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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Title | Carbohydrate and tryptophan induced increase in brain serotonin: biochemical and behavioral correlates |
Creator |
Crowther, Susan Eilers |
Date Issued | 1981 |
Description | Behavioral and biochemical correlates of the carbohydrate and tryptophan induced increase in brain serotonin were investigated in a series of 4 experiments. Experiment 1 was conducted to establish the nadir of brain tryptophan during the dark phase of the light cycle, Following a 16 hour fast, brains were removed, at 1600, 1800, 2000, 2.400, and 0400, hrs for 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 in brain tryptophan and serotonin were determined in Experiment 2. Rats were fasted from 0030 to 1730 and then offered a control diet and injected with saline or 50 mg/kg tryptophan, or offered a high carbohydrate, protein-free meal and injected with saline. One hour after treatment and hourly for the next 3 hours, brains were obtained for analysis of tryptophan and serotonin. Tryptophan injected rats exhibited a peak in brain tryptophan at 1 hour post injection and a fall in tryptophan to control levels by 2 hours. Carbohydrate fed animals exhibited an increase in brain tryptophan at all times observed. Elevated brain serotonin was found in both tryptophan and carbohydrate treated animals. Experiment 3 was conducted to establish a behavioral correlate of brain serotonin. Behaviors investigated included: latency to step-down and explore a novel chamber and acquisition and extinction of a passive avoidance response. Animals were fed ad libitum, and 1 hour (1700) prior to behavioral testing injected with saline or 50 mg/kg tryptophan. Animals did not differ on measures of passive avoidance acquisition or extinction. However, tryptophan injected animals were found to exhibit a longer latency to step-down and explore a novel chamber than controls. In Experiment 4, plasma corticosterone, latency to step-down, rearing, urination, and defecation in a novel chamber were assessed. Animals were fasted from 2400 to 1700 and injected and fed as in Experiment 2. One and 2 hours following treatment, behaviors were observed. Thereafter, brains were removed for determination of tryptophan and serotonin and blood obtained for plasma corticosterone analysis. In tryptophan administered rats, brain tryptophan was observed to peak at 1 hour post injection and to remain higher than controls at 2 hours post injection. Carbohydrate fed rats were found to exhibit higher levels of brain tryptophan than control animals at both times assayed. Brain serotonin was found to peak in tryptophan treated rats at 1 hour post injection and to remain elevated at 2 hours. No changes in brain serotonin were revealed in carbohydrate fed animals. No group differences were observed for any of the behavioral measures taken. However, increased plasma corticosterone was found in rats fed the high carbohydrate meal. These data revealed that injection of tryptophan resulted in an increased latency to step-down and explore a novel chamber when animals were fed ab libitum, whereas carbohydrate ingestion resulted in an increase in plasma corticosterone with no effect on behavior. Confirmation that serotonin mediated these biochemical and behavioral changes awaits further research. |
Subject |
Tryptophan Carbohydrates Serotonin |
Genre |
Thesis/Dissertation |
Type |
Text |
Language | eng |
Date Available | 2010-03-23 |
Provider | Vancouver : University of British Columbia Library |
Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
DOI | 10.14288/1.0095058 |
URI | http://hdl.handle.net/2429/22418 |
Degree |
Master of Science - MSc |
Program |
Human Nutrition |
Affiliation |
Land and Food Systems, Faculty of |
Degree Grantor | University of British Columbia |
Campus |
UBCV |
Scholarly Level | Graduate |
Aggregated Source Repository | DSpace |
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