CARBOHYDRATE AND TRYPTOPHAN INDUCED INCREASE IN BRAIN SEROTONIN BIOCHEMICAL AND BEHAVIORAL CORRELATES by SUSAN EILERS CROWTHER B.A., The U n i v e r s i t y of B r i t i s h Columbia, 1973 . S c . , The U n i v e r s i t y of Prince Edward I s l a n d , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 usan E i l e r s Crowther, 1980 In p r e s e n t i n g t h i s thesis an advanced degree at the L i b r a r y I further in p a r t i a l fulfilment of the requirements the U n i v e r s i t y of B r i t i s h Columbia, I agree s h a l l make it freely available for agree t h a t p e r m i s s i o n for e x t e n s i v e copying o f of this representatives. thesis for It financial that reference and study. this thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s for or i s understood that copying or p u b l i c a t i o n gain shall not be allowed without my written permission. Department of HofY\^T\ hlofnhon The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 J <Aod ^ ti*ni- B&X>n\tt%, - ii - ABSTRACT Behavioral and biochemical correlates of the carbohydrate and tryptophan induced increase in brain serotonin were investigated in a s e r i e s of 4 experiments. Experiment 1 was conducted to e s t a b l i s h the nadir of brain tryptophan during the dark phase of the l i g h t cycle, Follow- ing a 16 hour fast., brains were removed., at 1600,' 1800, 2000, 2.400, and 0400, hrs f o r tryptophan determination. Analyses indicated no differences in brain tryptophan throughout the dark period. The time course and peak concentrations of the carbohydrate and tryptophan induced increase i n brain tryptophan and serotonin were determined in Experiment 2. Rats were fasted from 0030 to 1730 and then offered a control d i e t and i n j e c t e d with s a l i n e or 50 mg/kg tryptophan, or offered a high carbohydrate, p r o t e i n - f r e e meal and i n j e c t e d with s a l i n e . One hour a f t e r treatment and hourly f o r the next 3 hours, brains were obtained f o r analysis of tryptophan and serotonin. Tryptophan i n j e c t e d rats exhibited a peak i n brain tryptophan at 1 hour post i n j e c t i o n and a f a l l in tryptophan to control l e v e l s by 2 hours. Carbohydrate fed animals exhibited an increase in brain tryptophan at a l l times observed. Ele- vated brain serotonin was found in both tryptophan and carbohydrate treated animals. Experiment 3 was conducted to e s t a b l i s h a behavioral c o r r e l a t e of brain s e r o t o n i n . Behaviors investigated included: latency to step-down and explore a novel chamber and a c q u i s i t i o n and e x t i n c t i o n of a passive avoidance response. Animals were fed ad l i b i t u m , and 1 hour (1700) p r i o r to behavioral t e s t i n g „ i n j e c t e d with s a l i n e or 50 mg/kg tryptophan. Animals did not d i f f e r on measures of passive avoidance a c q u i s i t i o n or - iii extinction. - However, t r y p t o p h a n i n j e c t e d a n i m a l s were found t o exhibit a l o n g e r l a t e n c y t o step-down and e x p l o r e a novel chamber than c o n t r o l s . In Experiment 4 , plasma c o r t i c o s t e r o n e , latency to step-down, r e a r i n g , u r i n a t i o n , and d e f e c a t i o n i n a novel chamber were a s s e s s e d . Animals were f a s t e d from 2400 t o 1700 and i n j e c t e d and f e d as i n Experiment 2. One and 2 hours f o l l o w i n g t r e a t m e n t , b e h a v i o r s were o b s e r v e d . brains Thereafter, were removed f o r d e t e r m i n a t i o n o f t r y p t o p h a n and s e r o t o n i n and blood o b t a i n e d f o r plasma c o r t i c o s t e r o n e a n a l y s i s . In t r y p t o p h a n r a t s , b r a i n t r y p t o p h a n was observed t o peak a t 1 hour p o s t administered injection and t o remain h i g h e r than c o n t r o l s a t 2 hours post i n j e c t i o n . Carbohydrate f e d r a t s were found t o e x h i b i t h i g h e r l e v e l s o f b r a i n t r y p t o p h a n t h a n c o n t r o l a n i m a l s a t both times a s s a y e d . B r a i n s e r o t o n i n was found to peak i n t r y p t o p h a n t r e a t e d r a t s a t 1 hour p o s t i n j e c t i o n and t o remain e l e v a t e d at 2 hours. fed animals. No changes i n b r a i n s e r o t o n i n were r e v e a l e d i n carbohydrate No group d i f f e r e n c e s were observed f o r any of the b e h a v i o r a l measures t a k e n . However, i n c r e a s e d plasma c o r t i c o s t e r o n e was found i n r a t s fed the h i g h c a r b o h y d r a t e m e a l . These data r e v e a l e d t h a t i n j e c t i o n o f t r y p t o p h a n r e s u l t e d i n an i n c r e a s e d l a t e n c y t o step-down and e x p l o r e a novel chamber when animals were f e d ab l i b i t u m , whereas c a r b o h y d r a t e i n g e s t i o n r e s u l t e d i n an i n c r e a s e plasma c o r t i c o s t e r o n e w i t h no e f f e c t on b e h a v i o r . Confirmation that sero- t o n i n mediated these b i o c h e m i c a l and b e h a v i o r a l changes a w a i t s research. in further - iv - TABLE OF CONTENTS CHAPTER Page Abstract. L i s t of Tables L i s t of F i g u r e s L i s t o f Appendix T a b l e s Acknowledgement I II INTRODUCTION 1 REVIEW OF LITERATURE 4 1. III IV ii vi vii viii ix Carbohydrate and Tryptophan Induced Changes in B r a i n Tryptophan and the Indoleamines 4 2. S e r o t o n i n D i s t r i b u t i o n and B i o s y n t h e s i s 5 3. 4. Regulation of Serotonin Synthesis Tryptophan M e t a b o l i s m and R e g u l a t i o n o f Tryptophan E n t r y A c r o s s the B l o o d B r a i n Barrier 7 10 5. S e r o t o n i n and B e h a v i o r 14 6. S e r o t o n i n and C o r t i c o s t e r o n e 20 7. Summary 22 8. Rationale 22 GENERAL METHODS . .' 23 1. Animals and R a t i o n s 23 2. Biochemical 23 EXPERIMENT 1: Determination BRAIN TRYPTOPHAN DURING THE DARK PHASE AFTER FASTING V 26 1. I n t r o d u c t i on 26 2. E x p e r i m e n t a l Procedure 26 3. R e s u l t s and D i s c u s s i o n EXPERIMENT 2: EFFECT OF TRYPTOPHAN ADMINISTRATION AND CARBOHYDRATE INGESTION ON BRAIN TRYPTOPHAN AND SEROTONIN 26 1. 29 Introduction 29 - V - CHAPTER VI Page 2. Experimental 3. Results 31 4. Discussion 34 EXPERIMENT 3: Procedure BEHAVIORAL EFFECTS OF TRYPTOPHAN ADMINISTRATION VII 29 35 1. E x p e r i m e n t a l Procedure 35 2. Results 37 3. Discussion 40 EXPERIMENT 4: TRYPTOPHAN AND CARBOHYDRATE INDUCED INCREASES IN BRAIN SEROTONIN: BIOCHEMICAL AND BEHAVIORAL CORRELATES 44 1. Introduction 44 2. E x p e r i m e n t a l Procedure 44 3. Results 46 4. Discussion 51 VIII SUMMARY 61 IX CONCLUSIONS 62 BIBLIOGRAPHY 63 APPENDIX 72 - vi - LIST OF TABLES TABLE I II III IV V VI Page B r a i n Tryptophan F o l l o w i n g a 16 Hour F a s t A c q u i s i t i o n o f an Avoidance Response 27 Following I n j e c t i o n o f S a l i n e o r Tryptophan 38 E f f e c t o f Tryptophan o r S a l i n e on E x t i n c t i o n 39 T o t a l Food and Carbohydrate I n t a k e o f Animals Fed C o n t r o l D i e t , Carbohydrate D i e t o r I n j e c t e d With Tryptophan E f f e c t of Carbohydrate I n g e s t i o n , C o n t r o l D i e t o r a Tryptophan I n j e c t i o n on R e a r i n g . . . . . 52 U r i n a t i o n or Defecation Following Intake of C a r b o h y d r a t e , C o n t r o l D i e t , or a Tryptophan Injection 54 55 -vii - LIST OF FIGURES FIGURE Page 1 Serotonin biosynthesis 2. Tryptophan metabolism 12 3 E f f e c t o f t r y p t o p h a n a d m i n i s t r a t i o n and c a r b o h y d r a t e i n g e s t i o n on b r a i n t r y p t o p h a n and s e r o t o n i n : Experimental design . . . . 30 B r a i n tryptophan f o l l o w i n g tryptophan a d m i n i s t r a t i o n or ingestion, of carbohydrate 32 5 Brain s e r o t o n i n a f t e r tryptophan a d m i n i s t r a t i o n carbohydrate ingestion 33 6 Behavioral e f f e c t s of tryptophan Experimental design 7 L a t e n c y to step-down f o l l o w i n g administration. 8 Tryptophan and c a r b o h y d r a t e induced i n c r e a s e s b r a i n s e r o t o n i n : B i o c h e m i c a l and b e h a v i o r a l correlates: Experimental design. . . 4 9 10 11 12 6 or administration: tryptophan B r a i n tryptophan f o l l o w i n g i n g e s t i o n of and t r y p t o p h a n a d m i n i s t r a t i o n . 41 in 45 carbohydrate 47 B r a i n s e r o t o n i n f o l l o w i n g i n g e s t i o n of or tryptophan a d m i n i s t r a t i o n carbohydrate Plasma c o r t i c o s t e r o n e a f t e r t r y p t o p h a n or c a r b o h y d r a t e i n g e s t i o n administration 49 L a t e n c y to step-down f o l l o w i n g c a r b o h y d r a t e i n g e s t i o n or tryptophan a d m i n i s t r a t i o n ) 36 50 53 -v i i i - LIST OF APPENDIX TABLES TABLE I II III IV V VI VII VIII Page P u r i f i e d C o n t r o l and Carbohydrate D i e t 72 B r a i n Tryptophan F o l l o w i n g I n g e s t i o n o f a C o n t r o l o r Carbohydrate Meal o r I n j e c t i o n o f Tryptophan . . . . 74 B r a i n S e r o t o n i n F o l l o w i n g Carbohydrate or Tryptophan A d m i n i s t r a t i o n 75 Ingestion Latency t o Step-Down F o l l o w i n g Tryptophan o r Saline Injections 76 B r a i n Tryptophan F o l l o w i n g Carbohydrate Intake o r Tryptophan A d m i n i s t r a t i o n 77 B r a i n S e r o t o n i n F o l l o w i n g I n g e s t i o n o f a High Carbohydrate Meal o r I n j e c t i o n o f Tryptophan 78 E f f e c t o f Carbohydrate Intake o r Tryptophan A d m i n i s t r a t i o n on Plasma C o r t i c o s t e r o n e 79 E f f e c t o f a Tryptophan I n j e c t i o n o r o f a CarboHydrate or C o n t r o l D i e t on Latency t o Step-Down . . . . 80 - ix - ACKNOWLEDGEMENT I would l i k e to thank my main a d v i s o r , Dr. P a t r i c i a G a l l o , s e r v i n g as a f i r s t this project. c l a s s model and f o r her c r i t i c a l for comments, throughout S i n c e r e a p p r e c i a t i o n i s extended t o D r s . Joanne Weinberg and R i c h a r d B e n i n g e r f o r t h e i r c o n t i n u e d moral s u p p o r t and i n v a l u a b l e a s s i s t a n c e i n the development o f the b e h a v i o r a l assay. The h e l p f u l s u g g e s t i o n s from committee members, D r s . C h r i s F i b i g e r and MeTvin Lee, are a l s o most a p p r e c i a t e d . Finally, thanks are e x p r e s s e d t o M r s . Naomi Woo f o r a n a l y z i n g plasma c o r t i c o s t e r o n e and to Ms. C h a r l o t t e Waddell f o r p r o d u c i n g .the f i g u r e s . - 1 - I, INTRODUCTION "Does e a t i n g i n f l u e n c e b r a i n f u n c t i o n ? To put the q u e s t i o n more s p e c i f i c a l l y , do changes i n b l o o d c h e m i s t r y t h a t f o l l o w the i n t a k e o f n u t r i e n t s p r o duce c o r r e s p o n d i n g changes i n t h e t i s s u e s o f the brain? I f s o , c o u l d such d i e t - i n d u c e d changes a f f e c t the f u n c t i o n a l a c t i v i t y o f the b r a i n ? " (Fernstrom and Wurtman, 1974). The t r a d i t i o n a l answer t o these q u e s t i o n s has u s u a l l y been no; when r e q u i r e d , the b r a i n e x t r a c t s oxygen, g l u c o s e , and o t h e r n u t r i e n t s the blood ( F e r n s t r o m and Wurtman, 1974). i n the c o n c e n t r a t i o n of n u t r i e n t s fluctuations i n plasma have not been thought to t h e b r a i n (Fernstrom and Wurtman, 1974). has r e v e a l e d some e x c e p t i o n s . Hence, temporary from However, more r e c e n t research F a s t e d r a t s f e d a high c a r b o h y d r a t e , t e i n - f r e e meal ( F e r n s t r o m and F a l l e r , affect pro- 1978; F e r n s t r o m , 1975b; Colmenares e t a l . , 1975; Jacoby e t a l . , 1975a; Madras e t a l . , 1974; F e r n s t r o m , 1971) o r i n j e c t e d w i t h t h e amino a c i d t r y p t o p h a n (Young e t a l . , 1978; Jacoby e t a l . , 1975b; Fernstrom and Wurtman, 1971) have been shown t o e l e v a t i o n s i n b r a i n t r y p t o p h a n and s e r o t o n i n . exhibit That t h i s same phenomenon c o u l d o c c u r i n humans has a l s o been s p e c u l a t e d ( F e r n s t r o m and Wurtman, 1974). Thus, the neurochemical response t o t r y p t o p h a n and c a r b o h y d r a t e has been r e p e a t e d l y demonstrated. However, the f u n c t i o n a l of e l e v a t e d b r a i n s e r o t o n i n has not been w e l l significance established. S e r o t o n i n has been a s s o c i a t e d w i t h many b e h a v i o r s . i n b r a i n s e r o t o n i n c o u l d have i m p o r t a n t p r a c t i c a l Thus, implications. increases Inhibi- t i o n o f a v o i d a n c e l e a r n i n g (Essman, 1 9 7 7 ) , memory (Essman, 1 9 7 7 ) , food i n t a k e ( B l u n d e l l and Latham, 1979), locomotor a c t i v i t y (Warbritton et a l . , 1 9 7 8 ) , and e x p l o r a t i o n ( F i l e and Pope, 1974) have been observed i n rats - 2 - with elevated levels of brain serotonin. Furthermore, b r a i n serotonin has been i m p l i c a t e d i n t h e r e g u l a t i o n o f the r a t ( Y u w i l l e r , 1 9 7 9 ) , and human ( M o d l i n g e r e t a l . , 1979) h y p o t h a l a m u s / p i t u i t a r y / a d r e n a l axis. In a d d i t i o n , s e v e r a l m e t a b o l i c c o n d i t i o n s are thought t o alterations i n serotonin metabolism. involve An e l e v a t e d c o n c e n t r a t i o n o f b r a i n s e r o t o n i n has been l i n k e d t o t h e e t i o l o g y o f c o n f u s i o n and coma, symptoms i n h e p a t i c encephalopathy traditional ( F i s c h e r e t a l . , 1978; S o u r k e s , 1 9 7 8 ) . The form o f t h e r a p y f o r t h i s c o n d i t i o n has been a high c a r b o - hydrate, protein-free d i e t . Thus, i t i s p o s s i b l e t h a t t h i s t r e a t m e n t could p o t e n t i a t e t h e d i s e a s e symptoms through f u r t h e r i n c r e a s i n g b r a i n s e r o tonin. In a d d i t i o n t o h e p a t i c coma, r a t neonatal i r o n d e f i c i e n c y has been shown t o i n c r e a s e b r a i n s e r o t o n i n ( M a c k l e r e t a l . , 1978). decreased b e h a v i o r a l Furthermore, responsiveness, often linked to elevated s e r o t o n i n , has been observed i n an open f i e l d t e s t o f 28 day o l d r a t s f e d an i r o n deficient diet.from birth (Weinberg e t a l . , 1979). Thus, ingestion of a c a r b o h y d r a t e , p r o t e i n - f r e e meal might be expected t o f u r t h e r responsiveness in i r o n d e f i c i e n t Finally, reduce rats. the b e h a v i o r a l and b i o c h e m i c a l consequences o f carbohydrate induced i n c r e a s e s i n b r a i n s e r o t o n i n are o f p a r t i c u l a r i n t e r e s t consumption o f r e f i n e d sugar i s high among North American because populations. In the U n i t e d S t a t e s s u c r o s e i n t a k e has been e s t i m a t e d a t 100 pounds per c a p i t a per y e a r (Bogart e t a l . , 1973). S i m i l a r l y , 68% o f c h i l d r e n i n a r u r a l B r i t i s h Columbia town were observed t o s n a c k , and 28% o f the snaeks consumed were high i n r e f i n e d sugar and low i n o t h e r (Onishi, nutrients 1980). From t h e above, the importance of d e t e r m i n i n g the f u n c t i o n a l signifi- cance o f the c a r b o h y d r a t e and t r y p t o p h a n induced i n c r e a s e i n b r a i n s e r o - - 3 - tonin is clear. Thus, the p r e s e n t study was conducted to determine the r i s e i n s e r o t o n i n f o l l o w i n g i n j e c t i o n s if of tryptophan or i n g e s t i o n of a high c a r b o h y d r a t e , p r o t e i n - f r e e m e a l , a f t e r f a s t i n g , has b e h a v i o r a l consequences. In a d d i t i o n , the e f f e c t o f the c a r b o h y d r a t e and t r y p t o p h a n i n d u c e d i n c r e a s e o f b r a i n s e r o t o n i n on plasma c o r t i c o s t e r o n e output was investigated. The s p e c i f i c o b j e c t i v e s o f t h i s study were: (1) To measure b r a i n t r y p t o p h a n , s e r o t o n i n , and plasma corticosterone i n f a s t e d male r a t s f e d a h i g h c a r b o h y d r a t e meal o r i n j e c t e d w i t h tryptophan. (2) To i n v e s t i g a t e whether the r i s e i n b r a i n s e r o t o n i n can be c o r r e l a t e d w i t h a change i n e x p l o r a t i o n , a b e h a v i o r which has been r e l a t e d to b r a i n s e r o t o n i n . (3) To determine whether t h e r e i s a c o r r e l a t i o n between plasma c o r t i c o s t e r o n e and b r a i n s e r o t o n i n . - 4 II. 1. REVIEW OF THE LITERATURE Carbohydrate and Tryptophan Induced Changes i n Brain Tryptophan and the I n d o l e a m i n e s . A h i g h c a r b o h y d r a t e p r o t e i n - f r e e meal f e d t o f a s t e d r a t s a t the b e g i n n i n g o f the l i g h t phase, has r e p e a t e d l y been shown t o i n c r e a s e t r y p t o p h a n , s e r o t o n i n , and 5 - h y d r o x y i n d o l e a c e t i c a c i d (5-HIAA) brain (Jacoby e t al., 1975; Colmenares e t a l . , 1975; F e r n s t r o m , 1975b , 1974; Fernstrom e t al., 1973; F e r n s t r o m , 1971). W i t h i n 2 hours a f t e r p r e s e n t a t i o n o f the c a r b o h y d r a t e , p r o t e i n - f r e e m e a l , t r y p t o p h a n and the i n d o l e a m i n e s were observed to r i s e and remain e l e v a t e d f o r 3 hours ( F e r n s t r o m , 1971). In a d d i t i o n , b r a i n t r y p t o p h a n was f u r t h e r e l e v a t e d when the c a r b o h y d r a t e meal was a l s o f a t - f r e e (Madras e t a l . , 1973). S i m i l a r f i n d i n g s were o b t a i n e d when a g l u c o s e s o l u t i o n was a d m i n i s t e r e d t o f a s t e d r a t s v i a e i t h e r a stomach tube o r gavage (De Montis e t a l . , 1978; Madras e t a l . , 1974, 1973). However, w i t h t h e s e l a t t e r methods o f a d m i n i s t r a t i o n , t h e peak i n t r y p t o phan o c c u r r e d 1'hour a f t e r g l u c o s e presentation. T r y p t o p h a n , s e r o t o n i n , and 5-HIAA have a l s o been assayed i n regions w i t h i n the c e n t r a l specific nervous system o f r a t s f e d a c a r b o h y d r a t e , p r o t e i n - f r e e meal (Colmenares e t a l . , 1975). While no i n c r e a s e i n trypto- phan and the 5 - h y d r o x y i n d o l e s was found i n the hypothalamus o r the corpus s t r i a t u m , e x a m i n a t i o n o f the b r a i n stem, s p i n a l c o r d , and the telencephalon ( n e o c o r t e x , o l f a c t o r y c o r t e x , and hippocampus) r e v e a l e d e l e v a t i o n s s e r o t o n i n , t r y p t o p h a n , and 5-HIAA (Colmenares e t a l . , 1 9 7 5 ) . the b r a i n stem i s b e l i e v e d t o c o n t a i n the p e r i k a r y a o f the neurons, w h i l e a l l in Because serotonergic o t h e r r e g i o n s o f the c e n t r a l nervous system are thought t o c o n t a i n the neuronal axons and t e r m i n a l s (Colmenares e t a l . , 1 9 7 5 ) , - 5 - these f i n d i n g s demonstrated t h a t t h e c a r b o h y d r a t e - i n d u c e d i n c r e a s e i n s e r o t o n i n o c c u r r e d i n both c e l l bodies and the p r e - s y n a p t i c t e r m i n a l s s e r o t o n i n - c o n t a i n i n g neurons. of T h e r e f o r e , upon d e p o l a r i z a t i o n , i t is p o s s i b l e t h a t more s e r o t o n i n c o u l d be r e l e a s e d i n t o the s y n a p t i c T h i s phenomenon i s e s s e n t i a l f o r the c a r b o h y d r a t e induced i n c r e a s e s e r o t o n i n t o be p h y s i o l o g i c a l l y s i g n i f i c a n t Intraperitoneal cleft. in (Colmenares e t a l . , 1975). i n j e c t i o n o f t r y p t o p h a n a l s o has been shown t o i n - c r e a s e b r a i n t r y p t o p h a n and s e r o t o n i n (Young e t a l . , 1978; Jacoby e t a l . , 1975b; Fernstrom and Wurtman, 1971). I n j e c t i o n s o f 12.5 t o 125 mg t r y p t o - phan/kg body w e i g h t r e s u l t e d i n dose r e l a t e d i n c r e a s e s i n b r a i n tryptophan 1 hour a f t e r i n j e c t i o n . serotonin However, t h e maximum i n c r e a s e i n b r a i n was o b t a i n e d w i t h t r y p t o p h a n doses o f 25-50 mg/kg (Young e t a l . , 1978; Jacoby e t a l . , 1975b; Fernstrom and Wurtman, 1971). Thus, i t has been r e p e a t e d l y demonstrated t h a t b r a i n t r y p t o p h a n , s e r o t o n i n , and 5-HIAA are i n c r e a s e d f o l l o w i n g c a r b o h y d r a t e i n g e s t i o n o r tryptophan dosing. 2. S e r o t o n i n D i s t r i b u t i o n and B i o s y n t h e s i s . B r a i n s e r o t o n i n , o r the monoamine 5 - h y d r o x y t r y p t a m i n e , has been found c o n f i n e d to a d i s t i n c t c l u s t e r o f n e u r o n s , the raphe ( F e r n s t r o m and Wurtman, 1974). The c e l l primarily nuclei bodies of the raphe n u c l e i are known t o be l o c a t e d i n the b r a i n stem and have f i b r e s a s c e n d i n g i n t o the remainder o f the b r a i n and descending through the s p i n a l c o r d (Fernstrom and Wurtman, 1974). S e r o t o n i n s y n t h e s i s has been f u l l y d e s c r i b e d ( A i r a k s i n e n and A i r a k s i n e n , 1978; F e r n s t r o m , 1978). 1978) i s d e p i c t e d i n F i g u r e 1. T h i s m e t a b o l i c pathway (Fernstrom, H H •C-C-NHj, i H COOH Tryptophan 1 VV-T«-^ h 2 vV W 5-Hydroxytryptophan Tryptophan h y d r o x y l a s e ; c o f a c t o r s : Vvrt^ f e r r o u s i r o n , reduced p t e r i d i n e 2 A r o m a t i c amino a c i d d e c a r b o x y l a s e ; c o f a c t o r : pyridoxal phosphate 3 Monoamine o x i d a s e ^ Aldehyde dehydrogenase F i g u r e 1. ( F e r n s t r o m , 1978) Serotonin biosynthesis 3 AD Serotonin cofactor Y>r - rt 4 >vV 5-HIAA - 7 - 3. Regulation of Serotonin Synthesis. A l t h o u g h s e r o t o n i n s y n t h e s i s has been e x t e n s i v e l y i n v e s t i g a t e d , regulation is s t i l l unclear. Tryptophan a v a i l a b i l i t y , rate of impulse f l o w a l o n g s e r o t o n e r g i c n e u r o n s , and p r o d u c t i n h i b i t i o n have a l l i m p l i c a t e d as f a c t o r s i n v o l v e d i n r e g u l a t i o n o f s e r o t o n i n normal c o n c e n t r a t i o n , i . e . ( F e r n s t r o m , 1978), t r y p t o p h a n a v a i l a b i l i t y been synthesis. Because the Km o f t r y p t o p h a n h y d r o x y l a s e f o r t r y p t o p h a n i s r e l a t i v e to i t s its high 50 uM versus 30 uM r e s p e c t i v e l y has been r e c o g n i z e d as an impor- t a n t f a c t o r i n the c o n t r o l o f s e r o t o n i n s y n t h e s i s . In a d d i t i o n , proce^ dures which a l t e r b r a i n t r y p t o p h a n c o n c e n t r a t i o n have a l s o been found to i n f l u e n c e b r a i n s e r o t o n i n o r 5-HIAA. Animals f e d a corn based d i e t , known to be d e f i c i e n t i n t r y p t o p h a n , w e r e observed t o e x h i b i t lower o f b r a i n t r y p t o p h a n and s e r o t o n i n ( L y t l e e t a l . , 1975). lower c o n c e n t r a t i o n s levels Furthermore, o f t r y p t o p h a n and 5-HIAA have been found i n rats o f f e r e d meals o f p u r i f i e d amino a c i d s known t o b l o c k t r y p t o p h a n e n t r y a c r o s s the b l o o d b r a i n b a r r i e r (Gessa e t a l . , 1974). In a d d i t i o n , p r e s e n t a - t i o n o f a low p r o t e i n d i e t t o w e a n l i n g r a t s has a l s o been shown to b r a i n t r y p t o p h a n , s e r o t o n i n , and 5-HIAA. lower These b r a i n parameters were observed t o r e t u r n t o normal a f t e r n u t r i t i o n a l r e h a b i l i t a t i o n f o r 7 days ( D i c k e r s o n and Pao, 1975). S i m i l a r l y , a m i n o p h y l l i n e , a drug b e l i e v e d t o i n c r e a s e b r a i n s e r o t o n i n , was observed t o e l e v a t e b r a i n t r y p t o p h a n (Curzon and K n o t t , 1974). In a d d i t i o n , b r a i n t r y p t o p h a n and s e r o t o n i n t u r n o v e r were found e l e v a t e d a f t e r 24 hours o f food d e p r i v a t i o n o r i m m o b i l i z a t i o n s t r e s s al., 1972). Finally, (Curzon e t consumption o f a h i g h c a r b o h y d r a t e meal (Fernstrom, 1971) o r i n j e c t i o n s o f t r y p t o p h a n ( F e r n s t r o m and Wurtman, 1971) were observed t o i n c r e a s e b r a i n t r y p t o p h a n , s e r o t o n i n ; and 5-HIAA. That t h i s - 8- i n c r e a s e i n b r a i n s e r o t o n i n r e f l e c t e d i n c r e a s e d s y n t h e s i s r a t h e r than lowered metabolism o r r e l e a s e was i n d i c a t e d by the p a r a l l e l (Wurtman and F e r n s t r o m , 1976). r i s e i n 5-HIAA Moreover, when animals were g i v e n a c a r b o - h y d r a t e meal a l o n g w i t h an a r o m a t i c amino a c i d d e c a r b o x y l a s e inhibitor, b r a i n 5 - h y d r o x y t r y p t o p h a n , the immediate p r e c u r s o r o f s e r o t o n i n , was s i g n i f i c a n t l y e l e v a t e d (Jacoby e t a l . , 1975a). From the above, i t i s c l e a r t h a t c h r o n i c and acute changes i n b r a i n t r y p t o p h a n a v a i l a b i l i t y appear t o be a major d e t e r m i n a n t o f b r a i n s e r o tonin synthesis. However, t r y p t o p h a n a v a i l a b i l i t y may not be c r i t i c a l the i n d u c t i o n o f b r a i n s e r o t o n i n and 5-HIAA d i u r n a l daily fluctuations rhythms. in Originally, o f both whole b r a i n (Wurtman and F e r n s t r o m , 1972) and b r a i n stem (Morgan e t a l . , 1975) t r y p t o p h a n were observed t o correlate with brain serotonin. positively However, more r e c e n t l y , c i r c a d i a n m o d i f i - c a t i o n s i n t r y p t o p h a n and s e r o t o n i n were shown t o be i n o p p o s i t e phase i n the f r o n t o - p a r i e t a l c o r t e x (Hery e t a l . , 1977). Furthermore, trypto- phan h y d r o x y l a s e a c t i v i t y and i t s rhythm were found t o vary depending on the s e r o t o n i n c o n t a i n i n g c e l l 1977). groups i n the r a t b r a i n stem (Kan e t a l . , Thus, the importance o f t r y p t o p h a n a v a i l a b i l i t y s e r o t o n i n o r 5-HIAA c i r c a d i a n r h y t h m i c i t y i s u n c l e a r . t h a t under normal p h y s i o l o g i c a l tryptophan a v a i l a b i l i t y in determining It is possible c o n d i t i o n s , the r e l a t i v e importance of i s a f u n c t i o n of b r a i n a r e a . In a d d i t i o n t o t r y p t o p h a n a v a i l a b i l i t y , i n f l u e n c e d by the r a t e o f neuronal f i r i n g s e r o t o n i n s y n t h e s i s may be ( B o a d l e - B i b e r , 1979a). Stimu- l a t i o n o f s e r o t o n e r g i c neurons o f the mid b r a i n raphe was shown t o i n crease s e r o t o n i n synthesis E c c l e s t o n , 1972). slices from r a d i o l a b e l l e d t r y p t o p h a n ( S h i e l d s Similarly, and i n - v i t r o d e p o l a r i z a t i o n o f r a t b r a i n stem i n a potassium e n r i c h e d i n c u b a t i o n medium was found to enhance - 9 - t r y p t o p h a n h y d r o x y l a s e a c t i v i t y as measured by i n c r e a s e d t r y p t o p h a n formation to 5-hydroxytryptophan (Boadle-Biber, (Hamon e t a l . , 1979). trans- Both c a l c i u m 1979a,b; E l k s e t a l . , 1979) and c y c l i c adenosine monophos- phate (CAMP) ( B o a d l e - B i b e r , 1980) have been i m p l i c a t e d i n the induction of tryptophan hydroxylase a c t i v i t y since manipulations that elevate w i t h i n nerve t i s s u e were observed t o i n c r e a s e t r y p t o p h a n activity. Similarly, hydroxylase procedures which removed c a l c i u m from t h e i n c u b a t i o n medium were found t o b l o c k enzyme a c t i v i t y f o l l o w i n g (Boadle-Biber, calcium 1979b; E l k s e t a l . , 1979). depolarization In a d d i t i o n , the a d d i t i o n CAMP has a l s o been shown to i n c r e a s e t r y p t o p h a n h y d r o x y l a s e a c t i v i t y vitro (Boadle-Biber, in- 1980). The r o l e o f c a l c i u m i n t h e i n d u c t i o n activity of i s not y e t u n d e r s t o o d . of tryptophan hydroxylase C a l c i u m has been shown t o c h e l a t e trypto- phan which c o u l d r e s u l t i n an i n c r e a s e i n t r y p t o p h a n uptake i n t o s n y a p t o somes ( B r u i n v e l s and Moleman, 1979). However, s i n c e a d m i n i s t r a t i o n o f a c a l c i u m b l o c k e r t o the i n c u b a t i o n medium was found to decrease tryptophan a c t i v i t y w i t h no e f f e c t on t r y p t o p h a n uptake ( E l k s e t a l . , 1 9 7 9 ) , mechanism appears u n l i k e l y . this C a l c i u m induced a c t i v a t i o n o f p r o t e i n kinase which i n t u r n s t i m u l a t e s t r y p t o p h a n h y d r o x y l a s e has a l s o been proposed (Boadle-Biber, 1979a; E l k s e t a l . , 1979). A l t h o u g h i t i s e v i d e n t t h a t the r a t e of neuronal f i r i n g may p l a y a r o l e i n the r e g u l a t i o n o f s e r o t o n i n s y n t h e s i s through i n d u c t i o n tryptophan hydroxylase a c t i v i t y , of a c t i v a t i o n o f t h i s enzyme has not always been f o l l o w e d by i n c r e a s e d s e r o t o n i n s y n t h e s i s . A d m i n i s t r a t i o n of pargy- l i n e , a drug which i n c r e a s e s s e r o t o n i n , was r e p o r t e d t o markedly reduce the s t i m u l a t o r y e f f e c t o f neuronal d e p o l a r i z a t i o n on t r y p t o p h a n conver- s i o n t o s e r o t o n i n (Hamon e t a l . , 1979). In a d d i t i o n , i n - v i t r o b r a i n s e r o - - 10 - t o n i n s y n t h e s i s was found i n h i b i t e d when s e r o t o n i n was added t o the i n c u b a t i o n medium (Young e t a l . , 1978). Such r e s u l t s i n d i c a t e t h a t i n h i b i t i o n may a l s o r e g u l a t e s e r o t o n i n s y n t h e s i s . i n h i b i t i o n i n the r e g u l a t i o n of s e r o t o n i n synthesis s a l l y supported. substrate However, feed back has not been u n i v e r - When r a t b r a i n s e r o t o n i n l e v e l s were e l e v a t e d pharma- c o l o g i c a l l y w i t h a monoamine o x i d a s e i n h i b i t o r and a n i m a l s then a l l o w e d t o consume a c a r b o h y d r a t e , p r o t e i n - f r e e m e a l , the subsequent i n c r e a s e b r a i n h y d r o x y i n d o l e l e v e l s was comparable t o those found i n c o n t r o l not g i v e n the drug (Jacoby e t a l . , 1975a). In a d d i t i o n , the normal i n c r e a s e i n s e r o t o n i n has been r e p o r t e d t o be 25%. in rats diurnal However, a 46% i n - c r e a s e has been r e p o r t e d when c a r b o h y d r a t e p l u s a monoamine i n h i b i t o r was a d m i n i s t e r e d (Jacoby e t a l . , 1975). Thus, t r y p t o p h a n a v a i l a b i l i t y , r a t e o f neuronal f i r i n g , a n d i n h i b i t i o n may be i n v o l v e d i n the, r e g u l a t i o n o f s e r o t o n i n synthesis. F u r t h e r m o r e , these f a c t o r s c o u l d work t o g e t h e r i n r e g u l a t i n g production. substrate serotonin The r e l a t i v e importance o f each o f these f a c t o r s may be c o n - t i n g e n t upon b r a i n r e g i o n . 4. Tryptophan M e t a b o l i s m and R e g u l a t i o n o f Tryptophan E n t r y A c r o s s the Blood B r a i n Of a l l the e s s e n t i a l Barrier. amino a c i d s , t r y p t o p h a n has been found t o o c c u r i n l o w e s t c o n c e n t r a t i o n i n p r o t e i n , o n l y 1.5% ( F e r n s t r o m , 1978). In c o n - t r a s t t o o t h e r amino a c i d s , 80%-90% o f t r y p t o p h a n has been observed t o c i r c u l a t e bound t o albumin w h i l e o n l y 10%-20% has been found to freely ( F e r n s t r o m , 1978). The m e t a b o l i c f a t e o f t r y p t o p h a n i s w i t h o n l y 1% u t i l i z e d f o r s e r o t o n i n s y n t h e s i s 1976). circulate complex (Fernstrom and Wurtman, The main m e t a b o l i c pathways f o r t r y p t o p h a n are d e p i c t e d in - 11 - F i g u r e 2 and i n c l u d e p r o t e i n s y n t h e s i s , n i c o t i n i c a c i d p r o d u c t i o n , s e r o t o n i n s y n t h e s i s , m e l a t o n i n f o r m a t i o n , and p r o d u c t i o n o f o t h e r inactive metabolites biologically ( A i r a k s i n e n and A i r a k s i n e n , 1 9 7 8 ) . The uptake o f t r y p t o p h a n i n t o the b r a i n i s thought t o be c o n t r o l l e d by t h r e e major f a c t o r s : amino a c i d s (1) the b l o o d c o n c e n t r a t i o n s o f the l a r g e neutral ( p h e n y l a l a n i n e , t y r o s i n e , l e u c i n e , i s o l e u c i n e , v a l i n e , and m e t h i o n i n e ) which compete f o r a common t r a n s p o r t c a r r i e r (Bender, 1978); (2) the c o n c e n t r a t i o n of the s m a l l f r a c t i o n o f t r y p t o p h a n not bound to albumin (Curzon and K n o t t , 1974); and (3) the time o f day (Hery e t a l . , 1972). C o m p e t i t i o n between plasma t r y p t o p h a n and the n e u t r a l amino a c i d s as a r e g u l a t o r y mechanism was shown when f a s t e d r a t s o f f e r e d a p r o t e i n - c o n t a i n i n g meal, i . e . l a b chow, o r a p u r i f i e d amino a c i d d i e t , e x h i b i t e d a s i g n i f i c a n t e l e v a t i o n i n plasma t r y p t o p h a n . t r y p t o p h a n was o b s e r v e d . B u t , no change i n However, i f t h e same p u r i f i e d d i e t , lacking t h e n e u t r a l amino a c i d s e x c e p t t r y p t o p h a n was f e d , e l e v a t i o n o f amino a c i d s consumed was found i n the plasma. phan, s e r o t o n i n , and 5-HIAA were a l s o e l e v a t e d . brain all those Furthermore, b r a i n trypto- In c o n t r a s t , when o n l y a s p a r t a t e and g l u t a m i n e , which share a d i f f e r e n t b r a i n t r a n s p o r t carrier from the n e u t r a l amino a c i d s , were o m i t t e d from the p u r i f i e d d i e t , the consumed amino a c i d s were i n c r e a s e d i n t h e plasma, w h i l e t r y p t o p h a n remained u n a f f e c t e d ( F e r n s t r o m e t a l . , 1973). all brain A high c o r r e l a - t i o n (.95) between b r a i n t r y p t o p h a n and the r a t i o o f serum tryptophan t o the sum o f the competing n e u t r a l amino a c i d s was s u b s e q u e n t l y demons t r a t e d ( F e r n s t r o m and F a l l e r , 1978). A much lower c o r r e l a t i o n was shown between b r a i n t r y p t o p h a n and plasma t r y p t o p h a n a l o n e et a l . , 1973). (.66) (Fernstrom - 12 - N i c o t i n i c acid >- NAD 1 Quinolinic acid I 3-Hydroxyanthranilic acid — • 6 t 3-Hydroxykynurenine B 5 f Be + NH3 + Hg) Xanthurenic acid Kynurenic acid Kynurenine 5,Bg Indole, skatole indoxylsulphate " — - M n t h r a n i l i c acid Formylkynurenine [4 "" Protein — Tryptophan 5-Methoxy-DMT /i 8 5- Methoxytryptamine 1 2 M 4-u u 4. N-methyltryptamine ! ^ 8 N,N-dimethyltryp ' m t i m i n e fi ™ 3 • Tryptamine — ^ I n d o l e a c e t i c acid ^"Indolepyruvic acid- 5-Hydroxytryptophan (5-HTP) Bg J2 5-Hydroxytryptamine g ^N-methyl-5-HT—-sBufotenine ( 5 - H T ) ^ Glucuronides and sulphates -Acetyl-5-HT. 7 5-Hydroxyi ndole-acetaldehyde Melatonin 5-Hydroxyindoleacetic acid (5-HIAA) 5-Hydroxytryptophol Figure 2 . Main metabolic pathways of tryptophan, including certain enzymes and the occurrence of pyridoxal (Bg) as the coenzyme: ( 1 ) tryptophan-5-hydroxylase (tryptophan-5-mono-oxygenase, E.C. 1 . 1 4 . 1 6 . 1 . ) ; ( 2 ) aromatic L-amino acid decarboxylase ( E . C . 4 . 1 . 1 . 2 8 ) ; ( 3 ) monoamine oxidase [MAO, Amine oxidase (flavin-containing), E.C. 1 . 4 . 3 . 4 ] ; ( 4 ) tryptophan pyrrolase (tryptophan oxidase, tryptophan-2,3-dioxygenase, E.D. 1 . 1 . 3 . 1 1 . 1 1 ) ; ( 5 ) . kynurenmase (E.C. 3 . 7 . 1 . 3 ) ; ( 6 ) kynurenine transminase, (E.C. 2 . 6 . 1 . 7 ) ; ( 7 ) Hydroxyindole-0-methyltransferase; ( 8 ) indole-ethylamine-N-methyltransferase-(+-aromatic-amine-N-methyltransferase?); Bg pyridoxal phosphate, as coenzyme. (Airaksinen and Airaksinen, 1 9 7 8 . ) - 13 - T h i s a l t e r a t i o n i n plasma n e u t r a l amino a c i d c o n c e n t r a t i o n i s believed t o a c c o u n t f o r t h e e l e v a t e d b r a i n t r y p t o p h a n c o n c e n t r a t i o n when a c a r b o h y d r a t e p r o t e i n - f r e e meal i s consumed (Fernstrom and F a l l e r , F e r n s t r o m , 1975b; Fernstrom e t a l . , 1973). Carbohydrate has been shown to i n c r e a s e b r a i n t r y p t o p h a n through i n s u l i n s e c r e t i o n . I n s u l i n was shown t o i n c r e a s e the r a t i o o f plasma t r y p t o p h a n t o the n e u t r a l which compete f o r uptake a c r o s s the b l o o d b r a i n b a r r i e r 1973). 1978; amino a c i d s (Fernstrom et a l . , Thus, when a h i g h c a r b o h y d r a t e meal i s consumed, plasma t r y p t o p h a n i s r e a d i l y t r a n s p o r t e d i n t o the b r a i n . In c o n t r a s t , when f a s t e d r a t s con- sumed r e g u l a r l a b chow o r a c a r b o h y d r a t e meal supplemented w i t h 18% c a s e i n , b r a i n t r y p t o p h a n was not found t o r i s e , d e s p i t e i n s u l i n ( F e r n s t r o m e t a l . , 1973). secretion With i n g e s t i o n o f p r o t e i n , the plasma concen- t r a t i o n of competing amino a c i d s was observed t o i n c r e a s e more than t r y p t o p h a n (Fernstrom e t a l . , 1 9 7 3 ) . . considerably S i m i l a r f i n d i n g s o f an i n c r e a s e d r a t i o o f plasma t r y p t o p h a n t o the amino a c i d s which compete w i t h i t f o r b r a i n uptake have been shown i n f a s t e d humans f e d a g l u c o s e solu- t i o n ( F e r n s t r o m and Wurtman, 1 9 7 4 ) . F u r t h e r e v i d e n c e f o r the e f f e c t o f c a r b o h y d r a t e on the plasma n e u t r a l amino a c i d p a t t e r n o f humans comes from the f o l l o w i n g . When human sub- j e c t s consumed h i g h c a r b o h y d r a t e , p r o t e i n - f r e e meals f o r 5 d a y s , the diurnal variations i n plasma n e u t r a l amino a c i d r a t i o s d i f f e r e d a b l y from i n d i v i d u a l s 1979). e a t i n g 150 g o f p r o t e i n d a i l y consider- ( F e r n s t r o m and Wurtman, When 0 g of p r o t e i n were consumed, the r a t i o o f t r y p t o p h a n t o c o m p e t i t o r s i n c r e a s e d throughout the day u n t i l a peak a t 1500 hrs then a gradual decrease t o n a d i r c o n c e n t r a t i o n s a t 0700 h r s . This its and rhythm was i n o p p o s i t e phase t o t h a t observed i n the group f e d 150 g p r o t e i n . F u r t h e r m o r e , the t r y p t o p h a n t o n e u t r a l amino a c i d r a t i o s were consistently - 14 - higher at a l l time p o i n t s assayed when the high c a r b o h y d r a t e meals a l o n e were consumed ( F e r n s t r o m and Wurtman, 1979). A l t h o u g h the r e l a t i v e c o n c e n t r a t i o n s o f n e u t r a l amino a c i d s p l a y an i m p o r t a n t r o l e i n the r e g u l a t i o n o f t r y p t o p h a n t r a n s p o r t a t i o n across the b l o o d b r a i n b a r r i e r , o t h e r mechanisms have a l s o been d e s c r i b e d . C l o f i b r a t e a d m i n i s t r a t i o n (Bloxam e t a l . , 1 9 8 0 ) , s t a r v a t i o n , and i m m o b i l i z a t i o n (Curzon e t a l . , 1972) were shown t o i n c r e a s e b r a i n tryptophan (Bloxam e t a l . , 1980; Curzon e t a l . , 1972) and s u b s e q u e n t l y s e r o t o n i n t u r n o v e r (Curzon e t a l . , 1972). Under these c i r c u m s t a n c e s , plasma f r e e f a t t y were shown t o r i s e and d i s p l a c e t r y p t o p h a n from albumin b i n d i n g hence r e n d e r i n g more f r e e t r y p t o p h a n a v a i l a b l e t o the b r a i n . c o n c e n t r a t i o n o f f r e e t r y p t o p h a n may p l a y a r o l e i n r e g u l a t i n g sites, Thus, the brain tryptophan f o l l o w i n g treatments that e l e v a t e f r e e f a t t y a c i d s . In a d d i - t i o n , time o f day has been demonstrated t o i n f l u e n c e the b r a i n ' s to t r a n s p o r t t r y p t o p h a n (Hery e t a l . , 1972). acids ability Hery e t a l . ( 1 9 7 2 ) , observed t h a t r a d i o l a b e l e d t r y p t o p h a n uptake i n t o b r a i n s l i c e s was h i g h e r during the l i g h t phase than d u r i n g the dark p e r i o d . Thus, t h r e e f a c t o r s appear t o r e g u l a t e t r y p t o p h a n e n t r y a c r o s s blood b r a i n b a r r i e r : r a t i o o f plasma t r y p t o p h a n t o the n e u t r a l the amino a c i d s , c o n c e n t r a t i o n o f f r e e t r y p t o p h a n , and time o f day. 5. S e r o t o n i n and B e h a v i o r . A l t h o u g h c o n s i d e r a b l e r e s e a r c h has c e n t e r e d around the r e g u l a t i o n of s e r o t o n i n s y n t h e s i s , the f u n c t i o n a l t o n i n has s t i l l s i g n i f i c a n c e of elevated brain sero- not been w e l l e s t a b l i s h e d . However, numerous b e h a v i o r s have been l i n k e d t o changes i n b r a i n s e r o t o n i n content. B e h a v i o r s m o t i v a t e d by n e g a t i v e r e i n f o r c e m e n t have been r e l a t e d to - 15 - serotonin. D e s t r u c t i o n o f s e r o t o n e r g i c neurons by i n t r a c i s t e r n a l t i o n o f 5,6 d i h y d r o x y t r y p t a m i n e lesions ( L i n e t a l . , 1978) o r ( K o h l e r and L o r e n s , 1978) electrolytic was observed t o f a c i l i t a t e o f a 2-way a c t i v e a v o i d a n c e s h u t t l e box t a s k . injec- learning P-chlorophenylalanine, (PCPA), a t r y p t o p h a n h y d r o x y l a s e i n h i b i t o r , was shown t o enhance Y-maze a v o i d a n c e a c q u i s i t i o n , w h i l e P-chloroamphetamine was found t o improve s h u t t l e box avoidance ( V o r h e e s , 1979). nucleus were shown t o f a c i l i t a t e tryptophan Oltmens, F u r t h e r m o r e , l e s i o n s o f the raphe a l e a r n e d t a s t e a v e r s i o n and 5-hydroxy- ' a d m i n i s t r a t i o n was found t o r e v e r s e t h i s e f f e c t (Lorden and 1978). In c o n t r a s t , 2 - w a y s h u t t l e box avoidance was u n a f f e c t e d and l e a r n i n g of an u n s i g n a l l e d 1-way avoidance response was i m p a i r e d w i t h PCPA a d m i n i s t r a t i o n ( K o h l e r and L o r e n s , 1978). Lack o f agreement w i t h o t h e r PCPA data on s h u t t l e box a v o i d a n c e may be r e l a t e d t o the s h o r t e r e x p e r i m e n t a l employed by K o h l e r and Lorens (1979) (2 d a y s ) . t h a t PCPA d i d not e x e r t i t s e f f e c t u n t i l Vorhees (1979) the t h i r d day o f period observed testing. In a d d i t i o n t o a v o i d a n c e t e s t i n g , s e r o t o n i n has been a s s o c i a t e d w i t h memory (Essman, 1977). S t i m u l a t i o n o f the d o r s a l raphe n u c l e i p a i r e d the memory o f a p a s s i v e - a v o i d a n c e response and PCPA b l o c k e d effect ( F i b i g e r e t a l . , 1978). Similarly, this PCPA markedly i n c r e a s e d e x - t i n c t i o n i n a punished step-down paradigm ( B e n i n g e r and P h i l l i p s , S e r o t o n i n has a l s o been i m p l i c a t e d i n s e v e r a l h o m e o s t a t i c particularly im- i n the r e g u l a t i o n o f food i n t a k e . 1979). behaviors, A d m i n i s t r a t i o n o f 5- h y d r o x y t r y p t o p h a n was observed t o decrease food consumption, lower meal s i z e , and depress r a t e o f e a t i n g per meal ( B l u n d e l l and Latham, 1979). Such r e s u l t s were i n a c c o r d w i t h t h e e f f e c t s o f f e n f l u r a m i n e and Leshem, 1975) and L i l l y (Blundell 110140 ( G o u d i e , 1976) drugs known t o p o t e n - - 16 - t i a t e serotonin action. Similarly, PCPA t r e a t m e n t was shown to r e s u l t hyperphagia and dose dependent i n c r e a s e s i n food i n g e s t i o n in (Mackenzie e t a l . , 1979; C o s c i n a e t a l . , 1978). In c o n t r a s t t o the above, t r y p t o p h a n i n j e c t i o n s were r e p o r t e d t o have no e f f e c t on food consumption i n d e p r i v e d r a t s 1979). (Weinberger e t a l . , S i m i l a r f i n d i n g s have been o b t a i n e d w i t h a methyl e s t e r o f PCPA, a serotonin depleter. In a d d i t i o n , i n t r a v e n t r i c u l a r i n j e c t i o n o f the amino a c i d methyl e s t e r s of l e u c i n e and t r y p t o p h a n was shown to result i n h y p e r p h a g i a , but no a s s o c i a t i o n was observed between over e a t i n g and a r e d u c t i o n i n b r a i n s e r o t o n i n (Mackenzie e t a l . , 1979). In a d d i t i o n t o s e r o t o n i n ' s intake, its p o s s i b l e r o l e i n the s u p p r e s s i o n o f food f u n c t i o n i n the r e g u l a t i o n o f e i t h e r p r o t e i n o r c a r b o h y d r a t e consumption has been d i s c u s s e d ( A n d e r s o n , 1979; Wurtman and Wurtman, 1979). In the s e l f - s e l e c t i n g r a t , an i n v e r s e r e l a t i o n s h i p , (r=-.96)>, was r e p o r t e d between p r o t e i n i n t a k e and the plasma t r y p t o p h a n c o n c e n t r a t i o n to i t s competitors. relative T h i s plasma amino a c i d p a t t e r n has been d i r e c t l y a s s o c i a t e d w i t h s e r o t o n i n c o n c e n t r a t i o n ( A n d e r s o n , 1979). Uncontrolled d i a b e t e s , a d i s e a s e t h a t causes both a marked decrease i n the r a t i o of t r y p t o p h a n t o the n e u t r a l amino a c i d s and whole b r a i n t r y p t o p h a n , was a l s o observed t o e l e v a t e p r o t e i n i n t a k e (Woodger e t a l . , 1979). PCPA a d m i n i s t r a t i o n , m i d - b r a i n raphe l e s i o n s , and 5,7 Conversely, dihydroxytryptamine d e s t r u c t i o n o f s e r o t o n e r g i c neurons were found t o decrease p r o t e i n intake. Reduced p r o t e i n consumption was shown to be d i r e c t l y a s s o c i a t e d w i t h depressed s e r o t o n i n and 5-HIAA ( A s h l e y e t a l . , 1979). thought to enhance s e r o t o n e r g i c t r a n s m i s s i o n , i . e . Finally, fenfluramine, drugs fluoxe- t i n e , o r MK 212, were observed t o s e l e c t i v e l y decrease consumption o f c a r b o h y d r a t e w i t h o u t i n f l u e n c i n g p r o t e i n i n t a k e (Wurtman and Wurtman, - 17 - 1979) . Thus, s e r o t o n e r g i c neurons appear, t o r e g u l a t e food i n t a k e but, t h e i r e x a c t f u n c t i o n has not been e s t a b l i s h e d . I n h i b i t i o n o f a g g r e s s i o n may be an a f f e c t i v e b e h a v i o r mediated by serotonin. C h l o r i m i p r a m i n e , a s e r o t o n i n r e - u p t a k e b l o c k e r , was shown t o i n h i b i t m u r i c i d e i n n a t u r a l k i l l e r r a t s but was not found t o p r e v e n t mouse killing i n r a t s w i t h l e s i o n s o f t h e a s c e n d i n g p r o j e c t i o n o f the d o r s a l and median raphe n u c l e i (Marks e t a l . , 1978). In a d d i t i o n , raphe lesions were observed t o induce a g g r e s s i v e b e h a v i o r s such as m u r i c i d e , an i n c r e a s e d f l i g h t response t o t a i l p i n c h i n g , a g r e a t e r s t r u g g l e response t o capturing w i t h a g l o v e d hand, and more s q u e a l i n g upon b e i n g caught (Yamaoto and U e k i , 1978). S e v e r a l o f these b e h a v i o r s were shown t o be i n h i b i t e d by t r y p t o p h a n , i m i p r a m i n e , and c h l o r o i m i p r a m i n e 5-hydroxy- (Yamaoto and U e k i , 1978). Shock induced f i g h t i n g was a l s o found t o be f a c i l i t a t e d in rats offered a t r y p t o p h a n f r e e d i e t , a l t h o u g h supplementing chow w i t h 0.5% t r y p t o p h a n had no e f f e c t on shock induced f i g h t i n g or m u r i c i d e (Kantak e t a l . , 1980) . In c o n t r a s t , m i c e o f f e r e d a p u r i f i e d 12% c a s e i n d i e t supplemented w i t h 0.25 o r 0.5% t r y p t o p h a n were found t o e x h i b i t i n c r e a s e d a g g r e s s i o n (Thurmond e t a l . , 1980). had no e f f e c t on t h i s b e h a v i o r . territorial However, a 1% t r y p t o p h a n supplement A f t e r 6 weeks on the t r y p t o p h a n s u p p l e - mented d i e t s , no d i e t s were observed t o a l t e r t e r r i t o r i a l i n c r e a s e d l e v e l s o f b r a i n t r y p t o p h a n and s e r o t o n i n . aggression, Interestingly, despite this same 12% c a s e i n d i e t supplemented w i t h l e u c i n e , r a t h e r than t r y p t o p h a n , was shown t o lower b r a i n t r y p t o p h a n , t y r o s i n e , and s e r o t o n i n , but not t o i n f l u e n c e a g g r e s s i v e b e h a v i o r (Thurmond, e t a l . , 1980). Although Thurmond's f i n d i n g s do not s u p p o r t the b e l i e f t h a t s e r o t o n i n inhibits a g g r e s s i v e b e h a v i o r , h i s c o n t r a s t i n g r e s u l t s c o u l d be due to the differ- - 18 - ences i n e x p e r i m e n t a l d e s i g n . species Both the type o f a g g r e s s i o n t e s t e d and the differed. In a d d i t i o n t o the above a f f e c t i v e b e h a v i o r , s e r o t o n i n has been a s s o c i a t e d w i t h s l e e p (Essman, 1978). Tryptophan a d m i n i s t r a t i o n t o humans (Hartman, 1978) was r e p o r t e d t o reduce s l e e p l a t e n c y and to reduce both s l e e p l a t e n c y and REM ( r a p i d eye movement) l a t e n c y i n r a t s Reyes, 1978). (Hill and R e c e n t l y , 4 g o f t r y p t o p h a n was shown t o i n c r e a s e the d u r a - t i o n o f stage 3 s l e e p i n humans, b u t , o n l y t r e n d s were found f o r awake a c t i v i t y , S t o n e , 1979). decreased i n c r e a s e d REM, and decreased drowsy s l e e p ( N i c h o l s o n and A l s o , t r y p t o p h a n was observed t o decrease slow wave s l e e p latency in r a t s . T h i s reduced l a t e n c y c o r r e l a t e d w i t h e l e v a t e d serotonin and 5-HIAA, and depressed dopamine and h o m o v a n i l l i c a c i d ( F o r n a l , e t a l . , 1 9 7 9 ) . However, when a t r y p t o p h a n analogue was a d m i n i s t e r e d , the e f f e c t on s l e e p l a t e n c y and the c a t e c h o l a m i n e s , i n c l u d i n g n o r e p i n e p h r i n e , was r e p e a t e d . S e r o t o n i n was o n l y s l i g h t l y i n c r e a s e d and no d i f f e r e n c e s o b t a i n e d ( F o r n a l e t a l . , 1979). i n 5-HIAA were T h e r e f o r e , the t r y p t o p h a n e f f e c t was thought t o be due t o a weakening o f c a t e c h o l a m i n e f u n c t i o n ( F o r n a l e t a l . , 1979). Locomotor a c t i v i t y has a l s o been r e l a t e d t o s e r o t o n i n et a l . , 1978). cerebral D i r e c t i n f u s i o n o f s e r o t o n i n i n t o the r i g h t (Warbritton lateral v e n t r i c l e o f r a t s was r e p o r t e d t o produce dose dependent decreases i n spontaneous l o c o m o t o r b e h a v i o r as measured by a 6-channel a c t i v i t y m o n i t o r ( W a r b r i t t o n e t a l . , 1978). electronic In a d d i t i o n , when r a t s ( F i b i g e r and C a m p b e l l , 1971) o r mice (Modigh, 1972) were i n j e c t e d w i t h 5 - h y d r o x y t r y p t o p h a n and p l a c e d i n a m o t i l i t y meter (Modigh,, 1972) or a stabilimeter was o b s e r v e d . ( F i b i g e r and C a m p b e l l , 1 9 7 1 ) , decreased locomotor Rats i n j e c t e d w i t h 5 - h y d r o x y t r y p t o p h a n , activity similarly - 19 - e x h i b i t e d decreased bar p r e s s i n g f o r food which c o r r e l a t e d w i t h increased b r a i n s e r o t o n i n ( A p r i s o n and H i n g t e n , 1962). In a c c o r d w i t h the above, procedures t h a t d e p l e t e s e r o t o n i n have been observed t o i n c r e a s e locomotor a c t i v i t y . t o n e r g i c neurons by s e l e c t i v e D e s t r u c t i o n o f the s e r o - l e s i o n s o f the raphe n u c l e i , were r e p o r t e d t o induce h y p e r a c t i v i t y when r a t s were t e s t e d i n a s t a b i l i m e t e r e t a l . , 1976; J a c o b s , 1 9 7 4 ) . T h i s e f f e c t was p o t e n t i a t e d amphetamine a d m i n i s t r a t i o n (Geyer e t a l . , 1976). (Geyer following In a d d i t i o n , when measured i n e i t h e r a s t a b i l i m e t e r o r a r u n n i n g w h e e l , dose dependent i n c r e a s e s i n locomotor a c t i v i t y were produced when r a t s were g i v e n PCPA (Mabry and C a m p b e l l , 1973; F i b i g e r and C a m p b e l l , 1 9 7 1 ) . PCPA has a l s o been r e p o r t e d t o p o t e n t i a t e the e f f e c t o f amphetamine on locomotor (Breese e t a l . , 1974). In a d d i t i o n , i n j e c t i o n s were shown t o r e v e r s e the h y p e r a c t i v i t y apomorphine, dopamine s t i m u l a n t s al., 5-hydroxytryptophan induced i n r a t s by amphetamine or (Baldessarini 1974; Mabry and C a m p b e l l , 1973). of activity e t a l . , 1975; Breese e t Moreover, a c a r b o h y d r a t e i n c r e a s e i n s e r o t o n i n a l s o r e v e r s e d the h y p e r a c t i v i t y induced i n induced rats i n j e c t e d w i t h c o c a i n e , and weakened the e f f e c t o f amphetamine ( T a y l o r and Ho, 1979). B r a i n s e r o t o n i n has a l s o been l i n k e d t o e x p l o r a t i o n . Rats injected w i t h PCPA were r e p o r t e d to make more head d i p s i n a hole board a p p a r a t u s , and both between and w i t h i n s e s s i o n h a b i t u a t i o n were found prolonged ( F i l e , 1977). In a d d i t i o n , c h l o r p r o m a z i n e , a drug which i n c r e a s e s brain t r y p t o p h a n and s e r o t o n i n , was shown t o decrease the number o f head d i p s and h o l e s e x p l o r e d i n the same apparatus ( F i l e and Pope, 1974). Further- more, mice i n t r a v e n t r i c u l a r ^ i n j e c t e d w i t h s e r o t o n i n were observed to walk and r e a r l e s s than mice i n j e c t e d w i t h v e h i c l e (Herman, 1975). More- - 20 - o v e r , v e r y low doses o f t r y p t o p h a n have been r e p o r t e d t o reduce locomot i o n o f r a t s i n an open f i e l d ( T r i c k l e b a n k e t a l . , 1978; T a y l o r , However, when m e t h i o n i n e , an amino a c i d , which competes w i t h 1976). tryptophan f o r uptake a c r o s s the b l o o d b r a i n b a r r i e r , was a d m i n i s t e r e d w i t h t r y p t o p h a n , no decrease i n l o c o m o t i o n was observed ( T a y l o r , 1976). Thus, s e r o t o n i n has been a s s o c i a t e d w i t h many b e h a v i o r s . Although r e s u l t s are c o n t r o v e r s i a l , i n g e n e r a l , s e r o t o n i n appears t o f u n c t i o n as an i n h i b i t o r o f most b e h a v i o r s 6. discussed. S e r o t o n i n and C o r t i c o s t e r o n e . The r o l e o f s e r o t o n i n i n the r e g u l a t i o n o f the h y p o t h a l a m u s / p i t u i tary/adrenal a x i s has been w i d e l y researched. Complete o r f r o n t a l d e a f f e r e n t a t i o n of the r a t hypothalamus, p r o b a b l e l o c u s o f the c o r t i c a l (Yuwiller, r e l e a s i n g hormone (CRH) neurons 1 9 7 9 ) , was r e p o r t e d t o d e p l e t e s e r o t o n i n and e l e v a t e c o s t e r o n e (Vermes e t a l . , 1973). e t h e r and s u r g i c a l the corti- In a d d i t i o n , the a d r e n a l response t o s t r e s s was found b l o c k e d due t o i m p l a n t a t i o n s e r o t o n i n i n t o the medial hypothalamus (Vermes and T e l e g d y , of 1972). R e s e r p i n e , a s e r o t o n i n b l o c k e r , was observed t o i n c r e a s e ACTH i n dogs (Egdahl e t a l . , 1956). E l e v a t e d plasma c o r t i c o s t e r o n e was a l s o found i n response to e t h e r s t r e s s when r a t s were a d m i n i s t e r e d PCPA (Vermes and T e l e g d y , 1973). Thus, the above s t u d i e s seem t o suggest t h a t s e r o t o n i n i n h i b i t s r e l e a s e o f hormones o f the h y p o t h a l a m u s / p i t u i t a r y / a d r e n a l r e s u l t s t o the c o n t r a r y have a l s o been r e p o r t e d . system. However, Intraperitoneal- injection o f 5 - h y d r o x y t r y p t o p h a n was found t o e l e v a t e plasma c o r t i c o s t e r o n e e t a l . , 1972). the (Popova F u r t h e r m o r e , i n c r e a s e d c o r t i c o s t e r o n e was a l s o observed - 21 - i n rats i n j e c t e d with 5-hydroxytryptophan ( F u l l e r and Snoddy, 1 9 7 9 ) , f l u o x e t i n e , an i n h i b i t o r o f s e r o t o n i n uptake ( F u l l e r and Snoddy, 1 9 7 9 ) , and q u i p a z i n e ( F u l l e r and Snoddy, 1979) and f e n f l u r a m i n e ( S c h e t t i n i al., 1979), s e r o t o n i n a g o n i s t s . Similarly, electrolytic n u c l e u s raphe median o r i n t r a v e n t r i c u l a r l e s i o n s of the i n j e c t i o n of 5,7 t r y p t a m i n e , a s e r o t o n i n n e u r o t o x i n , was shown t o b l o c k the induced i n c r e a s e i n plasma c o r t i c o s t e r o n e (Schettini et dihydroxyfenfluramine e t a l . , 1979). a d d i t i o n , m e t e r g a l i n e , a s e r o t o n i n a n t a g o n i s t , was found t o r e v e r s e In the i n c r e a s e d c o r t i c o s t e r o n e response produced by q u i p a z i n e ( F u l l e r and Snoddy, 1979). F i n a l l y , M o d l i n g e r e t a l . (1979, 1980) observed t h a t an o r a l dose o f 10 g t r y p t o p h a n i n humans i n c r e a s e d plasma C o r t i s o l . From the above, i t would appear t h a t s e r o t o n i n p l a y s a r o l e i n the regulation of corticosterone output. indicate that serotonin is corticosterone release. The h e t e r o g e n e i t y o f r e s u l t s may i n v o l v e d i n both i n h i b i t i o n and s t i m u l a t i o n In t h i s regard, i t of i s p o s s i b l e t h a t more than one system o f CRF neurons c o u l d e x i s t and t h a t they c o u l d respond to s e r o t o n i n i n d i f f e r e n t ways ( Y u w i l l e r , 1979). In a d d i t i o n , o t h e r systems have a l s o been r e p o r t e d to mediate the o u t p u t o f CRF ( Y u w i l l e r , Thus, the i n v e s t i g a t i o n o f s e r o t o n i n ' s hypothalamus/pituitary/adrenal r o l e i n the r e g u l a t i o n o f the a x i s i s very c o m p l i c a t e d . u n d e r s t a n d i n g may not be a c h i e v e d u n t i l 1979). A more complete the l o c u s o f the CRF neurons has been e s t a b l i s h e d and s e r o t o n i n pathways i n n e r v a t i n g the CRF neurons have been i d e n t i f i e d . - 22 - 7. Summary. F a s t e d r a t s f e d a h i g h c a r b o h y d r a t e meal o r injected with have been shown t o e x h i b i t an e l e v a t i o n i n b r a i n s e r o t o n i n . behavioral consequences o f t h i s neurochemical established. tryptophan However, the i n c r e a s e have not been w e l l In a d d i t i o n , the r o l e o f s e r o t o n i n i n the r e g u l a t i o n o f c o r t i c o s t e r o n e output i s s t i l l study was conducted t o : poorly understood. Thus, the p r e s e n t (1) i n v e s t i g a t e the s i g n i f i c a n c e o f the b r a i n s e r o t o n i n e l e v a t i o n r e l a t i v e t o e x p l o r a t i o n of a novel e n v i r o n m e n t , a b e h a v i o r r e l a t e d to b r a i n s e r o t o n i n c o n t e n t ; and (2) determine plasma corticosterone concentrations duced i n c r e a s e s 8. f o l l o w i n g t r y p t o p h a n and c a r b o h y d r a t e i n - in brain serotonin. Rationale. E l e v a t e d b r a i n s e r o t o n i n has been shown t o occur i n r a t s w i t h t r y p t o p h a n o r f e d a high c a r b o h y d r a t e m e a l . injected In a d d i t i o n , reduced e x p l o r a t o r y b e h a v i o r has been observed when s e r o t o n i n has been i n c r e a s e d . Thus, i t i s h y p o t h e s i z e d t h a t f a s t e d r a t s f e d a high c a r b o h y d r a t e meal or i n j e c t e d with tryptophan w i l l d i s p l a y decreased e x p l o r a t o r y r e l a t i v e t o c o n t r o l s and t h a t the r e d u c t i o n i n e x p l o r a t i o n w i l l t i v e l y correlated with increased serotonin. as both an i n h i b i t o r and s t i m u l a t o r o f the axis. However, i t behavior be p o s i - S e r o t o n i n has been i m p l i c a t e d hypothalamus/pituitary/adrenal i s expected t h a t the t r y p t o p h a n and c a r b o h y d r a t e duced i n c r e a s e i n s e r o t o n i n w i l l since elevated Cortisol i n c r e a s e plasma c o r t i c o s t e r o n e o u t p u t has been observed i n humans an o r a l dose o f t r y p t o p h a n . ( H a r t e t a l . , 1980). levels administered S i m i l a r l y , r a t s given a sucrose have a l s o been shown t o e x h i b i t an i n c r e a s e i n plasma in- solution corticosterone - 23 - III. 1. GENERAL METHODS Animals and R a t i o n s . Male W i s t a r r a t s w e i g h i n g 220-280 g were used i n a l l studies. Eight t o 10 days p r i o r t o each e x p e r i m e n t , r a t s were o f f e r e d water and a p u r i f i e d control d i e t (Appendix T a b l e I) ad l i b i t u m . A c c o r d i n g t o the N a t i o n a l Research C o u n c i l , t h i s d i e t meets the n u t r i e n t requirements o f growing rats ( N . R . C . , 1978). Animals were housed i n d i v i d u a l l y mesh cages and exposed t o l i g h t f o r 12 hrs Room temperature was m a i n t a i n e d a t 21 + 2°C. i n suspended w i r e d a i l y , 0600 to 1800 h r s . S t r e s s has been shown t o i n c r e a s e b r a i n s e r o t o n i n t u r n o v e r i n the r a t (Curzon e t a l . , 1972). Thus, animals were handled t w i c e d a i l y i n : o r d e r to a c c l i m a t i z e them t o the h a n d l i n g t h a t would occur on the day o f 2. Biochemical testing. Determinations. Whole b r a i n t r y p t o p h a n was measured u s i n g the methods o f Denkla and Dewey ( 1 9 6 7 ) , as m o d i f i e d by Bloxam and Warren ( 1 9 7 4 ) , and whole b r a i n s e r o t o n i n was assayed by the method o f Curzon and Green ( 1 9 7 0 ) . c o r t i c o s t e r o n e was measured by the method o f G l i c k e t Double tions. d i s t i l l e d w a t e r was used t o make up a l l Plasma al(1964). reagents and s o l u - A l l w a t e r was d e i o n i z e d through a p u r i f i c a t i o n cartridge ( B a r n s t e a d High C a p a c i t y , B a r n s t e a d Company, B o s t o n , Mass.) and was subsequently d i s t i l l e d . High p u r i t y HC1 (HC1 A r i s t a r , B r i t i s h Drug House, Vancouver, B.C.) was used i n the p r e p a r a t i o n o f a l l HC1 c o n t a i n i n g solu- tions. T r y p t o p h a n , c o r t i c o s t e r o n e , and s e r o t o n i n s t o c k s o l u t i o n s and the H S0 /ET0H m i x t u r e were s t o r e d a t 4°C. ? 4 Ortho-phthaldehyde (OPT) (Sigma all - 24 - Chemical Company, S a i n t L o u i s , M i s s o u r i ) was s t o r e d i n a dark b o t t l e -20°C. at S e r o t o n i n c r e a t i n i n e s u l p h a t e (Sigma Chemical Company) and c o r t i - c o s t e r o n e (Sigma Chemical Company) were kept i n a d e s s i c a t o r a t 4°C and all o t h e r c h e m i c a l s were m a i n t a i n e d a t room t e m p e r a t u r e . cysteine The OPT and ( B r i t i s h Drug House) s o l u t i o n s were prepared i m m e d i a t e l y before use. P r i o r t o a n a l y s e s , f r o z e n t i s s u e s were homogenized i n i c e c o l d butanol a c i d i f i e d w i t h 0.01 N HC1 and c e n t r i f u g e d (1000 x G) a t 4°C f o r 10 m i n . Four ml o f the butanol s u p e r n a t a n t were then added t o 5 ml c o l d heptane p l u s 0.5 ml o f 1% L - c y s t e i n e i n 0.1 N HC1. samples were a g a i n c e n t r i f u g e d as above. A f t e r v o r t e x i n g f o r 2.5 m i n , The b u t a n o l / h e p t a n e phase was s u b s e q u e n t l y a s p i r a t e d and a l i q u o t s o f the HC1 e x t r a c t were used f o r t r y p t o p h a n and s e r o t o n i n a n a l y s i s . brain Samples were kept i c e c o l d throughout processing. Brain Tryptophan. An a l i q u o t o f 0.1 ml o f the HC1. e x t r a c t was added t o 2 ml c o l d 10% TCA w i t h 0.2 ml o f 2% f o r m a l d e h y d e . FeCl^ ( 6 . 0 x 10 N e x t , 0.1 ml o f i n c o l d 10% TCA) was added and the tubes p l a c e d i n a b o i l i n g water b a t h . during t h i s heating procedure. immediately The f l u o r o p h o r e norharmen was produced Tubes were then c o o l e d , r e p l e n i s h e d t o 2.4 ml w i t h 10% TCA, and read f l u o r o m e t r i c a l l y a t wavelengths o f 370/452 mu ( F a r r a n d Manual S p e c t r o f l u o r o m e t e r , Farrand O p t i c a l Inc.). Brain tryptop- phan was e x p r e s s e d as ug/g b r a i n . The w o r k i n g t r y p t o p h a n ( J . T . B a k e r , Chemical C o . , N.J.) Phillipsburg, s t o c k s o l u t i o n was prepared by d i l u t i n g a 4 0 . 8 ug/ml solution ( 4 . 0 8 mg/100 ml 0.1 N NH^OH) t o 4.08 u g / m l . tryptophan A s t a n d a r d curve was read d a i l y a t c o n c e n t r a t i o n s o f 0.1-7 u g , 0.34 ug and 0.51 ug o f tryptophan/tube. - 25 - Brain Serotonin. To an 0.3 a l i q u o t o f the HC1 e x t r a c t was added 0.2 ml o f OPT (0.5% i n methanol) and 1.5 ml o f c o n c e n t r a t e d HC1. ing b r i e f l y , After vortex- samples were p l a c e d i n a b o i l i n g water bath f o r 10 m i n , c o o l e d , a n d r e p l e n i s h e d t o 2 ml w i t h c o n c e n t r a t e d HC1. B r a i n s e r o t o n i n was read f l u o r o m e t r i c a l l y a t wave l e n g t h s of 360/470 mu and expressed as ug/g brain. The s e r o t o n i n s t o c k s o l u t i o n , prepared from 40 mg o f serotonin c r e a t i n i n e s u l p h a t e i n 100 ml 0.01 N HC1, was s e r i a l l y d i l u t e d t o 40 u g / m l . A s t a n d a r d curve was read d a i l y from d u p l i c a t e s o f 4 u g / m l , 2 u g / m l , and 1 ug/ml of s e r o t o n i n . Plasma C o r t i c o s t e r o n e . To 0.05 ml plasma were added 0.50 ml d i s - t i l l e d w a t e r and 0.03 ml i s o - o c t a n e . Samples were then v o r t e x e d 15 sec and c e n t r i f u g e d 4 min a t (1000 x G ) . N e x t , the i s o - o c t a n e was a s p i r a t e d and 0.7 ml o f c h l o r o f o r m were added. and c e n t r i f u g e d as above. supernatant Samples were v o r t e x e d The s u p e r n a t a n t was a g a i n removed and 0.05 ml o f 0.1 N NaOH were added t o the r e m a i n i n g c h l o r o f o r m l a y e r . Tubes were s u b s e q u e n t l y v o r t e x e d , a n d c e n t r i f u g e d as p r e v i o u s l y d e s c r i b e d and the NaOH s u p e r n a t a n t a s p i r a t e d . N e x t , 0.35 ml o f a were added, v o r t e x e d and a g a i n c e n t r i f u g e d . s u p e r n a t a n t was removed, the r e m a i n i n g H2SO4/ETOH (13:7) F i n a l l y , the H2SO4/ETOH phase t o 1 ml g l a s s c u v e t t e s , a n d read f l u o r o m e t r i c a l l y mixture chloroform was transferred u s i n g a T u r n e r 110 f 1 uorometer,,with 47-B and 2A-12 primary and secondary f i l t e r s . Corti- c o s t e r o n e was expressed as ug/100 ml plasma. A w o r k i n g c o r t i c o s t e r o n e s t o c k s o l u t i o n was prepared from 1000 ug/ 100 ml i n ET0H. C o n c e n t r a t i o n s o f 10 ug/100 m l , 20 ug/100 m l , 30 ug/ 100 m l , and 40 ug/100 ml were read d a i l y t o produce a s t a n d a r d c u r v e . - 26 - IV. EXPERIMENT 1 BRAIN TRYPTOPHAN DURING THE DARK PHASE AFTER FASTING 1. Introduction. During the dark phase o f the l i g h t i n g c y c l e , a gradual i n c r e a s e in whole b r a i n t r y p t o p h a n has been demonstrated i n r a t s f e d ad l i b i t u m (Wurtman and F e r n s t r o m , 1972). However, the e f f e c t of f a s t i n g on t h i s d a i l y e l e v a t i o n h a s n o t been s t u d i e d . Thus, the o b j e c t i v e s o f the first experiment were: 1) to determine i f a 16 hour f a s t , t e r m i n a t e d a t v a r i o u s d u r i n g the dark c y c l e , would a l t e r b r a i n t r y p t o p h a n 2) times rhythmicity; t o e s t a b l i s h the n a d i r i n b r a i n t r y p t o p h a n d u r i n g the dark p e r i o d . These r e s u l t s would e s t a b l i s h the s t a r t i n g time o f the second e x p e r i - ment. 2. Experimental Procedure. T h i r t y r a t s were randomly a s s i g n e d t o 5 g r o u p s , o f f e r e d water ad l i b i t u m , but d e p r i v e d o f food a t e i t h e r 2400, 0200, 0400, 0800,.or 1200 hrs. S i x t e e n hours l a t e r , r a t s were d e c a p i t a t e d a t e i t h e r 1600, 1800, 2000, 2400, or 0400 hrs r e s p e c t i v e l y . B r a i n s were removed, immediately f r o z e n i n l i q u i d n i t r o g e n , and s t o r e d a t -70° f o r d e t e r m i n a t i o n o f b r a i n tryptophan. The data were a n a l y z e d by a 1-way a n a l y s i s o f v a r i a n c e (ANOVA). 3. R e s u l t s and D i s c u s s i o n . T a b l e I shows the mean c o n c e n t r a t i o n s o f whole b r a i n t r y p t o p h a n r a t s f a s t e d 16 h r s . in The ANOVA i n d i c a t e d t h a t d u r i n g the dark phase, b r a i n t r y p t o p h a n d i d not d i f f e r when r a t s were d e p r i v e d o f food f o r 16 h r s . - 27 - TABLE I BRAIN TRYPTOPHAN FOLLOWING A 16 HOUR FAST Time B r a i n Tryptophan (ug/g + SEM) 1600 ( 5 ) * 3.36 + 0.14 1800 (6) 3.30 + 0.12 2000 (6) 3.46 + 0.16 2400 (6) 3.24 + 0.08 0400 (5) 3.55 + 0.11 * Number o f animals/group - 28 - During the dark phase, r a t s fed ad l i b i t u m have been observed t o exh i b i t a gradual i n c r e a s e i n whole b r a i n (Wurtman and F e r n s t r o m , 1 9 7 2 ) , b r a i n stem (Morgan e t a l . , 1975), c e r e b r a l c o r t e x (Hery e t a l . , 1977), and. f r o n t o - p a r i e t a l fluctuations cerebral c o r t e x (Hery e t a l . , 1977) t r y p t o p h a n . Daily i n the r a t i o o f plasma t r y p t o p h a n t o the n e u t r a l amino a c i d s are b e l i e v e d t o generate the b r a i n t r y p t o p h a n rhythm ( F e r n s t r o m and Wurtman, 1979). cyclical Moreover, c o n s i d e r a b l e e v i d e n c e has i n d i c a t e d t h a t the consumption o f p r o t e i n c o n t r i b u t e s t o the d a i l y change i n plasma n e u t r a l amino a c i d s ( F e r n s t r o m e t a l . , 1979b). Thus, Fernstrom (1979) has proposed t h a t the d a i l y i n t a k e of p r o t e i n generates the rhythm i n b r a i n tryptophan. R e s u l t s from the p r e s e n t study s u p p o r t the p o s s i b i l i t y t h a t food i n take may c o n t r i b u t e t o the d a i l y i n c r e a s e i n t r y p t o p h a n d u r i n g the dark phase. However, t h i s i n t e r p r e t a t i o n must be viewed w i t h c a u t i o n , because an ad l i b i t u m c o n t r o l was not i n c l u d e d i n t h i s e x p e r i m e n t . These data f u r t h e r i n d i c a t e d t h a t b r a i n t r y p t o p h a n d i d not f l u c t u a t e d u r i n g the dark p e r i o d a f t e r a 16 hour fast. - 29 - V. EXPERIMENT 2 EFFECT OF TRYPTOPHAN ADMINISTRATION AND CARBOHYDRATE INGESTION ON BRAIN TRYPTOPHAN AND SEROTONIN 1. Introduction. B r a i n t r y p t o p h a n and s e r o t o n i n have r e p e a t e d l y been shown t o increase i n r a t s f a s t e d o v e r n i g h t and i n j e c t e d w i t h t r y p t o p h a n , o r o f f e r e d a high c a r b o h y d r a t e , p r o t e i n - f r e e meal a t the b e g i n n i n g o f the l i g h t phase. Whether t h i s same phenomenon o c c u r s when r a t s are f a s t e d p r i m a r i l y during the l i g h t c y c l e and then f e d c a r b o h y d r a t e a t the b e g i n n i n g of the dark p e r i o d has not been d e t e r m i n e d . Thus, the purpose o f the second e x p e r i - ment was t o determine the time c o u r s e and peak l e v e l s o f b r a i n and s e r o t o n i n i n f a s t e d r a t s f e d a c o n t r o l m e a l , a high tryptophan carbohydrate, p r o t e i n - f r e e m e a l , or i n j e c t e d w i t h t r y p t o p h a n e a r l y i n the dark phase. 2. Experimental Procedure. The e x p e r i m e n t a l d e s i g n i s summarized i n F i g u r e 3. A t 0030 hrs r a t s were randomly a s s i g n e d t o 4 groups and d e p r i v e d o f food but p e r m i t t e d access to water. At 1730 hrs water ad l i b i t u m and t r e a t e d as free the f a s t was t e r m i n a t e d and r a t s were o f f e r e d follows: Group 1 - d e c a p i t a t e d , b r a i n s removed, f r o z e n i n n i t r o g e n , and s t o r e d a t - 7 0 ° C ; liquid Group 2 - i n j e c t e d w i t h 0.9% s a l i n e , (pH 9 . 5 ) and o f f e r e d p u r i f i e d c o n t r o l d i e t ad l i b i t u m ; the Group 3 - i n j e c t e d w i t h 0.9% s a l i n e , (pH 9 . 5 ) and o f f e r e d the p r o t e i n - f r e e p u r i f i e d d i e t , i s o c a l o r i c t o the c o n t r o l d i e t with protein derived c a l o r i e s replaced with carbohydrate (Table 1 - appendix); Group 4 - i n j e c t e d w i t h L - t r y p t o p h a n , (50mg/kg) d i s s o l v e d i n s a l i n e (pH 9 . 5 ) and o f f e r e d the c o n t r o l d i e t ad 1ibitum. - 30 - ANIMALS FASTED 0030 1730 v FASTED" BASAL F i g u r e 3. * CONTROL & SALINE CARBOHYDRATE & SALINE & CONTROL TRYPTOPHAN 1830 }830* 1830 1930 1930* 1930* 2030 2030 2030* 2130 2130 E f f e c t o f t r y p t o p h a n a d m i n i s t r a t i o n and c a r b o h y d r a t e i n g e s t i o n on b r a i n t r y p t o p h a n and serotonin: Experimental d e s i g n . B r a i n s removed f o r a n a l y s i s o f b r a i n t r y p t o p h a n and s e r o t o n i n. - 31 - One hour p o s t t r e a t m e n t and h o u r l y f o r the next 3 hours, a n i m a l s from groups 2, 3 , and 4 were d e c a p i t a t e d and b r a i n s removed, f r o z e n i n n i t r o g e n , and s t o r e d a t -70°C. liquid B r a i n t r y p t o p h a n and s e r o t o n i n were a n a l y z e d by a Group (3) x Time (4) ANOVA. 3. Results. Brain Tryptophan. The time course and peak l e v e l s o f b r a i n tryptophan i n f a s t e d r a t s o f f e r e d a c o n t r o l m e a l , a high c a r b o h y d r a t e m e a l , o r w i t h t r y p t o p h a n are i l l u s t r a t e d i n F i g u r e 4 and Appendix T a b l e I I . r e v e a l e d a Group x Time i n t e r a c t i o n F ( 5 , 5 1 ) = 2 9 . 2 7 , p < . 0 0 1 . main e f f e c t s i n d i c a t e d a main e f f e c t o f group a t 1 h o u r , injected The ANOVA Tests f o r simple F(2,51)=101.75, p < . 0 1 , 2 hours F ( 2 , 5 1 ) = 4 . 2 9 , p < . 0 5 , 3 hours F(2,51 )=4.44, p < . 0 5 , and 4 hours F ( 2 , 5 1 ) = 3 . 3 1 , p < . 0 5 , p o s t t r e a t m e n t . Neuman Keuls p o s t hoc a n a l y s e s showed t h a t a n i m a l s i n j e c t e d w i t h t r y p t o p h a n had h i g h e r brain t r y p t o p h a n l e v e l s than c o n t r o l s , p < . 0 1 , and c a r b o h y d r a t e t r e a t e d rats, p < . 0 1 , a t 1 hour p o s t i n j e c t i o n . injected animals f e l l to c o n t r o l Brain tryptophan of tryptophan l e v e l s by 2 h o u r s . When compared t o c o n t r o l s , b r a i n t r y p t o p h a n was a l s o e l e v a t e d , p < . 0 5 , i n r a t s o f f e r e d the high c a r b o hydrate meal. A l t h o u g h the d i f f e r e n c e c o n t i n u e d f o r 4 hours p o s t treatment, p < . 0 5 , the g r e a t e s t d i f f e r e n c e o c c u r r e d 2 hours a f t e r food p r e s e n t a t i o n . Brain Serotonin. F i g u r e 5 and Appendix T a b l e I I I i l l u s t r a t e the time course o f b r a i n s e r o t o n i n i n f a s t e d r a t s fed e i t h e r a c o n t r o l d i e t , a high carbohydrate meal, or i n j e c t e d w i t h tryptophan. The ANOVA i n d i c a t e d a main e f f e c t o f group F ( 2 , 5 1 ) = 6 . 5 0 , p < . 0 0 1 , and Neumann Keuls p o s t hoc a n a l y s e s revealed t h a t b r a i n s e r o t o n i n i n r a t s i n j e c t e d with tryptophan or the h i g h c a r b o h y d r a t e , p r o t e i n - f r e e meal was e l e v a t e d over t h a t o f p < .05. offered controls, Fasted Basal Control Carbohydrate Tryptophan 2h Basal F i g u r e 4. One Hour Two Hours Three Hours Four Hours B r a i n tryptophan f o l l o w i n g tryptophan a d m i n i s t r a t i o n or i n g e s t i o n c a r b o h y d r a t e (mean + SEM) of F i g u r e 5: Brain serotonin a f t e r tryptophan a d m i n i s t r a t i o n or i n g e s t i o n (mean + SEM) carbohydrate - 34 - 4. Discussion. These d a t a are i n accordance w i t h the f i n d i n g s o f Fernstrom ( 1 9 7 1 , 1975b). F u r t h e r m o r e , the r e s u l t s demonstrate t h a t the c a r b o h y d r a t e induced i n c r e a s e i n b r a i n t r y p t o p h a n and s e r o t o n i n c o n t e n t o c c u r s d u r i n g the e a r l y s t a g e o f the dark c y c l e . As p r e v i o u s l y observed ( F e r n s t r o m , 1971), b r a i n t r y p t o p h a n i n t h i s s t u d y peaked 1 hour a f t e r t r y p t o p h a n i n j e c t i o n . Feed- i n g c a r b o h y d r a t e generated the g r e a t e s t change i n b r a i n s e r o t o n i n a t 2 and 3 hours a f t e r food i n t a k e . However, i n c o n t r a s t t o e a r l i e r r e s e a r c h , the p e r c e n t i n c r e a s e s b r a i n t r y p t o p h a n and s e r o t o n i n were s m a l l i n the p r e s e n t s t u d y . s u l t s are not s u r p r i s i n g . Reduced r a d i o l a b e l l e d in Such r e - t r y p t o p h a n uptake i n both hypothalamus and b r a i n stem s l i c e s has been observed d u r i n g the dark phase (Hery e t a l . , 1972). A l s o , reduced t r y p t o p h a n a v a i l a b i l i t y c o u l d the s m a l l e r p e r c e n t i n c r e a s e i n s e r o t o n i n o b t a i n e d i n t h i s s t u d y . explain How- e v e r , a g r e a t e r t u r n o v e r o f s e r o t o n i n t o 5-HIAA may have o c c u r r e d as w e l l , s i n c e i n c r e a s e d s e r o t o n i n t u r n o v e r has been demonstated d u r i n g the dark phase (Hery e t a l . , 1972). Thus, the r e s u l t s o f t h i s experiment i n d i c a t e d t h a t t r y p t o p h a n d o s i n g and c a r b o h y d r a t e i n g e s t i o n r e s u l t e d i n e l e v a t e d b r a i n t r y p t o p h a n and s e r o t o n i n d u r i n g the dark phase. 2 hours p o s t treatment. The g r e a t e s t change was observed a t 1 and - 35 - VI. EXPERIMENT 3 BEHAVIORAL EFFECTS OF TRYPTOPHAN ADMINISTRATION T h i s experiment was conducted t o e s t a b l i s h a b e h a v i o r a l correlate of increased brain serotonin concentration. Three b e h a v i o r s b e l i e v e d t o be a s s o c i a t e d w i t h s e r o t o n i n were a s s e s s e d : exploratory behavior, acquisi- t i o n o f a p a s s i v e - a v o i d a n c e r e s p o n s e , and e x t i n c t i o n . 1. Experimental The e x p e r i m e n t a l Procedure. procedure i s summarized i n F i g u r e 6. o f f e r e d p u r i f i e d c o n t r o l d i e t and w a t e r ad l i b i t u m . hrs Rats were From 1700 t o 2000 on the t r a i n i n g n i g h t , 48 r a t s were randomly s e l e c t e d and i n j e c t e d w i t h e i t h e r 0.9% s a l i n e (pH 9 . 5 ) o r 50 mg/kg t r y p t o p h a n i n 0.9% s a l i n e (pH 9 . 5 ) . On the n i g h t s o f e x t i n c t i o n , the 2 groups were f u r t h e r so t h a t h a l f the o r i g i n a l divided s a l i n e and t r y p t o p h a n a n i m a l s were a d m i n i s t e r e d s a l i n e and h a l f were i n j e c t e d w i t h t r y p t o p h a n . Thus, 4 groups o f 12 r a t s were e s t a b l i s h e d and i n j e c t e d from 1800 t o 1900 h r s . o c c u r r e d 1 hour p o s t i n j e c t i o n , Behavioral testing the time when b r a i n s e r o t o n i n was shown t o peak. Animals were t e s t e d i n a d a r k , sound c o n t r o l l e d room ( w h i t e n o i s e , 56db) a d j a c e n t t o the animal q u a r t e r s . The b e h a v i o r a l apparatus was a wooden box,25 x 29 x 40 cm,having one p l e x i g l a s s w a l l f o r observation and a copper g r i d f l o o r which c o u l d be e l e c t r i f i e d w i t h a scrambled mamp c u r r e n t . A p l a t f o r m , 7.5 x 25 x 7 cm, extended a c r o s s 1 w a l l 1.25 of the apparatus and a 20 w a t t i n c a n d e s c e n t 1 i g h t e v e n l y i l l u m i n a t e d the box. One hour a f t e r i n j e c t i o n , each r a t was i n d i v i d u a l l y p l a c e d on the narrow p l a t f o r m and the l a t e n c y t o step-down and e x p l o r e the e x p e r i m e n t a l - 36 - F i g u r e 6. Behavioral e f f e c t s of tryptophan administration: Experimental d e s i g n . - 37 - box on 2 - 1 min trials, t r i a l s was measured by 2 t r a i n e d o b s e r v e r s . Between r a t s were r e t u r n e d to a s m a l l p l a s t i c h o l d i n g cage f o r 1 m i n u t e . On the t h i r d t r i a l , the g r i d f l o o r was e l e c t r i f i e d and the following recorded: (1) l a t e n c y to escape shock; (2) total number o f step-downs (4 f e e t on f l o o r ) ; (3) total number o f step-down, attempts (4) t o t a l time to reach c r i t e r i o n consecutive minutes). During e x t i n c t i o n t r i a l s (1-3 f e e t on f l o o r ) ; (remain on the p l a t f o r m f o r 2 (3 min d u r a t i o n ) , run on the ,5 subse- quent n i g h t s , the f o l l o w i n g b e h a v i o r s were r e c o r d e d : (1) e x t i n c t i o n ( l a t e n c y t o step-down and remain on the f l o o r a c r i t e r i o n of 30 s e c o n d s ) ; (2) number o f step-down attempts (1-3 f e e t on f l o o r o r 4 f e e t on the f l o o r but f a i l u r e t o meet the 30 second c r i t e r i o n ) ; (3) number o f step-down attempts and t o t a l the f i r s t n i g h t of e x t i n c t i o n ; (4) total n i g h t s t o reach (5) total seconds t o e x t i n g u i s h ; (6) total step-down attempts p r i o r t o e x t i n c t i o n . time t o meet c r i t e r i o n on criterion; Rats were e l i m i n a t e d from the experiment once e x t i n c t i o n o c c u r r e d . Measures r e c o r d e d during" l e a r n i n g and the e x t i n c t i o n t r i a l s were a n a l y z e d by the S t u d e n t ' s t - t e s t . a n d 1-way ANOVA r e s p e c t i v e l y . A Group (2) x Time (2) ANOVA w i t h time as a r e p e a t e d measure was used t o t e s t the mean latencies 2. to step-down on 2 t r i a l s . Results. Acquisition/Extinction Testing. The group means f o r each measure taken d u r i n g a c q u i s i t i o n and e x t i n c t i o n are shown, i n T a b l e H a n d No d i f f e r e n c e s were observed f o r any o f the parameters tested. Table III. TABLE I I ACQUISITION OF AN AVOIDANCE RESPONSE FOLLOWING INJECTION OF SALINE OR TRYPTOPHAN Treatment Control (Saline) Escape l a t e n c y (23)* Tryptophan (24) 28.91 + - 4 . 4 3 34.89 + 10.57 * Number of animals/group t Mean seconds + SEM f Mean number o f step-down t Attempts Step-downs Total time (24) 1.33 +- 0.36* (24) 0 . 3 3 + 0.13 (24) 177.17 + 1 0 . 7 1 (24) 0.96 + 0.27 (24) 189.33 + 13.54 attempts/trial (24) 0.25 + 0.11 + ' TABLE III EFFECT OF TRYPTOPHAN OR SALINE ON EXTINCTION Treatment Training Extinction T o t a l Seconds Saline Saline* 249.17 + 9 1 . 0 0 ^yP " phan 196.83+66.06 - 1.85+0.60 - 119.00+20.47 - 1.33+0.48 1.75+0.35 220.08"+ 6 5 . 1 4 2.75+0.72 123.42+15.89 2.00+0.48 1 . 6 7 + 0.38 Saline phan " t 0 T S t o a 1 i n e Total Stepdown Attempts n 2.08 + 0 . 5 7 § T o t a l Seconds Night 1 115.92 + 1 9 . 1 4 + T o t a l SDA N i g h t .1 Total Nights 1.50 + 0.56* 1.83+0.44* & I Tryptophan Tryptophan 142.58+35.73 - 2.00+0.63 104.50+20.83 - * N = 12 a n i m a l s / g r o u p JI T o t a l seconds + SEM t o e x t i n g u i s h •-§ T o t a l step-down attempts + SEM over 5 n i g h t s of t Mean seconds + SEM f i r s t n i g h t of extinction extinction ^ Mean number o f step-down-attempts + SEM f i r s t n i g h t o f # T o t a l n i g h t s + SEM to e x t i n g u i s h extinction 1.25+0.39 - 1.42+0.39 - - 40 - L a t e n c y t o Step-Down. The ANOVA r e v e a l e d a main e f f e c t o f group F ( l , 4 5 ) = 4 . 6 4 , p < . 0 5 , and time F ( l , 4 5 ) = 12.41,' p < . 0 0 1 , f o r the t o step-down and e x p l o r e the novel chamber. illustrate latency F i g u r e 7 and Appendix Table IV t h a t t r y p t o p h a n i n j e c t e d animals took l o n g e r than c o n t r o l s step-down on both t r i a l s , p <-:.05. In a d d i t i o n , both the c o n t r o l t r y p t o p h a n groups stepped down f a s t e r on the second t r i a l to and than on the first t r i a l , p < .05. 3. Discussion. A c q u i s i t i o n o f an Avoidance Response. Depletion of b r a i n serotonin has r e p e a t e d l y been shown t o f a c i l i t a t e a c q u i s i t i o n o f an avoidance task ( V o r h e e s , 1979; L i n e t a l . , 1978; Koher and L o r e n s , 1978). Thus, i t was h y p o t h e s i z e d t h a t an e l e v a t i o n o f s e r o t o n i n , v i a t r y p t o p h a n administration, would i m p a i r l e a r n i n g o f a punished step-down response (passive-avoidance). A l t h o u g h the r e s u l t s o f t h i s experiment do not s u p p o r t the above hypothesis, they are i n a c c o r d w i t h the f i n d i n g s o f Engel and Modigh (1974) who observed t h a t 600-800 mg/kg t r y p t o p h a n i m p a i r e d a c q u i s i t i o n of shuttle box a c t i v e - a v o i d a n c e and t r y p t o p h a n doses o f l e s s than 600 mg/kg were i n effective i n a f f e c t i n g avoidance response l e a r n i n g . Thus, the tryptophan dose (50 mg/kg) used i n the p r e s e n t s t u d y , may have been too low t o l e a r n i n g o f the punished step-down r e s p o n s e . Engle and Modigh's affect (1974) r e s u l t s a l s o suggest t h a t the t r y p t o p h a n impairment o f s h u t t l e box a v o i d ance might not be r e l a t e d t o s e r o t o n i n , because much l o w e r doses o f trypto- phan (50 mg/kg) have been shown t o produce maximum i n c r e a s e s i n b r a i n s e r o t o n i n ( F e r n s t r o m and Wurtman, 1971; Young e t a l . , 1978). In a d d i t i o n t o the above, these f i n d i n g s may be due t o the b e h a v i o r studied, i.e. passive-avoidance. A l t h o u g h s e r o t o n i n has been r e l a t e d t o a c t i v e - a v o i d a n c e , a t a s k which i n v o l v e s i n i t i a t i o n o f a r e s p o n s e , serotonin 30 Control 25 Tryptophan 20 15 10 1 2 1 2 Trials F i g u r e 7. Latency to step-down f o l l o w i n g t r y p t o p h a n a d m i n i s t r a t i o n (mean + SEM). - 42 - may not f u n c t i o n i n the same manner i n p a s s i v e - a v o i d a n c e , a t a s k which r e q u i r e s response i n h i b i t i o n . In t h i s r e g a r d , these data " c o n f i r m e t a l . (1978) who observed t h a t e l e c t r i c a l s t i m u l a t i o n o f the d o r s a l n u c l e u s , a b r a i n r e g i o n r i c h i n s e r o t o n e r g i c p e r i k a r y a , d i d not a c q u i s i t i o n of a simple passive-avoidance Extinction. thoseofFibiq raphe affect task. S e r o t o n i n d e p l e t i o n w i t h PCPA has a l s o been shown t o i n c r e a s e e x t i n c t i o n time o f a punished step-down response ( B e n i n g e r and Phillips, 1980). Thus, i t was p r e d i c t e d t h a t i n : t h i s experiment extinc- t i o n o f the step-down b e h a v i o r would be reduced i f s e r o t o n i n was e l e v a t e d v i a a tryptophan i n j e c t i o n . However, these r e s u l t s were not i n agreement w i t h those o f B e n i n g e r and P h i l l i p s (1980). A g a i n , the dose (50 mg/kg) i n t h i s study may have been too low to i n f l u e n c e utilized extinction, even though 50 mg/kg t r y p t o p h a n i s known t o produce a maximum e l e v a t i o n i n b r a i n s e r o t o n i n ( F e r n s t r o m and Wurtman, 197,1-; Young e t a l . , 1978). In a d d i t i o n , m a n i p u l a t i o n s t h a t a l t e r s e r o t o n i n c o n t e n t , w i t h i n normal physiological r a n g e , may not be s u f f i c i e n t t o a f f e c t e x t i n c t i o n . Indeed, PCPA has been shown t o g r e a t l y d e p l e t e s e r o t o n i n through i n h i b i t i o n tryptophan hydroxylase a c t i v i t y Exploratory Behavior. ( B e n i n g e r and P h i l l i p s , of 1980). R e s u l t s from the p r e s e n t s t u d y suggest that t r y p t o p h a n i n j e c t e d r a t s d i s p l a y reduced e x p l o r a t o r y b e h a v i o r when e x posed to a novel chamber. These f i n d i n g s a r e i n agreement w i t h those o f and-Pope (1974) who a l s o demonstrated t h a t hole board e x p l o r a t o r y behavior was reduced when r a t s were a d m i n i s t e r e d c h l o r p r o m a z i n e , a drug which elevates brain serotonin. S i m i l a r l y , decreased w a l k i n g b e h a v i o r i n an open f i e l d was observed when r a t s were i n j e c t e d w i t h tryptophan ( T r i c k l e b a n k e t a l . , 1978) whereas, enhanced e x p l o r a t i o n i n a h o l e board apparatus was shown when s e r o t o n i n was d e p l e t e d w i t h PCPA ( F i l e , Fil 1977). - 43 - However, i n a d d i t i o n t o e x p l o r a t o r y b e h a v i o r , the l a t e n c y t o s t e p down may s i m p l y r e f l e c t the a n i m a l ' s a c t i v i t y l e v e l . Reduced locomotor b e h a v i o r has been observed i n r a t s w i t h e l e v a t e d c o n c e n t r a t i o n s o f b r a i n s e r o t o n i n ( T a y l o r , 1976). A l t h o u g h i t was d i f f i c u l t to separate these two b e h a v i o r s i n t h i s e x p e r i m e n t , the l a t e n c y s c o r e s d i d appear t o be correlated with serotonin levels. - 44 - VII. EXPERIMENT FOUR TRYPTOPHAN AND CARBOHYDRATE INDUCED INCREASES IN BRAIN SEROTONIN: BIO- CHEMICAL AND BEHAVIORAL CORRELATES 1. Introduction. The purpose of the f i n a l s t u d y was t o determine i f the c a r b o h y d r a t e induced i n c r e a s e i n b r a i n s e r o t o n i n would y i e l d b e h a v i o r a l p a r a b l e t o the t r y p t o p h a n induced e l e v a t i o n . r e s u l t s com- To h e l p c l a r i f y whether the l a t e n c y measure r e f l e c t s e x p l o r a t o r y b e h a v i o r o r locomotor activity, r e a r i n g b e h a v i o r on both t r i a l s was t a b u l a t e d as a n o t h e r component o f exploration. D e f e c a t i o n and u r i n a t i o n , b e h a v i o r s often negatively a t e d w i t h s e r o t o n i n (Kameyama e t a l . , 1980), were a l s o r e c o r d e d . plasma c o r t i c o s t e r o n e was assayed as a b i o c h e m i c a l i n d i c a t o r o f b r a i n s e r o t o n i n s i n c e t h i s n e u r o t r a n s m i t t e r has been shown t o the h y p o t h a l a m u s / p i t u i t a r y / a d r e n a l 2. Experimental The e x p e r i m e n t a l axis (Yuwiller, associFinally, increased regulate 1979). Procedure. procedure i s o u t l i n e d i n F i g u r e 8 . Seventeen hours p r i o r t o drug i n j e c t i o n s o r d i e t p r e s e n t a t i o n , r a t s were randomly assigned t o 5 groups and 80 of 88 animals were d e p r i v e d o f food but not w a t e r . The r e m a i n i n g 8 r a t s were g i v e n f r e e access t o both food and w a t e r . i n g was t e r m i n a t e d between 1700 and 1800 and animals were t r e a t e d as follows: Group 1 - ( n o n - f a s t e d group) - d e c a p i t a t e d , b l o o d and b r a i n s removed; Group 2 - d e c a p i t a t e d , b l o o d and b r a i n s removed; Group 3 - i n j e c t e d w i t h 0.9% s a l i n e and o f f e r e d the h i g h c a r b o h y d r a t e , p r o t e i n - f r e e meal ad l i b i t u m ; Fast- - 45 - ANIMALS FASTED 2400 NON-FASTED 1700 FASTED BASAL CONTROL SALINE F i g u r e 8. CARBOHYDRATE SALINE 1700 CONTROL TRYPTOPHAN 1800* 1800* 1800 1900* 1900" 1900* Tryptophan and c a r b o h y d r a t e induced i n c r e a s e s b r a i n s e r o t o n i n : _ b i o c h e m i c a l and b e h a v i o r a l correlates: Experimental d e s i g n . B r a i n s removed f o r a n a l y s i s o f b r a i n t r y p t o p h a n and s e r o t o n i n and b l o o d c o l l e c t e d f o r plasma c o r t i c o s t e r o n e . NON-FASTED BASAL in - 46 - Group 4 - i n j e c t e d w i t h 0.9% s a l i n e and o f f e r e d the c o n t r o l d i e t ad l i b i t u m ; Group 5 - i n j e c t e d w i t h 50 mg/kg t r y p t o p h a n and o f f e r e d the c o n t r o l d i e t ad l i b i t u m . A t 1 or 2 hours post t r e a t m e n t , r a t s from groups 3 , 4 and 5 were p l a c e d i n the novel chamber. individually Behaviors recorded i n c l u d e d : (1) l a t e n c y t o step-down on 2 t r i a l s ; (2) number o f r e a r s per t r i a l (3) t o t a l once on the floor; number o f b o l u s e s and u r i n a t i o n p o o l s a f t e r 2 t r i a l s . Immediately f o l l o w i n g b e h a v i o r a l t e s t i n g each r a t was d e c a p i t a t e d . b l o o d was c o l l e c t e d i n h e p a r i n i z e d t u b e s . A f t e r c e n t r i f u g a t i o n at x G) f o r 20 m i n , plasma was c o l l e c t e d and s t o r e d a t -20°C. Trunk (1000 B r a i n s were removed, r a p i d l y f r o z e n i n l i q u i d n i t r o g e n , and s t o r e d a t -70°C. Statistics. The S t u d e n t ' s t - t e s t was used t o a n a l y z e f a s t e d versus n o n - f a s t e d basal b r a i n t r y p t o p h a n , s e r o t o n i n , and plasma c o r t i c o s t e r o n e . B r a i n t r y p t o p h a n , s e r o t o n i n , plasma c o r t i c o s t e r o n e , latency to step-down, r e a r i n g , u r i n a t i o n , d e f e c a t i o n , and food i n t a k e , measures taken 1 and 2 h r s . p o s t t r e a t m e n t , were a n a l y z e d by a Group (3) x Time (2) ANOVA. 3. Results. Brain Tryptophan. B r a i n t r y p t o p h a n from f a s t e d b a s a l o r n o n - f a s t e d basal r a t s and the time course and peak c o n c e n t r a t i o n s o f b r a i n tryptophan i n animals f e d a h i g h c a r b o h y d r a t e , ' p r o t e i n - f r e e m e a l , i n j e c t e d w i t h trypto- phan, or f e d the c o n t r o l d i e t are p r e s e n t e d i n F i g u r e 9 and Appendix T a b l e V. The S t u d e n t ' s t-test i n d i c a t e d t h a t b r a i n t r y p t o p h a n was h i g h e r f a s t e d v e r s u s n o n - f a s t e d b a s a l animals T ( 1 3 ) = 4 . 1 2 , p < . 0 1 . in In a d d i t i o n , the ANOVA r e v e a l e d a Group x Time i n t e r a c t i o n F ( 2 , 6 4 ) = 1 1 9 , 2 6 , p < . 0 0 1 . T e s t s f o r s i m p l e main e f f e c t s i n d i c a t e d a main e f f e c t o f group a t 1 hour 18 • X 16 • Non-fasted basal Fasted basal 14 QO — I Q_ >- OH Control 12 Carbohydrate CD g> 10 Tryptophan CH CO 6 4 2 " Basal Figure 9 . One Hour Two Hours B r a i n t r y p t o p h a n f o l l o w i n g i n g e s t i o n o f c a r b o h y d r a t e and t r y p t o p h a n a d m i n i s t r a t i o n (mean + SEM) - 48 F ( 2 , 6 3 ) = 3 1 9 , 2 , p < . 0 1 , and 2 hours F ( 2 , 6 5 ) = 9 . 5 5 , p < . 0 1 , a f t e r food and drug a d m i n i s t r a t i o n . also found. A main e f f e c t o f time F ( 2 , 6 5 ) = 1 7 5 . 5 , p < . 0 1 , was Neuman-Keuls post hoc a n a l y s e s showed t h a t animal injected with trypotophan e x h i b i t e d higher b r a i n tryptophan concentrations than both c o n t r o l and c a r b o h y d r a t e t r e a t e d r a t s at 1 hour p o s t i n j e c t i o n , p < . 0 1 . By 2 h o u r s , b r a i n t r y p t o p h a n f e l l c o n c e n t r a t i o n s were s t i l l i n the t r y p t o p h a n i n j e c t e d a n i m a l s , but s i g n i f i c a n t l y more e l e v a t e d than c o n t r o l s , p < . 0 1 . P o s t hoc a n a l y s e s a l s o r e v e a l e d t h a t the c a r b o h y d r a t e f e d animals displayed an i n c r e a s e i n b r a i n t r y p t o p h a n over c o n t r o l s a t 1 and 2 hours a f t e r food presentation, p < .01. Brain Serotonin. F i g u r e 10 and Appendix T a b l e VI show f a s t e d and non- f a s t e d b a s a l c o n c e n t r a t i o n s o f b r a i n s e r o t o n i n and the time course and peak l e v e l s o f s e r o t o n i n i n animals f e d the c o n t r o l o r e x p e r i m e n t a l d i e t s o r i n jected with tryptophan. B r a i n s e r o t o n i n i n f a s t e d and n o n - f a s t e d r a t s was not d i f f e r e n t when a n a l y z e d by the S t u d e n t ' s t - t e s t . However, the ANOVA r e v e a l e d a Group x Time F ( 2 , 6 5 ) = 5 . 3 0 , p < . 0 1 , i n t e r a c t i o n f o r rats assayed 1 and 2 hours a f t e r t r y p t o p h a n i n j e c t i o n o r food p r e s e n t a t i o n . A main e f f e c t o f group was shown a t both 1,F(2,65)=50.1 , p < . 0 1 , and 2 , F ( 2 , 6 5 ) = 1 3 . 9 1 , p < . 0 1 , hours a f t e r t r e a t m e n t by the t e s t o f s i m p l e main e f f e c t s . Neuman-Keuls post hoc a n a l y s e s r e v e a l e d t h a t animals in- j e c t e d w i t h t r y p t o p h a n had a h i g h e r c o n c e n t r a t i o n o f b r a i n s e r o t o n i n , p < . 0 1 , than c a r b o h y d r a t e and c o n t r o l By 2 hours b r a i n s e r o t o n i n f e l l , controls, p < .01. f e d r a t s at 1 hour p o s t but c o n c e n t r a t i o n s were s t i l l Carbohydrate f e d animals were not shown t o injection. h i g h e r than differ from c o n t r o l s a t e i t h e r times a s s a y e d . Plasma C o r t i c o s t e r o n e . centrations The time course o f plasma c o r t i c o s t e r o n e i s i l l u s t r a t e d i n F i g u r e 11 and Appendix T a b l e V I I . r e v e a l e d a main e f f e c t o f group F ( 2 , 6 4 ) = 5 . 1 8 , p < . 0 1 . con- The ANOVA Neuman-Keuls post F i g u r e 10. B r a i n s e r o t o n i n f o l l o w i n g i n g e s t i o n of c a r b o h y d r a t e or a d m i n i s t r a t i o n (mean + SEM). tryptophan Figure 11. Plasma c o r t i c o s t e r o n e f o l l o w i n g c a r b o h y d r a t e i n g e s t i o n o r a d m i n i s t r a t i o n (mean + SEM). tryptophan - 51 - hoc a n a l y s e s i n d i c a t e d t h a t c a r b o h y d r a t e f e d r a t s e x h i b i t e d a s i g n i f i c a n t l y higher level o f c o r t i c o s t e r o n e than c o n t r o l s , p < . 0 1 . Food I n t a k e . T a b l e IV i n c l u d e s t h e amount o f f o o d and c a r b o h y d r a t e consumed by a n i m a l s e i t h e r i n j e c t e d w i t h t r y p t o p h a n , o f f e r e d the high c a r b o h y d r a t e m e a l , o r p r e s e n t e d the c o n t r o l d i e t . ANOVA r e v e a l e d that the animals o f f e r e d the high c a r b o h y d r a t e p r o t e i n - f r e e meal a t e less food than those g i v e n the c o n t r o l d i e t and i n j e c t e d w i t h e i t h e r trypto- phan o r s a l i n e F ( 2 , 6 2 ) = 1 4 . 5 7 , p < . 0 1 . However, c a r b o h y d r a t e consumption d i d not d i f f e r among g r o u p s . Latency To Step-Down/Rearing. Mean r e a r i n g and l a t e n c y t o s t e p - down f o r a n i m a l s i n j e c t e d w i t h t r y p t o p h a n , o f f e r e d the h i g h c a r b o h y d r a t e m e a l , o r g i v e n c o n t r o l d i e t i s g i v e n i n T a b l e V and F i g u r e 12 and Appendix, Table V I I I , r e s p e c t i v e l y . The ANOVA r e v e a l e d t h a t i n j e c t i o n o f tryptophan o r a d m i n i s t r a t i o n o f a h i g h c a r b o h y d r a t e meal d i d n o t a f f e c t r e a r i n g o r the l a t e n c y t o step-down on e i t h e r t r i a l , a t 1 o r 2 hours p o s t Urination i n Table V I . and B o l u s e s . treatment. U r i n a t i o n and b o l u s measures are presented No s i g n i f i c a n t e f f e c t o f group was o b t a i n e d when the u r i n a - t i o n and b o l u s d a t a were a n a l y z e d by ANOVA. f o r time was suggested f o r u r i n a t i o n . However, a t r e n d , . p< . 0 9 , Both the t r y p t o p h a n and c o n t r o l animals tended t o u r i n a t e l e s s 2 hours a f t e r i n j e c t i o n o r food consumption. 4. Discussion. Brain Tryptophan. The e l e v a t i o n s i n b r a i n t r y p t o p h a n observed in f a s t e d b a s a l r a t s o v e r t h a t of n o n - f a s t e d b a s a l r a t s i n t h i s study are i n a c c o r d w i t h the f i n d i n g s o f Curzon e t a l . ( 1 9 7 2 ) . These r e s u l t s were a n t i c i p a t e d because f a s t i n g has been observed t o i n c r e a s e t h e l e v e l of both plasma f r e e t r y p t o p h a n and s u b s e q u e n t l y b r a i n t r y p t o p h a n (Curzon e t al., 1972). As p r e v i o u s l y d i s c u s s e d , f a s t i n g has been shown t o increase - 52 - TABLE IV TOTAL FOOD AND CARBOHYDRATE INTAKE OF ANIMALS FED CONTROL DIET, CARBOHYDRATE MEAL, OR INJECTED WITH TRYPTOPHAN Treatment Food Intake (g + SEM) Carbohydrate Intake (g + SEM) Control (21) 4.74 + 0.23 2.56 + 0.12 Carbohydrate (24) 3.06 + 0.21 2.39 + 0.16 Tryptophan (23) 4.71 + 0.31 2.54 + 0.17 * number of animals/group c LATENCY TO STEP-DOWN seconds ro o ro o ro o -s o o c -s • • • * ro N o -a c+ DJ -s cr O o -a =3<< 3 -s Q> CL ro - eg - o o 13 c+ -s o TABLE V EFFECT OF CARBOHYDRATE INGESTION OR A TRYPTOPHAN INJECTION ON REARING Treatment One Hour Rear 1 Control (12)* 4.42 + 0 . 6 0 Carbohydrate (12) Tryptophan (11) Rear 2 Rear 1 Two Hours Rear 2 3.27+0.72 (12)4.83+0.96 3.42+1.00 4.50 + 0.86 2.75 + 0.59 (12) 3.17 + 0.58 3.50 + 0.73 3.64 + 0.65 3.09 + 0.63 (12) 2.67 + 0.56 2.83 + 0.76 * number o f animals/group t Mean number of r e a r s / t r i a l + SEM + TABLE VI URINATION OR DEFECATION FOLLOWING INTAKE OF CARBOHYDRATE, CONTROL DIET OR A TRYPTOPHAN INJECTION Treatment One Hour Urination Two Hours Bolus One Hour Two Hours Control 1.75 + 0.50 0.25 + 0.25 0.75 +• 0.41 0.00 + 0.00 Carbohydrate 0.42 + 0.23 0.67 + 0.31 0.08 + 0.83 0.50 + 0.29 Tryptophan 1.67 + 0.79 1 .08 + 0.31 0.50 + 0.50 0.58 + 0.40 * 12 a n i m a l s / g r o u p - 56 - f a t t y a c i d o u t p u t which i n t u r n e l e v a t e s plasma f r e e t r y p t o p h a n by d i s p l a c i n g t r y p t o p h a n from albumin b i n d i n g sites. The e f f e c t o f c a r b o h y d r a t e i n g e s t i o n and t r y p t o p h a n d o s i n g on b r a i n t r y p t o p h a n and i t s time course r e p l i c a t e d the f i n d i n g s o f the second e x p e r i m e n t . Brain Serotonin. I n j e c t i o n o f t r y p t o p h a n i n d u c e d an i n c r e a s e i n b r a i n s e r o t o n i n s i m i l a r to the e f f e c t observed i n Experiment 2. However, i n c o n t r a s t t o the p r e v i o u s s t u d y , b r a i n s e r o t o n i n d i d not i n r a t s f e d the high c a r b o h y d r a t e p r o t e i n - f r e e m e a l . corticosterone concentrations The e l e v a t e d plasma a l s o observed i n the c a r b o h y d r a t e fed r a t s , c o u l d o f f e r an e x p l a n a t i o n f o r these c o n t r a d i c t o r y r e s u l t s , i.e. the c a r b o h y d r a t e f e d group may have been more s t r e s s e d than the c o n t r o l Certain stressful rise rats. procedures have been shown t o i n c r e a s e the t u r n o v e r o f s e r o t o n i n t o 5-HIAA ( Y u w i l l e r , 1979). Thus, a l t h o u g h i n c r e a s e d serotonin s y n t h e s i s may have o c c u r r e d , the s t r e s s o f a p r o t e i n - f r e e meal a f t e r i n g , p l u s exposure t o the novel chamber c o u l d have been powerful t o cause an even g r e a t e r t u r n o v e r o f s e r o t o n i n . fast- enough C o n f i r m a t i o n c o u l d be o b t a i n e d by measuring l a b e l l e d 5 - h y d r o x y t r y p t o p h a n , an i n d i c a t o r o f i n creased s e r o t o n i n s y n t h e s i s . measure o f s e r o t o n i n In a d d i t i o n , 5-HIAA c o u l d be assayed as a turnover. Plasma C o r t i c o s t e r o n e . Although r a t s i n j e c t e d w i t h tryptophan e x h i b i t e d an i n c r e a s e i n b r a i n s e r o t o n i n , plasma c o r t i c o s t e r o n e d i d not d i f f e r levels from those of. c o n t r o l o r c a r b o h y d r a t e f e d a n i m a l s . These r e s u l t s were s u r p r i s i n g s i n c e they a r e hot i n agreement w i t h those o f M o d l i n g e r e t a l . (1979, 1980) who showed t h a t C o r t i s o l was i n c r e a s e d i n humans a d m i n i s t e r e d an o r a l dose o f t r y p t o p h a n . In a d d i t i o n , our d a t a are not i n a c c o r d w i t h the r a p i d l y growing body o f literature - 57 - which suggests s e r o t o n i n p l a y s a r o l e i n the r e g u l a t i o n o f the hypothalamus/ pituitary/adrenal axis. However, i t i s noteworthy t h a t when the c o r t i - costerone concentrations o f the t r y p t o p h a n versus c o n t r o l p o s t i n j e c t i o n ) were a n a l y z e d w i t h a t - t e s t , cant. Unfortunately, rats the d i f f e r e n c e s were s i g n i f i - the d e s i g n o f the p r e s e n t experiment the use o f t h i s s t a t i s t i c . (1 hour invalidates However, i t i s p o s s i b l e t h a t a change i n e x p e r i m e n t a l d e s i g n , t o i n c l u d e o n l y the t r y p t o p h a n and c o n t r o l group a t 1 hour post i n j e c t i o n , would y i e l d f i n d i n g s i n agreement w i t h those the in literature. In c o n t r a s t t o the t r y p t o p h a n e f f e c t , r a t s fed the h i g h c a r b o h y d r a t e , p r o t e i n - f r e e meal had s i g n i f i c a n t l y e l e v a t e d c o n c e n t r a t i o n s corticosterone. From t h i s s t u d y , s e r o t o n i n ' s r o l e in mediating response i s not c l e a r , e s p e c i a l l y s i n c e b r a i n s e r o t o n i n were not i n c r e a s e d . of plasma this concentrations However, s i n c e b r a i n t r y p t o p h a n was e l e v a t e d , i t p o s s i b l e t h a t s e r o t o n i n t u r n o v e r and r e l e a s e were i n c r e a s e d . is Confirmation o f the s e r o t o n i n mechanism must a w a i t f u r t h e r r e s e a r c h when 5-HIAA can be assayed o r the c o r t i c o s t e r o n e response t o c a r b o h y d r a t e can be a n a l y z e d f o l l o w i n g a d m i n i s t r a t i o n of a s e r o t o n i n blocker. P o s s i b l e e v i d e n c e t h a t s e r o t o n i n has no r o l e i n the carbohydrate induced i n c r e a s e i n plasma c o r t i c o s t e r o n e comes from a study i n which a s u c r o s e s o l u t i o n was a d m i n i s t e r e d t o r a t s (Hart et a l . , 1980). Although e l e v a t e d c o r t i c o i d s were shown, the time course appeared t o d i f f e r from t h a t n o r m a l l y found f o r s e r o t o n i n f o l l o w i n g c a r b o h y d r a t e i n g e s t i o n . Rats g i v e n a high c a r b o h y d r a t e , p r o t e i n - f r e e meal have been r e p o r t e d t o exh i b i t i n c r e a s e s i n s e r o t o n i n and 5-HIAA f o r 2-3 hours a f t e r food p r e s e n tation. However, H a r t e t a l . (1980) observed t h a t the c o r t i c o i d peaked a t 50 min and f e l l to c o n t r o l c o n c e n t r a t i o n s by 100 m i n . levels Thus, - 58 - some n o n - s e r o t o n e r g i c mechanism c o u l d have mediated the response found i n H a r t e t a l . ' s f o l l o w i n g carbohydrate corticosterone (1980) experiment and i n the p r e s e n t s t u d y ingestion. In a d d i t i o n , a c e t y l c h o l i n e has been i m p l i c a t e d i n the r e g u l a t i o n the h y p o t h a l a m u s / p i t u i t a r y / a d r e n a l system (Jones e t a l . , 1977). of Thus, it may have mediated the c o r t i c o s t e r o n e response a f t e r sugar c o n s u m p t i o n . However, t o d a t e , the c a r b o h y d r a t e e f f e c t on b r a i n a c e t y l c h o l i n e has not been s t u d i e d . A l t h o u g h the mechanism o f the c a r b o h y d r a t e induced i n c r e a s e in c o r t i c o s t e r o n e i s u n c l e a r , i t appears t h a t these a n i m a l s may have been s t r e s s e d more than c o n t r o l s . C o r t i c o s t e r o n e r e l e a s e has been shown t o o c c u r as p a r t o f the s t r e s s response ( Y u w i l l e r , 1979). However, because o f the d e s i g n o f t h i s s t u d y , the s p e c i f i c s t r e s s o r i s not c l e a r . Carbo- h y d r a t e per se c o u l d have generated the c o r t i c o i d r e s p o n s e . this But, seems u n l i k e l y s i n c e the amount of c a r b o h y d r a t e eaten was the same among groups. The more p r o b a b l e s t r e s s o r may have been the l a c k o f dietary protein. Food I n t a k e . The data from the p r e s e n t study r e v e a l t h a t fasted r a t s o f f e r e d a high c a r b o h y d r a t e , p r o t e i n - f r e e meal, consume l e s s than r a t s g i v e n the c o n t r o l d i e t o r i n j e c t e d w i t h t r y p t o p h a n . p r o t e i n - f r e e meal may have g r e a t e r s h o r t - t e r m s a t i e t y v a l u e , food Thus, the perhaps through i n d u c i n g a more pronounced e l e v a t i o n i n plasma g l u c o s e . Crapo e t a l . (1976) observed t h a t i n humans, consumption o f a s u c r o s e s o l u t i o n produced a g r e a t e r r i s e i n plasma g l u c o s e than o f the same sugar w i t h added p r o t e i n and f a t . ingestion The s i g n i f i c a n c e o f e l e - v a t e d plasma g l u c o s e i n s a t i e t y has a l s o been d e m o n s t r a t e d . Administration of 2 - d e o x y g l u c o s e , an i n h i b i t o r o f g l u c o s e m e t a b o l i s m , was observed to s t i m u - - 59 l a t e food i n t a k e (Houpt and Hance, 1 9 7 1 ) , whereas, duodenal and p o r t a l in- f u s i o n s o f g l u c o s e were found t o suppress f e e d i n g i n f a s t e d a n i m a l s (Russek, e t a l . , 1980). In a d d i t i o n , i n t a k e o f c a r b o h y d r a t e may have been r e g u l a t e d by s e r o tonin. S e v e r a l r e s e a r c h e r s have suggested t h a t s e r o t o n i n may i n f l u e n c e carbohydrate i n t a k e . Nance and K i l b e y (1973) observed t h a t s u c r o s e p r e - f e r e n c e was i n c r e a s e d when PCPA t r e a t e d r a t s were d e p l e t e d o f and t h a t t h i s b e h a v i o r was r e v e r s e d by 5 - h y d r o x y t r y p t o p h a n . serotonin Similarly, Wurtman and Wurtman ,(1979) showed t h a t drugs which e l e v a t e s e r o t o n i n c o n c e n t r a t i o n lowered c a r b o h y d r a t e i n t a k e but had no e f f e c t on p r o t e i n gestion. Both r e s e a r c h e r s suggested s e r o t o n i n may r e g u l a t e in- carbohydrate intake. A l t h o u g h the r e s u l t s o f the p r e s e n t study do not prove the above p r o p o s a l , e l e v a t e d s e r o t o n i n t u r n o v e r i n the c a r b o h y d r a t e f e d r a t s may have mediated the r e d u c t i o n i n food i n t a k e so t h a t t o t a l c a r b o h y d r a t e consumed would not exceed amounts i n g e s t e d by c o n t r o l Latency To Step-Down. N e i t h e r t r y p t o p h a n o r c a r b o h y d r a t e admin- istered rats exhibited differences fed animals. rats. i n l a t e n c y t o step-down from c o n t r o l That s e r o t o n i n a l s o was not e l e v a t e d i n the c a r b o h y d r a t e f e d r a t s c o u l d e x p l a i n the l a c k o f b e h a v i o r a l However, the i n a b i l i t y results in this to r e p e a t the t r y p t o p h a n e f f e c t on the s c o r e s i s more d i f f i c u l t to e x p l a i n . Rats were o f f e r e d food ad B u t , i n t h i s s t u d y , animals were f a s t e d 17 hours and then p r e s e n t e d food i m m e d i a t e l y a f t e r Bolles latency One p o s s i b i l i t y c o u l d be the un- a v o i d a b l e changes i n e x p e r i m e n t a l d e s i g n . l i b i t u m i n the p r e v i o u s e x p e r i m e n t . group. (1965) observed i n c r e a s e d home cage a c t i v i t y injection. in rats fasted 24 hours and then l i m i t e d t o 10-12 g food d a i l y f o r 15 d a y s , even on the f i r s t day o f t e s t i n g . In a d d i t i o n , the r a t s o f t h i s study were observed - 60 - t o be h i g h l y e x c i t a b l e when handled and exposed t o the novel chamber (when compared t o a n i m a l s of the p r e l i m i n a r y experiment). Thus, the p o s s i b l e e f f e c t s o f food d e p r i v a t i o n c o u l d e x p l a i n the nons i g n i f i c a n t r e s u l t s obtained i n t h i s study. - 61 - VIII. SUMMARY In t h i s series of s t u d i e s , behavioral and biochemical c o r r e l a t e s of the tryptophan and carbohydrate induced increase in brain serotonin were i n v e s t i gated. 1. Brain Tryptophan and Serotonin. Brain tryptophan and serotonin were c o n s i s t e n t l y found to peak in tryptophan administered rats at 1 hour post i n j e c t i o n . Both tryptophan and serotonin dropped to control l e v e l s by 2 hours a f t e r i n j e c t i o n . This time course was r e p l i c a t e d . Carbohydrate was found to induce an increase i n brain serotonin; however, when animals were also exposed to a novel chamber, the carbohydrate induced serotonin elevation was no longer present. 2. Latency to Step-Down. Aminals fed ad l i b i t u m and i n j e c t e d with tryptophan exhibited an i n creased latency to step-down and explore a novel chamber. However, these data were not r e p l i c a t e d in the f i n a l study when animals were fasted p r i o r to i n j e c t i o n and food presentation. 3. Plasma Corticosterone. Since brain serotonin has been implicated in the regulation of the hypothalamus/pituitary/adrenal a x i s , plasma corticosterone was analyzed as a biochemical i n d i c a t o r of increased serotonin a c t i v i t y . Tryptophan administration did not increase plasma corticosterone above control conc e n t r a t i o n s , but ingestion of a high carbohydrate, p r o t e i n - f r e e meal did produce an elevation in plasma c o r t i c o s t e r o n e . - IX. 62 - CONCLUSIONS The d a t a from t h e p r e s e n t s t u d i e s i n d i c a t e t h a t t r y p t o p h a n result injections i n an i n c r e a s e d l a t e n c y t o step-down and e x p l o r e a novel p r o v i d i n g the a n i m a l s are f e d ad l i b i t u m . However, a t r y p t o p h a n c o r t i c o i d response was not c l e a r l y demonstrated. In c o n t r a s t , chamber, induced carbo- h y d r a t e i n g e s t i o n a f t e r f a s t i n g d i d not a f f e c t step-down l a t e n c y under the c o n d i t i o n s o f t h i s e x p e r i m e n t , i . e . f a s t and b e h a v i o r a l corticosterone. behavioral food p r e s e n t a t i o n a f t e r a 17 hour t e s t i n g d u r i n g the dark phase, but d i d e l e v a t e plasma C o n f i r m a t i o n o f the r o l e o f s e r o t o n i n i n m e d i a t i n g the and b i o c h e m i c a l responses observed i n t h i s s t u d y , must a w a i t f u t u r e i n v e s t i g a t i o n when a n a l y s e s a r e conducted f o l l o w i n g of a s e r o t o n i n blocker. administration - 63 - BIBLIOGRAPHY A i r a k s i n e n , E.M. and M.M. A i r a k s i n e n . Tryptophan i n mental a b n o r m a l i t i e s . In: Serotonin i n Mental A b n o r m a l i t i e s , e d i t e d by B o u l l i n . New York: John W i l e y and Sons, 1978, pp. 183-223. 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Sci.._ Hung. 42: 4 9 - 5 9 , 1972. : Vorhees, C.V. F a c i l i t a t i o n o f a v o i d a n c e a c q u i s i t i o n i n r a t s produced by P - c h l o r o p h e n y l a l a n i n e on P-choroamphetamine. Pharmac. Biochem. Behav. 10: 569-576, 1979. W a r b r i t t o n , J . D . , R.M. S t e w a r t , and R . J . B a l d e s s a r i n i . Decreased l o c o motor a c t i v i t y and a t t e n u a t i o n o f amphetamine h y p e r a c t i v i t y w i t h " i n t r a v e n t r i c u l a r i n f u s i o n o f . s e r o t o n i n i n the r a t . B r a i n Res. 143: 373-382, 1978. Weinberg, J . A . , P.R. D a l l m a n , and S. L e v i n e . Iron d e f i c i e n c y d u r i n g e a r l y development i n the r a t : B e h a v i o r a l and p h y s i o l o g i c a l consequences. Pharmac. Biochem. Behav. 12: 493-502, 1980. Weinberger, S . B . , S. Knapp, and A . J . M a n d e l l . F a i l u r e of tryptophan l o a d induced i n c r e a s e s i n b r a i n s e r o t o n i n t o a l t e r food i n t a k e i n the rat. L i f e S c i . 22: 1595-1602, 1978. Woodger, T . I . , A. S i r i k , and G.H. A n d e r s o n . D i a b e t e s , d i e t a r y t r y p t o p h a n and p r o t e i n i n t a k e r e g u l a t i o n i n w e a n l i n g r a t s . Am. J . P h y s i o l . 236: R307-311, 1979. - 71 - Wurtman, J . J . and R . J . Wurtman. Drugs t h a t enhance c e n t r a l s e r o t o n e r g i c t r a n s m i s s i o n d i m i n i s h e l e c t i v e c a r b o h y d r a t e consumption i n r a t s . Life S c i . 24: 895-904, 1979. Wurtman, R. and J . D . F e r n s t r o m . C o n t r o l o f b r a i n n e u r o t r a n s m i t t e r s y n t h e s i s by p r e c u r s o r a v a i l a b i l i t y and n u t r i t i o n a l s t a t e . Biochem. Pharmac. 25: 1691-1696, 1976. Wurtman, R . J . and J . D . F e r n s t r o m . L - t r y p t o p h a n , L - t r y o s i n e , and the c o n t r o l o f b r a i n monoamine s y n t h e s i s . I n : P e r s p e c t i v e s i n Neuropharmacology, e d i t e d by S . H . Synder. New Y o r k : Oxford U n i v e r s i t y P r e s s , pp. 143193, 1972. Yamamoto, T. and S. U e k i . E f f e c t s o f drugs on h y p e r a c t i v i t y and a g g r e s s i o n induced by raphe l e s i o n s i n r a t s . Pharmac. Biochem. Behav. 9: 821 826, 1978. Young, R., E.M. Gal and A . D . Sherman. Tryptophan l o a d i n g : Consequent e f f e c t s o f the s y n t h e s i s o f k y n u r e n i n e and 5 - h y d r o x y i n d o l e s i n r a t brain. J . Neurochem. 3 1 : 237-244, 1978. Y u w i l l e r , A. S t r e s s and s e r o t o n i n . I n : S e r o t o n i n i n H e a l t h and D i s e a s e , V o l . :.V. The C e n t r a l Nervous System, e d i t e d by W a l t e r B. Essman. New Y o r k : Spectrum P u b l i c a t i o n s , I n c . , 1979, pp. 1-50. - 72 - APPENDIX TABLE 1 P u r i f i e d c o n t r o l and c a r b o h y d r a t e Control Kg/1 Kg Ingredients 2 diet* Carbohydrate Kg/1 Kg .240 - .185 .265 .185 .265 .165 .247 .150 .150 .035 .035 .027 .027 .007 .007 Vitamin m i x ^ .004 .004 Methionine^ .002 - Casein, vitamin 3 Dextrose free . 4 Dextrin 5 Sucrose Corn Agar* oil 7 o Mineral mix 9 • Choline Chloride Premix 1 1.000 Kg 1 2 M o d i f i e d from Fernstrom and F a l l e r , (1978). S u p p l i e d by T e k l a d T e s t D i e t s , ARS/Sprague-Dawley D i v i s i o n o f the Mogul C o r p o r a t i o n , M a d i s o n , W i s c o n s i n . 3 S u p p l i e d by Grand I s l a n d B i o l o g i c a l 4 1.000 Kg C o . , Grand I s l a n d , New Y o r k . S u p p l i e d by ICN N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O h i o . 5 S u p p l i e d by B.C. Sugar R e f i n e r y , Vancouver, B r i t i s h Columbia. ^ S u p p l i e d by B e s t Foods D i v . , The Canadian S t a r c h Co. L t d . , Montreal Quebec. 7 S u p p l i e d by ICN N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O h i o . - 73 - Appendix T a b l e 1 ( c o n t ' d ) Contains 16.10 g CaHP0 ; 6.82 g KC1; 1.98 g-MgS0 ; 1.54 g N a H P 0 ; 4 2 4 4 0.64 g C a C 0 ; 0.22 g FeCgH^xh^O (16% F e ) ; 0.15 MnS0 ; 3 4 0.02302 g Z n C 0 ; 0.01256 g C u S 0 ; 0.00221 g N a F l ; 3 4 0.001537 g C r C l 6 H 0 ; 0.0002188 g NaSe; 0.0001962 g K l ; 3 13 g s u c r o s e ; 2 M i n e r a l s s u p p l i e d by J . T . Baker Chemical C o . , P h i l l i p s b u r g , New J e r s e y . 9 Contains 2 g choline Chloride; 5 g sucrose. s u p p l i e d by ICN P h a r m a c e u t i a l s Choline chloride I n c . , L i f e Sciences Group, Cleveland, Ohio. ^ Contains 200 mg i n o s i t o l ; 120 mg v i t . E (50% d l - a - t o c o p h e r a l a c e t a t e ) : 40 mg n i c o t i n i c a c i d ; 20 mg P-aminobenzoic acid; 16 mg d l c a l c i u m p a n t o t h e n a t e ; 16 mg v i t . A a c e t a t e ; 12 mg p y r i d o x i n e HC1; 8 mg t h i a m i n e HC1; 6 mg r i b o f l a v i n ; 2 mg f o l i c a c i d ; 1 mg d - b i o t i n ; 1 mg menadione ( v i t . 0.1 mg v i t . B-| ; 0.05 mg v i t . 2 K); D (calciferol); 13 g s u c r o s e ; Vitamins.' s u p p l i e d by ICN P h a r m a c e u t i c a l s I n c . , L i f e S c i e n c e s Group, C l e v e l a n d , O h i o . 11 S u p p l i e d by ICN Pharmaceutals Ohio. I n c . , L i f e S c i e n c e s Group, C l e v e l a n d . APPENDIX TABLE II BRAIN TRYPTOPHAN FOLLOWING INGESTION OF A CONTROL OR CARBOHYDRATE MEAL OR'INJECTION OF TRYPTOPHAN Treatment One Hour Control (5)* 4.48 + 0 . 1 8 Carbohydrate (5) Tryptophan (4) Two Hours Three Hours Four Hours (6) 4.12 + 0.15 (6) 4.22 + 0.18 (6) 4.37 + 0.22 5.60 + 0.17 (6) 5.36 + 0.31 (6) 5.33 + 0.13 (6) 5.16 + 0.20 11.06+1.20 (6) 4.98+0.24 (6) 4.21+0.13 * Number of animals/group t Mean tryptophan (ug/g + SEM) + APPENDIX TABLE 1:11 BRAIN SEROTONIN FOLLOWING CARBOHYDRATE INGESTION OR TRYPTOPHAN ADMINISTRATION. Treatment One Hour Control (5)* .629 + . 0 3 8 Carbohydrate (5) Tryptophan (4) Two Hours Three Hours Four Hours (6) .616 + .030 (6) .589 + .022 (6) .585 + .030 .683 + .028 (6) .684 + .020 (6) .672 + .033 (6) .631 + .033 .845 + .061 (6) .675 + .031 (6) .657 + .021 * Number o f animals/group t Mean s e r o t o n i n (ug/g + SEM) + - 76 - APPENDIX TABLE IV LATENCY TO STEP-DOWN FOLLOWING TRYPTOPHAN OR SALINE INJECTIONS Mean L a t e n c y to Step-Down + SEM t Treatment Trial 1 Trial 2 Saline (24) 18.29 + 2.8 7.88 + 2.4 Tryptophan (23) 25.57 + 3.8 16.25 + 3.6 * number o f animals/group t Mean seconds - 77 - APPENDIX TABLE V BRAIN TRYPTOPHAN FOLLOWING CARBOHYDRATE INTAKE OR TRYPTOPHAN ADMINISTRATION Treatment B r a i n Tryptophan Non-fasted basal (8) Fasted basal (7) Treatment 4.38 + 0.15 5.42 + 0.37 One Hour Two Hours Control (12) 4.76 + 0.09 (12) 4.69 + 0.10 Carbohydrate (12) 6.29 + 0.21 (12) 6.29 + 0.17 Tryptophan (12) 16.18+0.80 (11) 6.73+0.25 * Number o f animals/group t Mean t r y p t o p h a n (ug/g + SEM) - 78 - APPENDIX TABLE VI BRAIN SEROTONIN FOLLOWING INGESTION OF A HIGH CARBOHYDRATE MEAL, OR INJECTION OF TRYPTOPHAN Treatment B r a i n ™tonin ug/g S e Non-fasted basal (7)* .702 + . 0 3 0 Fasted-basal (8) .731 + .019 Treatment + One Hour Two Hours Control (12) .632 + . 0 1 1 .(12) .653 + .014 Carbohydrate (12) .635 + .011 (12) .688 + 0.15 Tryptophan (12) .849 + .023 (11) .786 + .030 * Number o f animals/group f Mean s e r o t o n i n (ug/g + SEM) - 79 - APPENDIX TABLE V I I EFFECT OF CARBOHYDRATE INTAKE OR TRYPTOPHAN ADMINISTRATION ON PLASMA CORTICOSTERONE Treatment basai Plasma C o r t i c o s t e r o n e a S t e d Fasted b a s a l Treatment Control ( 8 ) * 1 4 (8) - 3 t 2 ' 5 9 + 15.4 + 2.72 One Hour Two Hours (11) 14.60+1.86 (11) 13.87+2.24 C a r b o h y d r a t e (12) 26.67 + 4.12 (12) 22.44 + 3.24 Tryptophan 23.48 + 3.95 (12) 15.45 + 2.66 Number o f + (12) animals/group Mean c o r t i c o s t e r o n e (ug/100 ml + SEM) APPENDIX TABLE V I I I EFFECT OF A TRYPOTOPHAN INJECTION OR INGESTION OF A CARBOHYDRATE OR CONTROL DIET ON LATENCY TO STEP-DOWN Treatment Latency t o Step-Down One Hour 1 1 Control (12)" 11.88 + 5 . 2 1 Carbohydrate (12) Tryptophan (12) 2 11.08 + 5.40 (12) 17.96 + 6.20 8.67 + 4.75 15.42+5.60 9.92+6.00 (12) 15.08 + 5.71 10.17+4.95 20.05+5.71 11.25+5.95 (11)16.00+5.67 10.00+5.06 number of. animals/group Mean seconds + SEM Latency t o Step-Down Two Hours +
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Carbohydrate and tryptophan induced increase in brain serotonin: biochemical and behavioral correlates Crowther, Susan Eilers 1981
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