e r i o d A n a l y s i s of v a r i a n c e of male and female GSI d a t a before and a f t e r  50 days of m e l a t o n i n  or s o l v e n t i n j e c t i o n IX  59  E f f e c t of m e l a t o n i n i n j e c t i o n on the GSI o f male and female g o l d f i s h f o l l o w i n g  VIII  .32  62  E f f e c t of m e l a t o n i n i n j e c t i o n on i n t e r r e n a l nuclear diameter,  r e l a t i v e thyroid epithelium  c e l l h e i g h t and gonadotroph diameter o f goldfish  '65  viii  Table  Page  X  Scope and t i m i n g of p i n e a l e c t o m y e x p e r i m e n t s .  XI  E f f e c t o f p i n e a l e c t o m y on the gonad s i z e o f  . . .77  male and female g o l d f i s h d u r i n g the p e r i o d J a n . 9 to May 3 XII  86  A n a l y s i s of v a r i a n c e of the GSI v a l u e s of 18 pinealectomized,  s h a m - p i n e a l e c t o m i z e d , and  unoperated c o n t r o l g o l d f i s h o f both sexes d u r i n g the p e r i o d J a n . 9 to May 3  • •  .87  ix  LIST OF FIGURES Figure 1.  Page D i s t r i b u t i o n of g o l d f i s h i n a l i g h t gradient following b l i n d i n g ,  pinealectomy,  or both 2.  23  Swimming a c t i v i t y o f normal g o l d f i s h or f o l l o w i n g p i n e a l e c t o m y , b l i n d i n g , or b o t h  3.  29  R e l a t i o n s h i p between mean t o t a l swimming a c t i v i t y and mean b r a i n s e r o t o n i n l e v e l i n v a r i o u s e x p e r i m e n t a l groups o f g o l d f i s h  k.  33  Seasonal v a r i a t i o n of the GSI i n male and female g o l d f i s h h e l d under l a b o r a t o r y conditions  5.  Seasonal v a r i a t i o n o f p h o t o p e r i o d e f f e c t the GSI i n male and female g o l d f i s h  83 on 84  X  ACKNOWLEDGMENTS  G r a t e f u l thanks a r e due torays u p e r v i s o r Dr. W i l l i a m S. Hoar f o r h i s generous help and f i n a n c i a l a s s i s t a n c e d u r i n g the i n v e s t i g a t i o n and throughout the p r e p a r a t i o n o f the manuscript. The author  would a l s o l i k e t o express h i s thanks to Dr.  Peter Ford, Dr. A.M. Perks, Dr. John E. P h i l l i p s and Dr. N.R. L i l e y f o r r e a d i n g the manuscript  and f o r o f f e r i n g h e l p f u l  suggestions d u r i n g the i n v e s t i g a t i o n . Dr. John L e a t h e r l a n d , Dr. Satyendra  I am a l s o indebted to  Pandey and Mr. Lynn Shar-  mon f o r t h e i r t i m e l y advice and a s s i s t a n c e on h i s t o l o g i c a l cedures and to Dr. Melvin Weisbart  pro-  f o r h i s help on i o n and osmo-  tic analysis. I would l i k e to express my a p p r e c i a t i o n to Mr. Alan Handley, Mr.  Kees Hogendyke, Mr.  Fred McConnell and Mr. Eugene  McCulloch  f o r t h e i r help with the day t o day problems, and t o Mrs. Jones f o r t y p i n g the manuscript.  Caren  F o r the f i n a n c i a l support by  the N a t i o n a l Research C o u n c i l o f Canada i n the form o f grantsi n - a i d of r e s e a r c h t o Dr. Hoar and the Studentship awarded to me  (1965-1968) I o f f e r my s i n c e r e a p p r e c i a t i o n . F i n a l l y , I thank my wife, J u l i e , the manuscript foreign  papers.  for her help i n p r e p a r i n g  and f o r her a s s i s t a n c e i n the t r a n s l a t i o n of  1  PREFACE  E x t e n s i v e and i l l u m i n a t i n g b o d i e s o f l i t e r a t u r e w i t h the ontogeny,  concerned  morphology, h i s t o l o g y and c y t o l o g y o f  f i s h p i n e a l organ are r e a d i l y a v a i l a b l e .  Nonetheless,  the  no b a s i c  or p r i m a r y f u n c t i o n has been u n e q u i v o c a l l y demonstrated  for  p i n e a l organ o f f i s h e s .  function  T h i s f a i l u r e to r e v e a l a b a s i c  of the p i n e a l i n f i s h e s p r o b a b l y stems d i r e c t l y from the o f p h y s i o l o g i c a l experiments c o n c e r n i n g i t s  the  paucity  role.  D e s p i t e the numerous r e p o r t s o f p h y s i o l o g i c a l e f f e c t s  of  p i n e a l e c t o m y i n o t h e r a n i m a l groups, r e l a t i v e l y few workers have examined the e f f e c t  o f p i n e a l a b l a t i o n on f i s h e s .  Further,  a l t h o u g h the hormone m e l a t o n i n has been l o c a l i z e d i n mammals, b i r d s , and amphibians,  and a l t h o u g h the enzyme necessary  for  its  s y n t h e s i s has been found i n the p i n e a l organs of r e p t i l e s and fishes,  nobody has demonstrated  the f i s h p i n e a l .  the presence o f m e l a t o n i n w i t h i n  Moreover, the e f f e c t s  of exogenous m e l a t o n i n  have not been d e s c r i b e d i n f i s h e s d e s p i t e g i c a l effects  o f t h i s substance on other  the marked p h y s i o l o vertebrates.  The aim of t h i s i n v e s t i g a t i o n , then has been to these problems and to extend our knowledge of the o f the f i s h p i n e a l o r g a n .  examine  function(s)  2  GENERAL INTRODUCTION  Although the p i n e a l organ of mammals has  assumed a p o s i t i o n  of r e s p e c t a b i l i t y among the endocrine organs (Thie'blot, 1 9 6 5 ) , t h i s same organ of f i s h e s i s viewed by many as a f u n c t i o n l e s s , evolutionary  vestige  of a p r i m i t i v e t h i r d  eye.  This  probably a r i s e s from.the obvious morphological  schism  differences  between the photosensory l i k e s t r u c t u r e of the fishes and  the  glandular  pineal  appearance of the mammalian p i n e a l organ.  The  of f i s h e s s t r o n g l y resembles a sensory s t r u c t u r e with zable photosensory c e l l s Riideberg,  1966)  and  (Studnicka,  1905;  1968a,b; a.o.).  Riideberg,  i n mammals has 1954;  Relkin,  innervation  only  r a r e l y i n the 1967),  a glandular 1966)  1894;  Kappers,  Altschule,  primary sensory c e l l s and  (Kappers, 1 9 6 5 ) .  The  efferent f i b e r s  (van de Kamer, 1955;  Lowenstein, 1956)  organs, the p i n e a l s of f i s h and  mammals (Quay, 1956;  described Ford,  same type  i n some mammals (Kap-  D e s p i t e the d i f f e r e n c e s i n the way  mation reaches the  efferent  Hafeez and  though p o s s i b l y f u n c t i o n a l , are probably of the  1965).  and  Conversely, t h i s same s t r u c t u r e  appearance ( K i t a y and  with no  f i s h pineal  (Hill,  of abberrant commissural f i b e r s d e s c r i b e d pers,  Holmgren, 1959a;  a f f e r e n t nerve f i b e r s running from the p i -  n e a l organ to other p a r t s of the b r a i n 1965;  recogni-  that the  infor-  (Grunewald-  Zweig et a l . ,  1966)  show responses to e x t e r n a l i l l u m i n a t i o n . Friedrick-Freksa and  Hafeez and  secretory  ( 1 9 3 2 ) , Rasquin ( 1 9 5 8 ) ,  Ford (1967) have now  Holmgren (1959b)  presented evidence that  a c t i v i t y of the f i s h p i n e a l organ occurs w i t h i n  the  the  3  sensory elements of the p i n e a l e p i t h e l i u m . though unable to educe any  Rudeberg  (1968a),  d e f i n i t i v e evidence f o r s e c r e t i o n  w i t h i n the sensory c e l l s of the p i n e a l organ of Sardina p i l chardus s a r d i n a  ( R i s s o ) , reported  d a l r o l e of these c e l l s .  some evidence f o r an  T h i s p o s s i b i l i t y of a sensory and  s e c r e t o r y f u n c t i o n of the same c e l l s was Roels (1967) who  autocoi-  supported by V i v i e n  observed c e r t a i n c e l l s w i t h i n the  secretory e f f e c t o r .  Thus, as pointed  ( 1 9 6 4 ) , i t i s p o s s i b l e that the phylogenetic glandular  trend toward a more  p i n e a l organ of f i s h e s , r e f l e c t s a change i n the way reaches the p i n e a l r a t h e r than a complete and I t was  t h e r e f o r e speculated  of f i s h i s both sensory and  from a  out by Roth  s t r u c t u r e i n the mammals, as opposed to the  i n function.  and  chelonian  e p i p h y s i s which possessed the a b i l i t y to change to and r e c e p t o r and  a  sensory information  unrelated  change  that the p i n e a l organ  s e c r e t o r y and  i s functionally simi-  l a r to the p i n e a l organ of mammals. In pursuance of these s u p p o s i t i o n s , l i s t e d below, and  discussed  the working hypotheses  i n t h e i r appropriate  s e c t i o n , were  tested. (i)  The  p i n e a l organ of f i s h e s , by v i r t u e of i t s photo-  s e n s i t i v i t y , mediates behavioural  responses to  en-  vironmental i l l u m i n a t i o n , (ii)  The  p i n e a l organ of f i s h e s s y n t h e s i z e s melatonin  and  t h i s substance has a hormonal f u n c t i o n , (iii)  The  p i n e a l organ of f i s h e s i s a s s o c i a t e d with the  docrine  system, the r e p r o d u c t i v e  system, or both,  t h i s a s s o c i a t i o n ( s ) i s i n f l u e n c e d by  environmental  enand  illumination.  5  SECTION I THE SENSORY ROLE OF THE PINEAL ORGAN IN GOLDFISH CARASSIUS ATTRACTS L. INTRODUCTION  The  p i n e a l organ o f the cyclostomes has been recognized as  a photoeceptor since Young ( 1 9 3 5 ) demonstrated that the marked d i u r n a l rhythm of c o l o r change i n lampreys was a b o l i s h e d i n ammocoetes and d i s t u r b e d extirpation. herr  i n a d u l t Lampetra f o l l o w i n g  pineal  Breder and Rasquin ( 1 9 5 0 ) , Hoar ( 1 9 5 5 ) and Schon-  ( 1 9 5 5 ) r e p o r t pigmentary d i s p e r s i o n  o c c l u s i o n or d e s t r u c t i o n to the t e l e o s t p i n e a l . t i a t e d t h i s conclusion  following  pineal  and a t t r i b u t e a photoreceptor Dodt ( 1 9 6 3 ) and M o r i t a ( 1 9 6 6 )  function substan-  by e l e c t r o p h y s i o l o g i c a l l y d e t e c t i n g a l -  t e r a t i o n s i n nervous a c t i v i t y i n the p i n e a l of Salmo i r i d e u s during  and a f t e r i l l u m i n a t i o n .  Studies on the e f f e c t s of p i n e a l o c c l u s i o n b l i n d i n g , or combinations thereof contradictory  have provided d i v e r s i f i e d and  r e s u l t s when b e h a v i o u r a l parameters were used.  Breder and Rasquin ( 1 9 4 7 ) reported Mexican cave characins reversed  and a b l a t i o n ,  that i n a s e r i e s of b l i n d  Anoptichthyes the s i g n of p h o t o t a x i s was  from p o s i t i v e to negative when an otherwise exposed  p i n e a l organ was covered, and that exposing an otherwise covered p i n e a l reversed  the s i g n o f p h o t o t a x i s from negative to p o s i t i v e .  These same authors reported  that the p h o t o t a x i s demonstrated,  whether p o s i t i v e or negative, depended upon the presence of  6  i n t a c t o p t i c c y s t s with nervous connection.  These f i n d i n g s  were not corroborated by s t u d i e s on other f i s h (Hoar,  1955;  Pang, 1 9 6 5 ) . Hoar (1955) found that the normal photo-negative response of sockeye salmon smolts Oncorhynchus nerka was not o b l i t e r a t e d by b l i n d i n g alone but that the f i s h d i d become i n d i f f e r e n t to l i g h t when the b l i n d e d animals had t h e i r p i n e a l areas damaged by probing.  The data from animals with i n t a c t v i s i o n but with  the p i n e a l area probed were e q u i v o c a l but d i d suggest an i n d i f ference to l i g h t on the p a r t of the animals.  Hoar (1955)  h e s i t a n t however and d i d not draw t h i s c o n c l u s i o n . has more r e c e n t l y r e p o r t e d that b l i n d Fundulus  was  Pang (1965)  heteroclitus  show a s i g n i f i c a n t l y greater preference f o r l i g h t than the l i g h t i n d i f f e r e n t b l i n d p l u s p i n e a l e c t o m i z e d Fundulus.  Unfortunately,  he p r o v i d e s no i n f o r m a t i o n about the normal p h o t o t a c t i c  response  of wholly i n t a c t animals under s i m i l a r experimental c o n d i t i o n s so that i t i s i m p o s s i b l e to draw c o n c l u s i o n s about the e f f e c t of b l i n d i n g alone, pinealectomy alone, or a combination of the on the p h o t o t a c t i c response of t h i s s p e c i e s .  two  Although these  s t u d i e s have i m p l i c a t e d the p i n e a l organ i n responses to i l l u mination, they have not examined the p o s s i b i l i t y that the d i f ferences r e s u l t e d from changes i n s e n s i t i v i t y to f a c t o r s other than l i g h t or to changes i n the general a c t i v i t y of the animals. Further, these workers have not recognized that p h o t o t a x i s depends upon two t h i n g s ; that i s , a p h o t o t a c t i c response depends on a preference f o r a p a r t i c u l a r degree of i l l u m i n a t i o n and the " r e a d i n e s s " of the animal to move from one c o n d i t i o n of  7  b r i g h t n e s s t o another.  Indeed, Janzen  (1933)  found that  when g o l d f i s h were given a choice between l i g h t and dark, some p r e f e r r e d the l i g h t ,  some p r e f e r r e d the dark, while o t h e r s  were i n t e r m e d i a t e i n t h e i r p r e f e r e n c e .  On the b a s i s of these  r e s u l t s he suggested t h a t two f e a t u r e s were concerned i n the p h o t o t a c t i c response; one, a preference to remain under the same degree of b r i g h t n e s s which he termed a " p e r s i s t e n c e t e n dency" and two, the seeking out of a p r e f e r r e d l e v e l of b r i g h t ness.  He a l s o showed that these two f e a t u r e s were a n a t o m i c a l l y  separated w i t h i n the b r a i n (Janzen, 1 9 3 3 ) • Recently, Wurtman (1967)  has suggested that the e f f e c t s  of p h o t i c i n f o r m a t i o n i n higher v e r t e b r a t e s may be d i v i d e d three c a t e g o r i e s :  (1)  into  the p s y c h o l o g i c a l e f f e c t s of a v i s u a l  f i e l d which may cause a c t i v e o r i e n t a t i o n or behaviour to a part i c u l a r v i s u a l image; (2)  short term n e u r o l o g i c a l r e f l e x e s over  which the animal has l i t t l e  or no c o n t r o l and which cause pas-  s i v e responses such as r e g u l a t i o n o f p u p i l l a r y s i z e and k i n e sis;  (~) and c e r t a i n l o n g term neuroendocrine responses such as  the maturation of gonads.  The p i n e a l organ of f i s h e s may be  i n v o l v e d i n any or a l l o f these e f f e c t s .  Therefore, the pre-  sent study was designed to t e s t the e f f e c t of pinealectomy on both f e a t u r e s of the p h o t o t a c t i c response, and on the a b i l i t y of the p i n e a l to operate as a photoreceptor governing a c t i v e or p a s s i v e responses to environmental i l l u m i n a t i o n .  In a d d i t i o n ,  the e f f e c t of pinealectomy on the response of g o l d f i s h to other environmental s t i m u l i and swimming a c t i v i t y was t e s t e d .  Fur-  ther, as the p i n e a l organ of f i s h e s (unpublished data) was  8  found to have the highest s e r o t o n i n c o n c e n t r a t i o n o f any r e g i o n of the b r a i n , and a s t h i s substance has been r e l a t e d t o the degree o f b e h a v i o u r a l  s e d a t i o n (Brodie and Bogdanski, 19&U;  Quay, 1965a; Weisman, 1967),  the r e l a t i o n s h i p between p i n e a l e c -  tomy, b l i n d i n g , t o t a l b r a i n s e r o t o n i n and swimming a c t i v i t y was studied. T h i s s e c t i o n r e p o r t s on the e f f e c t o f pinealectomy, b l i n d i n g and pinealectomy p l u s b l i n d i n g on the d i s t r i b u t i o n of g o l d f i s h i n a l i g h t g r a d i e n t , a b i l i t y t o use l i g h t and sound a s c o n d i tioned s t i m u l i , amount o f swimming a c t i v i t y , and t o t a l b r a i n serotonin l e v e l s .  Also i n v e s t i g a t e d were the e f f e c t o f p i n e a -  lectomy on the minimum e l e c t r i c a l v o l t a g e r e q u i r e d to j u s t  pro-  duce a d e t e c t a b l e response and the e f f e c t o f pinealectomy on the d i s t a n c e t h a t g o l d f i s h move f o l l o w i n g s t i m u l a t i o n with a constant  voltage.  The data are then d i s c u s s e d i n r e l a t i o n to  the p o s s i b l e sensory  r o l e o f the g o l d f i s h p i n e a l organ. The  p o s s i b l e r o l e o f the p i n e a l organ i n l o n g term neuroendocrine responses to photoperiod  w i l l be considered i n S e c t i o n I I I .  9  MATERIALS AND METHODS  G o l d f i s h ( C a r a s s i u s auratus, common v a r i e t y ; 7 . 5 - 1 0 cm s i z e c l a s s ) were obtained from the G o l d f i s h Supply Company, S t o u f f ville,  Ontario.  E x c l u d i n g the a c t u a l time of o b s e r v a t i o n , the  f i s h were maintained i n nylon-screen baskets approximately 20 cm square which were suspended i n a l a r g e h o l d i n g tank ( a p p r o x i mately 400 l i t e r  c a p a c i t y ) c o n t a i n i n g d e c h l o r i n a t e d water.  pH of the water i n the h o l d i n g tank v a r i e d from 6 . 4 h o l d i n g temperature ranged from 11 C to 12 C.  the  The  to 6 . 9 and An  artificial  photoperiod of 8 hours of l i g h t a l t e r n a t i n g with 16 hours of darkness was maintained a t a l l times; the l i g h t s came on a 8 : 0 0 AM and went o f f at 4 : 0 0 PM. Age dry f i s h food a t 9 : 0 0  The g o l d f i s h were fed C l a r k ' s New  AM d a i l y , with the exception that  fish  were not f e d on the day of t e s t i n g . Unless s p e c i f i c a l l y mentioned, compared i n each experiment:  s i x groups of animals were  p i n e a l e c t o m i z e d (P), sham-pineal-  ectomized ( S ) , and unoperated c o n t r o l s ( C ) , b l i n d  (B), b l i n d  p l u s p i n e a l e c t o m i z e d (BP), and b l i n d p l u s sham-pinealectomized (BS). to  The f i r s t  three c l a s s i f i c a t i o n s are o c c a s i o n a l l y r e f e r r e d  c o l l e c t i v e l y as the eyed groups and the remainder may be con-  s i d e r e d as the e y e l e s s groups.  Operative Procedures A.  General F i s h were a n a e s t h e t i z e d i n t r i c a n e methanesulphonate  222,  Sandoz,! 1 : 1 , 0 0 0 )  (MS  u n t i l i n c a p a b l e of r i g h t i n g r e a c t i o n s .  10  The  area of o p e r a t i o n  was  i l l u m i n a t e d with a b r i g h t microscope  lamp s u f f i c i e n t l y removed to prevent undue h e a t i n g of the Following  the operation,  fish.  the g i l l s were i r r i g a t e d with oxygen-  tated water u n t i l spontaneous g i l l movements reappeared.  B.  Pinealectomy and Following  paper towel. "U"  Sham-pinealectomy  n a r c o t i z a t i o n , the f i s h was The  area of the p i n e a l was  wrapped i n a damp  exposed by making a  shaped cut i n the p a r i e t a l bone with the  center of the  IT  b i s e c t i n g an imaginary l i n e connecting the p o s t e r i o r margins of the o r b i t s . of the U;  The  r e s u l t i n g bone f l a p was  t h i s was  destroyed.  p i n e a l gland was a glass pipette.  I f the  f l a p of bone broke free  detached with a gentle Only those  damaged during  discarded.  top  suction applied  on  the  Under a d i s s e c t i o n microscope ( 1 6 X ) ,  the  through  animals seen to l o s e t h e i r p i n e a l  organs were considered p i n e a l e c t o m i z e d . b r a i n was  towards the  done c a r e f u l l y to avoid b r e a k i n g the s k i n  the i n t a c t s i d e of the U. animal was  lifted  Animals i n which  the  the a s p i r a t i o n of the p i n e a l organ were  A f t e r p i n e a l a b l a t i o n the  to i t s o r i g i n a l p o s i t i o n and  f l a p of bone was  the wound was  returned  covered with a water  impermeable paste (Orabase, Squibb Home Drugs D i v i s i o n , Montreal,  Quebec).  T h i s paste adhered to the  f o r about 2.K hours and i n t o the  was  skull cavity.  p i n e a l t i s s u e was  No  of the head  thought to reduce leakage of water evidence f o r r e s i d u a l or regenerated  found i n any  of the pinealectomized g o l d f i s h  when examined h i s t o l o g i c a l l y or d u r i n g months p o s t - o p e r a t i v e l y .  surface  d i s s e c t i o n s up to  four  Sham operated c o n t r o l s were t r e a t e d i n  11  a s i m i l a r manner except that the p i n e a l organ was  C.  Blinding While n a r c o t i z e d , the f i s h was  towel.  Curved  wrapped i n a moist paper  f o r c e p s were used to p u l l the eye p a r t i a l l y  from i t s socket and the o c u l a r muscles, t i s s u e were severed with a s c a l p e l . blind  nerves and  free  connective  F o l l o w i n g the o p e r a t i o n ,  f i s h resumed f e e d i n g w i t h i n one h a l f of an hour and  appeared  D.  not removed.  to remain healthy.  P o s t - o p e r a t i v e Treatment F o l l o w i n g the v a r i o u s o p e r a t i o n s , the animals were held i n  a 0.2%  s o l u t i o n c o n s i s t i n g of equal p a r t s of C a C ^  which Terramycin  (Animal Formula, P f i z e r Co.,  and NaCl to  Montreal,  Quebec)  had been added to the extent of 250 mg to each 100 l i t e r s of solution.  A l l experimental animals and c o n t r o l s were h e l d under  t h i s c o n d i t i o n f o r a p e r i o d of one week before they were t r a n s f e r r e d to the o r i g i n a l  tanks.  Under the preceeding regimen, animals remained healthy and m o r t a l i t y was  Apparatus A.  l e s s than 2 % . i n any of the experimental  and Experimental  groups.  Procedures  Phototaxis The d i s t r i b u t i o n of g o l d f i s h i n a l i g h t  served i n three tanks. and 25 cm deep and was  Each tank was filled  224  gradient was  ob-  cm long, 25 cm wide,  to a depth of 10 cm with d e c h l o r -  i n a t e d water which contained 1 gm of N a C l / l i t e r .  The  tanks  12  were c o n s t r u c t e d of g a l v a n i z e d i r o n and were p a i n t e d grey.  Ex-  periments were conducted i n a darkened room; one end of each tank was i l l u m i n a t e d with a 60 watt incandescent l i g h t  set 30  cm  above the surface of the water. The water temperature w i t h i n the tanks v a r i e d between Ik C and 16: C but no temperature was  gradient  found a l o n g the l e n g t h of any one tank, nor between any  two  of the tanks. For  each t e s t group, 10 g o l d f i s h were used.  Observations  c o n s i s t e d of c o u n t i n g the number of g o l d f i s h i n that h a l f of the tank nearest to the l i g h t .  Ten counts were made d u r i n g one  day,  then the l i g h t was moved to the opposite end of the tank and ten counts were taken the f o l l o w i n g day. tinuously.  In t h i s way,  experimental treatment. the  B.  The l i g h t was l e f t on con-  20 o b s e r v a t i o n s were obtained f o r each F i s h were p l a c e d i n the trough d u r i n g  evening of the day p r e v i o u s to o b s e r v a t i o n .  C o n d i t i o n i n g to L i g h t and Sound G o l d f i s h were subjected to a c o n d i t i o n i n g s i t u a t i o n  to that d e s c r i b e d by Agranoff (1967).  The aquarium  used  similar was  c o n s t r u c t e d of c l e a r p l e x i g l a s with the s i d e s covered by b l a c k p l a s t i c to prevent the f i s h from o b s e r v i n g e x t e r n a l movements; the  f i s h were observed i n an overhanging m i r r o r .  was kk  cm long, 11 cm wide and Ik  partially floor.  The  aquarium  cm deep and the i n s i d e  was  d i v i d e d by a c e n t r a l b a r r i e r 10 cm high f i x e d to the  Water was added to the depth^of 12 cm and provided 2 cm  of water over the c e n t r a l b a r r i e r .  One  gram of NaCl was added  to each l i t e r of water to f a c i l i t a t e the conduction of e l e c t r i c a l  13  currents.  Each half of the tank was provided with a pair of  stainless steel electrodes (12 cm x 10 cm) and an unshaded 40 watt bulb was placed at either end of the tank. the  The bottom of  tank was covered with 2 cm of sand but i n the middle of the  tank the sand on both sides of the b a r r i e r was banked up to the top of the barrier.  This arrangement of the sand provided a  clue for the blind animals which otherwise had d i f f i c u l t y i n avoiding the central barrier during the conditioning experiments. A suitable series of switches permitted the l i g h t s to be turned on or o f f at either end of the tank and provided a means by which a series of e l e c t r i c a l impulses could be discharged between either pair of electrodes.  The series of e l e c t r i c a l shocks was  provided by a Grass S6C Stimulator (Grass Instruments, Quincey, Mass.) and consisted of seven pulses of 80 v o l t s per second for 20 seconds; each pulse had a duration of 1 msec.  A learning  t r i a l began with the illumination of the l i g h t located on the same side of the central barrier as the f i s h .  After 20 sec, a  series of e l e c t r i c a l shocks was i n i t i a t e d i n the same end, and were allowed to continue for a further 20 sec at which time both the l i g h t and the shocks were terminated and a 20 sec rest period ensued before a similar sequence was i n i t i a t e d at the opposite end.  Fish which did not swim over the barrier i n re-  sponse to the l i g h t alone or to the l i g h t plus the shocks were given a one minute rest period before the sequence was repeated. Each f i s h was given 50 t r i a l s , and five f i s h from each *, group were tested.  The t o t a l number of times that each f i s h  escaped the shock by swimming over the barrier i n response to  14  the l i g h t was  recorded.  C o n d i t i o n i n g to sound was to t h a t d e s c r i b e d above.  c a r r i e d out i n a manner s i m i l a r  Animals naive to the  experimental  s i t u a t i o n were used but i n place of a l i g h t , a d o o r b e l l provided as the c o n d i t i o n e d stimulus.  The b e l l was  was  turned o f f  as soon as the f i s h crossed the b a r r i e r . F i s h d i d not c r o s s the c e n t r a l b a r r i e r without the unconditioned  experiencing  or c o n d i t i o n e d stimulus as t h i s r e q u i r e d  f i s h to b r i n g t h e i r d o r s a l surface out of the water. the r e s u l t s obtained  were not due  the  Therefore,  to random movements over the  barrier. Both experiments were run i n a c o n t r o l l e d environment room at a temperature of 12 C and i n subdued l i g h t .  C.  Response to E l e c t r i c a l S t i m u l i Responses to e l e c t r i c a l  trough  s i m i l a r to that used by E l s o n ( 1 9 4 2 ) .  cm long, 12 cm wide, and plexiglas. was  s t i m u l a t i o n were measured i n a  15 cm deep and was  130  trough was  constructed  of  S u f f i c i e n t water c o n t a i n i n g 1 gm of NaCl per  liter  added to the tank to b r i n g the depth of water to 8 cm.  l o n g s i d e s of the trough were covered trough was  arranged  to observe the f i s h .  with black p l a s t i c and  l i n e was  light.  The bottom of the tank  The l i n e s were r e a d i l y seen i n -the m i r r o r , and assigned  the  Experiments were c a r r i e d out i n a room  marked with t r a n s v e r s e b l a c k l i n e s i n t o equal segments  cm wide.  The  so that an overhanging m i r r o r could be used  uniformly i l l u m i n a t e d with a dim was  The  a number from 1 to 52.  Stainless steel  2.5  each  15  e l e c t r o d e s measuring 8 cm by 10 cm were placed i n the water a t opposite ends of the trough.  A switch through which a c a p a c i -  t a t o r could be discharged was wired to a voltmeter so that the v o l t a g e used could be recorded  (Hoar et a l . , 1 9 5 5 ) .  Single f i s h  were placed i n the trough around 4 : 0 0 PM of one day and were first  t e s t e d a t 8:30 AM of the f o l l o w i n g day.  In t h i s way the  f i s h were given a short p e r i o d to become accustomed to the experimental c o n d i t i o n .  The problem of d i u r n a l v a r i a t i o n i n swim-  ming a c t i v i t y was averaged noon and 3 : 3 0  out by subsequent t e s t i n g a t 1 2 : 0 0  PM.  Commencing with a subthreshold v o l t a g e , s t i m u l a t i o n was c a r r i e d out a t 30 sec i n t e r v a l s while i n c r e a s i n g the v o l t a g e 1 v o l t a t a time u n t i l the f i s h showed i t s f i r s t  perceptible re-  sponse; t h i s v o l t a g e was recorded as the minimum response tage.  The procedure  was repeated  r e s t between each t r i a l .  vol-  f i v e times with a two minute  The stimulus s t r e n g t h was then i n -  creased to the v o l t a g e used as the unconditioned  stimulus i n  the l i g h t and sound c o n d i t i o n i n g experiments (80 v o l t s ) . f i s h , provided that i t was s t i l l ,  The  was then s t i m u l a t e d at one  minute i n t e r v a l s and the d i s t a n c e t h a t the f i s h moved d u r i n g the initial  d a r t was recorded.  Ten such r e c o r d i n g s were obtained  d u r i n g each of the three d a i l y t e s t p e r i o d s . day,  the 15 recorded minimum response  corded d i s t a n c e s were averaged minimum response  At the end of the  v o l t a g e s , and the 30 r e -  s e p a r a t e l y to give the average  v o l t a g e and the average d i s t a n c e moved f o r that  fish. E x t e n s i v e v a r i a b i l i t y was expected  from the experimental  16  procedure  employed (Elson, 1942;  reason, temperature  Hoar et a l . ,  and photoperiod were c o n t r o l l e d .  were t e s t e d i n the same apparatus, day.  D.  one  For  sham-pinealecto-  Amount of Swimming  c u l a r channel with an o u t s i d e diameter channel 10 cm wide and 20 cm deep. middle  of the channel was  307  cm.  observed i n a  of 116  each l i t e r  of water.  i z e d i r o n and was  The  The circumference a t the Water was  channel was  p a i n t e d grey.  marked with e i g h t e q u a l l y spaced ming a c t i v i t y was  cir-  cm and w i t h the  10 cm deep and f o r  the purpose of experimental u n i f o r m i t y 1 gm of NaCl was  et  each  f i v e i n t a c t c o n t r o l s were obtained i n t h i s manner.  The amount of swimming by g o l d f i s h was  to  this  A l l fish  f i s h being t e s t e d  Values from f i v e pinealectomized, f i v e  mized, and  1955)•  added  c o n s t r u c t e d of galvan-  The bottom of the channel transverse black l i n e s .  was Swim-  measured i n the same manner as used by Hoar  a l . (1955) by r e c o r d i n g the t o t a l number of l i n e s crossed by  three f i s h d u r i n g three 10 minute i n t e r v a l s .  To a d j u s t f o r  d i u r n a l v a r i a t i o n i n a c t i v i t y , r e c o r d i n g s were taken i n the morn ing  about 8:00  AM,  at noon, and i n the a f t e r n o o n about 4 : 0 0  F i s h were p l a c e d i n the channel at 4 3 0 :  the day of o b s e r v a t i o n . of One  PM the day preceeding ....  Observations were made on f i v e groups  three f i s h from each of the s i x experimental group of f i s h was  were turned on at 8:00  PM.  t e s t e d each day. AM and  classifications.  The l i g h t s i n the room  turned o f f at 4 : 0 0  PM.  17  S e r o t o n i n L e v e l s i n Whole B r a i n s A.  P r e p a r a t i o n of T i s s u e F i s h were k i l l e d by t r a n s e c t i o n of the s p i n a l cord  above the p o s t e r i o r border  of the o p e r c u l a .  Whole b r a i n s , minus  the p i n e a l organ and  o p t i c nerves,  the nearest 0 . 5  The b r a i n s were then placed i n a 10  mg.  were removed and weighed to  Thomas T i s s u e Hand Homogenizer (Arthur H. Thomas C , p h i a , Pa.) tassium  c o n t a i n i n g 0.1  with KC1  was  Philadel-  (EDTA-IC,, Cambridge Chemi-  S u f f i c i e n t 0.02  D e t r o i t , Mich.).  N HC1  saturated  added to b r i n g the f i n a l volume of f l u i d , i n c l u d i n g  the t i s s u e f l u i d ,  up to 3 ml.  to be 75% water and  judged  ml  ml of 3% a s c o r b i c a c i d i n 1% d i p o -  ethylenediamine-tetraacetate  c a l Products,  just  For t h i s purpose, the t i s s u e  the s e r o t o n i n assessed  water phase (Anden and Magnusson, 1 9 6 7 ) .  The  was  to be i n the  b r a i n s were homo-  genized with a t e f l o n p e s t l e d r i v e n by a power s t i r r i n g apparat u s and  the homogenized samples were s t o r e d s e p a r a t e l y i n p o l y -  ethylene b o t t l e s over s o l i d CO^  B.  f o r no longer than three days.  Fluorometry P r i o r to fluorometry,  the b r a i n s were thawed i n d i v i d u a l l y  and washed twice with d i e t h y l ether.  The  s e r o t o n i n l e v e l s were  then determined by measuring the n a t i v e f l u o r e s c e n c e of 5-HT 3N HC1  (Udenfriend et a l . , 1955;  Bogdanski et a l . , 1956)  i n g the d i f f e r e n t i a l e x t r a c t i o n of the amine (Quay, 1963; 1 9 6 7 a , b ) i n n-butanol. Model 111  Fluorometer  Fluorometry (G.K.  was  in  followWise,  performed with a Turner  Turner A s s o c i a t e s , Palo A l t o ,  f i t t e d with a #110-855 f a r u l t r a v i o l e t lamp and with  the  Calif.)  18  appropriate  primary and secondary f i l t e r s to permit a c t i v a t i o n  with 295 mu and measurement of the f l u o r e s c e n c e a t 550 mu, reduce the v a r i a b i l i t y ,  the f l u o r e s c e n c e  To  of each sample was  measured i n the same round, non-fluorescent,  quartz  cuvette  which had been so marked that i t was p o s s i b l e to place the cuve t t e i n the fluororaeter i n the same p o s i t i o n each time. t h i s arrangement, the p o l a r i z i n g f i l t e r s a) were not r e q u i r e d .  With  suggested by Wise (1967  The t o t a l b r a i n s e r o t o n i n l e v e l  was  determined from each of f i v e f i s h from each o f the s i x e x p e r i mental c o n d i t i o n s t e s t e d i n the swimming a c t i v i t y experiment. All  s e r o t o n i n l e v e l s were c a l c u l a t e d from a standard  t a i n e d by e x t r a c t i n g v a r i o u s c o n c e n t r a t i o n s  curve ob-  of serotonin  stan-  dard.  C.  R e p r o d u c i b i l i t y , E x t r a c t i o n E f f i c i e n c y and S p e c i f i c i t y of Serotonin The  Determinations  b r a i n homogenates o f 11 separate  g o l d f i s h were  mixed, and then d i v i d e d i n t o 11 equal volumes. of s e r o t o n i n  pooled,  A known amount  ( 0 . 2 0 ug) was added to four o f the samples.  Iden-  t i c a l amounts o f DL-5-hydroxytryptophane, 5 ~ h y d r o x y i n d o l e - 3 a c e t i c a c i d and N - a c e t y l s e r o t o n i n  were added to three  samples; one i n d o l e i n each sample. were l e f t i n t h e i r n a t i v e s t a t e .  The remaining four samples  A l l i n d o l e s were purchased  from the Mann Research L a b o r a t o r i e s , N.Y., chromatographically  other  and were t h i n l a y e r  pure.  0n».-each o f . f o u r s u c c e s s i v e days, one n a t i v e sample, and one  to which s e r o t o n i n had been added were analyzed.  The  19  s e r o t o n i n content of i d e n t i c a l l y t r e a t e d samples t e s t e d on d i f f e r e n t days were compared to e s t a b l i s h r e p r o d u c i b i l i t y ; technique.  o f the  The percent recovery o f s e r o t o n i n was c a l c u l a t e d by  s u b t r a c t i n g the amount o f s e r o t o n i n determined  i n the untreated  sample from the value obtained a f t e r a d d i t i o n o f s e r o t o n i n , d i v i d i n g the sum by the amount o f s e r o t o n i n added and m u l t i p l y i n g the q u o t i e n t by 1 0 0 .  F o r t h i s purpose, values obtained on  the same day were used i n the c a l c u l a t i o n s .  Related compounds  were a l l t e s t e d on the f i r s t day and percent r e c o v e r i e s were calculated.  The values so obtained were used to i n d i c a t e the  s p e c i f i c i t y o f the technique. The r e s u l t s from t h i s check are summarized i n Table I and i n d i c a t e s the e x c e l l e n t r e p r o d u c i b i l i t y obtained.  There was  s l i g h t v a r i a b i l i t y i n the e x t r a c t i o n e f f i c i e n c y (85%-95%) and the mean percent recovery of 91% was s l i g h t l y lower ported by Quay (1963)  or Wise ( 1 9 6 7 a ) .  than r e -  The a c t i v a t i o n / f l u o r e s -  cence maxima o f 2 9 5 / 5 4 5 niu i s c h a r a c t e r i s t i c of many 5-hydroxyand 5 - H i e t h o x y - i n d o l e compounds (Udenfriend et a l . , 1 9 6 3 ) , and i t i s probable  that some of the f l u o r e s c e n c e ob-  t a i n e d from the n a t i v e samples r e s u l t e d than s e r o t o n i n . though lower  1 9 5 5 ; Quay,  from compounds other  The 10% recovery of DL-5-hydroxytyptophane, a l -  than r e p o r t e d by Quay ( 1 9 6 3 ) , i s s t i l l  sufficient  to i n t r o d u c e some e r r o r i n t o the accuracy of the method. Nevertheless,  s e r o t o n i n comprises  the l a r g e s t f r a c t i o n of b r a i n  5-hydroxy- and 5-methoxy i n d o l e s when the above procedure i s used ( W i l h o f t and Quay, 1 9 6 5 ) .  F u r t h e r , the v a l u e s r e p o r t e d  h e r e i n f o r the g o l d f i s h approximate those r e p o r t e d  earlier  20  TABLE I R e p r o d u c i b i l i t y , E x t r a c t i o n E f f i c i e n c y and S p e c i f i c i t y of S e r o t o n i n Determinations.  Day  Sample (ug added)  1  brain  1  brain+5-HT  1  Recovery*  %  0.42 a 0.61  1  95  brain+DL-hydroxytryptophane ( 0 . 2 0 )  0.45  10  brain+5-hydroxy-indole3-acetic acid (0.20)  0.41  0  brain+N-acetylserotonin (0.20)  0.42  0  2  brain  2  brain+5-HT  3  brain  3  brain+5-HT  4  brain  4  5-HT Determined  0.39 (0.20)  0.56  85  0.41 (0.20)  brain+5-HT ( 0 . 2 0 )  *%•recovery equals b-a/c x 1 0 0 .  0.60  95  0.41 0.59  90  21  (Bogdanski et a l . , 1 9 6 3 ) .  Thus, the e r r o r i n t r o d u c e d by i n t e r -  f e r i n g compounds was considered sults valid.  small enough to render  To check the e x t r a c t i o n procedure,  the r e -  known concen-  t r a t i o n s of DL-5-hydroxytryptophane and blanks were c o n c u r r e n t l y and  s i m i l a r l y t r e a t e d with t i s s u e samples w i t h i n each  The  effective  s e n s i t i v i t y o f t h i s technique  series.  was 10 ng per sam-  p l e of standard s o l u t i o n though the accuracy and e f f i c i e n c y o f the technique  was greater at'thigher c o n c e n t r a t i o n s .  22  RESULTS  Pinealectomy, B l i n d i n g , and P h o t o t a x i s F i g u r e 1 summarizes the e f f e c t of pinealectomy, b l i n d i n g , or both on the p h o t o t a c t i c response of g o l d f i s h . c o n t r o l f i s h and sham-pinealectomized phototactic.  Unoperated  f i s h were n e g a t i v e l y  Removal of e i t h e r the p i n e a l organ or the eyes re-  s u l t e d i n a uniform d i s t r i b u t i o n i n the l i g h t g r a d i e n t .  If i t  i s considered that an o v e r l a p between the 95% confidence i n t e r v a l s of two groups i n d i c a t e s n o n s i g n i f i c a n c e of the d i f f e r e n c e between means, and that no o v e r l a p i n d i c a t e s a s i g n i f i c a n t ference at the v05 l e v e l , significant.  the d i f f e r e n c e s mentioned  dif-  above are  The apparent p o s i t i v e p h o t o t a c t i c response of the  b l i n d p l u s p i n e a l e c t o m i z e d animals i s not s i g n i f i c a n t l y f e r e n t from the b l i n d p l u s sham-pinealectomized not be considered as a p o s i t i v e response.  dif-  group and can  Although these data  c l e a r l y i m p l i c a t e the p i n e a l organ of g o l d f i s h i n the p h o t o t a c t i c response of t h i s s p e c i e s , they do not a d i r e c t p h o t o r e c e p t i v e r o l e of t h i s organ.  normal  demonstrate  But they do  indi-  cate that the eyes and the p i n e a l organ are both r e q u i r e d f o r p h o t o t a c t i c responses i n g o l d f i s h .  Pinealectomy, B l i n d i n g , and C o n d i t i o n i n g to L i g h t and Sound The preceeding experiment  suggests that the p i n e a l organ  i s r e l a t e d to p h o t o t a x i s but i s unable to produce a b e h a v i o u r a l response to l i g h t i n the absence of the eyes. ment was used to t e s t t h i s p o s s i b i l i t y .  A second  experi-  The v a r i o u s groups of  23  F i g u r e 1.  D i s t r i b u t i o n of g o l d f i s h i n a l i g h t gradient f o l l o w i n g B l i n d i n g , Pinealectomy of both. H o r i z o n t a l and v e r t i c a l l i n e s are means and 95% confidence i n t e r v a l s r e s p e c t i v e l y c a l c u l a t e d from 20 o b s e r v a t i o n s on ten f i s h per observation. (Overlap of confidence i n t e r v a l s taken as n o n s i g n i f i c a n c e at the .05 level.)  number IV)  T  in light half: of t a n k  w  -k  T  T  01  oo T  O  T  T  PINOXED SHAM-PINOXED NORMAL BLIND BLIND* SHAM-PINOXED BLIND- PINOXED m x m m > o —i rn o  24  g o l d f i s h l i s t e d i n Table I I were c o n d i t i o n e d to escape an e l e c trical  shock by swimming over a b a r r i e r i n response to a l i g h t  or to a r i n g i n g door b e l l . ments was as f o l l o w s .  The r a t i o n a l e behind these e x p e r i -  Animals which possessed both p i n e a l and  eyes would r e t a i n a negative p h o t o t a c t i c response and more o f t e n escape the e l e c t r i c a l shock by swimming away from the l i g h t and over the b a r r i e r than animals w i t h eyes but without a p i n e a l . B l i n d animals which possessed an i n t a c t p i n e a l organ could be "motivated" by an e l e c t r i c a l  shock to respond to the l i g h t ,  thereby p r o v i d i n g evidence f o r the p h o t o r e c e p t i v e a b i l i t y of the p i n e a l .  By r e p e a t i n g the experiment  u s i n g sound as the con-  d i t i o n e d stimulus, i t would be p o s s i b l e to judge whether the r e s u l t s from the l i g h t experiment sponse  were due to changes i n r e -  to l i g h t per se or r e s u l t e d  s e n s i t i v i t y to a l l environmental  from a general a l t e r a t i o n i n  stimuli.  Table I I combines the r e s u l t s of these two experiments. An a n a l y s i s of v a r i a n c e showed s i g n i f i c a n t d i f f e r e n c e s w-procedure, S t e e l and T o r r i e , I960) o f 7 . 1 2 periment and 3 . 9 2  f o r the sound experiment  f o r the l i g h t ex-  (P =  these v a l u e s to make a p p r o p r i a t e comparisons  (Tukey's  . 05).  Using  i n Table I I , i t i s  apparent that eyed animals,, as opposed to b l i n d animals, showed a s i g n i f i c a n t l y g r e a t e r response to l i g h t but that the r e v e r s e occurred when the c o n d i t i o n e d s t i m u l u s was sound. Pinealectomy d i d not a f f e c t the r e s p o n s i v e n e s s of eyed or b l i n d animals to sound, nor d i d i t a f f e c t the responsiveness of b l i n d animals to l i g h t .  On the other hand, pinealectomy r e -  s u l t e d i n a s i g n i f i c a n t l y g r e a t e r responsiveness to l i g h t when  25  TABLE I I E f f e c t of Pinealectomy, B l i n d i n g , or both on the cond i t i o n i n g of g o l d f i s h to l i g h t and sound. The tabul a t e d v a l u e s represent the mean number of c o n d i t i o n e d responses out of 50 t r i a l s . *  Treatment  Light  Sound  Blind  Eyed  Blind  Eyed  Pinealectomized  3.4  32.6  14.4  9.2  Sham-pinealec tomized  3.8  24.2  13.6  9.6  Unoperated  4.4  19.8  14.4  9.8  controls  * S i g n i f i c a n t d i f f e r e n c e s of Tukey's w at the 0 . 0 5 l e v e l of s i g n i f i c a n c e were c a l c u l a t e d as 7.12 f o r the l i g h t e x p e r i ment and 3.92 f o r the sound experiment.  26  the p i n e a l e c t o m i z e d animals r e t a i n e d t h e i r eyes.  This result  i s d i a m e t r i c a l l y opposed to the expected as these f i s h were p r e v i o u s l y found to have l o s t t h e i r negative p h o t o t a c t i c r e sponse and  should be l e s s i n c l i n e d to swim away from the l i g h t  than the wholly i n t a c t animals which remain  photo-negative.  The i n a b i l i t y of b l i n d g o l d f i s h with a p i n e a l to use l i g h t  as  a c o n d i t i o n e d s t i m u l u s more o f t e n than b l i n d g o l d f i s h without a p i n e a l i s taken as f u r t h e r evidence that the r o l e of the photor e c e p t i v e f u n c t i o n of the p i n e a l organ depends upon the presence of normal The  vision.  f a i l u r e of pinealectomy to produce  any changes i n the  responsiveness to sound i n d i c a t e s that the removal of the p i n e a l gland does not i n f l u e n c e responsiveness to a l l e n v i r o n mental  stimuli.  Pinealectomy and Response to E l e c t r i c a l S t i m u l a t i o n Pinealectomy d i d not i n f l u e n c e the d i s t a n c e that  goldfish  moved when s t i m u l a t e d with the unconditioned stimulus from the preceeding experiment  (80  v o l t s ) ; that i s , the d i f f e r e n c e s noted  i n the c o n d i t i o n i n g experiment  were the r e s u l t of changes i n  response to the l i g h t and not the e l e c t r i c a l shocks (Table I I I ) . But p i n e a l e c t o m i z e d f i s h d i d have a lower minimum response tage r e l a t i v e to the sham-pinealectomized  and i n t a c t  groups, the d i f f e r e n c e s being s i g n i f i c a n t at the . 0 5 (Tukey s W 1  n q  = 2.68).  vol-  control level  Although the i m p l i c a t i o n s of t h i s  find-  i n g are not immediately apparent, i t does i n d i c a t e a general i n c r e a s e i n a c t i v i t y or e x c i t a b i l i t y of the p i n e a l e c t o m i z e d  27  TABLE I I I E f f e c t of pinealectomy on the response to e l e c t r i c a l s t i m u l a t i o n .  of g o l d f i s h  Values given are the lowest v o l t a g e s which w i l l j u s t cause a p e r c e p t a b l e r e a c t i o n or the u n i t s ( 2 . 5 cm) moved i n response to a s i n g l e shock of 80 v o l t s . A l l v a l u e s are averages of morning, noon and afternoon values taken from f i v e separate f i s h t e s t e d on d i f f e r e n t days.*  Treatment  Minimum E f f e c t i v e Voltage  Distance Moved to 80 v o l t s  7.93  4-93  Sham-pinealectomized  10.79  4.11  Intact controls  10.23  k.2.9  Pinealectomized  • S i g n i f i c a n t d i f f e r e n c e of Tukey's w a t 0 . 0 5 l e v e l o f s i g n i f i c a n c e was c a l c u l a t e d as 2 . 6 8 f o r minimum e f f e c t i v e voltage.  28  group.  T h i s p o s s i b i l i t y w i l l be considered i n the d i s c u s s i o n .  Pinealectomy, B l i n d i n g , and Swimming A c t i v i t y The e f f e c t of pinealectomy, b l i n d i n g , or both, on the mean number of t o t a l l i n e s crossed by three g o l d f i s h d u r i n g three 10 minute i n t e r v a l s i s presented i n F i g u r e 2 .  The data from  which these means were c a l c u l a t e d were subjected to a two  way  a n a l y s i s of v a r i a n c e from which a s i g n i f i c a n t d i f f e r e n c e of was  obtained (Tukey's W procedure, P < . 0 1 ) .  93.k  Table IV p r e s e n t s  data to show d i u r n a l v a r i a t i o n i n swimming a c t i v i t y and  the  e f f e c t of v a r i o u s treatments. The b l i n d groups showed s i g n i f i c a n t l y more swimming a c t i v i t y than the eyed groups.  Within the eyed groups, the p i n e a l -  ectomized f i s h were more a c t i v e than e i t h e r the  sham-pinealec-  tomized or the i n t a c t c o n t r o l group; the r e v e r s e was the e y e l e s s groups. at the .01  The d i f f e r e n c e s mentioned  t r u e among  are s i g n i f i c a n t  level.  A marked d i u r n a l rhythm i n a c t i v i t y was Among the eyed groups, a c t i v i t y dropped  found (Table I V ) .  from a peak l e v e l i n  the morning to a low l e v e l a t noon and then r o s e s l i g h t l y i n g the a f t e r n o o n . a c t i v i t y at noon. activity  dur-  The b l i n d groups d i s p l a y e d peak swimming Pinealectomy d i d not a p p r e c i a b l y a f f e c t the  cycles.  E f f e c t of Pinealectomy, B l i n d i n g , or Both on B r a i n Serotonin Levels Pinealectomy produced  no a p p r e c i a b l e a l t e r a t i o n i n whole  29  Figure 2 .  Swimming a c t i v i t y of normal g o l d f i s h or f o l lowing pinealectomy, b l i n d i n g , or both. Mean number of l i n e s crossed i n a c i r c u l a r channel d u r i n g three 10 minute i n t e r v a l s (AM, noon, and PM). The top of the bars r e p r e s e n t the mean number o f l i n e s crossed by f i v e groups of three g o l d f i s h from each of the treatments. Pinealectomized, P; sham-pinealectomized, S; unoperated c o n t r o l s , C; b l i n d and p i n e a l e c tomized, BP; b l i n d and sham-pinealectomized, BS; and b l i n d , B.  activity ->  ro  units to  per 100 &  <j]  O)  30  TABLE IV Swimming a c t i v i t y of g o l d f i s h showing d i u r n a l rhythm and e f f e c t of p i n e a l e c t o m y , b l i n d i n g , or b o t h . A c t i v i t y u n i t s are a v e r a g e number of l i n e s c r o s s e d by t h r e e g o l d f i s h i n a c i r c u l a r , channel d u r i n g a 10 m i n ute p e r i o d . Values are a v e r a g e s of f i v e 10 minute i n t e r v a l s taken on d i f f e r e n t days u s i n g d i f f e r e n t groups of t h r e e f i s h .  Average f o r 5 days  Total Units f o r 5 groups ,of 3 - f i s h  Treatment  am  noon  pm  Pinealectomized  131.8  31.0  38.8  1008  Sham-pinealectomized  66.8  11.6  6.2  424  Intact  69.8  11.0  13.0  469  Blind  191.3  233.4  168.6  Blind plus pinealectomized  112.8  157.0  149.0  Blind plus sham-pinealectomized  191.6  228.8  140.6  controls  2967 2094 2805  31  brain  s e r o t o n i n content (Table V).  s i g n i f i c a n t decrease i n b r a i n l e v e l s found i n the  But  blinding resulted  s e r o t o n i n when compared to  eyed groups (Tukeys W „  = 0.09).  in a the  When the  mean s e r o t o n i n l e v e l s f o r each group of g o l d f i s h were compared to the corresponding mean swimming a c t i v i t i e s (Figure negative c o r r e l a t i o n was s i g n i f i c a n t at the Correlation,  .05  obtained ( r  which was  a just  l e v e l (Spearmans C o e f f i c i e n t of Rank  r , Siegal, 1956).  be viewed with c a u t i o n since only about the  = 0.83)  3)>  However, t h i s r e l a t i o n s h i p must  a c o r r e l a t i o n p r o v i d e s evidence  j o i n t r e l a t i o n s h i p between two  not  e s t a b l i s h cause and  not  refute  effect.  variables  and  does  Nevertheless, t h i s f i n d i n g does  the p o s s i b i l i t y of a f u n c t i o n a l r e l a t i o n s h i p between  s e r o t o n i n l e v e l s and  swimming a c t i v i t y .  32  TABLE V E f f e c t of pinealectomy, b l i n d i n g , or both on the s e r o t o n i n content i n g o l d f i s h b r a i n s . Each tabul a t e d value r e p r e s e n t s a mean of f i v e d e t e r m i n a t i o n s on f i v e f i s h . *  Serotonin Content (ug/gm of wet Eyed Groups  Treatment  tissue) B l i n d Groups  Pinealectomized  0.36  0.24  Sham-pinealectomized  0.34  0.21  Control  O.Mf  0.19  *Tukey's w  n q  =  0.09.  33  Figure 3 .  R e l a t i o n s h i p between mean t o t a l swimming a c t i v i t y and mean b r a i n s e r o t o n i n i n v a r i o u s experimental groups of g o l d f i s h . Pinealectomized, P; sham-pinealectomized, SP; unoperated c o n t r o l s , C; b l i n d and pinealectomized, BP; b l i n d and shampinealectomized, BS; and b l i n d , B.  1  2  activity  3  4  units  5 per  6 100  34  DISCUSSION  Evidence f o r a p h o t o r e c e p t i v e r o l e of the f i s h  pineal  organ i s d e r i v e d from d i r e c t n e u r o p h y s i o l o g i c a l s t u d i e s (Dodt, 1963;  Morita, 1966),  m o r p h o l o g i c a l s t u d i e s (Rivas, 1 9 5 3 ) ,  i n d i r e c t i n v e s t i g a t i o n s i n v o l v i n g the h i s t o l o g i c a l 1959 5 Eakin, 1963; a  ( K e l l y , 1962;  Rudeberg,  Rudeberg,  1966)  1968a,b) and appearance  and  (Holmgren,  ultrastructural  of t h i s organ.  Valu  r  able i n f o r m a t i o n c o n c e r n i n g i t s f u n c t i o n has been obtained by t e s t s i n v o l v i n g c o l o r changes 1950;  Hoar, 1955;  (Young, 1935;  and Schonherr, 1 9 5 5 ) ,  sponses (Breder and Rasquin, 1947;  Breder and Rasquin,  and b e h a v i o u r a l r e -  Hoar, 1955;  and Pang, 1 9 6 5 ) .  Despite the d i s p a r a t e experimental approaches, a l l of these s t u d i e s i n d i c a t e a d i r e c t photosensory r o l e of the f i s h  pineal  organ. The present i n v e s t i g a t i o n shows that the p i n e a l organ of g o l d f i s h i s i n v o l v e d i n p h o t o t a x i s and r e s p o n s i v e n e s s to l i g h t s t i m u l i but o f f e r s no evidence that the p i n e a l organ has photoreceptive a b i l i t i e s .  On the other hand, from p r e v i o u s r e p o r t s ,  there i s no unequivocal evidence f o r e f f e r e n t nerve f i b e r s to the  f i s h p i n e a l organ, l i g h t  has been found to a f f e c t the h i s -  tology of the p i n e a l organ, and i l l u m i n a t i o n of the p i n e a l r e s u l t s i n a l t e r e d nervous a c t i v i t y w i t h i n t h i s organ.  So•in  s p i t e of the r e s u l t s of t h i s experiment, the evidence i s s t r o n g l y in  favour of the h y p o t h e s i s t h a t the p i n e a l organ of f i s h e s i s  photosensitive.  I f t h i s i s t r u e , how  organ of g o l d f i s h i s unable to e l i c i t  i s i t that the p i n e a l a response by the animal  35 to  i l l u m i n a t i o n when the eyes a r e absent? Among f i s h with i n t a c t l a t e r a l v i s i o n , pinealectomy r e s u l t s  i n a l o s s of p h o t o t a x i s , and an apparent i n c r e a s e i n the respons i v e n e s s to l i g h t when l i g h t i s used as a c o n d i t i o n e d s t i m u l u s . However, f i s h without eyes but with a p i n e a l show the same r e sponses to i l l u m i n a t i o n as b l i n d f i s h without a p i n e a l .  A  s i m i l a r s i t u a t i o n was r e p o r t e d e a r l i e r by Breder and Rasquin ( 1 9 4 7 ) who found that i n the b l i n d Mexican cave f i s h Anoptichthys p h o t o t a x i s depended upon the presence of the remnant o p t i c although the s i g n o f p h o t o t a x i s was r e l a t e d to the p i n e a l  cysts organ.  These f i n d i n g s together suggest that the r o l e o f the p i n e a l o r gan i n p h o t o r e c e p t i o n i s to a l t e r the responses e l i c i t e d by the photic i n f o r m a t i o n r e c e i v e d by the eyes so that the normal  photo-  t a c t i c response depends upon the f u n c t i o n of the eyes as w e l l as the  p i n e a l organ. To understand more f u l l y how the p i n e a l can be a photore-  ceptor and s t i l l be unable to produce b e h a v i o u r a l responses to i l l u m i n a t i o n i t i s necessary to remember two of the d i f f e r e n t e f f e c t s of l i g h t .  The f i r s t  e f f e c t i s to produce  the complex  p h y s i o l o g i c a l - p s y c h o l o g i c a l experience of v i s i o n to which an animal may be b e h a v i o u r a l l y r e s p o n s i v e . the  s t i m u l a t o r y e f f e c t on autonomic  The second e f f e c t i s  r e f l e x e s which the animal  may or may not experience and over which the animal has l i t t l e or  no c o n t r o l .  I f t h i s second e f f e c t , or autonomic  to  modulate the response to the f i r s t  reflex, i s  e f f e c t , or experience of  v i s u a l s t i m u l i , then the i n t e g r a t e d response r e s u l t i n g from both e f f e c t s w i l l not occur i n the absence  of e i t h e r e f f e c t .  In other  36  words, i f the p h o t o r e c e p t i v e a b i l i t y of the p i n e a l organ of g o l d f i s h i s a s s o c i a t e d with an autonomic r e f l e x which modulates the response to v i s u a l s t i m u l i r e c e i v e d v i a the eyes, then r e moval of e i t h e r the p i n e a l or the eyes w i l l r e s u l t i n a l o s s of that response.  Thus, with regard to p h o t o t a x i s , the p i n e a l  organ of the g o l d f i s h i s a s s o c i a t e d with the r e c e p t i o n of p h o t i c s t i m u l i which the animal does not experience, and the informat i o n so r e c e i v e d produces no e f f e c t i n the absence of the l a t e r a l eyes. The data from the c o n d i t i o n i n g experiments supports t h i s conclusion.  B l i n d animals with a p i n e a l organ could not be  e f f e c t i v e l y t r a i n e d to use l i g h t as a c o n d i t i o n e d stimulus to avoid an e l e c t r i c a l shock even though a l l of the b l i n d could be t r a i n e d to respond to sound.  groups  Conversely, animals  with eyes but without a p i n e a l showed a g r e a t e r responsiveness to the l i g h t and avoided the shock more o f t e n than animals with both p i n e a l organ and As mentioned  eyes.  i n the i n t r o d u c t i o n to t h i s s e c t i o n ,  photo-  t a x i s depends upon the p o s s e s s i o n of a preference f o r a p a r t i c u l a r degree of b r i g h t n e s s but the a c t u a l response i s d i s p l a y e d only when the f i s h has a r e a d i n e s s to move from one c o n d i t i o n of b r i g h t n e s s to another.  Pinealectomy e l i m i n a t e d the negative  p h o t o t a c t i c response of g o l d f i s h but a l s o r e s u l t e d i n an  appar-  ent i n c r e a s e d responsiveness to l i g h t as evidenced by the i n creased number of c o n d i t i o n e d responses i n which the animal swam from the l i g h t h a l f of the tank to the dark h a l f . suggests that the p i n e a l organ i s a s s o c i a t e d with the  This  37  " w i l l i n g n e s s " of the f i s h to change the c o n d i t i o n of b r i g h t n e s s under which the f i s h f i n d s i t s e l f r a t h e r than the p r e f e r e n c e f o r any p a r t i c u l a r degree o f b r i g h t n e s s . The d i f f e r e n c e s i n response to l i g h t  f o l l o w i n g pinealectomy  were not due t o changes i n s e n s i t i v i t y to the unconditioned stimulus.  The d i s t a n c e that pinealectomized g o l d f i s h swam when  s t i m u l a t e d with the unconditioned s t i m u l u s was the same as the d i s t a n c e swam by wholly i n t a c t and sham operated f i s h .  Further,  although pinealectomy r e s u l t e d i n a reduced minimum response v o l t a g e and an i n c r e a s e i n swimming a c t i v i t y , i t had no e f f e c t on the number o f c o n d i t i o n e d responses e l i c i t e d by the r i n g i n g doorbell.  From these data i t was concluded that the sensory  r o l e of the p i n e a l organ i n g o l d f i s h i s s p e c i f i c a l l y r e l a t e d to photic  stimuli.  The e f f e c t s of pinealectomy and b l i n d i n g on the p h o t o t a c t i c response of v a r i o u s f i s h e s have been explored p r e v i o u s l y .  Hoar  (1955) r e p o r t e d the presence of p h o t o t a x i s i n b l i n d e d salmon smolts with i n t a c t p i n e a l s , and t h e r e f o r e d i f f e r e d i n h i s conc l u s i o n s from those r e p o r t e d here.  The d i f f e r e n c e i n the r e -  s u l t s of these experiments may be explained by the age of the animals used, or i n the i n t e n s i t y of l i g h t (1955) suggests that developmental  employed.  Hoar  d i f f e r e n c e s i n the degree of  negative p h o t o t a x i s shown by young sockeye  salmon i s r e l a t e d to  the degree of p i n e a l development.  the p i n e a l organ of  Indeed,  f i s h e s becomes i n c r e a s i n g l y i n v a g i n a t e d with age (Hoar, 1 9 5 5 ) , and undergoes a decrease i n sensory c e l l number with continued development (Hafeez and Ford, 1967) together v/ith a marked  38  d e g e n e r a t i o n of nervous elements (Kappers, 1 9 6 5 ) .  Thus, the  p i n e a l body of f i s h e s may change from the o n l y photosensory s t r u c t u r e i n v o l v e d i n p h o t o t a x i s i n young f i s h to a s u b s i d i a r y photosensory s t r u c t u r e i n the o l d e r f i s h where i t can n o s l o n g e r autonomously produce a p h o t o t a c t i c r e s p o n s e .  On the o t h e r hand,  Hoar (1955) performed h i s experiments under n a t u r a l l i g h t where the l i g h t i n t e n s i t y was many times g r e a t e r the p r e s e n t (1935)  study.  than t h a t employed i n  P r e v i o u s l y , von F r i s c h (1911a,b) and Young  suggested a g e n e r a l l i g h t s e n s i t i v i t y of the d i e n c e p h a l i c  r o o f and s i n c e the s m o l t s used by Hoar (1955) had t h i n  skulls,  the p h o t o t a x i s demonstrated by h i s b l i n d f i s h c o u l d have r e s u l t e d from the e f f e c t o f h i g h l i g h t i n t e n s i t y on the b r a i n i t s e l f . N e v e r t h e l e s s , the p o s s i b i l i t y o f s p e c i e s d i f f e r e n c e s i n p i n e a l f u n c t i o n can not be d i s r e g a r d e d . These d a t a ,  then, l e a d to the c o n c l u s i o n t h a t the  r o l e of the p i n e a l organ o f the a d u l t g o l d f i s h i s r e l a t e d to p h o t i c s t i m u l i .  sensory  specifically  F u r t h e r , t h a t t h i s r o l e depends  upon, and i s s u b s i d i a r y t o , i n t a c t l a t e r a l v i s i o n so t h a t w i t h r e g a r d s t o d i s t r i b u t i o n a l responses to l i g h t ,  the eyes and the  p i n e a l organ f u n c t i o n t o g e t h e r .  When the r e s u l t s from the l i -  t e r a t u r e are c o n s i d e r e d as w e l l ,  the f o l l o w i n g h y p o t h e s i s emer-  ges.  I n young f i s h ,  the p i n e a l organ autonomously r e g u l a t e s  the p h o t o t a c t i c response by o p e r a t i n g as the r e c e p t o r s i t e  for  s t i m u l i g o v e r n i n g the p e r s i s t e n c e tendency of Janzen ( 1 9 3 3 ) . In o l d e r a n i m a l s , the p i n e a l r e t a i n s c o n t r o l over the p e r s i s tence tendency but the l a t e r a l eyes a c q u i r e the f u n c t i o n o f c o n t r o l l i n g the b r i g h t n e s s p r e f e r e n c e .  The importance o f the  39  photosensory r o l e of the f i s h p i n e a l organ decreases with advanc i n g age.  T h i s decrease p a r a l l e l s the l o s s of the p i n e a l  p h o t o r e c e p t i v e a b i l i t y seen between f i s h and mammals and might t h e r e f o r e r e p r e s e n t another example of ontogeny r e c a p i t u l a t i n g phylogeny. No p r e v i o u s r e p o r t s have appeared  on the e f f e c t of the  p i n e a l gland on the general a c t i v i t y of f i s h .  This i s surpri-  s i n g i n view of the t h y r o i d a l hypertrophy and suggested i n c r e a s e i n gonadal endocrine a c t i v i t y seen i n young L e b i s t e s  (Pflug-  f e l d e r , 1 9 5 3 , 1954) and the hyperthyroidism of g o l d f i s h f e l d e r , 1964) f o l l o w i n g pinealectomy.  (Pflug-  Hoar et a l . (1955) have  shown that t h y r o x i n e and gonadal s t e r o i d s when administered exogenously,  r e s u l t i n i n c r e a s e d locomotory a c t i v i t y i n g o l d -  f i s h as w e l l as a decrease i n the s t r e n g t h of e l e c t r i c a l  stimuli  r e q u i r e d to cause g o l d f i s h to move a standard d i s t a n c e .  Simi-  l a r r e s u l t s were obtained i n t h i s study f o l l o w i n g pinealectomy of animals with i n t a c t l a t e r a l v i s i o n .  The p o s s i b i l i t y of such  endocrine e f f e c t s w i l l be d i s c u s s e d i n the General D i s c u s s i o n at the end of the t h e s i s . Another p o s s i b l e reason f o r the changes noted i n swimming a c t i v i t y f o l l o w i n g pinealectomy was i n v e s t i g a t e d .  Serotonin i s  found i n high c o n c e n t r a t i o n s i n the p i n e a l gland (Quay, I964, 1965) tivity  and can cause marked s e d a t i v e e f f e c t s on the l e v e l o f ac(Weisman, 1 9 6 7 ) .  Thus, i f the p i n e a l organ o f f i s h i s  a major source of b r a i n s e r o t o n i n , or can i n c r e a s e b r a i n serot o n i n l e v e l s , then removal o f the p i n e a l organ might r e s u l t i n a decreased b r a i n s e r o t o n i n l e v e l , i n c r e a s e d e x c i t a b i l i t y , and  40  i n c r e a s e d swimming a c t i v i t y of the f i s h .  The r e s u l t s obtained  from t h i s study do not support t h i s hypothesis (Table IV and Fig. 2 ) .  Pinealectomy  serotonin.  d i d not a l t e r the l e v e l of whole b r a i n  However, as the r e s u l t s reported h e r e i n are f o r  whole b r a i n s and as s e r o t o n i n i s known to be unevenly  distri-  buted between the anatomical r e g i o n s of the b r a i n (Quay and Wilhoft,  1964)>  the p o s s i b i l i t y that pinealectomy  caused  zed changes i n s e r o t o n i n l e v e l s can not be r u l e d out.  locali-  At  any  r a t e , F i g u r e 3 suggests a negative r e l a t i o n s h i p between the l e v e l of b r a i n s e r o t o n i n and  the l e v e l of swimming a c t i v i t y .  At present, there i s i n s u f f i c i e n t the f i s h p i n e a l organ  evidence to e x p l a i n how  functions i n photoreception.  There i s ,  however, ample evidence that the p i n e a l organ of the g o l d f i s h i s a s s o c i a t e d with b e h a v i o u r a l responses to l i g h t . the e f f e c t s of pinealectomy mum  Further,  on the swimming a c t i v i t y and m i n i -  e f f e c t i v e v o l t a g e s to cause a response i n g o l d f i s h  a second vity.  suggests  f u n c t i o n of the p i n e a l organ r e l a t e d to n e u r a l a c t i -  Whether these two  f u n c t i o n s are separate, or whether  i s the r e s u l t of the other i s not known.  Nevertheless, the  p i n e a l organ of the g o l d f i s h does have s u b t l e but b a s i c and important f u n c t i o n s .  one  41  SECTION I I DEMONSTRATION AND EFFECT OF MELATONIN IN FISH INTRODUCTION  Though the question has been r a i s e d K i r s c h s t e i n , 1 9 6 7 ; Rudeberg, 1 9 6 8 ) ,  (Quay, 1965; Oksche' and  i t i s not known whether  melatonin (N-acetyl-5-methoxytryptamine, Learner et a l . , 1958) which has been found present i n the p i n e a l organs of mammals (Wurtman and Axelrod, 1 9 6 5 ) , phibians (Charlton,  birds  (Ralph et a l . , 1967) and am-  1964; Van de Veerdonk, 1967) i s present i n  the p i n e a l organ of f i s h e s .  Melatonin o f t e n  (Wurtman et a l . ,  1963;  Chu e t a l . , 1 9 6 4 ; Adams e t a l . , 1 9 6 5 ; F r a s c h i n i et a l . ,  1968)  though not c o n s i s t e n t l y  ( R e i t e r , 1967) e x e r t s a n t i g o n a d a l  e f f e c t s i n mammals s i m i l a r to those of bovine p i n e a l ( K i t a y and A l t s c h u l e ,  extracts  1 9 5 4 ; Wurtman et a l . , 1 9 5 9 ; Mayer et a l . ,  I 9 6 I ) and n e u t r a l i z e s some of the e f f e c t s of pinealectomy (Motta et a l . , 1 9 6 7 ; F r a s c h i n i e t _ a l . , 1 9 6 8 ) . support the s p e c u l a t i o n  These  findings  that melatonin i s a p i n e a l hormone i n  mammals (Wurtman and Axelrod, 1 9 6 5 ) ,  and that the p i n e a l organ  of mammals i s a f u n c t i o n a l endocrine organ ( T h i e b l o t ,  1965)•  A s i m i l a r f u n c t i o n o f the p i n e a l organ i n f i s h e s has been suggested.  Krochert  ( 1 9 3 6 a , b ) found that the growth r a t e and  the appearance of secondary sexual c h a r a c t e r i s t i c s were i n young guppies L e b i s t e s Pflugfelder  (1953, 1954),  fed dessicated  bull pineal  retarded  glands.  a l s o working with guppies, found  that  pinealectomy induced a s l i g h t a c c e l e r a t i o n i n the appearance  42  of secondary sexual c h a r a c t e r i s t i c s i n young males.  Recently,  Quay (1965t>) l o c a l i z e d the enzyme necessary f o r melatonin synt h e s i s w i t h i n the p i n e a l organ of f i s h e s .  Together,  these  f i n d i n g s suggest that the p i n e a l organs of mammals and  fishes  are f u n c t i o n a l l y s i m i l a r v/ith regard to s y n t h e s i s of melatonin and i t s ensuing i n h i b i t i o n on the r e p r o d u c t i v e system. The acceptance or r e j e c t i o n of t h i s hypothesis depends to a l a r g e extent upon the demonstration of a p i n e a l melatonin component i n the f i s h e s and the establishment o f some e f f e c t of melatonin on the r e p r o d u c t i v e or endocrine system of f i s h .  Further, e v i -  dence f o r the p a r t i c i p a t i o n of melatonin i n the r e p r o d u c t i v e ? process of f i s h might be a c q u i r e d i f a r e l a t i o n s h i p between p i n e a l melatonin s t o r e s and the s t a t e of r e p r o d u c t i v e maturity could be demonstrated  under n a t u r a l c o n d i t i o n s .  Quay (1965b) a l s o found a r e t i n a l component of the methylat i n g enzyme, hydroxy-indole-O-methyl  t r a n s f e r a s e (HIOMT), the  enzyme which c o n v e r t s N - a c e t y l - s e r o t o n i n to melatonin, i n f i s h e s , and suggested the p o s s i b i l i t y of compensatory i n c r e a s e s i n r e t i n a l melatonin p r o d u c t i o n f o l l o w i n g decreased p r o d u c t i o n i n the pineal.  For t h i s reason, and f o r the purpose  of i n t e r p r e t i n g  experiments i n v o l v i n g pinealectomy of g o l d f i s h , the r e l a t i o n s h i p between p i n e a l and r e t i n a l melatonin l e v e l s was examined. To t h i s end, melatonin, and i t s p r e c u r s o r s e r o t o n i n , were examined q u a l i t a t i v e l y and q u a n t i t a t i v e l y i n the p i n e a l  organ  and r e t i n a s of the l a r g e P a c i f i c salmon, the chinook or s p r i n g . T h i s animal was choosen  f o r the l a r g e s i z e of i t s p i n e a l  organ  p l u s the f a c t t h a t t h i s s p e c i e s e x h i b i t s a marked and prolonged  43  reproductive  c y c l e which enabled immature and mature salmon of  s i m i l a r s i z e s to be compared.  G o l d f i s h were used to study the  e f f e c t of exogenous melatonin on the r e p r o d u c t i v e selected  endocrines.  system and  These animals are e a s i l y acquired,  main-  tenance i s minimal, and they are hardy enough to withstand d a i l y injections. T h i s s e c t i o n r e p o r t s on q u a l i t a t i v e and  quantitative  s t u d i e s f o r r e t i n a l and p i n e a l melatonin i n mature as w e l l as immature salmon and attempts to r e l a t e the f i n d i n g s to the r e p r o d u c t i v e c y c l e of t h i s s p e c i e s .  The e f f e c t s of i n t r a p e r i -  t o n e a l i n j e c t i o n s of melatonin on the g o l d f i s h gonads, i n t e r r e n a l n u c l e a r diameter, t h y r o i d a l t i s s u e and p i t u i t a r y gonadotrophs are d e s c r i b e d cussed.  and the p o s s i b l e mode of a c t i o n i s d i s -  44  MATERIALS AND METHODS  Melatonin I n j e c t i o n S t u d i e s G o l d f i s h , common v a r i e t y ( 7 . 5 - 10 cm s i z e c l a s s ) , were procured from the G o l d f i s h Supply Company, S t o u f v i l l e , O n t a r i o , and a r r i v e d a t the l a b o r a t o r y on 21 December, 1 9 6 7 .  They were  p l a c e d i n a h o l d i n g trough of approximately 400 l i t e r c a p a c i t y c o n t a i n i n g d e c h l o r i n a t e d water (pH 6 . 4 - 6 . 9 ) .  The l i g h t  regimen w i t h i n the h o l d i n g tank was maintained a t 8 hours o f l i g h t a l t e r n a t i n g with 16 hours of darkness and the temperature of the water ranged  from 11 C t o 12 C.  A l l f i s h , both stock and  experimentals, were fed C l a r k ' s New Age dry f i s h food once daily. On the 2 0 t h of February, 1 9 6 8 , 48 f i s h were t r a n s f e r r e d to each of two experimental tanks s e t up i n a c o n t r o l l e d e n v i r o n ment room maintained a t 10 C.  The tanks were f i l l e d with 300  l i t e r s of water c o n t a i n i n g equal p a r t s of NaCl and C a C ^ to a f i n a l c o n c e n t r a t i o n of 0.2%; Vancouver c i t y water i s low i n d i s solved s o l i d s and the s a l t s a l s o prevented S a p r o l e g n i a i n f e c t i o n of the f i s h .  To f u r t h e r prevent t h i s i n f e c t i o n , one drop  of 1% aqueous s o l u t i o n of Malachite Green was added t o each 100 l i t e r s of water.  The water was held a t 20 C + 0 . 0 5 C with  s u i t a b l e thermostats, r e l a y s , and h e a t i n g c o i l s .  Photoperiods  w i t h i n the tanks were achieved by f i x i n g three 40 ¥/att i n c a n descent bulbs to the r o o f of the l i g h t t i g h t covers f i t t e d t o each tank.  The l i g h t s were a c t i v a t e d through a t i m i n g d e v i c e .  T h i s system y i e l d e d 25 foot candles o f i l l u m i n a t i o n a t the  45  s u r f a c e o f the water while the l i g h t s were on and e l i m i n a t e d p e r c e p t i b l e l i g h t when the l i g h t s were o f f . The l i g h t  regimens  w i t h i n the separate tanks were: 1 hour of l i g h t a l t e r n a t i n g with 23 hours of darkness (abbreviated I L ) ; and 8 hours of l i g h t a l t e r n a t i n g w i t h 16 hours of darkness (abbreviated 8 L ) .  After  35 days, 12 f i s h were removed from each tank and k i l l e d .  Body  weights and gonad weights were taken and the gonosomatic index (GSI = GONAD WEIGHT/BODY WEIGHT X 100) was c a l c u l a t e d f o r each fish.  The remaining 36 f i s h i n each tank were then d i v i d e d  into  groups of 12 f i s h and each group was placed i n a separate f l o a t ing,  nylon-screen basket e n c l o s i n g 12,000 cm^ of water.  The  photoperiod w i t h i n both tanks was s e t a t 16 hours of l i g h t a l t e r n a t i n g w i t h 8 hours of darkness (abbreviated 16L).  On the  same day, the f i s h i n one basket from each tank were i n j e c t e d with melatonin, the f i s h i n another basket were i n j e c t e d with the  aqueous c a r r i e r only, while the f i s h i n the remaining basket  were l e f t a s untreated c o n t r o l s . day f o r 50 days. ted  The f i s h were i n j e c t e d once a  A l l f i s h were then k i l l e d ,  GSI's were c a l c u l a -  f o r each f i s h and the gonads, head kidneys c o n t a i n i n g the  i n t e r r e n a l t i s s u e , t h y r o i d a l t i s s u e from the t h r o a t r e g i o n and whole heads were f i x e d f o r h i s t o l o g i c a l examination. no d i f f e r e n c e s between the gonad and 8L p r i o r to i n j e c t i o n ,  There were  s i z e of animals exposed to I L  or between f i s h that had r e c e i v e d  s i m i l a r treatment under 16L.  T h e r e f o r e , the data from the two  tanks were pooled p r i o r to s t a t i s t i c a l  analysis.  46  A.  I n j e c t i o n o f Melatonin Each f i s h r e c e i v e d 20 ug o f melatonin (Mann Research La-  b o r a t o r i e s , N.Y.) per day d i s s o l v e d i n 0 . 2 5 ml of freshwater t e l e o s t Ringer's s o l u t i o n (Hoar and Hickman, 1 9 6 7 ) . i n j e c t e d animals r e c e i v e d only the Ringer's s o l u t i o n . d a i l y i n j e c t i o n a l t e r n a t e d from the l e f t  The solvent The  to the r i g h t side of  the animal. For the i n j e c t i o n s , a short, #30 gauge needle f i t t e d to a 0.25  ml t u b e r c u l i n s y r i n g e was used.  F i s h were not anaesthe-  t i z e d , but were held i n a bare wet hand. serted i n t o the abdominal  The needle was i n -  c a v i t y i n an area 2 mm below the l a -  t e r a l l i n e and 5 mm forward of an imaginary p e r p e n d i c u l a r pass i n g through the vent.  The needle was d i r e c t e d forward and  s l i g h t l y ventrad and the p o i n t of the needle was kept near to the body w a l l to prevent puncture o f the v i s c e r a l organs.  Be-  tween the d a i l y s e r i e s of i n j e c t i o n s , the needle and s y r i n g e were stored i n 99% e t h a n o l . day approximates  The dose of 20 ug o f melatonin per  the dose used i n mammals (Adams et a l . , 1 9 6 5 ;  Panda and Turner, 1 9 6 8 ) were the mammals of e q u i v a l e n t s i z e to the f i s h employed i n t h i s study.  B.  Histological  Procedures  Gonads and head kidneys c o n t a i n i n g the i n t e r r e n a l t i s s u e were f i x e d i n Bouin's p i c r i c a c i d - f o r m a l - a c e t i c a c i d (75*-25:5)«  solution  Thyroid t i s s u e from the r e g i o n of the throat and  whole heads were f i x e d i n m o d i f i e d Bouin's s o l u t i o n with the a c e t i c a c i d r e p l a c e d by formic a c i d to d e c a l c i f y any contained  Wl  bone.  P i t u i t a r y glands were d i s s e c t e d from the heads f o l l o w i n g  decalcification.  A l l t i s s u e s were embedded i n p a r a f f i n and  sectioned a t 6 \i. The t i s s u e s , with the exception of the p i t u i t a r y , were s t a i n e d with haemotoxylin and e o s i n .  The p i t u i t a r i e s  were s t a i n e d with A l c i a n b l u e - p e r i o d i c a c i d S c h i f f  (PAS)-orange  G.  C  *  A n a l y s i s of H i s t o l o g i c a l R e s u l t s Q u a n t i f i a b l e measurements v/ere not made on the h i s t o l o g i c a l  appearance of the t e s t i s or o v a r i e s . on f i v e animals were recorded  General  o b s e r v a t i o n s based  of each sex from each experimental  to a s c e r t a i n the s t a t e of maturation  condition of the gonads.  Apparent t h y r o i d a l a c t i v i t y was based on the a s s i g n i n g of i n t e g e r s from one to f i v e to represent the r e l a t i o n s h i p between cell  height and nuclear diameter where 1 i n d i c a t e s a nuclear  diameter g r e a t e r than the average c e l l height; 2 , a nuclear  dia-  meter equal to the average c e l l height; 3> a c e l l height greater than, but l e s s than twice, the n u c l e a r diameter;  4, a cell  between two and three times the n u c l e a r diameter;  and 5> a c e l l  height g r e a t e r than three times the n u c l e a r diameter. f i v e r e c o r d i n g s were made from each f i s h , section.  f i v e solvent injected f i s h  f i v e from every  tenth  micrometer.  F i v e melatonin  injected  were analyzed i n t h i s way.  I n t e r r e n a l n u c l e a r diameters  and  Twenty-  The r e c o r d i n g s from a s i n g l e f i s h were averaged to  produce a s i n g l e value f o r that f i s h . and  height  were measured with an o c u l a r  When the n u c l e i were not round, the sum of the l o n g  short a x i s was halved to provide a s i n g l e value.  Twenty-  48  f i v e n u c l e i from each f i s h were measured; f i v e measurements were made on every f i f t h s e c t i o n .  The 25 n u c l e a r diameters from each  f i s h were averaged to o b t a i n a s i n g l e value f o r each f i s h .  Five  melatonin i n j e c t e d and f i v e s o l v e n t i n j e c t e d f i s h were compared. Ocular u n i t s v/ere converted to microns p r i o r to s t a t i s t i c a l testing. The c e l l diameters of p i t u i t a r y gonadotrophs were measured i n the same way as the i n t e r r e n a l n u c l e i but the s t a t i s t i c s were performed d i r e c t l y on the o c u l a r u n i t s .  Ten gonadotrophs were  measured i n each o f f i v e melatonin i n j e c t e d and f i v e s o l v e n t i n j e c t e d animals.  As there were no s i g n i f i c a n t d i f f e r e n c e s be-  tween i n d i v i d u a l f i s h w i t h i n the same group, v a l u e s from one f i s h were not averaged but were considered independent observations. to  D.  For comparison purposes, the sample s i z e was considered  be 5 0 .  S t a t i s t i c a l Methods One way a n a l y s e s of v a r i a n c e were performed on the male and  female GSI data s e p a r a t e l y .  I n d i v i d u a l d i f f e r e n c e s between  means were t e s t e d w i t h the l e a s t  significant difference (lsd)  procedure as m o d i f i e d f o r unequal r e p l i c a t i o n I960).  ( S t e e l and T o r r i e ,  But t - t e s t s were used to compare d i f f e r e n c e s i n t h y r o i d a l  c e l l height, gonadotroph diameter and i n t e r r e n a l n u c l e a r diamet e r s from melatonin and s o l v e n t i n j e c t e d animals.  49  Q u a l i t a t i v e and Q u a n t i a t i v e E s t i m a t i o n of Melatonin and Serotonin A.  General During the course of t h i s study i t became evident that  slight  changes i n procedure or chemicals r e s u l t e d i n marked a l -  t e r a t i o n s i n the e x t r a c t i o n and fluorometry of i n d o l e s .  For  t h i s reason, the source of important chemicals and a b r i e f resume of some standard procedures are p r o v i d e d . A l l water used was d e i o n i z e d by passage through a Bantam Model BD-1  d e m i n e r a l i z e r (Barnstead S t i l l and S t e r i l i z e r  Co.,  Boston, Mass.) and d i s t i l l e d i n ah a l l g l a s s s t i l l (Corning, Model AG 1A).  Ascorbic a c i d was obtained from the N u t r i t i o n a l  Biochemical Corp., C l e v e l a n d , Ohio, EDTA-K^ and EDTA-Na the  2  from  Cambridge Chemical Products Inc., D e t r o i t , Mich., and the  b o r i c a c i d was purchased from A l l i e d Chemical and Dye Corp. of New  York.  Reagent  grade NaCl, NaOH, and KOH  the  N i c h o l s Chemical Co., of Montreal, P.Q.  from the Eastman Organic Co., Rochester N.Y. HC1,  KC1,  were procured from Heptane was  bought  Diethyl-ether,  d i - p o t a s s i u m hydrogen orthophosphate, and cyclohexane  were obtained from the B r i t i s h Drug Houses, Poole England, and the  n-butanol and p-cymene were bought  Co., Fairlawn, N.J. the  from F i s h e r  Scientific  A l l i n d o l e standards were purchased from  Mann Research L a b o r a t o r i e s , N.Y.,  and were t h i n - l a y e r  chromatographically pure (Mann Assayed). Organic s o l v e n t s were d i s t i l l e d twice p r i o r to use.  In  a d d i t i o n , the p-cymene was washed twice i n each of IN NaOH, IN HC1,  and d i s t i l l e d water p r i o r to d i s t i l l a t i o n .  The  initial  50  p-cymene d i s t i l l a t e  was d i s c a r d e d ; the subsequent  I78C) was c o l l e c t e d f o r use (Quay and Baker,  fraction  (176C-  1965).  Concentrated standard s o l u t i o n s o f the i n d o l e s were prepared by d i s s o l v i n g the i n d o l e i n a small amount of ethanol (when r e q u i r e d ) and d i l u t i n g with s u f f i c i e n t 0.1N HC1 or 0.1N HC1, 0.5% a s c o r b i c a c i d to b r i n g the c o n c e n t r a t i o n of i n d o l e to 250 mg per ml o f s o l u t i o n . checked  Working standards were prepared and  daily.  A l l glassware was washed with C a l g o n i t e as t h i s type of soap does not l e a v e f l u o r e s c e n t r e s i d u e s (Udenfriend, 1 9 6 2 ) . When necessary, the cuvette used d u r i n g fluorometry was washed by b o i l i n g i n 50% HNO^ a f t e r the method of Udenfriend  (1962).  The b u f f e r used f o r s e r o t o n i n e x t r a c t i o n s was made a c c o r d i n g to Wise ( 1 9 6 7 b ) . phate  C r y s t a l l i n e di-potassium hydrogen  ( 2 5 2 . 2 gm) was d i s s o l v e d i n 400 ml of water.  orthophosThe pH was  adjusted to 1 0 . 0 0 with concentrated KOH s o l u t i o n and the r e s u l t i n g s o l u t i o n was s a t u r a t e d with KC1.  The volume of the buf-  f e r was then i n c r e a s e d to 500 ml with water.  A small amount of  KC1 was l e f t a t the bottom of the b u f f e r which was s t o r e d a t room temperature.  F r e s h b u f f e r was prepared every two weeks.  For melatonin e x t r a c t i o n , the borate b u f f e r (pH 1 0 . 0 0 ) Quay (1963) was employed.  used by  I t was prepared by d i s s o l v i n g 5 9 . 6  gm o f b o r i c a c i d and 4 1 . 7 gm of NaOH i n two l i t e r s of water. The borate b u f f e r was saturated with NaCl and p-cymene p r i o r to use.  51  B.  Source of B i o l o g i c a l M a t e r i a l S e x u a l l y mature salmon were obtained  from f r e s h water at  the Washington State Department of F i s h e r i e s  1  L i t t l e White and 100  Spring Creek salmon h a t c h e r i e s l o c a t e d approximately above the mouth of the Columbia R i v e r .  miles  Only r e p r o d u c t i v e l y  mature salmon were used: the c r i t e r i o n f o r maturity was  the  t u a l use of the animal as a source  immature  of roe or m i l t .  The  ac-  salmon were taken by commercial t r a w l e r s i n the P a c i f i c Ocean o f f the coast of Vancouver I s l a n d .  No attempt was  made to d e t e r -  mine i f the mature and immature salmon represented spawning p o p u l a t i o n , nor was  a similar  the sex of the animals  recorded.  F i s h were k i l l e d by c u t t i n g a core of t i s s u e , 2 . 5  cm i n  diameter, from the head i n the r e g i o n of the b r a i n as d e s c r i b e d by Tsuyuki  et a l . (.1964).  P i n e a l organs were immediately d i s -  sected from the core of b r a i n t i s s u e and were stored i n p o l y ethylene  b o t t l e s over s o l i d CO2.  Whole eyes were stored i n the  same manner f o l l o w i n g removal from the o r b i t s .  Retinas were  removed as r e q u i r e d from p a r t i a l l y f r o z e n eyes.  C.  P r e p a r a t i o n of E x t r a c t s f o r TCL s e p a r a t e l y to y i e l d 2  P i n e a l s or r e t i n a s were pooled and  5 S  The  t i s s u e was  m  7 p i n e a l s or 12  retinas).  ml of i c e - c o l d 0.1M  anhydrous  r e s p e c t i v e l y (approximately homogenized i n 15  sodium phosphate (monobasic).  The  homogenate was  extracted  twice with two volumes of i c e - c o l d e t h y l acetate and t r a c t s were pooled. to the pooled  gm  Anhydrous sodium sulphate  (30  e x t r a c t s to remove excess water and  the  gm)  was  exadded  the mixture  52  was allowed to stand one hour.  T h i s mixture was then  through a medium c i n t e r e d g l a s s f i l t e r sulphate and any p i e c e s of t i s s u e . and  filtered  to remove the sodium  F i n a l l y , both the f i l t e r  the sodium sulphate were washed with s e v e r a l ml of e t h y l  a c e t a t e which were then added to the p r e v i o u s f i l t r a t e . f i l t r a t e was evaporated  The  to dryness by f l a s h evaporation a t 20 C  under an atmosphere of n i t r o g e n .  The r e s i d u e was taken up i n 2  ml of a mixture c o n s i s t i n g of absolute ethanol and 0.1N (4:1), and the r e s u l t i n g s o l u t i o n was evaporated n i t r o g e n atmosphere to 0 . 5 ml.  HC1  under a reduced  Test i n d o l e s o l u t i o n s were p r e -  pared by d i s s o l v i n g commercial i n d o l e s d i r e c t l y i n the e t h a n o l 0.1N HC1 s o l u t i o n .  The e x t r a c t i o n procedure  was checked by  e x t r a c t i n g water, s e r o t o n i n and melatonin standards. t r a c t s produced  Water ex-  no spots when run chromatographically.  Both the  s e r o t o n i n and the melatonin standards were found to be chromat o g r a p h i c a l l y pure; that i s , each i n d o l e y i e l d e d only one spot f o l l o w i n g development and d e t e c t i o n of i n d o l e s . s e r o t o n i n and melatonin and approached 100%.  Recovery of  from standard s o l u t i o n s was e x c e l l e n t  E x t r a c t s were chromatographed on the same  day as prepared.  D.  T h i n Layer Chromatography Chromatographic s e p a r a t i o n s were performed  on g l a s s p l a t e s  ( 2 0 cm x 20 cm) coated on one s i d e to a depth of 0 . 2 5 mm the absorbant  Silica  Gel G (Merck).  P l a t e s were a i r d r i e d and  stored i n a d e s i c c a t o r over anhydrous calcium c h l o r i d e . to  with  Prior  use the p l a t e s were sprayed with a s o l u t i o n of 0.5% a s c o r b i c  53  a c i d i n absolute methanol to enhance the s u r v i v a l and recovery of the 5-hydroxy and 5-methoxy compounds (Quay and Bagnara, 1964).  Ascending, two dimensional s e p a r a t i o n o f the i n d o l e s . standard covered  development was used f o r the  The d e v e l o p i n g chambers were of the  r e c t a n g u l a r trough type with the i n s i d e of three w a l l s with a l a y e r o f t h i c k f i l t e r paper to enhance e q u i l i -  b r a t i o n of the s o l v e n t systems.  The chambers were allowed to  e q u i l i b r a t e f o r a minimum of two h o u r s . f o l l o w i n g a d d i t i o n o f 100 ml o f the s o l v e n t system. to  The f o l l o w i n g s o l v e n t systems proved  be the most u s e f u l f o r t h i s study A l k a l i n e system:  methyl a c e t a t e - i s o p r o p a n o l 25%  Acidic  system:  1965)*  (Stahl,  NH  3  (45:35:20)  chloroform-96%  acetic  acid  (95:5)  For the a c t u a l s e p a r a t i o n s , 0 . 1 ml of the t i s s u e e x t r a c t was  spotted on the p l a t e a t a p o i n t 2 . 5 cm e q u i d i s t a n t from two  sides.  Along both of these  s i d e s and 2 . 5 cm from the edge of  the p l a t e , an i d e n t i c a l s e r i e s of standards spot was 2 cm d i s t a n t from another.  was spotted;  each  S u f f i c i e n t standard ex-  t r a c t was a p p l i e d to each spot t o b r i n g the t o t a l amount of i n dole spotted t o 0 . 1 ug.  The p l a t e was then placed i n the cham-  ber c o n t a i n i n g the a l k a l i n e system and the s o l v e n t f r o n t was allowed  to r i s e to a d i s t a n c e of 10 cm above the o r i g i n a l  of spots. NHj  F o l l o w i n g development i n the f i r s t  direction,  was removed by p l a c i n g the p l a t e over concentrated  a d e s i c c a t o r , evacuating  to 1 5 - 2 0 mm o f Hg, f i l l i n g the  line excess  B^SO^ i n  54  d e s i c c a t o r with N , r e e v a c u a t i n g ( t h i s was done s e v e r a l times), 2  and then a l l o w i n g the p l a t e to stand i n the reduced phere over B^SO^ f o r one hour.  atmos-  The p l a t e was then removed  from  the d e s i c c a t o r , r o t a t e d 9°° t o the d i r e c t i o n of the f i r s t development, and developed  i n the a c i d i c system u n t i l the s o l v e n t  f r o n t had r i s e n to a p o i n t 10 cm above the spots a l o n g the second  E.  side.  Demonstration  and I d e n t i f i c a t i o n o f Indoles  For the d e t e c t i o n and i d e n t i f i c a t i o n of the v a r i o u s i n d o l e s , the f o l l o w i n g r e a c t i o n s ( S t a h l , 1965) were employed and the Rf v a l u e s (Rf = d i s t a n c e of spot from s t a r t i n g p o i n t (cm)/height of s o l v e n t f r o n t above s t a r t i n g p o i n t x 100) obtained i n both  sol-  vent systems were compared with those o f standard s e r o t o n i n and melatonin e x t r a c t s run at the same time on the same p l a t e . General D e t e c t i o n of Simple  Indoles  sprayed w i t h a f r e s h l y prepared mixture  The p l a t e s were c o n s i s t i n g o f 10 ml of  formaldehyde (35%, aqueous), 10 ml o f 25% H C 1 , and 20 ml o f absolute ethanol.  The p l a t e was then exposed to the vapours o f  aqua r e g i a and heated  f o r f i v e minutes at 100 C.  This reaction  (Prochazka r e a c t i o n ) g i v e s only yellow r e a c t i o n products which f l u o r e s c e s t r o n g l y i n a l o n g wave u l t r a v i o l e t l i g h t . t h i s i s an extremely  Although  s e n s i t i v e technique f o r demonstrating i n -  doles, and i s u s e f u l f o r the purpose of c a l c u l a t i n g Rf values of i n d o l e s present i n low c o n c e n t r a t i o n s , i t i s not s p e c i f i c i n i t s color reactions. D e t e c t i o n of Serotonin  The p l a t e was sprayed  with  55  n i n h y d r i n (0.25%) i n a s o l u t i o n of acetone c o n t a i n i n g 10% of acetic acid.  T h i s i s an extremely  sensitive test f o r serotonin  (Jepson and Stevens, 1953) hut a l s o r e a c t e d withp'Other i n d o l e compounds.  However, s e r o t o n i n y i e l d s a blue-green f l u o r e s c e n c e  when the p l a t e i s heated  f o r two or three minutes a t 90 C-100 C.  D e t e c t i o n of Melatonin  The most s p e c i f i c of the t e c h -  niques f o r the i d e n t i f i c a t i o n of simple i n d o l e d e r i v a t i v e s ( S t a h l , 1965) and one which i s p a r t i c u l a r l y u s e f u l f o r demonr-" s t r a t i n g melatonin i s the van Urk's r e a c t i o n .  The p l a t e i s  sprayed c o p i o u s l y (approximately 10 ml f o r a 20 cm x 20 cm ;p p l a t e ) immediately  f o l l o w i n g development with a reagent (van  Urk's) prepared by d i s s o l v i n g 1 gm of i+-dimethylaminobenzaldehyde i n 50 ml of HC1 and adding 50 ml o f a b s o l u t e ethanol to the r e s u l t i n g s o l u t i o n .  A f t e r the p l a t e was thoroughly  wetted  with van Urk's reagent, i t was exposed b r i e f l y to the vapours of aqua r e g i a .  Melatonin appears as a blue spot almost imme-  diately. Although no s i n g l e technique employed f o r the demonstration of the i n d o l e s was s p e c i f i c enough to e s t a b l i s h the presence of s e r o t o n i n or melatonin, a combination of the two Rf values and the c o l o r r e a c t i o n s with the d i f f e r e n t techniques of d e t e c t i o n enabled the accurate i d e n t i f i c a t i o n of these i n d o l e s . chromatography permitted estimates to be made about  Though  the r e l a t i v e  q u a n t i t i e s o f s e r o t o n i n and melatonin i n d i f f e r e n t e x t r a c t s , i t d i d not y i e l d accurate measures of c o n c e n t r a t i o n .  56  F.  Fluorometric  Techniques  The method employed f o r the f l u o r o m e t r i c a n a l y s i s of serot o n i n was d e s c r i b e d i n S e c t i o n I and w i l l not be repeated here. Melatonin l e v e l s were measured f l u o r o m e t r i c a l l y f o l l o w i n g  ex-  t r a c t i o n with p-cymene from an a l k a l i n e s o l u t i o n as d e s c r i b e d by Quay ( 1 9 6 3 ) and Quay and Baker ( 1 9 6 5 ) .  Reproducibility,  ex-  t r a c t i o n e f f i c i e n c y and s p e c i f i c i t y obtained i n the melatonin determinations were estimated i n the same manner as d e s c r i b e d f o r s e r o t o n i n ( S e c t i o n I) with the exception that 5-hydroxytryptophane was r e p l a c e d by 5-methoxytryptamine. b i l i t y was  Reproduci-  e x c e l l e n t , the recorded melatonin l e v e l s from the  same sample t e s t e d on d i f f e r e n t days v a r i e d w i t h i n a few tage p o i n t s . and 89%.  Recovery  percen-  of added melatonin ranged between 73%  For t h i s reason, melatonin l e v e l s i n t i s s u e  extracts  were c a l c u l a t e d from a standard curve obtained by  extracting  melatonin standards of d i f f e r e n t c o n c e n t r a t i o n s .  Because ex-  t r a c t i o n of 5-methoxytryptamine standards by the melatonin  ex-  found to y i e l d a 6% r e c o v e r a b i l i t y ,  the  t r a c t i o n procedure was  s p e c i f i c i t y of the technique was  judged i n a c c u r a t e f o r estab-  l i s h i n g the presence of melatonin.  However, when melatonin  present i n reasonably h i g h q u a n t i t i e s (100  ng per sample),  was the  technique y i e l d e d p r e c i s e measures of melatonin c o n c e n t r a t i o n s . Using these techniques, the l e v e l s of s e r o t o n i n and  melatonin  were measured i n the p i n e a l organs of mature and immature s a l mon  and the l e v e l of s e r o t o n i n only was measured i n the r e t i n a s  of the same animals.  57  RESULTS  Demonstration  of Serotonin and Melatonin i n F i s h  Melatonin was  found i n the p i n e a l s of both immature and  mature salmon but could not be found i n the r e t i n a l Serotonin was  tissue.  found i n the p i n e a l and the r e t i n a s of immature  and mature salmon. A spot was  found on the chromatoplates f o l l o w i n g TLC of  e i t h e r r e t i n a l or p i n e a l e x t r a c t s with Rf v a l u e s of 64 and 2 i n the a l k a l i n e and a c i d i c systems r e s p e c t i v e l y . 64 d i f f e r s by one p o i n t ( 6 5 )  The Rf value of  from that r e p o r t e d by S t a h l  f o r s e r o t o n i n developed i n the same manner.  (1965)  F u r t h e r , both Rf  v a l u e s were i d e n t i c a l to those obtained from s e r o t o n i n standards developed  simultaneously with the t i s s u e e x t r a c t s , and  from other i n d o l e s t e s t e d .  By way  of i d e n t i f i c a t i o n ,  differed t h i s spot  turned grey with van Urk's reagent, y e l l o w with Prochazka r e a gent, and f l u o r e s c e d blue-green under u l t r a v i o l e t f o l l o w i n g treatment with the n i n h y d r i n reagent.  radiation Since a l l of  these r e a c t i o n s are c h a r a c t e r i s t i c of s e r o t o n i n e x t r a c t s , the spot was i d e n t i f i e d as s e r o t o n i n . The r e l a t i v e c o n c e n t r a t i o n s of s e r o t o n i n i n the p i n e a l sue appeared  tis-  to be four or f i v e times that seen i n an e q u i v a l e n t  amount of r e t i n a l t i s s u e .  No d i f f e r e n c e s i n the r e t i n a l or  p i n e a l s e r o t o n i n l e v e l s were found between mature and immature salmon. The d e c i s i o n f o r the presence of melatonin i n the p i n e a l t i s s u e was  based on the f a c t that the p i n e a l e x t r a c t s , when  58 chromatographed, y i e l d e d a spot with Rf values o f 85 i n the a l k a l i n e system and 16 i n the a c i d i c  system, and that both of ,  these v a l u e s c o i n c i d e d e x a c t l y with the Rf v a l u e s obtained e x t r a c t e d standard melatonin run a t the same time. i n d o l e t e s t e d had the same two Rf v a l u e s .  from  No other  F u r t h e r , when the  p l a t e was sprayed with van Urk's reagent, the spot turned a blue c o l o r which could be i n t e n s i f i e d by exposure to aqua r e g i a vapours.  Although other i n d o l e s produced  the same r e a c t i o n , the  blue c o l o r d i d not appear as q u i c k l y as when melatonin was the reacting indole.  When estimated by t h i s technique, the r e l a t i v e  amount of melatonin i n the p i n e a l o f immature salmon appeared to be three or four times that seen i n the p i n e a l s of mature s a l mon.  Melatonin was not found i n other p a r t s of the b r a i n , the  blood, or muscle t i s s u e .  S e r o t o n i n and Melatonin L e v e l s i n Immature and Mature Salmon P i n e a l and r e t i n a l l e v e l s of s e r o t o n i n , and p i n e a l l e v e l s of melatonin, i n mature and immature salmon, are summarized i n Table VI.  Melatonin was not i d e n t i f i e d i n the r e t i n a l  tissue  by chromatography and was t h e r e f o r e not measured f l u o r o m e t r i cally i n this  tissue.  The t a b u l a t e d values i n Table VI represent mean l e v e l s of the i n d o l e s c a l c u l a t e d from f i v e pooled samples o f p i n e a l or retinal tissue.  Although the l e v e l of s e r o t o n i n found i n the  p i n e a l glands was approximately  four times as great as that  found i n the r e t i n a s , there were no d i f f e r e n c e s i n the l e v e l o f s e r o t o n i n between mature and immature salmon i n e i t h e r  tissue.  59  TABLE VI Serotonin and melatonin content of mature and immature salmon p i n e a l s and r e t i n a l t i s s u e . *  Pineal s Indole  Immature  Mature  Serotonin  2.08+0.12  2.21+0.08  Melatonin  4.^9+0.72  0.68+0.08  Retinas P t-test  Immature  Mature  NS  0.29+0.09  0.27+0.10  .005**  not t e s t e d  *The f i g u r e s a r e means + S.E.M. based on f i v e pooled e x t r a c t i o n s each; the t a b u l a t e d v a l u e s are i n ug/g of wet t i s s u e . * * 0 n e t a i l e d t e s t and t h e r e f o r e the mean l e v e l of p i n e a l melat o n i n i n the immature salmon i s s i g n i f i c a n t l y ( P < . 0 0 5 ) g r e a t e r than the mean l e v e l o f melatonin i n t h e mature salmon.  60  These r e s u l t s c o r r o b o r a t e those r e p o r t e d from the TLC s t u d i e s . On the other hand, as suggested l e v e l o f melatonin  by the q u a l i t a t i v e s t u d i e s , the  found i n the p i n e a l glands of immature  mon was s i g n i f i c a n t l y  sal-  (P<.005) higher than t h a t found i n the  p i n e a l glands of mature salmon.  The approximate amount of mela-  t o n i n i n s i n g l e p i n e a l glands was 180 ng i n the mature f i s h as opposed to 1200 ng i n the immature f i s h .  The s i m i l a r i t y i n the  r e l a t i o n s h i p s between the amount of s e r o t o n i n and melatonin i n the p i n e a l s of immature and mature salmon as estimated by t h i n l a y e r chromatography and fluorometry was taken a s f u r t h e r dence t h a t the i n d o l e s were c o r r e c t l y  E f f e c t s of Melatonin The female  evi-  identified.  Injection  e f f e c t s of melatonin i n j e c t i o n on the GSI of male and  g o l d f i s h are summarized i n Table V I I .  v a l u e s are mean GSI's of male and female  The t a b u l a t e d  g o l d f i s h p r i o r to  melatonin i n j e c t i o n and h e l d under short photoperiod  (1L or 8L)  and a f t e r f i f t y  (l6L) with  days of exposure to l o n g photoperiod  or without melatonin  or s o l v e n t i n j e c t i o n s .  were r u n on the male and female  A n a l y s i s of v a r i a n c e  data s e p a r a t e l y (Table V I I I ) .  The l e a s t s i g n i f i c a n t d i f f e r e n c e s >for s i n g l e comparisons were c a l c u l a t e d from the a p p r o p r i a t e a n a l y s i s of v a r i a n c e and were used to t e s t the s i g n i f i c a n c e of d i f f e r e n c e s between mean GSI v a l u e s from the d i f f e r e n t experimental  groups.  Melatonin i n h i b i t e d the i n c r e a s e i n the GSI o f male and f e male g o l d f i s h exposed to l o n g photoperiod.  Male and female  g o l d f i s h which were not i n j e c t e d or had r e c e i v e d i n j e c t i o n s of  61  TABLE VII E f f e c t o f melatonin i n j e c t i o n on the GSI of male and female g o l d f i s h f o l l o w i n g exposure to l o n g photoperiod (16L).  Sex  Initial Controls  Final Controls  Solvent Injected  Melatonin Injected  Male  0.37(10)  1.53*(14)  1.59*(8)  0.50(12)  Female  1.37(13)  4.31*(10)  4.90*(l6)  1.64(12)  1  2  2  Values are means (Number of o b s e r v a t i o n s ) . "^"Pooled v a l u e s of animals held under I L and 8L f o r 3 5 days but before exposure to 16L. 2  A f t e r 5 0 days of 1 6 L .  •Represents a s i g n i f i c a n t i n c r e a s e from mean GSI o f i n i t i a l controls  (P<.05).  Represents a s i g n i f i c a n t l y l a r g e r GSI when compared to the melatonin i n j e c t e d groups ( P < . 0 5 ) . ;  2  62  TABLE VIII A n a l y s i s of v a r i a n c e of male and female GSI data before and a f t e r 50 days o f melatonin or s o l v e n t injection.  Males: Source  SS  df  MS  F 6.35  Treatment  13.72  3  4-57  Error  28.78  40  .72  F  0 > 0 1  (3,40)=4.31  Therefore treatment i s s i g n i f i c a n t  Females: Source Treatment  SS 131.21  df  MS  3  43-74  F 4.49 F  Error  457.46  47  9.73  Therefore treatment i s s i g n i f i c a n t  o i 3,47)=4.08 °* (  0  63 the s o l v e n t only had  significantly  ( P - < . 0 5 ) l a r g e r gonads a f t e r  50 days exposure to l o n g photoperiod  than the male or  f i s h sampled p r i o r to exposure to l o n g photoperiod.  female The mela-  t o n i n i n j e c t e d animals d i d not show a s i g n i f i c a n t i n c r e a s e i n the s i z e of t h e i r GSI's d u r i n g the 50 days of exposure to l o n g photoperiod.  F a i l u r e to f i n d a s i g n i f i c a n t d i f f e r e n c e between  the u n i n j e c t e d female  c o n t r o l s and the melatonin i n j e c t e d f e -  males (Table V I I ) , p l u s the f a c t that both the male and melatonin i n j e c t e d  female  groups showed s l i g h t i n c r e a s e s i n t h e i r  values d u r i n g the p e r i o d of i n j e c t i o n , b i t i o n of the gonads by melatonin was  suggests  that the  incomplete,  s o l u t e d i f f e r e n c e s were not found,  in  injection.  h i s t o l o g i c a l o b s e r v a t i o n s on the t e s t i s s u b s t a n t i a t e d  the data from the male GSI v a l u e s .  for  causing  I f the l a t t e r were t r u e , we would expect a de-  crease i n the gonad s i z e f o l l o w i n g melatonin The  inhi-  and that the  melatonin i n i n h i b i t i n g an i n c r e a s e r a t h e r than d i r e c t l y a regression.  GSI  reduced  filling  Despite the f a c t t h a t abthere was  a marked tendency  of the t u b u l e s by spermatozoa and  the t e s t i s of melatonin i n j e c t e d males.  stages of spermatogenesis  Nevertheless, a l l  were present i n a l l groups of males.  The h i s t o l o g i c a l appearance of the i n i t i a l served, but the reduced  spermatids  s i z e and reduced  c o n t r o l s was  not  ob-  amount of m a t e r i a l  w i t h i n the t u b u l e s of the melatonin i n j e c t e d males i n d i c a t e s an i n h i b i t i o n of spermatogenesis. spermatogenesis  The  presence  of a l l stages of  shows that the i n h i b i t i o n was not  complete.  The h i s t o l o g i c a l appearance of the o v a r i e s v a r i e d c o n s i d e r a b l y w i t h i n any one  experimental  group but no d i f f e r e n c e s were  64  observed  between any two  groups.  A l l stages of maturation were  present w i t h i n the o v a r i e s of each group.  Therefore, although  melatonin i n h i b i t e d the o v e r a l l growth of the o v a r i e s , i t d i d not s p e c i f i c a l l y i n h i b i t any one step i n ovogenesis or completely i n h i b i t the process of maturation. The  e f f e c t s of melatonin i n j e c t i o n on the i n t e r r e n a l  c l e a r diameter,  the r e l a t i v e height of the t h y r o i d a l e p i t h e l i u m ,  and the diameter i n Table IX.  of the p i t u i t a r y gonadotrophs are summarized  Melatonin had no e f f e c t on the r e l a t i v e height of  the t h y r o i d a l e p i t h e l i u m . had  significantly  ficantly  nu-  (P<.01)  (P<.025)  But the melatonin i n j e c t e d  animals  smaller i n t e r r e n a l n u c l e i and  signi-  l a r g e r gonadotrophs than the s o l v e n t i n j e c t e d  group. These f i n d i n g s , together with those presented e a r l i e r ,  sug-  gest that the i n h i b i t o r y a c t i o n of melatonin on the gonads i s exerted by way  of the p i t u i t a r y gland.  The i n c r e a s e d s i z e of  the gonadotrophs could i n d i c a t e that these c e l l s are producing but not s e c r e t i n g gonadotropic hormone. may  Conversely, melatonin  be i n h i b i t i n g the a c t i o n of gonadotropin on the gonads with  the r e s u l t that the gonadotrophs become h y p e r a c t i v e . Decreased  i n t e r r e n a l n u c l e a r diameters f o l l o w i n g melatonin  i n j e c t i o n shows that the a c t i o n of melatonin i s not l i m i t e d to the r e p r o d u c t i v e system but a l s o a f f e c t s the e l e c t r o l y t e through the i n t e r r e n a l t i s s u e .  system  Nevertheless, melatonin d i d not  a f f e c t the t h y r o i d a l t i s s u e and i s t h e r e f o r e not a general p i tuitary  inhibitor.  65  TABLE IX E f f e c t of melatonin i n j e c t i o n on i n t e r r e n a l nuclear diameter, r e l a t i v e • t h y r o i d e p i t h e l i a l c e l l height and gonadotroph diameter of g o l d f i s h . Tabulated values are means.  Solvent Injected  Tissue Interrenal  5.52  Thyroid e p i t h e l i u m  1.40  Gonadotroph diameter (ocular u n i t s ) *  5.92  ocular  Significance t - t e s t (df)  nuclei  (microns)  *1  Melatonin Injected  u n i t = .92  microns  5.15  P<.025  (4)  2.04  NS  (4)  6.93  P<.01  (49)  66  DISCUSSION  The l o c a l i z a t i o n of melatonin within the pineal organ of f i s h corroborates the r e s u l t s of other workers. found that the enzyme hydroxyindole-O-methyl  Quay ( 1 9 6 5 b )  transferase, re-  quired for melatonin synthesis, i s present i n the pineal gland of fishes.  Recently, Oguri et a l . ( 1 9 6 8 ) demonstrated that  Re-  labelled 5-hydroxytryptophane i s taken up by the pineal organ of trout i n greater quantities than any other tissue studied.  As  t h i s amine i s a precursor of serotonin, and therefore a precursor of melatonin, they concluded that the photoreceptive pineal organ of the trout Salmon i r i d e u s (Dodt, 1 9 6 3 ; also produces melatonin.  Morita, 1966)  Quay (1965) also found HIOMT i n the  r e t i n a of the f i s h but i n the present study melatonin could not be l o c a l i z e d i n t h i s tissue taken from salmon.  Whether t h i s  means that the r e t i n a produces melatonin but does not store i t , or whether the presence of the enzyme HIOMT doesijnot always result i n melatonin synthesis i s not known.  Nevertheless, the  high l e v e l s of melatonin found i n the pineal organ of the s a l mon indicate that i n t h i s species the pineal gland i s a p r i n c i pal source of melatonin. The l e v e l of melatonin found i n mature salmon ( 0 . 6 8  ug/g)  approximates the l e v e l found i n rats (O.kO ug/g) by Prop and Arie'ns Kappers ( 1 9 6 1 ) but exceeds those reported i n the pineal of cows (Learner et a l . , i 9 6 0 ) , (Ralph et a l . , 1 9 6 7 ) .  pigeons (Quay, 1966)  arid weavers  In immature salmon, the pineal melatonin  store i s much greater (k.k9 ug/g) than i n the pineals of mature  67  salmon, and may  be r e l a t e d to the i n h i b i t i o n of maturation.  Be  that as i t may,  the marked changes i n p i n e a l melatonin s t o r e s  betv/een mature and immature salmon might a l s o be r e l a t e d to the aquatic environment.  The higher melatonin l e v e l was  found i n  the p i n e a l s of ocean d w e l l i n g salmon and the lower l e v e l  was  found i n mature salmon which had moved i n t o f r e s h water.  Mela-  t o n i n has p r e v i o u s l y been i m p l i c a t e d i n a d r e n a l p h y s i o l o g y ( F r a s c h i n i et a l . , 1 9 6 8 ) , and r e s u l t s i n a r e d u c t i o n of a d r e n a l weight when implanted i n t o the median eminence of male r a t s . S i m i l a r r e s u l t s were obtained i n the g o l d f i s h (Table I X ) .  Mela-  t o n i n i n j e c t e d animals had s m a l l e r i n t e r r e n a l n u c l e i than solvent i n j e c t e d animals.  How  melatonin a f f e c t s the i n t e r n a l n u c l e i i s  not c l e a r , but the p o s s i b i l i t y that melatonin l e v e l s are r e l a t e d to the osmotic environment  of f i s h e s must be considered.  Notwithstanding the p o s s i b i l i t y that the lower p i n e a l melatonin s t o r e i n mature as opposed to immature salmon i s r e l a t e d to the change from sea water to f r e s h water,  the decrease  corresponds with what one would expect i f melatonin i s a c t i n g as a gonadal*- i n h i b i t o r . The data from g o l d f i s h i n j e c t e d with melatonin shows a d i r ect r e l a t i o n s h i p between melatonin and  the r e p r o d u c t i v e system,  and t h e r e f o r e c o r r o b o r a t e s the a l t e r a t i o n s i n melatonin l e v e l between immature and mature salmon.  The e f f e c t of melatonin  was  to i n h i b i t the i n c r e a s e i n gonad s i z e of male and female g o l d f i s h when they were exposed  to i n c r e a s e d day l e n g t h .  The  inhi-  b i t i o n of the gonads was accompanied by an i n c r e a s e i n the s i z e of the p i t u i t a r y gonadotrophs,  and, i n the male, a decrease i n  68  the q u a n t i t y of spermatozoa  and spermatids w i t h i n the t u b u l e s of  the t e s t i s . Previous r e p o r t s from mammals concur with the f i n d i n g s r e Wurtman et a l . (1963) have r e p o r t e d a delay i n the  ported here.  v a g i n a l opening and a r e d u c t i o n of o v a r i a n weight  following  melatonin treatment i n r a t s , and Wurtman and Axelrod (1965b) found a decreased i n c i d e n c e o f e s t r u s i n melatonin t r e a t e d a d u l t rats.  In pursuance  of the same problem,  Adams, Wan and Sohler  (1965) i n h i b i t e d the weight i n c r e a s e i n the o v a r i e s o f r a t s between days 35 n d k9 p o s t n a t a l and found that these animals had a  smaller p i t u i t a r i e s with higher l e v e l s of l u t e i n i z i n g hormone (LH).  T h i s l a t t e r f i n d i n g i s supported here by the l a r g e r  gonadotropic c e l l s found i n the melatonin i n j e c t e d  goldfish.  Unfortunately, data p r i o r to melatonin i n j e c t i o n was not c o l l e c t e d so that i t i s not known i f the l a r g e r gonadotrophs i n the melatonin i n j e c t e d animals r e s u l t e d  from an a c t u a l i n c r e a s e  i n s i z e , or a f a i l u r e to decrease f o l l o w i n g exposure t o l o n g photoperiod.  Nevertheless, the p r i n c i p a l e f f e c t of melatonin  i n mammals i s suggested to be t h a t of i n h i b i t i o n of LH s e c r e t i o n (Motta et a l . , 1967; F r a s c h i n i et a l . ,  1968).  Among male mammals, melatonin s e l e c t i v e l y decreases the weight of the p r o s t a t e and seminal v e s i c l e s which are l a r g e l y under the c o n t r o l of LH but does not reduce the weight o f the FSH dependant t e s t i s (Wurtman et a l . , 1 9 6 3 ; Kappers, Moskowska, 1 9 6 5 ) .  196k;  As an LH l i k e hormone i s thought to.be the  major, i f not the only f i s h gonadotropin (Yamazaki  and Donald-  son, 1 9 6 8 ) , the e f f e c t of melatonin i n j e c t i o n s on the p i t u i t a r y  69  gonadotrophs and the marked e f f e c t on the gonads i s probably not f o r t u i t o u s .  I t i s tempting to suggest that the data ob-  t a i n e d i n t h i s study supports the hypothesis that a s i n g l e  fish  gonadotropic hormone serves the f u n c t i o n c a r r i e d out by two hormones i n mammals, and that the f i s h gonadotropic hormone has c h a r a c t e r i s t i c s common to both mammalian hormones. The data r e p o r t e d here, as w e l l as that a v a i l a b l e i n the literature  ( F r a s c h i n i et a l . , 1968)  i n d i c a t e that melatonin  a c t s by i n h i b i t i n g the s t i m u l a t i o n of the gonads r a t h e r than d i r e c t l y producing r e g r e s s i o n of them.  Although melatonin pre-  vented a s i g n i f i c a n t i n c r e a s e i n the s i z e of the gonads d u r i n g exposure of the g o l d f i s h to l o n g photoperiod, the gonads d i d not decrease i n s i z e .  From t h i s f a c t , as w e l l as the e f f e c t of  the melatonin on the gonadotrophs, i t i s concluded t h a t the act i o n of the melatonin i s centered i n the p i t u i t a r y gland or the hypothalamic  c e n t e r s governing p i t u i t a r y  function.  From s t u d i e s with mammals, a d i r e c t a c t i o n of melatonin on the p i t u i t a r y appears u n l i k e l y .  Moszkowska  (1965)  found  that melatonin alone was unable to produce any e f f e c t on i n v i t r o p i t u i t a r y c u l t u r e s but that i t d i d reduce the a c t i v a t i o n of  t h i s t i s s u e by hypothalamic  extracts.  Recently, F r a s c h i n i  et a l . ( 1 9 6 8 ) r e p o r t that melatonin, implanted d i r e c t l y i n t o the p i t u i t a r y t i s s u e of r a t s , does not change the l e v e l of p i t u i t a r y LH but" that i m p l a n t a t i o n of melatonin i n t o the median eminence or r e t i c u l a r substance of the midbrain r e s u l t s i n a r e d u c t i o n of p i t u i t a r y LH s t o r e s and a decrease i n the l e v e l of c i r c u l a t i n g LH.  Hence, the e f f e c t s o f melatonin r e p o r t e d here are  70  probably the r e s u l t of some a c t i o n on the hypothalamic  centers  which r e g u l a t e the s y n t h e s i s or s e c r e t i o n of p i t u i t a r y gonadotropin.  The l a r g e r s i z e of the gonadotrophs i n the p i t u i t a r y  glands of melatonin.rinjected g o l d f i s h i n d i c a t e s an i n c r e a s e i n gonadotropin s y n t h e s i s or s t o r a g e .  The i n c r e a s e i n s y n t h e s i s  appears u n l i k e l y however, as melatonin implants decrease  the  l e v e l of LH i n the p i t u i t a r y glands of mammals ( F r a s c h i n i et a l . , 1968).  F u r t h e r , i n h i b i t i o n of r e l e a s e of gonadotropin i s i n d i -  cated by the f a c t that melatonin t r e a t e d r a t s do not undergo a decrease i n p i t u i t a r y LH s t o r e s d u r i n g puberty.  Whatever the  mechanism, i t appears as though melatonin i n h i b i t s the s t i m u l a t o r y e f f e c t of l o n g photoperiod on the gonads. F a i l u r e to d e t e c t any e f f e c t of melatonin on the t h y r o i d a l a c t i v i t y i n g o l d f i s h might r e s u l t employed.  The h i s t o l o g i c a l approach  from the technique  used i s thought by some to  by an u n r e l i a b l e - c r i t e r i o n of t h y r o i d a l a c t i v i t y Eales, 1 9 6 3 ) .  apparent  (Matty,  I960;  However, as the p r i n c i p a l c r i t i c i s m of t h i s t e c h -  nique i s a l a g between the t h y r o i d a l c e l l height and the s e c r e tory a c t i v i t y , and as the animals t e s t e d i n t h i s study were allowed 5 ° days f o r changes to occur, the r e s u l t s obtained were taken as i n d i c a t i o n s of t h y r o i d a l a c t i v i t y .  The r e s u l t s of the  study on the e f f e c t of melatonin i n j e c t i o n on the t h y r o i d a l sue are at v a r i a n c e with those of Panda and Turner  (1968)  tis-  who  r e p o r t that i n r a t s , melatonin has an a c t i o n s i m i l a r to a goitrogen and produces  a decrease i n t h y r o i d a l s e c r e t o r y r a t e ,  but agree with the f i n d i n g s of T h i e b l o t et a l . ,  (1966).  The  i m p l i c a t i o n ' s that i n the g o l d f i s h , the a c t i o n of melatonin i s  71  s p e c i f i c f o r c e r t a i n p i t u i t a r y f u n c t i o n s and does not g e n e r a l l y i n h i b i t hypophyseal f u n c t i o n s . In c o n c l u s i o n , the f o l l o w i n g p i c t u r e emerges. present  w i t h i n the p i n e a l of both mammals and f i s h .  of melatonin and  Melatonin i s The l e v e l  i s r e l a t e d to the s t a t e o f r e p r o d u c t i v e  exogenous melatonin  groups of animals.  maturity  can i n h i b i t gonadal growth i n both  I t i s t h e r e f o r e p o s t u l a t e d that with  regards  to the r e p r o d u c t i v e system, the p i n e a l s of mammals and f i s h e s are f u n c t i o n a l l y s i m i l a r .  Melatonin,  present i n high  t i o n s i n the p i n e a l of immature salmon, may prevent t i o n of the gonads by a c t i n g on c e r t a i n hypothalamic which r e g u l a t e s gonadotropin  release.  r e p r o d u c t i v e c y c l e , the melatonin  tion.  the maturacenters  At some time d u r i n g the  l e v e l i n the p i n e a l decreases  r e s u l t i n g i n an i n c r e a s e i n gonadotropin stimulate  concentra-  s e c r e t i o n which i n t u r n  an i n c r e a s e i n gonad s i z e and r e p r o d u c t i v e matura-  The melatonin,  or r a t h e r the decrease i n p i n e a l  melatonin  s t o r e d , then a c t s as a t r i g g e r by r e l e a s i n g greater q u a n t i t i e s or a l l o w i n g enhanced productio n of gonadotropic  hormone.  72  SECTION I I I EFFECT OF PINEALECTOMY ON  THE  REPRODUCTIVE  AND  ENDOCRINE SYSTEMS' IN GOLDFISH INTRODUCTION  A secretory  r o l e of the f i s h p i n e a l organ has  Holmgren ( 1 9 5 8 ) , Rasquin ( 1 9 5 8 ) , and  ted.  scribed a secretory Ford, 1 9 6 7 ) .  Altner  been sugges-. (1965)  r o l e based on apocrine s e c r e t i o n  (Hafeez  1958;  g o l g i complex (Riideberg,  Holmgren, 1959a) and  a w e l l developed  1968b) i n f i s h p i n e a l c e l l s .  Though  most authors agree that t h i s s e c r e t i o n enters the t h i r d (Rasquin, 1958;  Friedrick-Freksa  Holmgren, 1959b; Hafeez and  (1932) and  few  Holmgren (1959) suggest that some of  i n v e s t i g a t i o n s on the p o s s i b l e  p i n e a l s e c r e t i o n have been reported, s t u d i e s are c o n t r a d i c t o r y Pflugfelder  ven^  Ford, 1 9 6 7 ) ,  the p i n e a l s e c r e t i o n passes d i r e c t l y i n t o the blood But r e l a t i v e l y  and  C y t o l o g i c a l s t u d i e s have i n d i c a t e d a h i g h c e l l u l a r  metabolism (Palayer,  tricle  de-  and  (1955, 1954,  and  capillaries.  function  of  the r e s u l t s of these  inconclusive. 1956a) suggested that w i t h i n  the  teleosts a pineal-pituitary relationship exists.  His observa-  t i o n s , based on pinealectomized guppies L e b i s t e s ,  indicated  hypertrophy of the adenohypophyses and  t h y r o i d , decreased growth  r a t e , a s l i g h t a c c e l e r a t i o n i n the appearance of secondary c h a r a c t e r i s t i c s i n young males, an i n c r e a s e the i n t e r r e n a l c e l l s , and t i n g i n s p i n a l curvature.  a disturbed  i n the a c t i v i t y  sex of  calcium metabolism r e s u l -  Subsequently, the  same author  73  ( P f l u g f e l d e r , 1956a)  found that the t h y r o i d a l hypertrophy  was  diminished by epiphysan or t h y r o x i n and that pinealectomized g o l d f i s h had a l t e r a t i o n s i n the pars d i s t a l i s and t h y r o i d sue ( P f l u g f e l d e r ,  tis-  1964).  Holmgren (1959b) r e p o r t e d s k e l e t a l a b n o r m a l i t i e s i n pinealectomized Fundulus h e t e r o c l i t u s which were attenuated by adm i n i s t e r a t i o n of beef p i n e a l e x t r a c t s .  In the same study, he  found that pinealectomy a f f e c t e d a decreased r a d i o c a l c i u m uptake while an i n c r e a s e was apparent f o l l o w i n g p i n e a l e x t r a c t ment.  treat-  Weisbart and Fenwick (1966) found no d i f f e r e n c e s i n  blood plasma l e v e l s of calcium f o l l o w i n g pinealectomy of goldfish. Contrary to the r e p o r t by P f l u g f e l d e r  ( 1 9 6 4 ) , Pang (1968)  found a decreased t h y r o i d a l c e l l height i n p i n e a l e c t o m i z e d Fundulus and Rasquin  (1958),  Holmgren (1959b) and Peter (1968)  found that pinealectomy had no e f f e c t on the t h y r o i d a l t i s s u e of various f i s h .  Peter (1968) a l s o r e p o r t s no e f f e c t of p i n e a l e c -  tomy on the i n t e r r e n a l c e l l s of p i n e a l e c t o m i z e d g o l d f i s h . Schohherr  ( 1 9 5 5 ) , Rasquin (1958) and Peter (1968) a l s o found  that pinealectomy had no e f f e c t on the gonads of f i s h .  Pang  ( 1 9 6 7 ) , however, d e s c r i b e d a delayed appearance of n u p t i a l c o l o r a t i o n i n p i n e a l e c t o m i z e d Fundulus but r e p o r t e d that t r o l s had\a s m a l l e r gonad s i z e .  con-  Whether t h i s means t h a t the  c o n t r o l s underwent gonadal atrophy, or that h i s pinealectomized f i s h showed gonadal hypertrophy i s not c l e a r .  Nevertheless, the  p o s s i b l e i n t e r n a l s e c r e t i o n of the p i n e a l c e l l s and the d i v e r s e e f f e c t s f o l l o w i n g pinealectomy i n some f i s h suggests that the  7k p i n e a l organ of the f i s h i s an endocrine organ. P r e v i o u s s t u d i e s on the e f f e c t of pinealectomy on the gonads of f i s h have not taken i n t o c o n s i d e r a t i o n the marked r e p r o d u c t i v e c y c l e s o f the experimental animals.  Pang (1967)  found d i f f e r e n c e s i n the degree that pinealectomy  affected  growth r a t e and gonad s i z e when pinealectomy was performed  dur-  i n g the i n i t i a l phase o f growth and sexual maturation i n Fundulus as opposed to when the same animals were pinealectomized d u r i n g the h e i g h t of growth and r e p r o d u c t i o n .  This indicates  that the f u n c t i o n s o f the p i n e a l organ i n f i s h v a r i e s between d i f f e r e n t times o f the r e p r o d u c t i v e c y c l e , among animals o f d i f f e r e n t ages, or both.  Therefore, i n t e s t i n g the e f f e c t of  pinealectomy on the r e p r o d u c t i v e system of f i s h ,  the annual  y e a r l y c y c l e must be considered and the animals should be t e s t e d at v a r i o u s stages throughout the c y c l e . The p r i n c i p a l aim of t h i s study was to examine the e f f e c t of pinealectomy on the gonads i n g o l d f i s h .  To achieve t h i s ,  the normal r e p r o d u c t i v e c y c l e of the g o l d f i s h was estimated under l a b o r a t o r y c o n d i t i o n s .  S t u d i e s were then undertaken to  f i n d when the r e p r o d u c t i v e c y c l e was most a f f e c t e d by i n c r e a s i n g day l e n g t h .  Finally,  the e f f e c t of pinealectomy was t e s t e d at  d i f f e r e n t times of the r e p r o d u c t i v e c y c l e and under d i f f e r e n t photoperiods. S u b s i d i a r y s t u d i e s were performed  to t e s t the e f f e c t of  pinealectomy on the t h y r o i d a l t i s s u e , i n t e r r e n a l t i s s u e , blood plasma osmotic c o n c e n t r a t i o n and blood plasma l e v e l s of C a , + +  C l ~ , and N a . +  75  MATERIALS AND METHODS  A l l g o l d f i s h used, together with t h e i r source and maintenance p r i o r to the s t a r t of experiments  have been d e s c r i b e d i n  S e c t i o n I, but i n the present experiment  the f i s h were allowed  to swim f r e e l y w i t h i n the h o l d i n g tank.  The o p e r a t i v e pro-  cedure o f pinealectomy i s to be found i n the same s e c t i o n . The h i s t o l o g i c a l procedures and q u a n t i f i c a t i o n of h i s t o l o g i c a l r e s u l t s are d e s c r i b e d i n S e c t i o n I I .  Unless s p e c i f i c a l l y men-  t i o n e d , the f i s h were maintained i n water h e l d a t 20 C d u r i n g the experiments and were subjected to c o n t r o l l e d l i g h t r e gimens.  s  E s t i m a t i o n of Reproductive Cycle To estimate the seasonal v a r i a t i o n i n gonad s i z e of goldf i s h held under l a b o r a t o r y c o n d i t i o n s , a minimum of 10 male and 10 female g o l d f i s h were sampled d u r i n g each of s i x pres e l e c t e d months.  The photoperiod to which these f i s h were  exposed v a r i e d throughout approximated  the year and a t any given time  that of the n a t u r a l d a i l y photoperiod.  E f f e c t of Photoperiod on the Gonad Size a t D i f f e r e n t Times of the" Year To e s t a b l i s h the e f f e c t of photoperiod a t v a r i o u s times of the year, groups of f i s h were exposed t o photoperiods o f IL (one hour of l i g h t a l t e r n a t i n g with 2 3 hours of darkness), 8L ( e i g h t hours of l i g h t a l t e r n a t i n g with 16 hours of darkness),  76  16L (16 hours of l i g h t a l t e r n a t i n g with 8 hours of darkness), or  24L (continuous l i g h t ) d u r i n g the i n t e r v a l s Dec. 23 -  March 21, A p r i l 5 - May 20, Aug. 1 to 10, and Oct. 28 - Dec. 14.  The f i s h were k i l l e d and the mean gonad  s i z e (GSI =  gonad weight/body weight x 100) f o r male and female g o l d f i s h were c a l c u l a t e d s e p a r a t e l y .  The mean GSI v a l u e s c a l c u l a t e d  were based on an average sample  s i z e of 11 (Range:  4-I8).  E f f e c t of Pinealectomy on the GSI Table X l i s t s the d u r a t i o n and t i m i n g of experiments t e s t i n g the  e f f e c t o f pinealectomy on the GSI of male and female gold-  f i s h a t d i f f e r e n t times of the year and under d i f f e r e n t photop e r i o d s and l i s t s other parameters when t e s t e d . of  Since many  the d a t a r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s between the  treatments they w i l l not be i n c l u d e d i n the r e s u l t s . the  s i z e and scope of each experiment together with i n d i v i d u a l  sample out  However,  s i z e s are given (Table X ) . A l l experiments were c a r r i e d  a t the photoperiods i n d i c a t e d i n Table X.  Experiments 1,  2, and 3 were performed i n s t a n d i n g water at 20 C and E x p e r i ment 4 was performed i n water a t 13 C +1.0 C i n t o which  fresh  water was allowed to flow (overflow was removed by a standpipe a t one end). pinealectomized,  Each experiment c o n s i s t e d of groups of sham-pinealectomized and unoperated c o n t r o l s .  The c o n t r o l and experimental f i s h f o r any one experiment and photoperiod were maintained together i n a q u a r i a of approximately 350 l i t e r s f i l l e d  to c a p a c i t y with d e c h l o r i n a t e d water.  Equal p a r t s of NaCl and C a C l  P  were added i n s u f f i c i e n t  .TABLE X Experiment number and Duration  Scope and timing of pinealectomy experiments. Hours of -, light-  Treai ment  £  Osmotic Pressure  GSI  JL _ i P S N P S  8 25/6/66  to  8/8/66 16 -  2  1  28/10/66  to  14/12/66  8  16  24  if-  P s N P S N P S N  13 15  Number of f i s h tested Histology Plasma Ca Interrenal^ Thyroid^" Gonads  £_ 11  15  5  5  5  16 11 16  11  5  5  5  16 12 17  10  5  5  5  13 14 13  14  5  5  5  11 13 13  ll  5  5  5  5  5  5  •12  -8 13  8  6 13  5  5  5  7  5  5  8  9  5  5  5  4  5  4  6  8  3  5  5  5  5  4  11  8  5  5  k 18  4  5  8  9  5  5  6  P S  9  5  5  7 15  5  5  N  6  5  5  9  TABLE X (Cont.) Experiment Number and Duration  Number o f f i s h Hours of , Light 1  1 5A/67  to  20/5/67  8  16  24  I  5  9/1/68  to 3/5/68  8  Treatment^  GSI  JL  Osmotic Pressure o* ?  Plasma Ca <T ?  tested  Histology 3 4 Interrenal-^ T h y r o i d ^ Gonads <?  ?  P  n  S  12 17  N  20  P  17 13  S  17 11  N  16 15  P  11 18  5  5  S  13 16  5  5  N  12 17  5  5  P  15 14  S  16 12  N  14 17  P  18 18  5  5  5  5  S  18 18  5  5  5  5  N  18 18  5  5  5  5  17 7  Hours of l i g h t the animals were exposed to each day. Treatment; P, p i n e a l e c t o m i z e d , S, sham-pinealectomized, N, unoperated  controls.  I n t e r r e n a l n u c l e a r diameters were measured. R e l a t i v e height of t h y r o i d a l e p i t h e l i u m was determined. Temperature  of water was 15 C as opposed  to 20 C f o r a l l other experiments.  79 q u a n t i t y to b r i n g the f i n a l s a l t c o n c e n t r a t i o n to 0.2%. the case of Experiment k,  In  the s a l t s were added d a i l y to r e -  p l a c e l o s s through the overflow p i p e .  Treatment with  salt,  together with the a d d i t i o n o f Malachite Green (one drop o f 1% aqueous s o l u t i o n to each 100 l i t e r s of water), adjusted f o r the low l e v e l of d i s s o l v e d s o l i d s i n Vancouver c i t y water, and prevented  Saprolegnia i n f e c t i o n of the g o l d f i s h .  In a d d i t i o n to the i n v e s t i g a t i o n s l i s t e d i n Table X, 10 pinealectomized,  10 sham-pinealectomized and 10 unoperated  c o n t r o l f i s h were placed i n 50% sea water and e q u i v a l e n t numbers of the same groups were placed i n f r e s h water. ++ weeks the plasma osmolar c o n c e n t r a t i o n of Ca  A f t e r two  , CI  + and Na  were determined together with the m e l t i n g p o i n t s of the plasma from the d i f f e r e n t groups. A. C o l l e c t i o n of Plasma G o l d f i s h were anaesthetized as d e s c r i b e d e a r l i e r , d r i e d l i g h t l y with a paper towel, and descaled from the r e g i o n post e r i o r to the vent.  The caudal peduncle was severed p o s t e r i o r  to the anal f i n and the blood was allowed h e p a r i n i z e d c a p i l l a r y tube.  The tubes were sealed with a mix-  ture of p a r a f f i n wax and petroleum at 600X g f o r two minutes.  to run down a  jelly  ( 1 : 1 ) and c e n t r i f u g e d  The c a p i l l a r y tubes c o n t a i n i n g  the blood were cut at the j u n c t i o n between the plasma and the c e l l s and small samples of plasma were taken.  8o  B.  A n a l y s i s of Plasma M e l t i n g p o i n t s of plasma were determined  p o i n t determination apparatus designed (1955)*  with a m e l t i n g  by Ramsay and Brown  Small samples of the plasma were drawn i n t o f i n e (#40090, C e n t r a l S c i e n t i f i c Co.,  l a r y tubes  Vancouver).  samples were enclosed by p a r a f f i n o i l to prevent and were kept over s o l i d CO2 of blood were determined  u n t i l analysis.  on the day  Concentrations of the c a t i o n Na  capilThese  evaporation  Melting points  of c o l l e c t i o n . +  were analysed by  emission  flame photometry u s i n g a Unicam S P 9 0 0 A Spectrophotometer (Unicam Instruments  L t d . , Cambridge, England).  The plasma sample  was  d i l u t e d 5000X i n polyethylene v i a l s ( 1 0 ml) u s i n g Drummond Microcaps  (Drummond S c i e n t i f i c Co.,  Broomall, Pa.).  The  were s t o r e d over s o l i d CQ^ p r i o r to sample a n a l y s i s . taken to analyse only a f t e r the sample had reached ture.  vials  Care  was  room tempera-  As a check on the q u a n t i t a t i v e accuracy of t h i s c a t i o n  determination, d i l u t i o n s of Harleco Serum C o n t r o l (#64098 Harlman-Leddon Co.,  P h i l a d e l p h i a , Pa) were a l s o analysed  by  flame photometry. C h l o r i d e c o n c e n t r a t i o n s were measured p o t e n t i o m e t r i c a l l y on 1 m i c r o l i t e r  ( 0 . 0 0 1 ml) plasma samples d i l u t e d i n 20  micro-  l i t e r s of 50% a c e t i c a c i d a f t e r the method of Ramsay ( 1 9 5 5 ) .  A  Radiometer pH Meter 25SE (Radiometer, Copenhagen, Denmark) and a Misco V i b r a t i n g S t i r r e r  (#1385 Microchemical  Berkeley, C a l i f o r n i a ) with a 60 former and  the s o l e n o i d was  ohm  Specialities  Co.,  r e s i s t o r between the t r a n s -  used.  Calcium c o n c e n t r a t i o n s i n plasma were measured  fluoro-  81  m e t r i c a l l y on 2 0 m i c r o l i t e r samples of plasma.  The determina-  t i o n s were made on a Turner Model I I I Fluorometer  (G.K. Turner  A s s o c i a t e s , Palo A l t o , C a l i f o r n i a ) u s i n g the method d e s c r i b e d i n the Turner manual with the exception that Harleco Serum Cont r o l was used to o b t a i n standard curves.  S t a t i s t i c a l Methods With the exception of the h i s t o l o g i c a l appearance of the gonads, on which no q u a n t i f i a b l e o b s e r v a t i o n s were made, a l l groups of data obtained on any parameter from any one experiment were t e s t e d s e p a r a t e l y by a n a l y s i s of v a r i a n c e .  When s i g n i f i c a n t  d i f f e r e n c e s (P < . 0 5 ) were found, i n d i v i d u a l d i f f e r e n c e s between means were t e s t e d with Tukey*s w procedure.  82  RESULTS  Seasonal V a r i a t i o n i n Gonad Size G o l d f i s h e x h i b i t e d marked seasonal d i f f e r e n c e s i n gonad size (Figure 4 ) «  The maximum GSI value f o r both males and f e -  males was found d u r i n g the month of May, and c o i n c i d e d with the normal spawning p e r i o d of these animals (Yamazaki, 1 9 6 5 ) . nature,  In  t h i s peak would d e c l i n e r a p i d l y d u r i n g o v u l a t i o n or  spermiation  (Yamazaki, 1 9 6 5 ) .  Under l a b o r a t o r y c o n d i t i o n s the  r a p i d d e c l i n e f a i l e d "to appear; the gonads.regressed  slowly  d u r i n g the summer, s t a b i l i z e d a t a small s i z e d u r i n g the winter, and  underwent renewed growth i n March.  Thus, although  seasonal  v a r i a t i o n s i n the GSI of g o l d f i s h held under a r t i f i c i a l ' , t i o n s only p a r t i a l l y r e f l e c t the normal annual general trend approximates that seen i n nature. experimental  condi-  c y c l e , the Further, the  r e s u l t s a l l o w i n f e r e n c e s to be drawn about the  general s t a t e of the animal's r e p r o d u c t i v e physiology  at the time  of the v a r i o u s pinealectomy experiments.  Seasonal V a r i a t i o n o f the E f f e c t of Photoperiod  on the Gonad  Size Figure 5 summarizes the e f f e c t of d i f f e r e n t  photoperiods  on the GSI's of male and female g o l d f i s h d u r i n g d i f f e r e n t of t h e i r annual r e p r o d u c t i v e c y c l e .  times  With few exceptions, the  y e a r l y c y c l e d e s c r i b e d p r e v i o u s l y p e r s i s t s r e g a r d l e s s of the photoperiod  under which the animals were h e l d .  Further,  only  d u r i n g the p e r i o d A p r i l 5 to May 20 ( F i g . 5) was a marked and  83  F i g u r e 4.  Seasonal v a r i a t i o n of the GSI i n male and female g o l d f i s h under l a b o r a t o r y c o n d i t i o n s . Values are means + 95% confidence i n t e r v a l , each mean i s based on 10 animals.  •—• o—o  _J  Jan  l  i  I  Feb MarchApril  I  I  J  May June J u l y  I  Aug  females males  i  i  Sept Oct  i  i  Nov  Dec  84  Figure 5.  Seasonal v a r i a t i o n of photoperiod e f f e c t on the gonad s i z e of male and female g o l d f i s h r e p r e s e n t i n g gonads as percent of body weight (GSI). (IL, 1 hour of l i g h t a l t e r n a t i n g with 23 hours of darkness; 8L, 8 hours of l i g h t a l t e r n a t i n g with 16 hours of darkness; 16L s i x t e e n hours of l i g h t a l t e r n a t i n g with 8 hours of darkness; 2UL, continuous light). Animals were held under the s p e c i f i e d photoperiod f o r a minimum of 48 days.  GS I  —r—  1—  CJ1  —r-  "T"  O  : :  6 q,  O  i |  —T"  • • • • i j  i  i |  6 •i • • to+o+o-rO  PJ (U PJ PJ pj pj pj <-+• r+ r+ r-4- r+ r+  O ) oo . o > oo -> r ~ r ~ r ~ r ~ r~ r r  85  c o n s i s t e n t i n c r e a s e i n the gonad s i z e evident with i n c r e a s i n g day l e n g t h .  Females a l s o showed a c o n s i s t e n t i n c r e a s e i n ovary  s i z e with i n c r e a s e d photoperiod d u r i n g the p e r i o d from Dec. to the  March 21 while the males d i d not.  23  T h i s i n c r e a s e was not of  same magnitude as that seen l a t e r i n the s p r i n g .  Thus, i n -  creased l i g h t exposure a c t s as a gonadal stimulus d u r i n g only p a r t of the year. In  summary, the s i z e of the gonads i n male and female gold-  f i s h i s under at l e a s t a dual c o n t r o l . genous rhythm  The f i r s t i s an endo-  which i s not dependant upon environmental cues  such as temperature and photoperiod and f u n c t i o n s c o n t i n u o u s l y throughout the year.  The second shows a c y c l i c a l r e s p o n s i v e -  ness to environmental photoperiods, the peak responsiveness c o i n c i d i n g with the peak gonadal growth under the endogenous cycle.  E f f e c t of  Pinealectomy on the Gonad Size  Throughout 14,  the p e r i o d s June 25 to Aug. 8,  and A p r i l 5 to May  20,  Oct. 28 to Dec.  pinealectomy had no e f f e c t on the  gonad s i z e i n male or female g o l d f i s h , r e g a r d l e s s of the photop e r i o d under which they were h e l d . of  Jan. 9 to May 3 ,  During the longer i n t e r v a l  pinealectomy enhanced the s i z e of the go-  nads i n both male and female g o l d f i s h .  The d a t a from t h i s  l a t t e r experiment are summarized i n Tables XI and X I I . XI l i s t s  Table  the mean GSI v a l u e s f o r groups of 18 male or female  pinealectomized, sham-pinealectomized  and unoperated  g o l d f i s h , and Table XII p r e s e n t s a two way  control  a n a l y s i s of v a r i a n c e  86  TABLE XI E f f e c t of pinealectomy on the gonad s i z e of male and female g o l d f i s h d u r i n g the p e r i o d Jan. 9 to May 3 . *  Sex  Treatment  Males  Pinealectomized  2.56  4.28  Sham-pinealectomized  1.08  2.18  Unoperated  0.91  1.50  controls  Females  *Each mean i s taken from 18 f i s h held at 13 C + 1 C and an eight hour photoperiod. Tukey*s w , = 1 . 3 2 . n  under  87  TABLE XII A n a l y s i s of v a r i a n c e of the GSI v a l u e s of 18 p i n e a l ectomized, sham-pinealectomized, and unoperated cont r o l g o l d f i s h o f both sexes d u r i n g the p e r i o d Jan. 9 to May 3 .  Source  df  SS  MS  F  Sex  1  35.20  35.20  27.93  F  > 0 1  (1,102)  6.96  Treat— ment  2  99.40  49-70  39.44  F  > 0 1  (2,102)  4.93  Interaction  2  5.74  2.87  2.28  102  128.72  1.26  Error  Therefore Sex and Treatment are h i g h t l y I n t e r a c t i o n i s not s i g n i f i c a n t .  significant.  88 on the data from which the means were taken. ference at the 99% l e v e l was procedure).  A significant  c a l c u l a t e d at 1.32  dif-  (Tukey s w 1  Using t h i s value to t e s t the d i f f e r e n c e s between  d i f f e r e n t groups of males and females i t was p i n e a l e c t o m i z e d males and  found that the  females had s i g n i f i c a n t l y  (P<.01)  l a r g e r GSI v a l u e s than the corresponding sham-operated or unoperated c o n t r o l s . significant  The sham o p e r a t i o n d i d not produce  any  effect.  E f f e c t of Pinealectomy on the H i s t o l o g y of Reproductive and Endocrine T i s s u e Pinealectomy d i d not e f f e c t any changes i n the diameter of the i n t e r r e n a l n u c l e i , c e l l height of t h y r o i d a l e p i t h e l i u m , or the general h i s t o l o g i c a l appearance  of the t e s t i s or o v a r i e s  i n any of the experiments i n which these t i s s u e s were s t u d i e d (Table X).  E f f e c t of Pinealectomy on the E l e c t r o l y t e System The m e l t i n g p o i n t of blood plasma, t i o n s Na , +  the l e v e l s of the ca-  and C a , and the l e v e l of the anion C l ~ were not + +  a l t e r e d by pinealectomy under any of the c o n d i t i o n s t e s t e d . O c c a s i o n a l l y v a r i a t i o n s i n these parameters r e s u l t e d f e r e n c e s i n the environment  from  dif-  (photoperiod, s a l i n i t y ) but the  v a r i a t i o n s were not a f f e c t e d by  pinealectomy.  89  DISCUSSION  Pinealectomy of mammals (Wurtman, A l t s c h u l e and 1959;  Holmgren,  Motta, F r a s c h i n i and M a r t i n i , 1 9 6 7 ) , b i r d s (Izawa,  S h e l l a b a r g e r and  Breneman, 1 9 5 0 ) , and l i z a r d s (Stebbins, I960)  produces gonadal hypertrophy. r e s u l t was  1923;  Under s p e c i f i c c o n d i t i o n s , t h i s  d u p l i c a t e d i n the present  study on  goldfish.  Reported e f f e c t s of pinealectomy on the r e p r o d u c t i v e  system  P f l u g f e l d e r (1956a) demonstrated  of f i s h are c o n t r a d i c t o r y .  a r e l a t i o n s h i p between the p i n e a l organ and reproductive, maturat i o n i n male L e b i s t e s r e t i c u l a t u s , but h i s f i n d i n g s have not been s u b s t a n t i a t e d i n Gasterosteus Astyanax mexicanus (Rasquin, f i s h C a r a s s i u s auratus  1958)  (Schonnherr, 1 9 5 5 ) ,  aculeatus  or p r e v i o u s l y i n the gold-  (Peter, 1 9 6 8 ) .  T h i s discrepancy  may  be  accounted f o r by s p e c i e s d i f f e r e n c e s i n the f u n c t i o n of the p i n e a l organ or by d i f f e r e n c e s i n experimental In the present  study  design.  the g o l d f i s h were found to have an  endogenous c y c l e i n gonad s i z e which p e r s i s t e d under a l l photoperiods tested.  Further, a marked i n c r e a s e i n gonad s i z e  f o l l o w i n g prolonged  d a i l y l i g h t exposure was  evident only  during  the i n t e r v a l w i t h i n which the gonads would endogenously i n c r e a s e in size.  Based on these  f i n d i n g s , i t was  e f f e c t of pinealectomy on the r e p r o d u c t i v e f i s h would vary d u r i n g the course  p o s t u l a t e d that the system of the g o l d -  of the endogenous c y c l e or  a l t e r the endogenous c y c l e . The  GSI  ectomized and  values obtained  from pinealectomized,  sham-pineal-  c o n t r o l animals held under d i f f e r e n t  photoperiods  90  at v a r i o u s times of the r e p r o d u c t i v e p i n e a l gland  c y c l e i n d i c a t e that the  does not c o n t r o l the endogenous c y c l e ; that i s ,  removal of the p i n e a l gland d i d not a l t e r the t i m i n g of the whole endogenous c y c l e .  Pinealectomized  g o l d f i s h had maximum  and  minimum GSI values a t the same time as the sham operated  and  c o n t r o l f i s h and the o p e r a t i o n  the year.  had no e f f e c t d u r i n g most of  Pinealectomy d i d , however, enhance the s i z e of the  gonads when the o p e r a t i o n  was performed j u s t p r i o r (Jan. 9) to  the time when the gonads endogenously i n c r e a s e d . s u l t s were not obtained  from animals pinealectomized  time ( A p r i l 5) when the gonads were already genously.  Similar r e d u r i n g the  i n c r e a s i n g endo-  The time at which pinealectomy was e f f e c t i v e a l s o  c o i n c i d e d with the p e r i o d during which the i n c r e a s i n g photop e r i o d i s capable of s t i m u l a t i n g gonadal enlargement.  These  r e s u l t s i n d i c a t e that the p i n e a l gland i s a s s o c i a t e d with the t i m i n g of the responsiveness of the hypothaiamous, p i t u i t a r y or gonads .to i n c r e a s i n g day length, gland lowers the threashold  so that removal o f the p i n e a l  of day l e n g t h r e q u i r e d  s t i m u l a t i o n or enhances the photoperiodic  f o r gonadal  efficiency.  Thus,  the data i n d i c a t e a f u n c t i o n a l r e l a t i o n s h i p between the p i n e a l organ, photoperiod, and r e p r o d u c t i v e  development.  This rela-  t i o n s h i p has been demonstrated i n mammals (Hoffman and R e i t e r , 1966;  Roth, 1 9 6 4 ; R e i t e r and Hester, The  lower GSI values  1966).  of a l l c o n t r o l groups held a t 13 C  as opposed to those held at 20 C under eight hours of l i g h t per day  and a t the same time of year suggests a temperature  I t i s w e l l documented ( P i c k f o r d and Atz, 1 9 5 7 ) that  effect.  temperature  91  has a marked e f f e c t on the r e p r o d u c t i v e system i n f i s h . ( c i t e d i n P i c k f o r d and Atz, 1957) ;  Butler  found that g o l d f i s h held at f\;  9 C possessed t e s t e s i n a r e s t i n g s t a t e while those a t 21 C had all  stages of spermatogenesis present w i t h i n t h e i r t e s t e s .  the r e s u l t s obtained by pinealectomy of f i s h at 13 C may  Thus  indicate  a change i n the thermal requirements f o r gonadal growth.  How-  ever, as p i n e a l e c t o m i z e d male and female g o l d f i s h had l a r g e r values (2.56  and k.2.8  at 20 C ( 1 . 9 0  r e s p e c t i v e l y ) at 13 C than normal  GSI  animals  and 3• 86'..'for males and females) at the same time  of year and under the same photoperiod, the i n c r e a s e i n the  GSI  f o l l o w i n g pinealectomy i s more than j u s t a change i n thermal r e quirements. Data concerning the e f f e c t s of pinealectomy on the e l e c t r o l y t e system and the i n t e r r e n a l t i s s u e shows no between the p i n e a l gland and t h i s system and ate the d i s t u r b e d calcium metabolism ( P f l u g f e l d e r , 1963;  Holmgren, 1 9 5 9 h ) .  relationship  f a i l s to c o r r o b o r -  seen f o l l o w i n g pinealectomy These authors based  their  o p i n i o n s on s k e l e t a l a b n o r m a l i t i e s or changes i n uptake of r a d i o a c t i v e calcium, both of which could occur without t i o n s i n the plasma C a  + +  altera-  levels.  F a i l u r e to f i n d any a l t e r a t i o n i n the i n t e r r e n a l conforms with the f i n d i n g s of P f l u g f e l d e r  nuclei  (1964) and Peter (1968)  on the g o l d f i s h , and Pang (1967) on the k i l l i f i s h Ftlndulus h e t e r o c l i t u s , but i s at v a r i a n c e w i t h the r e p o r t e d i n c r e a s e d a c t i v i t y of i n t e r r e n a l c e l l s i n p i n e a l e c t o m i z e d guppies felder, 1953).  (Pflug-  However, the r e s u l t s agreed with those obtained  from m e l t i n g p o i n t s and i o n i c l e v e l s of the plasma s i n c e they  92  a l s o f a i l e d to respond to pinealectomy. The  hyperthyroidism  g o l d f i s h and  reported by P f l u g f e l d e r (1964) on  guppy ( P f l u g f e l d e r , 1953,  pinealectomy was  1954,  the  1956a) f o l l o w i n g  not s u b s t a n t i a t e d by t h i s study.  This result  agrees with those of Rasquin (1958) on Astyanax mexicanus, Pang (1967) on Fundulus h e t e r o c l i t u s and Peter A l l data considered,  (1968) on  goldfish.  the p i n e a l gland of the g o l d f i s h  appears to exert an i n h i b i t o r y i n f l u e n c e s p e c i f i c a l l y on reproductive  system.  The  the  endogenous rhythm e s t a b l i s h e s the  phase of s e n s i t i v i t y to photoperiodic  illumination.  p e r i o d s e t s the l e v e l of photic s t i m u l a t i o n . p i n e a l gland which s e t s the  The  photo-  But i t i s the  threshold' l e v e l of i l l u m i n a t i o n  d u r i n g the s e n s i t i v e phase of the endogenous rhythm. During much of the year,  the phase of the endogenous c y c l e  i s such t h a t the r e p r o d u c t i v e system i s i n s e n s i t i v e to i n c r e a s ing daily light  exposure.  At t h i s time, the i n h i b i t o r y a c t i o n  of the p i n e a l organ i s superfluous. c y c l e enters the  However, as the endogenous  photosensitive,phase  i n f l u e n c e becomes o p e r a t i o n a l .  the p i n e a l ' s i n h i b i t o r y  T h i s i n h i b i t o r y a c t i o n may  slow  down the development of the gonads r e s u l t i n g from the i n c r e a s i n g photoperiods  which begin i n mid-winter and maintain  at a low l e v e l of development u n t i l the photoperiod i t s effective  the gonads has reached  threshold:./  Such a system would account f o r the a c c e l e r a t e d i n c r e a s e i n the s i z e of the gonads d u r i n g the month of A p r i l as opposed to the slow i n c r e a s e i n the preceeding the photoperiod  i s increasing.  The  three months d u r i n g which  p i n e a l does not a l t e r  the  93  phase of the endogenous c y c l e , but i t does i n h i b i t the response to the i n c r e a s i n g photoperiod. T h i s s y n c h r o n i z a t i o n of the gonadal development with the photoperiod could have a marked  e f f e c t on the e f f i c i e n c y of the  r e p r o d u c t i o n of the p o p u l a t i o n as a whole.  I f the endogenous  c y c l e and the photoperiod operated alone, i n d i v i d u a l  variations  i n the t i m i n g of the phases of s e n s i t i v i t y would produce i n d i v i d u a l responses f e r e n t times.  to photoperiod and animals would mature at d i f -  By adding the p i n e a l gland to the system, two  s y n c h r o n i z i n g mechanisms are at work:  (1)  the endogenous  c y c l e must be i n the phase which i s s e n s i t i v e to i n c r e a s i n g photoperiods, and (2)  the photoperiod must i n c r e a s e to a c e r -  t a i n t h r e s h o l d to d e a c t i v a t e the p i n e a l ' s i n h i b i t o r y  influence.  94  GENERAL DISCUSSION  The  p i n e a l organ of f i s h e s has (Young, 1935;  sensitive structure Dodt, 1963)  intimately associated  (Breder and  Rasquin, 1947;  been d e s c r i b e d  Breder and  A l t n e r , 1965)  ( F r i e d r i c k - F r e k s a , 1932;  Friedrick-Freksa  (1932) and  I t has  of undetermined f u n c t i o n .  Hafeez and  the  gren, 1959a; and  cerebrospinal  Ford, 1967)  1 9 5 6 a , 1964; by  although  vessels.  p h o t o r e c e p t i o n and  f i s h p i n e a l organ (Palayer,  Rudeberg, 1968b) and  are r e l a t e d to the  that one  secre-  1958;  Holm-  or both of these  endocrine system ( P f l u g f e l d e r , 1953,  Pang, 1967)  some (Rasquin, 1958;  The  Holmgren (1959a) suggested that some  i t appears that both f u n c t i o n s ,  t i o n , occur w i t h i n the  also  organ (Holmgren, 19l8a,b; Holm-  of the p i n e a l s e c r e t i o n i s taken up by p i n e a l blood Now  1950;  behaviour  Pang, 1 9 6 6 ) .  s e c r e t i o n i s g e n e r a l l y regarded as e n t e r i n g fluid  as a photo-  Rasquin,  with p h o t o t a c t i c  Hoar, 1955;  as a s e c r e t o r y  gren, 1958b; and  been d e s c r i b e d  1954,  though such a r e l a t i o n s h i p i s denied Holmgren, 1959b; Peter,  1968;  Peter  and  Gorbman, 1 9 6 8 ) . Within two  c l o s e l y r e l a t e d s p e c i e s of P a c i f i c  corhynchus nerka and  0.  salmon  tshawytscha) the p i n e a l organ was  to f u n c t i o n as a photoreceptor (Hoar, 1955)  and,  to produce the hormone melatonin.  i n various  Further,  (Onfound  i n t h i s study, studies  on the rainbow t r o u t Salmo g a i r d n e r i i i r i d e u s , Dodt (1963) Morita could  and  (1966) found a c t i o n p o t e n t i a l s i n the p i n e a l body which be a l t e r e d by l i g h t .  Quay (1965h) l o c a l i z e d w i t h i n  p i n e a l organ the enzyme r e s p o n s i b l e  the  f o r melatonin s y n t h e s i s ,  and  95  Oguri et a l . (1968) reported that r a d i o a c t i v e phane, a p r e c u r s o r of melatonin,  was  taken up i n greater  amounts i n t o the p i n e a l t i s s u e than any The  present  5-hydroxytrypto-  other t i s s u e s t u d i e d .  study i n d i c a t e s that the p i n e a l body of the  g o l d f i s h i s i n v o l v e d i n n e u r a l phenomena as e x e m p l i f i e d by e f f e c t of pinealectomy and  on v a r i o u s b e h a v i o u r a l responses to l i g h t  on swimming a c t i v i t y .  have endocrine  the  F u r t h e r , the p i n e a l gland appears to  e f f e c t s s i n c e i t s removal d u r i n g part of the  year r e s u l t s i n an i n c r e a s e i n the s i z e of the gonads.  This  l a t t e r e f f e c t appears to be r e l a t e d to the p h o t o s e n s i t i v e  nature  of the p i n e a l organ or to responses to p h o t i c i l l u m i n a t i o n . Pinealectomy only produces a response of the gonads d u r i n g time when photoperiod F u r t h e r , melatonin,  i s capable  the  of s t i m u l a t i n g the gonads.  the presumed p i n e a l hormone, i s capable  i n h i b i t i n g t h i s l i g h t induced  i n c r e a s e i n gonad s i z e .  This  l e a d s to the c o n c l u s i o n that the p i n e a l organ of the f i s h e s two  of  has  f u n c t i o n s which are under a s i n g l e c o n t r o l ; that i s , the  p i n e a l has n e u r a l as w e l l as endocrine are r e l a t e d to the p h o t o r e c e p t i v e The  b e h a v i o u r a l e f f e c t s and  f u n c t i o n s , both of which  nature  of the p i n e a l organ.  the e n d o c r i n e , e f f e c t s of the  :  p i n e a l are both a s s o c i a t e d with i t s p h o t o r e c e p t i v e  ability.  Within the mammals, the p i n e a l has l o s t i t s d i r e c t  photosensory  role  (Kappers, 1965)  t u r e s ( K e l l y , 1962) Nevertheless,  and  i s devoid  present  of the p h o t o r e c e p t i v e  struc-  w i t h i n the f i s h p i n e a l organs.  the p i n e a l organ of mammals remains i n p a r t under  the c o n t r o l of environmental  i l l u m i n a t i o n ( F i s k e et a l . , 1962;  Quay, 1 9 6 3 ^ , c ; Snyder, Zweig and  Axelrod,  1964;  Zweig et a l . ,  96  1966) and  by way of the eyes (Wurtman, 1 9 6 7 ; R e i t e r and Hester, 1 9 6 6 ) sympathetic nervous system (Snyder et a l . . 1 9 6 6 ; R e i t e r and  Hester, 1966)and. as such i s f u n c t i o n a l l y s i m i l a r to that o f the f i s h p i n e a l organ.  The d i f f e r e n c e s between the d i r e c t e f f e c t s  of l i g h t on the p i n e a l gland of f i s h e s and the i n d i r e c t e f f e c t s of l i g h t on the p i n e a l gland of mammals represent  only a change  i n the pathway by which l i g h t a f f e c t s the glands r a t h e r than a d i f f e r e n c e i n the f u n c t i o n o f the g l a n d s themselves. The  data from the present i n v e s t i g a t i o n i n d i c a t e that the  e f f e c t s of the p i n e a l on the r e p r o d u c t i v e  system are mediated by  melatonin a c t i n g on the p i t u i t a r y gland.  From p r e v i o u s  studies  on mammals ( F r a s c h i n i et a l . , 1 9 6 8 ) i t i s suggested that the e f f e c t s of melatonin on the p i t u i t a r y are due to the presence o f receptors tonin.  w i t h i n the hypothalamus which are responsive to"mela-  Melatonin a d m i n i s t r a t i o n  i n mammals, a s i d e  from the endo-  c r i n e e f f e c t s (Wurtman et a l . , 1 9 6 1 ) a l s o produces neural  effects  (Fiske and Huppert, 1 9 6 8 ) i n c l u d i n g the e l e v a t i o n of the serotonin concentration cortex,  i n the mid b r a i n but not i n the c e r e b r a l  o l f a c t o r y bulbs or t u b e r c l e  (Anton-Tay et a l . , 1 9 6 8 ) .  As noted e a r l i e r ( F i g . 2 ) removal of the p i n e a l organ of goldf i s h resulted i n increased whole b r a i n s e r o t o n i n l e v e l  swimming a c t i v i t y but d i d not a l t e r (Table V).  But the p o s s i b i l i t y of  l o c a l i z e d changes i n s e r o t o n i n l e v e l was mentioned.  I f the  changes i n s e r o t o n i n l e v e l i n the mid b r a i n r e s u l t from melat o n i n , then removal of the p i n e a l , which may be the p r i n c i p a l source of melatonin, could r e s u l t i n a decreased l e v e l i n the mid b r a i n of g o l d f i s h , which could  serotonin then a l t e r  local  97  n e u r a l a c t i v i t y and r e s u l t -in the i n c r e a s e d swimming a c t i v i t y . On the other hand, the i n c r e a s e d swimming a c t i v i t y could have r e s u l t e d from an i n c r e a s e d p r o d u c t i o n of gonadal s t e r o i d s lowing pinealectomy.  fol-  T h i s seems u n l i k e l y , however, as i t would  not account f o r the even g r e a t e r i n c r e a s e i n swimming a c t i v i t y seen i n b l i n d e d g o l d f i s h . activity  F u r t h e r , the i n c r e a s e i n swimming  f o l l o w i n g pinealectomy was observed i n g o l d f i s h which  were p i n e a l e c t o m i z e d d u r i n g that part of the year when p i n e a l ectomy was found to have no e f f e c t on the s i z e of the gonads. Thus, the f u n c t i o n s of the p i n e a l gland, both n e u r a l and endocrine may depend upon i t s p r o d u c t i o n of melatonin.  Fur-  ther, the n e u r a l e f f e c t s may be d i r e c t l y r e l a t e d to the endocrine effects.  Melatonin, by a l t e r i n g the l e v e l of s e r o t o n i n , could  e f f e c t changes i n the neuroendocrine a c t i v i t y i n the midbrain (Anton-Tay  et a l . , 1 9 6 8 ) which might  the hypothalamo-hypophyseal tuitary  then a l t e r the a c t i v i t y of  complex r e s u l t i n g i n a l t e r e d p i -  function.  In reexamining the working hypothesis of p i n e a l f u n c t i o n s of f i s h e s i n the g e n e r a l i n t r o d u c t i o n i t i s found that, with c e r t a i n l i m i t a t i o n s , no reasons were found to r e j e c t them and the evidence was s t r o n g l y i n favour of a c c e p t i n g them.  The p i -  neal gland of g o l d f i s h does mediate b e h a v i o u r a l responses to environmental i l l u m i n a t i o n .  I t c o n t a i n s melatonin i n the s a l -  mon and melatonin has the hormonal l i k e q u a l i t y of i n h i b i t i n g gonadal growth i n the g o l d f i s h .  F u r t h e r , removal o f the p i n e a l  organ i n the g o l d f i s h , which presumably  c o n t a i n s melatonin,  enhances the s i z e of the gonads when the o p e r a t i o n i s performed  98  p r i o r to the onset of the endogenous gonadal i n the e a r l y p a r t of the year.  growth i n i t i a t e d  But pinealectomy  on the gonads at other times of the year.  has no  effect  Therefore, the p i -  neal gland i s a s s o c i a t e d with the r e p r o d u c t i v e system and s e r ves as one of the channels by which the r e p r o d u c t i v e system i s i n f l u e n c e d by the environmental  illumination.  99  SYNOPSIS  Now i t appears that the p i n e a l body of f i s h e s i s not a functionless, evolutionary On the contrary,  v e s t i g e of a p r i m i t i v e t h i r d eye.  the evidence suggests that i t performs a dual  f u n c t i o n and that both f u n c t i o n s depend upon l i g h t and p o s s i b l y melatonin s y n t h e s i s .  reception  Melatonin, produced  the f i s h p i n e a l organ governs l o c a l neural  within  a c t i v i t y w i t h i n the  b r a i n which i n turn c o n t r o l s neuroendocrine a c t i v i t y , behavio u r a l responses, or both.  In the mammals, the p i n e a l has l o s t  i t s d i r e c t p h o t o s e n s i t i v i t y but remains i n p a r t under the cont r o l of environmental i l l u m i n a t i o n and i s i n t h i s way s i m i l a r to the p i n e a l organ of f i s h e s .  Further,  both the mammalian  p i n e a l organ and the f i s h p i n e a l organ produce melatonin and t h i s substance has a n t i g o n a d a l e f f e c t s i n both groups.  Re-  moval of the p i n e a l organ i n mammals or g o l d f i s h r e s u l t s i n gonadal hypertrophy although i n the f i s h t h i s e f f e c t i s l i m i ted to c e r t a i n times of the r e p r o d u c t i v e  cycle.  The l e v e l of  melatonin i n the p i n e a l of f i s h e s and mammals i s r e l a t e d to the reproductive  system and i s d i r e c t l y i n f l u e n c e d  genous c y c l e , c o n d i t i o n s  of l i g h t , or both.  organ of f i s h i s both a sensory and s e c r e t o r y s e c r e t i o n a f f e c t s both neural  by the endo-  Thus, the p i n e a l structure. I t s  and endocrine a c t i v i t y so that  f u n c t i o n a l l y , the p i n e a l glands of mammals and f i s h e s are similar.  100  SUMMARIES  SECTION I 1. all  Pinealectomy, b l i n d i n g , or both had the same e f f e c t i n that three treatments r e s u l t e d i n the l o s s of the photonegative  response c h a r a c t e r i s t i c of wholly i n t a c t animals. 2.  G o l d f i s h with i n t a c t eyes but without a p i n e a l d i s p l a y e d  more swimming a c t i v i t y than i n t a c t animals.  Those f i s h without  eyes were more a c t i v e than f i s h with eyes. 3.  Although a c a u s a l r e l a t i o n s h i p was not e s t a b l i s h e d , the  amount of swimming a c t i v i t y i n g o l d f i s h was lowest i n those groups which possessed the h i g h e s t l e v e l of whole b r a i n serotonin.  Pinealectomy d i d not a f f e c t the l e v e l of whole b r a i n  serotonin. 4.  Pinealectomized g o l d f i s h with i n t a c t eyes were more respon-  s i v e to l i g h t as a c o n d i t i o n e d s t i m u l u s than were wholly i n tact controls.  B l i n d g o l d f i s h , with or without a p i n e a l , were  not e f f e c t i v e l y c o n d i t i o n e d to l i g h t although they were c o n d i tioned to sound. 5.  Removal of the p i n e a l organ r e s u l t e d i n a decrease i n the  voltage r e q u i r e d to j u s t produce a response but d i d not a f f e c t the response to the constant v o l t a g e used i n the c o n d i t i o n i n g experiments. 6.  Pinealectomy d i d not a f f e c t the c o n d i t i o n i n g of g o l d f i s h  101  to sound.  SECTION I I 1. and  Melatonin was l o c a l i z e d i n the p i n e a l organ o f the salmon was found i n higher c o n c e n t r a t i o n s  i n the p i n e a l of immature  salmon than i n the p i n e a l of mature salmon. 2.  Melatonin i n j e c t i o n i n g o l d f i s h i n h i b i t e d the endogenous  increase  i n gonad s i z e which occurs d u r i n g the s p r i n g and i n -  h i b i t e d the s t i m u l a t o r y  e f f e c t of i n c r e a s i n g daylength on the  gonads. 3.  G o l d f i s h i n j e c t e d w i t h melatonin had l a r g e r p i t u i t a r y  gonadotrophs and smaller  i n t e r r e n a l n u c l e i than u n i n f e c t e d or  solvent i n j e c t e d controls. k.  Melatonin i n j e c t i o n e f f e c t e d no changes i n t h y r o i d a l  tis-  sue.  SECTION I I I 1.  G o l d f i s h , held under n a t u r a l photoperiods and a t a con-  stant temperature, e x h i b i t e d a marked c y c l e i n gonad s i z e with both males and females having the l a r g e s t gonads d u r i n g May. 2.  Increasing  the photoperiod under which the g o l d f i s h were  held  stimulated  the gonads only d u r i n g  gonads would normally i n c r e a s e . the  the i n t e r v a l when the  Therefore,  i t d i d not e f f e c t  timing of the c y c l e as much as i t augmented the normal  increase.  102  3.  Pinealectomy i n c r e a s e d  the gonad s i z e only when performed •  near the time when the gonads would normally s t a r t to i n c r e a s e . k.  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