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CARBOHYDRATE AND TRYPTOPHAN INDUCED INCREASE IN BRAIN SEROTONIN BIOCHEMICAL AND BEHAVIORAL CORRELATES by SUSAN EILERS CROWTHER B.A., The Univers i ty of B r i t i s h Columbia, 1973 .Sc. , The Univers i ty of Prince Edward Is land, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Div is ion of Human Nutr i t ion School of Home Economics We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 usan E i l e rs Crowther, 1980 In present ing th is thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of HofY\^T\ hlofnhonJ <Aod ^ ti*ni- B&X>n\tt%, The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i i -ABSTRACT Behavioral and biochemical correlates of the carbohydrate and tryptophan induced increase in brain serotonin were invest igated in a ser ies of 4 experiments. Experiment 1 was conducted to estab l ish the nadir of brain tryptophan during the dark phase of the l i gh t cycle, Follow-ing 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 per iod. The time course and peak concentrations of the carbohydrate and tryptophan induced increase in brain tryptophan and serotonin were deter-mined in Experiment 2. Rats were fasted from 0030 to 1730 and then offered a control d iet and injected with sa l ine or 50 mg/kg tryptophan, or offered a high carbohydrate, prote in-free meal and injected with sa l i ne . One hour a f ter 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 in jec t ion and a f a l l in tryptophan to control leve ls by 2 hours. Carbohydrate fed animals exhibited an increase in brain tryptophan at a l l times observed. E le-vated brain serotonin was found in both tryptophan and carbohydrate treated animals. Experiment 3 was conducted to estab l ish a behavioral correlate of brain serotonin. Behaviors invest igated included: latency to step-down and explore a novel chamber and acqu is i t ion and ext inct ion of a passive avoidance response. Animals were fed ad l i b i tum, and 1 hour (1700) pr io r to behavioral tes t ing„ in jected with sa l ine or 50 mg/kg tryptophan. Animals did not d i f f e r on measures of passive avoidance acqu is i t ion or - i i i -e x t i n c t i o n . However, t ryptophan i n j e c t e d animals were found to e x h i b i t a longer l a tency to step-down and exp lo re 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 , l a tency to step-down, r e a r i n g , u r i n a t i o n , and de feca t i on i n a novel chamber were assessed . Animals were f a s t ed from 2400 to 1700 and i n j e c t e d and fed as i n Experiment 2. One and 2 hours f o l l ow i ng t reatment , behav iors were observed. The rea f t e r , b ra ins were removed f o r de te rminat ion of t ryptophan and se ro ton in and blood obta ined f o r plasma co r t i c o s t e r one a n a l y s i s . In t ryptophan admin i s te red r a t s , b r a i n t ryptophan was observed to peak at 1 hour post i n j e c t i o n and to remain h igher than con t r o l s a t 2 hours post i n j e c t i o n . Carbohydrate fed ra t s were found to e x h i b i t h igher l e v e l s o f b ra i n t ryptophan than con t ro l animals a t both t imes assayed. B ra in se ro ton in was found to peak i n t ryptophan t r ea t ed ra t s a t 1 hour post i n j e c t i o n and to remain e l eva ted a t 2 hours . No changes i n b ra i n se ro ton in were revea led i n carbohydrate fed an ima ls . No group d i f f e r ence s were observed f o r any of the behav io ra l measures taken . However, inc reased plasma co r t i c o s t e r one was found i n r a t s fed the high carbohydrate meal . These data revea led tha t i n j e c t i o n of t ryptophan r e su l t e d i n an i n -creased l a tency to step-down and exp lo re a novel chamber when animals were fed ab l i b i t u m , whereas carbohydrate i n ge s t i o n r e su l t e d i n an inc rease i n plasma c o r t i c o s t e r one w i th no e f f e c t on behav io r . Conf i rmat ion tha t se ro -ton in mediated these b iochemica l and behav io ra l changes awaits f u r t h e r r e sea r ch . - i v -TABLE OF CONTENTS CHAPTER Page A b s t r a c t . i i L i s t of Tables v i L i s t of F igures v i i L i s t o f Appendix Tables v i i i Acknowledgement i x I INTRODUCTION 1 II REVIEW OF LITERATURE 4 1. Carbohydrate and Tryptophan Induced Changes i n B ra in Tryptophan and the Indoleamines 4 2. Sero ton in D i s t r i b u t i o n and B i o syn thes i s 5 3. Regu la t ion of Se ro ton in Synthes i s 7 4. Tryptophan Metabol ism and Regu la t ion of Tryptophan Entry Across the Blood B ra in B a r r i e r 10 5. Sero ton in and Behav ior 14 6. Sero ton in and Co r t i c o s t e rone 20 7. Summary 22 8. Ra t i ona l e 22 I I I GENERAL METHODS . .' 23 1. Animals and Rat ions 23 2. B iochemica l Determinat ion 23 IV EXPERIMENT 1: BRAIN TRYPTOPHAN DURING THE DARK PHASE AFTER FASTING 26 1. I n t r oduc t i on 26 2. Exper imenta l Procedure 26 3. Resu l t s and D i s cuss ion 26 V EXPERIMENT 2: EFFECT OF TRYPTOPHAN ADMINISTRATION AND CARBOHYDRATE INGESTION ON BRAIN TRYPTOPHAN AND SEROTONIN 29 1. I n t r oduc t i on 29 - V -CHAPTER Page 2. Exper imental Procedure 29 3. Resu l t s 31 4. D i s cuss i on 34 VI EXPERIMENT 3: BEHAVIORAL EFFECTS OF TRYPTOPHAN ADMINISTRATION 35 1. Exper imenta l Procedure 35 2. Resu l t s 37 3. D i s cuss i on 40 VII EXPERIMENT 4: TRYPTOPHAN AND CARBOHYDRATE INDUCED INCREASES IN BRAIN SEROTONIN: BIOCHEMICAL AND BEHAVIORAL CORRELATES 44 1. I n t r oduc t i on 44 2. Exper imenta l Procedure 44 3. Resu l t s 46 4 . D i s cuss ion 51 VI I I SUMMARY 61 IX CONCLUSIONS 62 BIBLIOGRAPHY 63 APPENDIX 72 - v i -LIST OF TABLES TABLE Page I B ra in Tryptophan Fo l l ow ing a 16 Hour Fast 27 II A c q u i s i t i o n of an Avoidance Response Fo l l ow ing I n j e c t i o n of Sa l i ne or Tryptophan 38 I I I E f f e c t of Tryptophan or S a l i n e on E x t i n c t i o n 39 IV Tota l Food and Carbohydrate Intake of Animals Fed Contro l D i e t , Carbohydrate D ie t or In j ec ted With Tryptophan 52 V E f f e c t of Carbohydrate I nge s t i o n , Cont ro l D ie t or a Tryptophan I n j e c t i o n on Rea r i ng . . . . . 54 VI U r i n a t i o n or Defecat ion Fo l l ow ing Intake of Carbohydrate , Cont ro l D i e t , or a Tryptophan I n j e c t i o n 55 - v i i -LIST OF FIGURES FIGURE Page 1 Sero ton in b i o syn thes i s 6 2. Tryptophan metabol ism 12 3 E f f e c t of t ryptophan adm in i s t r a t i o n and carbohydrate i nge s t i on on b r a i n t ryptophan and s e r o t on i n : Exper imenta l des ign . . . . 30 4 B ra i n t ryptophan f o l l ow i ng tryptophan adm in i s t r a -t i o n or i nges t i on , o f carbohydrate 32 5 B ra in se ro ton i n a f t e r t ryptophan adm in i s t r a t i on or carbohydrate i nge s t i on 33 6 Behav iora l e f f e c t s o f t ryptophan adm i n i s t r a t i o n : Exper imental des ign 36 7 Latency to step-down f o l l ow i ng t ryptophan a d m i n i s t r a t i o n . 41 8 Tryptophan and carbohydrate induced inc reases i n b r a i n s e r o t on i n : B iochemica l and behav io ra l c o r r e l a t e s : Exper imenta l des i gn . . . 45 9 B ra i n t ryptophan f o l l ow i ng i n ge s t i o n of carbohydrate and tryptophan adm in i s t r a t i o n . 47 10 B ra in se ro ton in f o l l ow i ng i nge s t i on of carbohydrate or t ryptophan adm in i s t r a t i o n 49 11 Plasma c o r t i c o s t e r one a f t e r t ryptophan adm i n i s t r a t i o n or carbohydrate i nge s t i on 50 12 Latency to step-down f o l l ow i ng carbohydrate i n ge s t i o n or t ryptophan adm in i s t r a t i o n 53 ) - v i i i -LIST OF APPENDIX TABLES TABLE Page I P u r i f i e d Contro l and Carbohydrate D ie t 72 II B ra i n Tryptophan Fo l low ing Inges t ion of a Contro l or Carbohydrate Meal or I n j e c t i o n of Tryptophan . . . . 74 I I I B ra i n Sero ton in Fo l low ing Carbohydrate Inges t ion or Tryptophan Adm in i s t r a t i on 75 IV Latency to Step-Down Fo l l ow ing Tryptophan or S a l i n e I n j e c t i on s 76 V B ra i n Tryptophan Fo l low ing Carbohydrate Intake or Tryptophan Adm in i s t r a t i on 77 VI B ra i n Sero ton in Fo l low ing Inges t ion of a High Carbohydrate Meal or I n j e c t i o n of Tryptophan 78 VII E f f e c t of Carbohydrate Intake or Tryptophan Adm in i s t r a t i on on Plasma Co r t i c o s t e rone 79 V I I I E f f e c t o f a Tryptophan I n j e c t i o n or of a Carbo-Hydrate or Contro l D ie t on Latency to Step-Down . . . . 80 - i x -ACKNOWLEDGEMENT I would l i k e to thank my main adv i s o r , Dr. P a t r i c i a G a l l o , f o r se rv i ng as a f i r s t c l a s s model and f o r her c r i t i c a l comments, throughout t h i s p r o j e c t . S incere app re c i a t i on i s extended to Drs . Joanne Weinberg and R ichard Beninger f o r t h e i r cont inued moral support and i n va l uab l e a s s i s t ance in the development of the behav io ra l assay. The he l p f u l suggest ions from committee members, Drs . Chr i s F i b i g e r and MeTvin Lee, are a l s o most app rec i a t ed . F i n a l l y , thanks are expressed to Mrs. Naomi Woo f o r ana l y z i ng plasma co r t i c o s t e r one and to Ms. Cha r l o t t e Waddell f o r producing .the f i g u r e s . - 1 -I, INTRODUCTION "Does ea t i ng i n f l u ence b r a i n f unc t i on? To put the ques t ion more s p e c i f i c a l l y , do changes i n b lood chemist ry t ha t f o l l ow the in take of nu t r i e n t s p ro-duce corresponding changes i n the t i s s ue s of the b ra in? I f so , cou ld such d i e t - i nduced changes a f f e c t the f unc t i ona 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 to these quest ions has u sua l l y been no; when r equ i r ed , the b r a i n e x t r a c t s oxygen, g l ucose , and o ther nu t r i e n t s from the blood (Fernstrom and Wurtman, 1974). Hence, temporary f l u c t u a t i o n s i n the concen t ra t i on of n u t r i e n t s i n plasma have not been thought to a f f e c t the b r a i n (Fernstrom and Wurtman, 1974). However, more recent research has revea led some excep t i ons . Fasted ra t s fed a high carbohydrate , pro-t e i n - f r e e meal (Fernstrom and F a l l e r , 1978; Fernstrom, 1975b; Colmenares et a l . , 1975; Jacoby e t a l . , 1975a; Madras e t a l . , 1974; Fernstrom, 1971) o r i n j e c t e d w i th the amino a c i d t ryptophan (Young e t a l . , 1978; Jacoby e t a l . , 1975b; Fernstrom and Wurtman, 1971) have been shown to e x h i b i t e l e v a t i on s i n b ra i n t ryptophan and s e r o t on i n . That t h i s same phenomenon cou ld occur i n humans has a l s o been specu la ted (Fernstrom and Wurtman, 1974). Thus, the neurochemical response to t ryptophan and carbohydrate has been repeated ly demonstrated. However, the f un c t i o na l s i g n i f i c a n c e of e l eva ted b r a i n se ro ton i n has not been we l l e s t a b l i s h e d . Sero ton in has been assoc i a ted w i th many behav io r s . Thus, inc reases i n b ra i n s e ro ton i n cou ld have important p r a c t i c a l i m p l i c a t i o n s . I n h i b i -t i o n of avoidance l ea rn i ng (Essman, 1977), memory (Essman, 1977), food in take (B l unde l l and Latham, 1979), locomotor a c t i v i t y (Warbr i t ton e t a l . , 1978), and e xp l o r a t i o n ( F i l e and Pope, 1974) have been observed i n r a t s - 2 -w i th e l eva ted l e v e l s o f b r a i n s e r o t on i n . Furthermore, b r a i n se ro ton in has been imp l i c a t ed i n the r egu l a t i o n o f the r a t ( Y u w i l l e r , 1979), and human ( M o d l i n g e r e t a l . , 1979) hypo tha lamus/p i tu i t a ry /ad rena l a x i s . In a d d i t i o n , seve ra l metabo l i c cond i t i ons are thought to i n vo l ve a l t e r a t i o n s i n s e ro ton i n metabol ism. An e l eva ted concen t ra t i on of b r a i n se ro ton in has been l i n k ed to the e t i o l o g y of confus ion and coma, symptoms i n hepat i c encephalopathy ( F i s che r e t a l . , 1978; Sourkes, 1978). The t r a d i t i o n a l form o f therapy f o r t h i s c ond i t i on has been a high carbo-hydra te , p r o t e i n - f r e e d i e t . Thus, i t i s po s s i b l e t ha t t h i s t reatment cou ld po t en t i a t e the d i sease symptoms through f u r t h e r i n c r ea s i ng b ra i n se ro -t o n i n . In a dd i t i o n to hepa 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 to i nc rease b r a i n se ro ton in (Mackler et a l . , 1978). Furthermore, decreased behav io ra l respons iveness , o f ten l i n ked to e l eva ted s e r o t on 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 ra t s fed an i r o n d e f i c i e n t d i e t . f r om b i r t h (Weinberg e t a l . , 1979). Thus, i nge s t i on of a carbohydra te , p r o t e i n - f r e e meal might be expected to f u r t h e r reduce respons iveness i n i r o n d e f i c i e n t r a t s . F i n a l l y , the behav io ra l and b iochemica l consequences of carbohydrate induced inc reases i n b r a i n se ro ton in are of p a r t i c u l a r i n t e r e s t because consumption of r e f i n ed sugar i s high among North American popu l a t i ons . In the Uni ted S ta tes sucrose i n take has been est imated at 100 pounds per c ap i t a per year (Bogart et a l . , 1973). S i m i l a r l y , 68% o f c h i l d r e n in a r u r a l B r i t i s h Columbia town were observed to snack, and 28% of the snaeks consumed were high i n r e f i n ed sugar and low i n other nu t r i en t s (On i s h i , 1980). From the above, the importance of determin ing the f un c t i o na l s i g n i f i -cance of the carbohydrate and tryptophan induced inc rease i n b r a i n se ro -- 3 -t on i n i s c l e a r . Thus, the present study was conducted to determine i f the r i s e i n s e ro ton i n f o l l ow i ng i n j e c t i o n s of t ryptophan or i nge s t i on of a high carbohydra te , p r o t e i n - f r e e mea l , a f t e r f a s t i n g , has behav io ra l consequences. In a d d i t i o n , the e f f e c t o f the carbohydrate and tryptophan induced inc rease of b r a i n se ro ton in on plasma c o r t i c o s t e r one output was i n v e s t i g a t e d . The s p e c i f i c ob j e c t i v e s of t h i s study were: (1) To measure b r a i n t ryp tophan , s e r o t o n i n , and plasma co r t i c o s t e r one i n f a s ted male ra t s fed a high carbohydrate meal or i n j e c t e d wi th t r yp tophan . (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 ro ton i n can be co r r e -l a t e d w i th a change i n e x p l o r a t i o n , a behav ior 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 there i s a c o r r e l a t i o n between plasma c o r t i -costerone and b ra i n s e r o t on i n . - 4 -I I . REVIEW OF THE LITERATURE 1. Carbohydrate and Tryptophan Induced Changes i n B ra in  Tryptophan and the Indoleamines. A high carbohydrate p r o t e i n - f r e e meal fed to f a s t ed ra t s a t the beg inn ing of the l i g h t phase, has repeated ly been shown to inc rease b r a i n t r yp tophan , s e r o t o n i n , and 5 -hyd roxy indo leace t i c a c i d (5-HIAA) (Jacoby e t a l . , 1975; Colmenares e t a l . , 1975; Fernstrom, 1975b , 1974; Fernstrom e t a l . , 1973; Fernstrom, 1971). Wi th in 2 hours a f t e r p resen ta t i on of the carbohydra te , p r o t e i n - f r e e meal , t ryptophan and the indoleamines were observed to r i s e and remain e leva ted f o r 3 hours (Fernst rom, 1971). In a d d i t i o n , b r a i n t ryptophan was f u r t h e r e leva ted when the carbohydrate 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 ng s were obta ined when a g lucose s o l u t i o n was admin i s te red to f a s t ed ra t s v i a e i t h e r a stomach tube or gavage (De Montis et a l . , 1978; Madras e t a l . , 1974, 1973). However, w i t h these l a t t e r methods of a d m i n i s t r a t i o n , the peak i n t r y p t o -phan occurred 1'hour a f t e r g lucose p r e sen t a t i on . Tryptophan, s e r o t on i n , and 5-HIAA have a l s o been assayed i n s p e c i f i c reg ions w i t h i n the c en t r a l nervous system of r a t s fed a carbohydra te , p r o t e i n - f r e e meal (Colmenares et a l . , 1975). Whi le no inc rease i n t r y p t o -phan and the 5-hydroxy indo les was found i n the hypothalamus or the corpus s t r i a t u m , examinat ion of the b r a i n stem, sp i na l c o r d , and the te lencepha lon (neocor tex , o l f a c t o r y c o r t e x , and hippocampus) revea led e l e va t i on s i n s e r o t on i n , t r yp tophan , and 5-HIAA (Colmenares e t a l . , 1975). Because the b r a i n stem i s be l i e ved to con ta in the pe r i ka r ya of the se ro tone rg i c neurons, wh i l e a l l o ther reg ions of the cen t r a l nervous system are thought to conta in the neuronal axons and te rm ina l s (Colmenares et a l . , 1975), - 5 -these f i n d i ng s demonstrated tha t the carbohydrate- induced inc rease i n se ro -ton in occurred i n both c e l l bodies and the p re - s ynap t i c t e rm ina l s of s e r o t on i n - c on t a i n i ng neurons. There fo re , upon d e p o l a r i z a t i o n , i t i s po s s i b l e t ha t more se ro ton in cou ld be re l eased i n t o the s ynap t i c c l e f t . Th is phenomenon i s e s s e n t i a l f o r the carbohydrate induced inc rease i n se ro ton in to 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 (Colmenares et a l . , 1975). I n t r ape r i t o nea l i n j e c t i o n of t ryptophan a l so has been shown to i n -crease b r a i n t ryptophan and se ro ton in (Young e t a l . , 1978; Jacoby et a l . , 1975b; Fernstrom and Wurtman, 1971). I n j e c t i o n s o f 12.5 to 125 mg t r y p t o -phan/kg body weight r e su l t e d i n dose r e l a t e d inc reases i n b r a i n t ryptophan 1 hour a f t e r i n j e c t i o n . However, the maximum inc rease i n b r a i n se ro ton in was obta ined w i th t ryptophan doses o f 25-50 mg/kg (Young e t a l . , 1978; Jacoby et a l . , 1975b; Fernstrom and Wurtman, 1971). Thus, i t has been repeated ly demonstrated tha t b r a i n t r yp tophan , s e r o t o n i n , and 5-HIAA are inc reased f o l l ow i ng carbohydrate i nge s t i on or t ryptophan dos i ng . 2. Se ro ton in D i s t r i b u t i o n and B i o s yn t he s i s . B ra i n s e r o t o n i n , or the monoamine 5-hydroxyt ryptamine, has p r i m a r i l y been found conf ined to a d i s t i n c t c l u s t e r of neurons, the raphe nuc l e i (Fernstrom and Wurtman, 1974). The c e l l bodies of the raphe nuc l e i are known to be l o ca ted i n the b r a i n stem and have f i b r e s ascending i n t o the remainder of the b r a i n and descending through the sp i na l cord (Fernstrom and Wurtman, 1974). Sero ton in syn thes i s has been f u l l y desc r ibed (A i r ak s i nen and A i r a k s i n e n , 1978; Fernstrom, 1978). Th is metabo l i c pathway (Fernst rom, 1978) i s dep i c ted i n F igure 1. i hVV-T«-^ 2 Vvrt^ 3 rtY>r -vV W A D 4 >vV 5-Hydroxytryptophan Sero ton in 5-HIAA 1 Tryptophan hydroxy lase; c o f a c t o r s : fe r rous i r o n , reduced p t e r i d i n e c o f a c t o r 2 Aromat ic amino ac id decarboxy lase; c o f a c t o r : pyr idoxa l phosphate 3 Monoamine ox idase ^ Aldehyde dehydrogenase F igure 1. Seroton in b i o s yn the s i s (Fernstrom, 1978) H H •C-C-NHj, H COOH Tryptophan - 7 -3. Regu la t ion of Sero ton in Syn thes i s . Although se ro ton in syn thes i s has been e x t en s i v e l y i n v e s t i g a t e d , i t s r e gu l a t i o n i s s t i l l un c l ea r . Tryptophan a v a i l a b i l i t y , r a te o f impulse f low a long se ro tone rg i c neurons, and product i n h i b i t i o n have a l l been imp l i c a t ed as f a c t o r s i nvo l ved i n r egu l a t i o n of se ro ton in s y n t h e s i s . Because the Km of t ryptophan hydroxy lase f o r t ryptophan i s high r e l a t i v e to i t s normal c on cen t r a t i o n , i . e . 50 uM versus 30 uM r e s p e c t i v e l y (Fernst rom, 1978), t ryptophan a v a i l a b i l i t y has been recogn ized as an impor-tan t f a c t o r i n the con t r o l o f s e ro ton in 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 ryptophan concen t ra t i on have a l s o been found to i n f l u ence b r a i n se ro ton in or 5-HIAA. Animals fed a corn based d i e t , known to be d e f i c i e n t i n tryptophan,were observed to e x h i b i t lower l e v e l s o f b r a i n t ryptophan and se ro ton in ( L y t l e et a l . , 1975). Furthermore, lower concent ra t i ons of t ryptophan and 5-HIAA have been found i n r a t s o f f e r ed meals of p u r i f i e d amino ac ids known to b lock t ryptophan ent ry across the blood 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 resen ta -t i o n of a low p ro t e i n d i e t to weanl ing r a t s has a l s o been shown to lower b r a i n t r yp tophan , s e r o t o n i n , and 5-HIAA. These b ra i n parameters were observed to re tu rn to 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 ickerson and Pao, 1975). S i m i l a r l y , am inophy l l i n e , a drug be l i e ved to inc rease b r a i n se ro -t o n i n , was observed to e l eva te b r a i n t ryptophan (Curzon and Knot t , 1974). In a d d i t i o n , b r a i n t ryptophan and se ro ton i n turnover were found e leva ted a f t e r 24 hours of food dep r i v a t i on or immob i l i z a t i on s t r e s s (Curzon e t a l . , 1972). F i n a l l y , consumption of a h igh carbohydrate meal (Fernst rom, 1971) or i n j e c t i o n s o f t ryptophan (Fernstrom and Wurtman, 1971) were observed to i nc rease b r a i n t ryp tophan , s e r o t on i n ; and 5-HIAA. That t h i s - 8 -i nc rease i n b ra i n se ro ton in r e f l e c t e d inc reased syn thes i s r a the r than lowered metabol ism or re l ease was i n d i c a t e d by the p a r a l l e l r i s e i n 5-HIAA (Wurtman and Fernstrom, 1976). Moreover, when animals were g iven a carbo-hydrate meal a long w i th an aromat ic amino a c i d decarboxy lase i n h i b i t o r , b ra i n 5-hydroxyt ryptophan, the immediate precursor of s e r o t on i n , was s i g n i f i c a n t l y e l eva ted (Jacoby e t a l . , 1975a). From the above, i t i s c l e a r t ha t ch ron i c and acute changes i n b r a i n t ryptophan a v a i l a b i l i t y appear to be a major determinant of b r a i n s e ro -t on i n s y n t h e s i s . However, t ryptophan a v a i l a b i l i t y may not be c r i t i c a l i n the i nduc t i on of b r a i n se ro ton in and 5-HIAA d i u rna l rhythms. O r i g i n a l l y , d a i l y f l u c t u a t i o n s of both whole b ra i n (Wurtman and Fernstrom, 1972) and b ra in stem (Morgan et a l . , 1975) t ryptophan were observed to p o s i t i v e l y c o r r e l a t e w i th b ra i n s e r o t o n i n . 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 -ca t i ons i n t ryptophan and se ro ton in were shown to be i n oppos i te phase i n the f r o n t o - p a r i e t a l co r tex (Hery e t a l . , 1977). Furthermore, t r y p t o -phan hydroxy lase a c t i v i t y and i t s rhythm were found to vary depending on the se ro ton i n con ta i n i ng c e l l groups i n the r a t b r a i n stem (Kan et a l . , 1977). Thus, the importance of t ryptophan a v a i l a b i l i t y i n determin ing se ro ton in or 5-HIAA c i r c a d i a n r hy thm i c i t y i s unc l ea r . I t i s po s s i b l e t ha t under normal p h y s i o l o g i c a l c o n d i t i o n s , the r e l a t i v e importance of t ryptophan a v a i l a b i l i t y i s a f un c t i on of b r a i n a r ea . In a dd i t i o n to t ryptophan a v a i l a b i l i t y , se ro ton in syn thes i s may be i n f l uenced by the ra te o f neuronal f i r i n g (Boad l e -B i be r , 1979a). S t imu-l a t i o n of se ro tone rg i c neurons of the mid b r a i n raphe was shown to i n -crease se ro ton in syn thes i s from r a d i o l a b e l l e d tryptophan (Sh ie lds and E c c l e s t on , 1972). S i m i l a r l y , i n - v i t r o d e p o l a r i z a t i o n of r a t b r a i n stem s l i c e s i n a potassium enr i ched i n cuba t i on medium was found to enhance - 9 -t ryptophan hydroxy lase a c t i v i t y as measured by inc reased t ryptophan t r a n s -format ion to 5-hydroxytryptophan (Hamon et a l . , 1979). Both ca lc ium (Boad le -B ibe r , 1979a,b; E lks et a l . , 1979) and c y c l i c adenosine monophos-phate (CAMP) (Boad le -B ibe r , 1980) have been imp l i c a t ed i n the i nduc t i on of t ryptophan hydroxy lase a c t i v i t y s i nce man ipu la t ions tha t e l eva te ca l c ium w i t h i n nerve t i s s u e were observed to inc rease tryptophan hydroxy lase a c t i v i t y . S i m i l a r l y , procedures which removed ca l c ium from t h e i ncuba t i on medium were found to b lock enzyme a c t i v i t y f o l l ow i ng d e p o l a r i z a t i o n (Boad le -B ibe r , 1979b; E lks e t a l . , 1979). In a d d i t i o n , the add i t i o n of CAMP has a l s o been shown to inc rease t ryptophan hydroxy lase a c t i v i t y i n -v i t r o (Boad l e -B i be r , 1980). The r o l e of ca l c ium i n the i nduc t i on of t ryptophan hydroxy lase a c t i v i t y i s not y e t understood. Calc ium has been shown to che l a te t r y p t o -phan which cou ld r e s u l t i n an inc rease i n t ryptophan uptake i n t o snyapto-somes (B ru i nve l s and Moleman, 1979). However, s i nce adm in i s t r a t i o n of a ca l c ium b l o cke r to the i n cuba t i on medium was found to decrease t ryptophan a c t i v i t y w i th no e f f e c t on t ryptophan uptake (E lks et a l . , 1979), t h i s mechanism appears u n l i k e l y . Calc ium induced a c t i v a t i o n of p r o t e i n k inase which i n tu rn s t imu l a te s t ryptophan hydroxy lase has a l so been proposed (Boad le -B ibe r , 1979a; E lks e t a l . , 1979). Al though i t i s ev iden t tha t the ra te of neuronal f i r i n g may p lay a r o l e i n the r egu l a t i o n of se ro ton in syn thes i s through i nduc t i on of t ryptophan hydroxy lase a c t i v i t y , a c t i v a t i o n of t h i s enzyme has not always been fo l l owed by inc reased se ro ton in s y n t h e s i s . Adm in i s t r a t i o n o f pargy-l i n e , a drug which inc reases s e r o t o n i n , was repor ted to markedly reduce the s t imu l a t o r y e f f e c t o f neuronal d epo l a r i z a t i o n on tryptophan conver-s i on to se ro ton in (Hamon et a l . , 1979). In a d d i t i o n , i n - v i t r o b ra i n s e ro -- 10 -ton in syn thes i s was found i n h i b i t e d when se ro ton in was added to the incuba-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 ha t subs t ra te i n h i b i t i o n may a l s o regu la te se ro ton i n s y n t h e s i s . However, feed back i n h i b i t i o n i n the r egu l a t i o n of se ro ton in syn thes i s has not been un i ve r -s a l l y suppor ted. When r a t b ra i n se ro ton in l e v e l s were e l eva ted pharma-c o l o g i c a l l y w i th a monoamine ox idase i n h i b i t o r and animals then a l lowed to consume a carbohydrate , p r o t e i n - f r e e mea l , the subsequent inc rease i n b ra i n hydroxy indo le l e v e l s was comparable to those found i n con t ro l r a t s not g iven the drug (Jacoby e t a l . , 1975a). In a d d i t i o n , the normal d i u r na l i nc rease i n se ro ton in has been repor ted to be 25%. However, a 46% i n -crease has been repor ted when carbohydrate plus a monoamine i n h i b i t o r was admin i s te red (Jacoby e t a l . , 1975). Thus, t ryptophan a v a i l a b i l i t y , ra te of neuronal f i r i n g , a n d subs t ra te i n h i b i t i o n may be i nvo l ved i n the, r e gu l a t i o n of se ro ton in s y n t h e s i s . Furthermore, these f a c t o r s cou ld work together i n r egu l a t i n g se ro ton in p roduc t i on . The r e l a t i v e importance of each o f these f a c t o r s may be con-t i ngen t upon b r a i n r e g i o n . 4. Tryptophan Metabol ism and Regu la t ion of Tryptophan Entry Across  the Blood B ra in B a r r i e r . Of a l l the e s s en t i a l amino a c i d s , t ryptophan has been found to occur i n lowest concen t ra t i on i n p r o t e i n , on ly 1.5% (Fernstrom, 1978). In con-t r a s t to o ther amino a c i d s , 80%-90% of t ryptophan has been observed to c i r c u l a t e bound to albumin wh i l e on ly 10%-20% has been found to c i r c u l a t e f r e e l y (Fernst rom, 1978). The metabo l i c f a t e of t ryptophan i s complex w i th on ly 1% u t i l i z e d f o r se ro ton in syn thes i s (Fernstrom and Wurtman, 1976). The main metabo l i c pathways f o r t ryptophan are dep i c ted i n - 11 -F igure 2 and i nc l ude 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 roduc t i on , se ro -ton in s y n t h e s i s , melaton in f o rma t i on , and produc t ion of o ther b i o l o g i c a l l y i n a c t i v e metabo l i t es (A i r a k s i nen and A i r a k s i n e n , 1978). The uptake of t ryptophan i n t o the b r a i n i s thought to be c o n t r o l l e d by three major f a c t o r s : (1) the blood concent ra t ions of the l a rge neut ra l amino ac ids (pheny l a l an ine , 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 methionine) which compete f o r a common t r anspo r t c a r r i e r (Bender, 1978); (2) the concen t ra t i on of the smal l f r a c t i o n of t ryptophan not bound to albumin (Curzon and Knot t , 1974); and (3) the time of day (Hery e t a l . , 1972). Compet i t ion between plasma t ryptophan and the neut ra l amino ac ids as a r egu l a to r y mechanism was shown when f a s t ed ra t s o f f e r ed a p r o t e i n - c on -t a i n i n g mea l , i . e . lab chow, o r a p u r i f i e d amino a c i d d i e t , e xh 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 ryp tophan . But , no change i n b ra i n t ryptophan was observed. However, i f the same p u r i f i e d d i e t , l a c k i ng a l l the neu t ra l amino ac ids except t ryptophan was f e d , e l e v a t i o n of those amino ac ids consumed was found i n the plasma. Furthermore, b r a i n t r y p t o -phan, s e r o t on i n , and 5-HIAA were a l s o e l e va t ed . In c on t r a s t , when on ly aspa r ta te and g lu tamine , which share a d i f f e r e n t b ra in t r anspo r t c a r r i e r from the neut ra l amino a c i d s , were omit ted from the p u r i f i e d d i e t , a l l the consumed amino ac ids were inc reased i n the plasma, wh i l e b r a i n t ryptophan remained unaf fec ted (Fernstrom et a l . , 1973). A high c o r r e l a -t i o n (.95) between b r a i n t ryptophan and the r a t i o of serum tryptophan to the sum of the competing neu t ra l amino ac ids was subsequent ly demon-s t r a t e d (Fernstrom and F a l l e r , 1978). A much lower c o r r e l a t i o n (.66) was shown between b r a i n t ryptophan and plasma tryptophan alone (Fernstrom e t a l . , 1973). - 12 -Indole, skatole indoxylsulphate Protein 5-Methoxy-DMT /i 8 5-Nicotinic acid >- NAD 1 Quinolinic acid I 3-Hydroxyanthranilic acid — • + NH3 + Hg) B6 t 5 3-Hydroxykynurenine f Be Xanthurenic acid Kynurenine Kynurenic acid 5,Bg "—-Mnthranilic acid Formylkynurenine [4 "" M 4-u u 4. 8 N,N-dimethyltryp N-methyltryptamine ' t i m i n e fim™ 2 ! ^ 3 — Tryptophan • Tryptamine —^Indoleacetic acid 1 ^"Indolepyruvic acid-5-Hydroxytryptophan (5-HTP) Methoxytryptamine Bg J2 g ^N-methyl-5-HT—-sBufotenine 5-Hydroxytryptamine ( 5-HT)^ Glucuronides and sulphates -Acetyl-5-HT. 7 5-Hydroxyi ndole-acetaldehyde Melatonin 5-Hydroxyindoleacetic acid 5-Hydroxytryptophol (5-HIAA) 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 . 28 ) ; (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 trans-minase, (E.C. 2 . 6 . 1 . 7 ) ; (7 ) Hydroxyindole-0-methyltransferase; (8) indole-ethylamine-N-methyltransferase-(+-aromatic-amine-N-methyltrans-ferase?); Bg pyridoxal phosphate, as coenzyme. (Airaksinen and Airaksinen, 1978 . ) - 13 -Th is a l t e r a t i o n i n plasma neut ra l amino a c i d concen t ra t i on i s be l i e ved to account f o r the e leva ted b ra i n t ryptophan concen t ra t i on when a carbo-hydrate p r o t e i n - f r e e meal i s consumed (Fernstrom and F a l l e r , 1978; Fernstrom, 1975b; Fernstrom e t a l . , 1973). Carbohydrate has been shown to i nc rease b r a i n t ryptophan 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 to inc rease the r a t i o of plasma tryptophan to the neut ra l amino ac ids which compete f o r uptake across the b lood b r a i n b a r r i e r (Fernstrom et a l . , 1973). Thus, when a high carbohydrate meal i s consumed, plasma tryptophan i s r e a d i l y t r anspo r ted i n t o the b r a i n . In c on t r a s t , when f a s ted r a t s con-sumed regu l a r lab chow or a carbohydrate meal supplemented w i th 18% c a s e i n , b r a i n t ryptophan was not found to r i s e , desp i t e i n s u l i n s e c r e t i o n (Fernstrom e t a l . , 1973). With i nge s t i on of p r o t e i n , the plasma concen-t r a t i o n of competing amino ac ids was observed to inc rease cons ide rab l y more than t ryptophan (Fernstrom et a l . , 1973). . S i m i l a r f i n d i ng s of an increased r a t i o of plasma t ryptophan to the amino ac ids which compete w i th i t f o r b r a i n uptake have been shown i n f a s t ed humans fed a g lucose s o l u -t i o n (Fernstrom and Wurtman, 1974). Fur ther ev idence f o r the e f f e c t of carbohydrate on the plasma neu t ra l amino ac i d pa t te rn of humans comes from the f o l l o w i n g . When human sub-j e c t s consumed high carbohydra te , p r o t e i n - f r e e meals f o r 5 days, the d i u rna l v a r i a t i o n s i n plasma neut ra l amino a c i d r a t i o s d i f f e r e d cons i de r -ab ly from i n d i v i d u a l s ea t i ng 150 g of p ro t e i n d a i l y (Fernstrom and Wurtman, 1979). When 0 g of p r o t e i n were consumed, the r a t i o of t ryptophan to i t s compet i tors inc reased throughout the day u n t i l a peak a t 1500 hrs and then a gradual decrease to nad i r concen t ra t i ons a t 0700 h r s . Th is rhythm was i n oppos i te phase to t ha t observed i n the group fed 150 g p r o t e i n . Furthermore, the tryptophan to neut ra l amino a c i d r a t i o s were c o n s i s t e n t l y - 14 -h igher at a l l t ime po in t s assayed when the high carbohydrate meals a lone were consumed (Fernstrom and Wurtman, 1979). Al though the r e l a t i v e concen t ra t i ons of neut ra l amino ac ids p lay an important r o l e i n the r egu l a t i o n of t ryptophan t r a n spo r t a t i o n across the b lood b r a i n b a r r i e r , o ther mechanisms have a l s o been des c r i bed . C l o f i b r a t e adm in i s t r a t i o n (Bloxam et a l . , 1980), s t a r v a t i o n , and immobi l -i z a t i o n (Curzon e t a l . , 1972) were shown to inc rease b r a i n t ryptophan (Bloxam et a l . , 1980; Curzon et a l . , 1972) and subsequent ly se ro ton in turnover (Curzon e t a l . , 1972). Under these c i r cumstances , plasma f r ee f a t t y ac ids were shown to r i s e and d i s p l a c e t ryptophan from albumin b ind ing s i t e s , hence render ing more f r ee t ryptophan a v a i l a b l e to the b r a i n . Thus, the concen t ra t i on of f r ee t ryptophan may p lay a r o l e i n r egu l a t i n g b r a i n t ryptophan f o l l ow i ng treatments tha t e l eva te f r ee f a t t y a c i d s . In add i -t i o n , t ime of day has been demonstrated to i n f l u ence the b r a i n ' s a b i l i t y to t r anspo r t t ryptophan (Hery et a l . , 1972). Hery e t a l . (1972), observed tha t r a d i o l a b e l e d t ryptophan uptake i n t o b r a i n s l i c e s was h igher dur ing the l i g h t phase than dur ing the dark pe r i od . Thus, three f a c t o r s appear to regu la te t ryptophan ent ry across the b lood b r a i n b a r r i e r : r a t i o of plasma tryptophan to the neu t ra l amino a c i d s , concen t ra t i on of f r ee t ryp tophan , and time of day. 5. Sero ton in and Behav io r . Al though cons ide rab l e research has centered around the r e gu l a t i o n of se ro ton in s y n t h e s i s , the f un c t i o na l s i g n i f i c a n c e of e leva ted b ra i n se ro -t on i n has s t i l l not been we l l e s t a b l i s h e d . However, numerous behav iors have been l i n k ed to changes i n b r a i n s e ro ton i n con ten t . Behav iors mot ivated by negat ive re in forcement have been r e l a t e d to - 15 -s e r o t on i n . Des t ruc t i on of se ro tone rg i c neurons by i n t r a c i s t e r n a l i n j e c -t i o n of 5,6 d ihydroxyt ryptamine ( L i n et a l . , 1978) o r e l e c t r o l y t i c l e s i on s (Koh ler and Lorens , 1978) was observed to f a c i l i t a t e l ea rn i ng of a 2-way a c t i v e avoidance s h u t t l e box t a s k . P - ch lo ropheny la l an ine , (PCPA), a t ryptophan hydroxy lase i n h i b i t o r , was shown to enhance Y-maze avoidance a c q u i s i t i o n , wh i l e P-chloroamphetamine was found to improve s h u t t l e box avoidance (Vorhees, 1979). Furthermore, l e s i on s of the raphe nucleus were shown to f a c i l i t a t e a learned t a s t e ave rs ion and 5-hydroxy-tryptophan ' a dm in i s t r a t i o n was found to reverse t h i s e f f e c t (Lorden and Oltmens, 1978). In cont ras t ,2 -way s h u t t l e box avoidance was unaf fec ted and l ea rn i ng of an uns i gna l l ed 1-way avoidance response was impai red w i th PCPA admin is -t r a t i o n (Koh ler and Lorens , 1978). Lack of agreement w i th o ther PCPA data on s h u t t l e box avoidance may be r e l a t e d to the sho r t e r exper imenta l per iod employed by Kohler and Lorens (1979) (2 days ) . Vorhees (1979) observed tha t PCPA d id not exe r t i t s e f f e c t u n t i l the t h i r d day of t e s t i n g . In add i t i o n to avoidance t e s t i n g , se ro ton in has been assoc i a ted w i th memory (Essman, 1977). S t imu l a t i o n of the dorsa l raphe nuc l e i im-pa i red the memory of a pass ive-avo idance response and PCPA b locked t h i s e f f e c t ( F i b i g e r e t a l . , 1978). S i m i l a r l y , PCPA markedly inc reased ex-t i n c t i o n i n a punished step-down paradigm (Beninger and P h i l l i p s , 1979). Se ro ton in has a l s o been imp l i c a t ed i n seve ra l homeostat ic behav i o r s , p a r t i c u l a r l y i n the r egu l a t i o n of food i n t a k e . Adm in i s t r a t i on of 5-hydroxytryptophan was observed to decrease food consumption, lower meal s i z e , and depress ra te of ea t i ng per meal (B lunde l l and Latham, 1979). Such r e s u l t s were i n accord w i th the e f f e c t s o f f en f lu ramine (B lunde l l and Leshem, 1975) and L i l l y 110140 (Goudie, 1976) drugs known to poten-- 16 -t i a t e se ro ton in a c t i o n . S i m i l a r l y , PCPA treatment was shown to r e s u l t i n hyperphagia and dose dependent inc reases i n food i nge s t i on (Mackenzie e t a l . , 1979; Cosc ina e t a l . , 1978). In con t r a s t to the above, t ryptophan i n j e c t i o n s were repor ted to have no e f f e c t on food consumption i n depr ived ra t s (Weinberger e t a l . , 1979). S i m i l a r f i nd i ng s have been obta ined w i th a methyl e s t e r of PCPA, a se ro ton in dep l e t e r . 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 of the amino ac i d methyl e s t e r s of l euc i ne and tryptophan was shown to r e s u l t i n hyperphag ia , but no a s s o c i a t i o n was observed between over ea t i ng and a reduc t i on i n b r a i n se ro ton in (Mackenzie e t a l . , 1979). In a dd i t i o n to s e r o t on i n ' s po s s i b l e r o l e i n the suppress ion of food i n t a k e , i t s f un c t i on i n the r egu l a t i o n of e i t h e r p r o t e i n or carbohydrate consumption has been d i scussed (Anderson, 1979; Wurtman and Wurtman, 1979). In the s e l f - s e l e c t i n g r a t , an inve rse r e l a t i o n s h i p , (r=-.96)>, was repor ted between p ro t e i n i n take and the plasma tryptophan concen t ra t i on r e l a t i v e to i t s compet i t o r s . This plasma amino a c i d pa t te rn has been d i r e c t l y a s soc i a t ed w i th se ro ton in concen t ra t i on (Anderson, 1979). Uncon t ro l l ed d i abe te s , a d i sease tha t causes both a marked decrease i n the r a t i o of t ryptophan to the neu t ra l amino ac ids and whole b ra i n t r yp tophan , was a l so observed to e l eva te p ro t e i n i n t ake (Woodger e t a l . , 1979). Conve rse l y , PCPA adm in 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 d ihydroxyt ryptamine de s t r u c t i on of se ro tone rg i c neurons were found to decrease p r o t e i n i n t a k e . Reduced p r o t e i n consumption was shown to be d i r e c t l y a s soc i a ted w i th depressed se ro ton i n and 5-HIAA (Ashley e t a l . , 1979). F i n a l l y , drugs thought to enhance se ro tone rg i c t r an sm i s s i on , i . e . f en f l u r am ine , f l u o x e -t i n e , or MK 212, were observed to s e l e c t i v e l y decrease consumption of carbohydrate w i thout i n f l u e n c i n g p ro t e i n i n take (Wurtman and Wurtman, - 17 -1979) . Thus, s e ro tone rg i c neurons appear, to regu la te food in take bu t , t h e i r exact f un c t i on has not been e s t a b l i s h e d . I n h i b i t i o n of aggress ion may be an a f f e c t i v e behav ior mediated by s e r o t o n i n . Ch lo r im ip ramine , a se ro ton in re-uptake b l o c ke r , was shown to i n h i b i t mur i c ide i n na tu ra l k i l l e r r a t s but was not found to prevent mouse k i l l i n g i n r a t s w i th l e s i on s of the ascending p r o j e c t i o n of the dorsa l and median raphe nuc le i (Marks e t a l . , 1978). In a d d i t i o n , raphe l e s i ons were observed to induce aggress i ve behaviors such as mu r i c i d e , an inc reased f l i g h t response to t a i l p i n ch i ng , a g rea te r s t rugg l e response to cap tu r ing w i th a g loved hand, and more squea l i ng upon being caught (Yamaoto and Uek i , 1978). Severa l o f these behav iors were shown to be i n h i b i t e d by 5-hydroxy-t ryp tophan , imipramine, and ch loro imipramine (Yamaoto and Uek i , 1978). Shock induced f i g h t i n g was a l s o found to be f a c i l i t a t e d i n r a t s o f f e r ed a t ryptophan f r ee d i e t , a l though supplementing chow w i th 0.5% tryptophan had no e f f e c t on shock induced f i g h t i n g or mur i c ide (Kantak et a l . , 1980) . In con t ra s t ,m i ce o f f e r ed a p u r i f i e d 12% case in d i e t supplemented w i th 0.25 or 0.5% tryptophan were found to e x h i b i t i nc reased t e r r i t o r i a l aggress ion (Thurmond e t a l . , 1980). However, a 1% tryptophan supplement had no e f f e c t on t h i s behav io r . A f t e r 6 weeks on the t ryptophan supp le -mented d i e t s , no d i e t s were observed to a l t e r t e r r i t o r i a l agg res s i on , desp i t e inc reased l e v e l s o f b r a i n t ryptophan and s e r o t on i n . I n t e r e s t i n g l y , t h i s same 12% case in d i e t supplemented w i th l e u c i n e , r a the r than t ryp tophan , was shown to lower b ra in t ryp tophan , t y r o s i n e , and s e r o t o n i n , but not to i n f l u ence aggress i ve behav ior (Thurmond, et a l . , 1980). A l though Thurmond's f i n d i ng s do not support the b e l i e f t ha t se ro ton in i n h i b i t s aggress ive behav io r , h i s c on t r a s t i n g r e s u l t s cou ld be due to the d i f f e r -- 18 -ences i n exper imenta l des i gn . Both the type of aggress ion t e s t ed and the spec ies d i f f e r e d . In a dd i t i o n to the above a f f e c t i v e behav io r , s e ro ton in has been a s soc i a t ed w i th s l eep (Essman, 1978). Tryptophan adm in i s t r a t i o n to humans (Hartman, 1978) was repor ted to reduce s leep la tency and to reduce both s leep l a tency and REM ( rap id eye movement) l a tency i n ra t s ( H i l l and Reyes, 1978). Recen t l y , 4 g of t ryptophan was shown to inc rease the dura -t i o n of stage 3 s leep i n humans, bu t , on ly trends were found f o r decreased awake a c t i v i t y , i nc reased REM, and decreased drowsy s leep (N icho lson and Stone, 1979). A l s o , t ryptophan was observed to decrease slow wave s leep l a t ency i n r a t s . Th is reduced l a tency c o r r e l a t e d w i th e l eva ted se ro ton in and 5-HIAA, and depressed dopamine and homovan i l l i c a c i d ( Fo rna l , et a l . , 1 9 7 9 ) . However, when a t ryptophan analogue was adm in i s t e red , the e f f e c t on s leep l a tency and the catecho lamines , i n c l ud i ng norep inephr ine , was repeated . Sero ton in was on ly s l i g h t l y inc reased and no d i f f e r en ce s i n 5-HIAA were obta ined (Forna l e t a l . , 1979). The re fo re , the tryptophan e f f e c t was thought to be due to a weakening of catecholamine f unc t i on (Forna l et 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 to se ro ton in (Warbr i t ton e t a l . , 1978). D i r e c t i n f u s i o n of se ro ton in i n t o the r i g h t l a t e r a l ce rebra l v e n t r i c l e of r a t s was repor ted to produce dose dependent decreases i n spontaneous locomotor behav ior as measured by a 6-channel e l e c t r o n i c a c t i v i t y moni tor (Warbr i t ton e t a l . , 1978). In a d d i t i o n , when r a t s ( F i b i g e r and Campbel l , 1971) or mice (Modigh, 1972) were i n j e c t e d w i th 5-hydroxytryptophan and p laced i n a m o t i l i t y meter (Modigh,, 1972) or a s t a b i l i m e t e r ( F i b i g e r and Campbel l , 1971), decreased locomotor a c t i v i t y was observed. Rats i n j e c t ed w i th 5-hydroxyt ryptophan, s i m i l a r l y - 19 -e xh i b i t ed decreased bar p ress ing f o r food which c o r r e l a t e d wi th inc reased b r a i n se ro ton in (Apr i son and H ing ten , 1962). In accord w i th the above, procedures t h a t dep le te se ro ton in have been observed to i nc rease locomotor a c t i v i t y . Des t ruc t i on of the se ro -t one rg i c neurons by s e l e c t i v e l e s i on s of the raphe n u c l e i , were repor ted to induce h y p e r a c t i v i t y when ra t s were t e s t ed i n a s t a b i l i m e t e r (Geyer e t a l . , 1976; Jacobs , 1974). Th is e f f e c t was po ten t i a t ed f o l l ow i ng amphetamine adm i n i s t r a t i o n (Geyer e t a l . , 1976). 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 or a running whee l , dose dependent i n -creases i n locomotor a c t i v i t y were produced when ra t s were g iven PCPA (Mabry and Campbel l , 1973; F i b i g e r and Campbel l , 1971). PCPA has a l so been repor ted to po t en t i a t e the e f f e c t o f amphetamine on locomotor a c t i v i t y (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 of 5-hydroxytryptophan were shown to reverse the h y p e r a c t i v i t y induced i n r a t s by amphetamine or apomorphine, dopamine s t imu lan t s ( Ba l d e s s a r i n i e t a l . , 1975; Breese et a l . , 1974; Mabry and Campbel l , 1973). Moreover, a carbohydrate induced inc rease i n se ro ton in a l s o reversed the h y p e r a c t i v i t y induced in r a t s i n j e c t e d w i th coca ine , and weakened the e f f e c t of amphetamine (Tay lo r and Ho, 1979). B ra in se ro ton in has a l s o been l i n ked to e x p l o r a t i o n . Rats i n j e c t e d w i th PCPA were repor ted to make more head d ips i n a hole board apparatus, and both between and w i t h i n sess i on hab i t ua t i on were found prolonged ( F i l e , 1977). In a d d i t i o n , ch lo rpromaz ine , a drug which inc reases b ra i n t ryptophan and s e r o t o n i n , was shown to decrease the number of head d ips and holes exp lo red i n the same apparatus ( F i l e and Pope, 1974). Fu r the r -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 th se ro ton in were observed to walk and rea r l e s s than mice i n j e c t e d w i th v eh i c l e (Herman, 1975). More-- 20 -over , very low doses of t ryptophan have been repor ted to reduce locomo-t i o n of r a t s i n an open f i e l d (T r i ck lebank e t a l . , 1978; T a y l o r , 1976). However, when meth ion ine , an amino ac id , which competes w i th t ryptophan f o r uptake across the b lood b r a i n b a r r i e r , was admin i s te red w i th t r yp tophan , no decrease i n locomot ion was observed (Tay l o r , 1976). Thus, se ro ton in has been a s soc i a t ed w i th many behav io r s . Al though r e s u l t s are c o n t r o v e r s i a l , i n g ene r a l , se ro ton in appears to f un c t i on as an i n h i b i t o r of most behav iors d i s cu s sed . 6. Se ro ton in and Co r t i c o s t e r one . The r o l e of se ro ton in i n the r egu l a t i o n of the hypo tha lamus/p i tu i -t a ry /ad rena l a x i s has been w ide l y researched. Complete or f r o n t a l d ea f f e r en t a t i on of the r a t hypothalamus, the probable locus of the c o r t i c a l r e l e a s i n g hormone (CRH) neurons ( Y u w i l l e r , 1979), was repor ted to dep le te se ro ton in and e l eva te c o r t i -costerone (Vermes e t a l . , 1973). In a d d i t i o n , the adrenal response to e ther and s u r g i c a l s t r e s s was found b locked due to imp lan ta t i on of se ro ton in i n t o the medial hypothalamus (Vermes and Te legdy, 1972). Reserp ine , a se ro ton in b l o c ke r , was observed to inc rease ACTH i n dogs (Egdahl e t a l . , 1956). E levated plasma c o r t i c o s t e r one was a l s o found i n response to e ther s t r e s s when r a t s were admin i s te red PCPA (Vermes and Te legdy, 1973). Thus, the above s tud i e s seem to suggest tha t se ro ton in i n h i b i t s the re l ease of hormones of the hypo tha lamus/p i tu i t a ry /ad rena l system. However, r e s u l t s to the con t ra ry have a l s o been repo r t ed . In t raper i tonea l - i n j e c t i o n of 5-hydroxytryptophan was found to e l eva te plasma co r t i c o s t e r one (Popova e t a l . , 1972). Furthermore, inc reased c o r t i c o s t e r one was a l s o observed - 21 -i n r a t s i n j e c t e d w i th 5-hydroxytryptophan ( F u l l e r and Snoddy, 1979), f l u o x e t i n e , an i n h i b i t o r of se ro ton in uptake ( F u l l e r and Snoddy, 1979), and qu ipaz ine ( F u l l e r and Snoddy, 1979) and fen f lu ramine ( S c he t t i n i e t a l . , 1979), se ro ton in agon i s t s . S i m i l a r l y , e l e c t r o l y t i c l e s i on s of the nucleus raphe median or 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 of 5,7 d ihydroxy-t ryp tamine , a se ro ton in neu ro tox i n , was shown to b lock the fen f lu ramine induced inc rease i n plasma c o r t i c o s t e r one ( S c he t t i n i e t a l . , 1979). In a d d i t i o n , me te rga l i ne , a se ro ton in an t agon i s t , was found to reverse the inc reased c o r t i c o s t e r one response produced by qu ipaz ine ( F u l l e r and Snoddy, 1979). F i n a l l y , Mod l inger e t a l . (1979, 1980) observed tha t an o ra l dose of 10 g t ryptophan i n humans inc reased plasma C o r t i s o l . From the above, i t would appear tha t se ro ton in p lays a r o l e i n the r egu l a t i o n of c o r t i c o s t e r one output . The he te rogene i ty of r e s u l t s may i n d i c a t e tha t se ro ton in i s i nvo l ved i n both i n h i b i t i o n and s t imu l a t i o n of c o r t i c o s t e r one r e l e a s e . In t h i s r ega rd , i t i s po s s i b l e t ha t more than one system of CRF neurons cou ld e x i s t and tha t they cou ld respond to se ro ton in 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 ther systems have a l s o been repor ted to mediate the output of CRF ( Y u w i l l e r , 1979). Thus, the i n v e s t i g a t i o n o f s e r o t on i n ' s r o l e i n the r e gu l a t i o n of the hypo tha lamus/p i tu i t a ry /ad rena l ax i s i s very comp l i ca ted . A more complete understanding may not be achieved u n t i l the locus of the CRF neurons has been e s t ab l i s hed and se ro ton in pathways i nne r va t i ng the CRF neurons have been i d e n t i f i e d . - 22 -7. Summary. Fasted r a t s fed a high carbohydrate meal or i n j e c t e d w i th t ryptophan have been shown to e x h i b i t an e l e v a t i o n i n b ra i n s e r o t o n i n . However, the behav io ra l consequences of t h i s neurochemical i nc rease have not been we l l e s t a b l i s h e d . In a d d i t i o n , the r o l e of se ro ton in i n the r egu l a t i o n of c o r t i c o s t e r one output i s s t i l l poor l y understood. Thus, the present study was conducted t o : (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 of the b ra i n se ro ton in e l e v a t i o n r e l a t i v e to e xp l o r a t i o n of a novel environment, a behav ior r e l a t e d to b r a i n se ro ton i n con ten t ; and (2) determine plasma c o r t i c o s t e r one concent ra t ions f o l l ow i ng t ryptophan and carbohydrate i n -duced inc reases i n b r a i n s e r o t on i n . 8. R a t i o na l e . E leva ted b ra i n se ro ton in has been shown to occur i n r a t s i n j e c t e d w i th t ryptophan or fed a high carbohydrate meal . In a d d i t i o n , reduced exp l o r a t o r y behav ior has been observed when se ro ton in has been i n c r ea sed . Thus, i t i s hypothes ized tha t f a s t ed ra t s fed a high carbohydrate meal or i n j e c t e d w i th t ryptophan w i l l d i s p l a y decreased exp l o ra to r y behav ior r e l a t i v e to con t r o l s and tha t the reduc t i on i n e xp l o r a t i o n w i l l be p o s i -t i v e l y c o r r e l a t e d w i th inc reased s e r o t on i n . Sero ton in has been imp l i c a t ed as both an i n h i b i t o r and s t imu l a t o r of the hypo tha lamus/p i tu i t a ry /ad rena l a x i s . However, i t i s expected tha t the t ryptophan and carbohydrate i n -duced inc rease i n se ro ton in w i l l i nc rease plasma co r t i c o s t e r one l e v e l s s i nce e l eva ted C o r t i s o l output has been observed i n humans admin i s te red an o ra l dose of t r yp tophan . S i m i l a r l y , r a t s g iven a sucrose s o l u t i o n have a l s o been shown to e x h i b i t an i nc rease i n plasma co r t i c o s t e r one (Hart e t a l . , 1980). - 23 -I I I . GENERAL METHODS 1. Animals and Ra t i ons . Male Wis ta r r a t s weighing 220-280 g were used i n a l l s t u d i e s . E igh t to 10 days p r i o r to each exper iment , r a t s were o f f e r ed water and a p u r i -f i e d con t ro l d i e t (Appendix Table I) ad l i b i t u m . Accord ing to the Nat iona l Research C o u n c i l , t h i s d i e t meets the nu t r i e n t requirements of growing r a t s (N .R .C . , 1978). Animals were housed i n d i v i d u a l l y i n suspended w i re mesh cages and exposed to l i g h t f o r 12 hrs d a i l y , 0600 to 1800 h r s . Room temperature was mainta ined a t 21 + 2°C. S t ress has been shown to inc rease b r a i n se ro ton in turnover i n the r a t (Curzon et a l . , 1972). Thus, a l l animals were handled twice d a i l y i n : o r de r to a c c l ima t i z e them to the hand l ing tha t would occur on the day of t e s t i n g . 2. B iochemica l Dete rminat ions . Whole b r a i n t ryptophan was measured us ing the methods of Denkla and Dewey (1967), as mod i f i ed by Bloxam and Warren (1974) , and whole b ra i n se ro ton in was assayed by the method of Curzon and Green (1970). Plasma co r t i c o s t e r one was measured by the method of G l i c k e t a l ( 1 9 6 4 ) . Double d i s t i l l e d water was used to make up a l l reagents and s o l u -t i o n s . A l l water was de ion i zed through a p u r i f i c a t i o n c a r t r i d g e (Barnstead High Capac i t y , Barnstead Company, Boston, Mass.) and was sub-sequent ly d i s t i l l e d . High pu 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 in the p repa ra t i on of a l l HC1 con ta i n i ng s o l u -t i o n s . Tryptophan, c o r t i c o s t e r o n e , and se ro ton in stock s o l u t i o n s and the H ?S0 4/ET0H mixture were s to red at 4°C. Ortho-phthaldehyde (OPT) (Sigma - 24 -Chemical Company, Sa i n t Lou i s , M i s sou r i ) was s to red i n a dark b o t t l e a t -20°C. Se ro ton in c r e a t i n i n e su lphate (Sigma Chemical Company) and c o r t i -costerone (Sigma Chemical Company) were kept i n a de s s i c a t o r a t 4°C and a l l o ther chemica ls were mainta ined a t room temperature. The OPT and cy s te i ne ( B r i t i s h Drug House) s o l u t i o n s were prepared immediate ly before use . P r i o r to ana l y se s , f rozen t i s s ue s were homogenized in i c e c o l d butanol a c i d i f i e d w i th 0.01 N HC1 and cen t r i f uged (1000 x G) a t 4°C f o r 10 min. Four ml of the butanol supernatant were then added to 5 ml co ld heptane p lus 0.5 ml of 1% L -cys te ine i n 0.1 N HC1. A f t e r vo r t ex i ng f o r 2.5 min, samples were aga in c en t r i f uged as above. The butanol/heptane phase was subsequent ly a sp i r a t ed and a l i q uo t s of the HC1 e x t r a c t were used f o r b ra i n t ryptophan and se ro ton in a n a l y s i s . Samples were kept i c e co ld throughout p roces s i ng . B ra in Tryptophan. An a l i q u o t of 0.1 ml of the HC1. e x t r a c t was added to 2 ml c o l d 10% TCA w i th 0.2 ml o f 2% formaldehyde. Next , 0.1 ml of FeCl^ (6.0 x 10 i n co l d 10% TCA) was added and the tubes immediately p laced i n a b o i l i n g water ba th . The f l uo rophore norharmen was produced dur ing t h i s heat ing procedure. Tubes were then coo l ed , r ep l en i shed to 2.4 ml w i th 10% TCA, and read f l u o r o m e t r i c a l l y a t wavelengths of 370/452 mu (Farrand Manual Spec t ro f l uo romete r , Farrand Op t i c a l I n c . ) . B ra in t r yp t op -phan was expressed as ug/g b r a i n . The work ing t ryptophan ( J . T . Baker, Chemical Co . , P h i l l i p s b u r g , N.J . ) s tock s o l u t i o n was prepared by d i l u t i n g a 40.8 ug/ml t ryptophan s o l u t i o n (4.08 mg/100 ml 0.1 N NH^OH) to 4.08 ug/ml. A standard curve was read d a i l y a t concent ra t ions o f 0.1-7 ug, 0.34 ug and 0.51 ug of t ryptophan/ tube. - 25 -B ra in Se ro t on i n . 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 of OPT (0.5% i n methanol) and 1.5 ml of concent ra ted HC1. A f t e r vo r tex -ing b r i e f l y , samples were p laced i n a b o i l i n g water bath f o r 10 min, coo led,and rep l en i shed to 2 ml w i th concentrated HC1. B ra i n se ro ton in was read f l u o r o m e t r i c a l l y a t wave lengths of 360/470 mu and expressed as ug/g b r a i n . The se ro ton i n stock s o l u t i o n , prepared from 40 mg of se ro ton in c r e a t i n i n e su lphate i n 100 ml 0.01 N HC1, was s e r i a l l y d i l u t e d to 40 ug/ml. A standard curve was read d a i l y from dup l i c a t e s of 4 ug/ml , 2 ug/ml , and 1 ug/ml of s e r o t on i n . Plasma Co r t i c o s t e r one . To 0.05 ml plasma were added 0.50 ml d i s -t i l l e d water and 0.03 ml i s o - o c t ane . Samples were then vortexed 15 sec and cen t r i f uged 4 min a t (1000 x G) . Next, the i so -oc tane supernatant was a sp i r a t ed and 0.7 ml of ch lo ro fo rm were added. Samples were vortexed and c en t r i f uged as above. The supernatant was aga in removed and 0.05 ml of 0.1 N NaOH were added to the remaining ch lo ro fo rm l a y e r . Tubes were subsequent ly vor texed,and c en t r i f uged as p r ev i ou s l y desc r ibed and the NaOH supernatant a s p i r a t e d . Next , 0.35 ml of a H2SO4/ETOH (13:7) mixture were added, vortexed and again c en t r i f u g ed . F i n a l l y , the ch loroform supernatant was removed, the remaining H2SO4/ETOH phase was t r a n s f e r r e d to 1 ml g lass cuvet tes ,and read f l u o r o m e t r i c a l l y us ing a Turner 110 f 1 uorometer,,with 47-B and 2A-12 pr imary and secondary f i l t e r s . C o r t i -costerone was expressed as ug/100 ml plasma. A working c o r t i c o s t e r one stock s o l u t i o n was prepared from 1000 ug/ 100 ml i n ET0H. Concent ra t ions of 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 to produce a standard curve . - 26 -IV. EXPERIMENT 1 BRAIN TRYPTOPHAN DURING THE DARK PHASE AFTER FASTING 1. I n t r odu c t i o n . During the dark phase of the l i g h t i n g c y c l e , a gradual inc rease i n whole b r a i n t ryptophan has been demonstrated i n r a t s fed ad l i b i t u m (Wurtman and Fernstrom, 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 hasno t been s t ud i e d . Thus, the ob j e c t i v e s of the f i r s t experiment were: 1) to determine i f a 16 hour f a s t , terminated at var ious t imes dur ing the dark c y c l e , would a l t e r b r a i n t ryptophan r h y t hm i c i t y ; 2) to e s t a b l i s h the nad i r i n b ra i n t ryptophan dur ing the dark pe r i od . 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 t ime of the second e x p e r i -ment. 2. Exper imental Procedure. T h i r t y r a t s were randomly ass igned to 5 groups, o f f e red water ad l i b i t u m , but depr ived of food a t e i t h e r 2400, 0200, 0400, 0800,.or 1200 h r s . S i x teen hours l a t e r , r a t s were decap i ta ted 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 . Bra ins were removed, immediately f rozen i n l i q u i d n i t r o gen , and s to red a t -70° f o r de te rminat ion of b r a i n t ryp tophan. The data were analyzed by a 1-way ana l y s i s o f va r i ance (ANOVA). 3. Resu l t s and D i s cu s s i on . Table I shows the mean concent ra t i ons of whole b r a i n t ryptophan i n r a t s f a s ted 16 h r s . The ANOVA i nd i c a t ed tha t dur ing the dark phase, b r a i n t ryptophan d id not d i f f e r when r a t s were depr ived of food f o r 16 h r s . - 27 -TABLE I BRAIN TRYPTOPHAN FOLLOWING A 16 HOUR FAST Time B ra in 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 of an imals/group - 28 -During the dark phase, r a t s fed ad l i b i t u m have been observed to ex-h i b i t a gradual i nc rease i n whole b r a i n (Wurtman and Fernstrom, 1972), b r a i n stem (Morgan e t a l . , 1975), ce reb ra l co r tex (Hery e t a l . , 1977), and. f r o n t o - p a r i e t a l ce rebra l cor tex (Hery e t a l . , 1977) t ryptophan. Da i l y f l u c t u a t i o n s in the r a t i o of plasma tryptophan to the neut ra l amino ac ids are be l i e ved to generate the b r a i n t ryptophan rhythm (Fernstrom and Wurtman, 1979). Moreover, cons ide rab l e ev idence has i nd i c a t ed tha t the c y c l i c a l consumption of p r o t e i n con t r i bu t e s to the d a i l y change i n plasma neut ra l amino ac ids (Fernstrom et a l . , 1979b). Thus, Fernstrom (1979) has proposed tha t the d a i l y i n take of p r o t e i n generates the rhythm in b r a i n t ryp tophan. Resu l t s from the present study support the p o s s i b i l i t y tha t food i n -take may con t r i bu t e to the d a i l y i nc rease i n t ryptophan dur ing 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 th c a u t i o n , because an ad l i b i t u m con t ro l was not i nc luded i n t h i s exper iment. These data f u r t h e r i n d i c a t ed tha t b r a i n t ryptophan d id not f l u c t u a t e dur ing the dark per iod a f t e r a 16 hour f a s t . - 29 -V. EXPERIMENT 2 EFFECT OF TRYPTOPHAN ADMINISTRATION AND CARBOHYDRATE INGESTION ON BRAIN TRYPTOPHAN AND SEROTONIN 1. I n t r odu c t i o n . B ra i n t ryptophan and se ro ton in have repeated ly been shown to inc rease i n r a t s f a s ted over n igh t and i n j e c t e d w i th t r yp tophan , or o f f e red a high ca rbohydra te , p r o t e i n - f r e e meal a t the beg inn ing of the l i g h t phase. Whether t h i s same phenomenon occurs when r a t s are f a s ted p r i m a r i l y dur ing the l i g h t c y c l e and then fed carbohydrate a t the beg inn ing of the dark pe r iod has not been determined. Thus, the purpose o f the second e x pe r i -ment was to determine the t ime course and peak l e v e l s o f b r a i n t ryptophan and se ro ton in i n f a s ted r a t s fed a con t ro l meal , a high carbohydrate , p r o t e i n - f r e e mea l , or i n j e c t e d w i th t ryptophan e a r l y i n the dark phase. 2. Experimental Procedure. The exper imenta l des ign i s summarized i n F igure 3. At 0030 hrs r a t s were randomly ass igned to 4 groups and depr ived o f food but permi t ted f r ee access to water . At 1730 hrs the f a s t was terminated and r a t s were o f f e red water ad l i b i t u m and t r ea ted as f o l l o w s : Group 1 - d e cap i t a t ed , b ra ins removed, f rozen i n l i q u i d n i t r o gen , and s to red a t -70°C; Group 2 - i n j e c t e d wi th 0.9% s a l i n e , (pH 9.5) and o f f e r ed the p u r i f i e d con t ro l d i e t ad l i b i t u m ; Group 3 - i n j e c t e d w i th 0.9% s a l i n e , (pH 9.5) and o f f e red 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 to the con-t r o l d i e t w i th p ro t e i n de r i ved c a l o r i e s rep laced w i th carbohydrate (Table 1 - append ix ) ; Group 4 - i n j e c t e d w i th L - t ryp tophan, (50mg/kg) d i s s o l v ed in s a l i n e (pH 9.5) and o f f e r ed the con t ro l d i e t ad 1 i b i t um. - 30 -A N I M A L S F A S T E D 0030 1730 F A S T E D " B A S A L v C O N T R O L & S A L I N E C A R B O H Y D R A T E & S A L I N E C O N T R O L & T R Y P T O P H A N 1830 }830* 1830 1930 1930* 1930* 2030 2030 2030* 2 1 3 0 2130 F igure 3. E f f e c t of t ryptophan adm in i s t r a t i o n and carbo-hydrate i nge s t i on on b r a i n t ryptophan and s e r o t on i n : Exper imental des i gn . * Bra ins removed f o r ana l y s i s of b ra i n t ryptophan and se ro ton i n. - 31 -One hour post t reatment and hour ly f o r the next 3 hours , animals from groups 2, 3 , and 4 were decap i ta ted and b ra ins removed, f rozen i n l i q u i d n i t r o gen , and s to red a t -70°C. B ra i n t ryptophan and se ro ton in were analyzed by a Group (3) x Time (4) ANOVA. 3. Re su l t s . B ra i n Tryptophan. The t ime course and peak l e v e l s of b r a i n t ryptophan i n f a s t ed r a t s o f f e r ed a con t ro l meal , a high carbohydrate mea l , or i n j e c t ed wi th t ryptophan are i l l u s t r a t e d i n F igure 4 and Appendix Table I I . The ANOVA revea led a Group x Time i n t e r a c t i o n F(5,51)=29.27, p < .001. Tests f o r s imple main e f f e c t s i n d i c a t ed a main e f f e c t of group a t 1 hour, F(2,51)=101.75, p < . 01 , 2 hours F(2,51)=4.29, p < .05 , 3 hours F(2,51 )=4.44, p < .05 , and 4 hours F(2 ,51)=3.31, p < .05 , post t reatment . Neuman Keuls post hoc ana lyses showed tha t animals i n j e c t ed w i th t ryptophan had h igher b r a i n t ryptophan l e v e l s than c o n t r o l s , p < . 01 , and carbohydrate t r ea ted r a t s , p < . 01 , a t 1 hour post i n j e c t i o n . B ra i n t ryptophan of t ryptophan i n j e c t e d animals f e l l to con t ro l l e v e l s by 2 hours . When compared to c o n t r o l s , b r a i n t ryptophan was a l s o e l e v a t ed , p < .05 , i n r a t s o f f e red the high carbo-hydrate meal . A l though the d i f f e r e n c e cont inued f o r 4 hours post t rea tment , p < .05 , the g rea te s t d i f f e r e n c e occurred 2 hours a f t e r food p r e sen t a t i on . B ra i n Se ro ton i n . F igure 5 and Appendix Table I I I i l l u s t r a t e the time course of b r a i n se ro ton in i n f a s ted r a t s fed e i t h e r a con t ro l d i e t , a high carbohydrate meal , or i n j e c t e d w i th t ryp tophan. The ANOVA i nd i c a t ed a main e f f e c t o f group F(2,51)=6.50, p < .001, and Neumann Keuls post hoc analyses revea led tha t b r a i n se ro ton in i n r a t s i n j e c t e d w i th t ryptophan or o f f e r ed the high carbohydra te , p r o t e i n - f r e e meal was e leva ted over tha t of c o n t r o l s , p < .05 . 2 h Fasted Basal Cont ro l Carbohydrate Tryptophan Basal One Hour Two Hours Three Hours Four Hours F igure 4. B ra in tryptophan f o l l ow i ng tryptophan adm in i s t r a t i on or i nge s t i on of carbohydrate (mean + SEM) F igure 5: Bra in se ro ton in a f t e r tryptophan adm in i s t r a t i on or carbohydrate i nges t i on (mean + SEM) - 34 -4. D i s c u s s i on . These data are i n accordance w i th the f i n d i ng s of Fernstrom (1971, 1975b). Furthermore, the r e s u l t s demonstrate tha t the carbohydrate induced inc rease i n b r a i n t ryptophan and se ro ton in content occurs dur ing the e a r l y stage of the dark c y c l e . As p r ev i ou s l y observed (Fernst rom, 1971), b r a i n t ryptophan i n t h i s study peaked 1 hour a f t e r t ryptophan i n j e c t i o n . Feed-ing carbohydrate generated the g rea te s t change i n b r a i n se ro ton in a t 2 and 3 hours a f t e r food i n t a ke . However, i n con t r a s t to e a r l i e r r e sea r ch , the percent inc reases i n b r a i n t ryptophan and seroton in were smal l i n the present s tudy. Such r e -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 t ryptophan uptake in both hypothalamus and b r a i n stem s l i c e s has been observed dur ing the dark phase (Hery e t a l . , 1972). A l s o , reduced tryptophan a v a i l a b i l i t y cou ld e xp l a i n the sma l l e r percent i nc rease in se ro ton in obta ined in t h i s s tudy . How-ever , a g rea te r tu rnover of se ro ton in to 5-HIAA may have occurred as w e l l , s i n ce increased se ro ton in turnover has been demonstated dur ing the dark phase (Hery e t a l . , 1972). Thus, the r e s u l t s of t h i s experiment i nd i c a t ed tha t t ryptophan dos ing and carbohydrate i nge s t i on r e su l t e d i n e leva ted b r a i n t ryptophan and se ro -t on i n dur ing the dark phase. The g rea tes t change was observed a t 1 and 2 hours post t reatment . - 35 -V I . EXPERIMENT 3 BEHAVIORAL EFFECTS OF TRYPTOPHAN ADMINISTRATION Th is experiment was conducted to e s t a b l i s h a behav io ra l c o r r e l a t e of inc reased b r a i n se ro ton in concen t r a t i on . Three behav iors be l i e ved to be a s soc i a t ed w i th se ro ton in were assessed: exp l o r a to r y behav io r , a c q u i s i -t i o n of a pass ive-avo idance response, and e x t i n c t i o n . 1. Exper imental Procedure. The exper imenta l procedure i s summarized i n F igure 6. Rats were o f f e r ed p u r i f i e d con t ro l d i e t and water ad l i b i t u m . From 1700 to 2000 hrs on the t r a i n i n g n i g h t , 48 r a t s were randomly se l e c t ed and i n j e c t ed w i th e i t h e r 0.9% s a l i n e (pH 9.5) or 50 mg/kg tryptophan i n 0.9% s a l i n e (pH 9 . 5 ) . On the n ights of e x t i n c t i o n , the 2 groups were f u r t h e r d i v i ded so tha t h a l f the o r i g i n a l s a l i n e and tryptophan animals were admin i s te red s a l i n e and h a l f were i n j e c t e d w i th t ryp tophan. Thus, 4 groups of 12 r a t s were e s t ab l i s hed and i n j e c t ed from 1800 to 1900 h r s . Behav iora l t e s t i n g occurred 1 hour post i n j e c t i o n , the time when b r a i n se ro ton in was shown to peak. Animals were tes ted i n a dark , sound c o n t r o l l e d room (white no i s e , 56db) ad jacent to the animal qua r t e r s . The behav io ra l apparatus was a wooden box,25 x 29 x 40 cm,having one p l e x i g l a s s wa l l f o r observa t ion and a copper g r i d f l o o r which cou ld be e l e c t r i f i e d w i th a scrambled 1.25 mamp cu r r en t . A p l a t f o rm , 7.5 x 25 x 7 cm, extended across 1 wa l l o f the apparatus and a 20 watt incandescent 1 i gh t evenly i l l um i na t ed 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 laced on the narrow p l a t f o rm and the l a t ency to step-down and exp lo re the exper imenta l - 36 -F igure 6. Behav iora l e f f e c t s of t ryptophan a d m i n i s t r a t i o n : Exper imental des i gn . - 37 -box on 2 - 1 min t r i a l s was measured by 2 t r a i ned observe rs . Between t r i a l s , r a t s were re turned to a smal l p l a s t i c ho ld ing cage f o r 1 minute. 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 f o l l ow i ng recorded: (1) l a t ency to escape shock; (2) t o t a l number of step-downs (4 f e e t on f l o o r ) ; (3) t o t a l number of step-down, attempts (1-3 f e e t on f l o o r ) ; (4) t o t a l t ime to reach c r i t e r i o n (remain on the p l a t fo rm f o r 2 consecut i ve m inu tes ) . During e x t i n c t i o n t r i a l s (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 ow i ng behav iors were recorded: (1) e x t i n c t i o n ( l a t ency to step-down and remain on the f l o o r a c r i t e r i o n of 30 seconds) ; (2) number of step-down attempts (1-3 f e e t on f l o o r or 4 f e e t on the f l o o r but f a i l u r e to meet the 30 second c r i t e r i o n ) ; (3) number of step-down attempts and t o t a l t ime to meet c r i t e r i o n on the f i r s t n igh t of e x t i n c t i o n ; (4) t o t a l n igh ts to reach c r i t e r i o n ; (5) t o t a l seconds to e x t i n gu i s h ; (6) t o t a l step-down attempts p r i o r to e x t i n c t i o n . Rats were e l im ina t ed from the experiment once e x t i n c t i o n occu r red . Measures recorded during" l e a rn i ng and the e x t i n c t i o n t r i a l s were analyzed by the S tudent ' 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 th time as a repeated measure was used to t e s t the mean l a t en c i e s to step-down on 2 t r i a l s . 2. Re su l t s . A c q u i s i t i o n / E x t i n c t i o n T e s t i n g . The group means f o r each measure taken dur ing 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 Table H a n d Table I I I . No d i f f e r en ce s were observed f o r any of the parameters t e s t e d . TABLE II ACQUISITION OF AN AVOIDANCE RESPONSE FOLLOWING INJECTION OF SALINE OR TRYPTOPHAN Treatment Escape la tency Attempts Step-downs Tota l t ime Contro l (23)* 28.91 + - 4 . 4 3 t (24) 1.33 +- 0.36* (24) 0.33 + 0.13 (24) 177.17 + 10 .71 + ( Sa l i n e ) Tryptophan (24) 34.89 + 10.57 (24) 0.96 + 0.27 (24) 0.25 + 0.11 (24) 189.33 + 13.54 * Number of animals/group t Mean seconds + SEM f Mean number of step-down a t t emp t s / t r i a l ' TABLE I I I EFFECT OF TRYPTOPHAN OR SALINE ON EXTINCTION Treatment Tota l Step- To ta l Seconds Tota l SDA Tota l T r a i n i ng E x t i n c t i o n Tota l Seconds down Attempts Night 1 Night .1 Nights Sa l i n e Sa l i n e* 249.17 + 91 . 00 n 2.08 + 0 .57 § 115.92 + 19 .14 + 1.50 + 0.56* 1 . 8 3 + 0 . 4 4 * Sa l i n e T ^ y P t o " 1 96 . 8 3+66 . 0 6 1 . 8 5 + 0 . 6 0 1 1 9 . 0 0 + 2 0 . 4 7 1 . 3 3 + 0 . 4 8 1 . 7 5 + 0 . 3 5 phan - - -p h a n t 0 " S a 1 i n e 220.08"+ 65 . 14 2 . 7 5 + 0 . 7 2 1 2 3 . 4 2 + 1 5 . 8 9 2 . 0 0 + 0 . 4 8 1 .67+ 0.38 & I Tryp to - T ryp to - 1 4 2 . 5 8 + 3 5 . 7 3 2 . 0 0 + 0 . 6 3 1 0 4 . 5 0 + 2 0 . 8 3 1 . 2 5 + 0 . 3 9 1 . 4 2 + 0 . 3 9 phan phan - - - -* N = 12 animals/group JI Tota l seconds + SEM to ex t i ngu i sh •-§ Tota l step-down attempts + SEM over 5 n ights of e x t i n c t i o n t Mean seconds + SEM f i r s t n ight of e x t i n c t i o n ^ Mean number of step-down-attempts + SEM f i r s t n ight o f e x t i n c t i o n # Tota l n ights + SEM to ex t i ngu i sh - 40 -Latency to Step-Down. The ANOVA revea led a main e f f e c t of group F ( l , 4 5 ) = 4 .64 , p < .05 , and time F ( l , 4 5 ) = 12.41, ' p < .001, f o r the la tency to step-down and exp lo re the novel chamber. F igure 7 and Appendix Table IV i l l u s t r a t e tha t t ryptophan i n j e c t e d animals took longer than con t r o l s to step-down on both t r i a l s , p <-:.05. In a d d i t i o n , both the con t ro l and tryptophan groups stepped down f a s t e r on the second t r i a l than on the f i r s t t r i a l , p < .05. 3. D i s c u s s i o n . A c q u i s i t i o n of an Avoidance Response. Dep le t ion of b r a i n se ro ton in has repeated ly been shown to f a c i l i t a t e a c q u i s i t i o n of an avoidance task (Vorhees, 1979; L i n e t a l . , 1978; Koher and Lorens , 1978). Thus, i t was hypothes ized tha t an e l e v a t i o n of s e r o t o n i n , v i a t ryptophan a d m i n i s t r a t i o n , would impa i r l ea rn i ng of a punished step-down response (pass i ve -avo idance ) . Although the r e s u l t s of t h i s experiment do not support the above hypothe-s i s , they are i n accord w i th the f i n d i ng s of Engel and Modigh (1974) who observed tha t 600-800 mg/kg tryptophan impaired a c q u i s i t i o n of s h u t t l e box ac t i ve -avo idance and t ryptophan doses of l e s s than 600 mg/kg were i n -e f f e c t i v e 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 present s tudy , may have been too low to a f f e c t l e a rn i ng of the punished step-down response. Engle and Modigh's (1974) r e s u l t s a l s o suggest that the tryptophan impairment of s h u t t l e box avo i d -ance might not be r e l a t e d to s e r o t o n i n , because much lower doses of t r y p t o -phan (50 mg/kg) have been shown to produce maximum inc reases in b r a i n se ro ton in (Fernstrom and Wurtman, 1971; Young et a l . , 1978). In add i t i o n to the above, these f i n d i ng s may be due to the behav ior s t u d i e d , i . e . pass i ve -avo idance . Although se ro ton in has been r e l a t ed to a c t i v e - avo i dance , a task which i nvo l ves i n i t i a t i o n of a response, se ro ton in 30 25 20 15 10 Cont ro l Tryptophan 1 2 1 2 T r i a l s F igure 7. Latency to step-down f o l l ow i ng tryptophan adm in i s t r a t i o n (mean + SEM). - 42 -may not f un c t i on i n the same manner i n pass i ve -avo idance , a task which r e -qu i res response i n h i b i t i o n . In t h i s r ega rd , these data "conf i rm t h o s e o f F i b i q e t a l . (1978) who observed tha t e l e c t r i c a l s t imu l a t i o n of the dorsa l raphe nuc leus , a b r a i n reg ion r i c h i n se ro tone rg i c p e r i k a r y a , d i d not a f f e c t a c q u i s i t i o n of a s imple pass ive-avo idance task . E x t i n c t i o n . Sero ton in dep l e t i on w i th PCPA has a l s o been shown to inc rease e x t i n c t i o n time o f a punished step-down response (Beninger and P h i l l i p s , 1980). Thus, i t was p red i c t ed tha t i n : t h i s experiment e x t i n c -t i o n of the step-down behav ior would be reduced i f se ro ton in was e l eva ted v i a a t ryptophan 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 th those of Beninger and P h i l l i p s (1980). Aga in , the dose u t i l i z e d (50 mg/kg) i n t h i s study may have been too low to i n f l u ence e x t i n c t i o n , even though 50 mg/kg tryptophan i s known to produce a maximum e l e v a t i o n i n b ra i n se ro ton in (Fernstrom and Wurtman, 197,1-; Young et a l . , 1978). In a d d i t i o n , man ipu la t ions t ha t a l t e r se ro ton in con ten t , w i t h i n normal p h y s i o l o g i c a l range, may not be s u f f i c i e n t to a f f e c t e x t i n c t i o n . Indeed, PCPA has been shown to g r e a t l y dep le te se ro ton in through i n h i b i t i o n of t ryptophan hydroxy lase a c t i v i t y (Beninger and P h i l l i p s , 1980). Exp lo ra to ry Behav io r . Resu l t s from the present study suggest t ha t t ryptophan i n j e c t e d r a t s d i s p l a y reduced exp l o r a t o r y behav ior when ex-posed to a novel chamber. These f i nd i ng s are i n agreement w i th those of F i l and-Pope (1974) who a l so demonstrated tha t hole board exp l o r a to r y behav ior was reduced when ra t s were admin i s te red ch lo rp romaz ine , a drug which e leva tes b r a i n s e r o t on i n . S i m i l a r l y , decreased wa lk ing behav ior 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 th t ryptophan (T r i ck l ebank e t a l . , 1978) whereas, enhanced e xp l o r a t i o n i n a ho le board apparatus was shown when se ro ton in was dep le ted wi th PCPA ( F i l e , 1977). - 4 3 -However, i n add i t i o n to exp l o r a to r y behav io r , the l a tency to s tep -down may s imply r e f l e c t the an ima l ' s a c t i v i t y l e v e l . Reduced locomotor behav ior has been observed i n r a t s w i th e leva ted concent ra t i ons of b r a i n se ro ton in (Tay l o r , 1976). Although i t was d i f f i c u l t to separate these two behav iors i n t h i s exper iment , the l a tency scores d i d appear to be c o r r e l a t e d w i th se ro ton in l e v e l s . - 44 -V I I . EXPERIMENT FOUR TRYPTOPHAN AND CARBOHYDRATE INDUCED INCREASES IN BRAIN SEROTONIN: BIO-CHEMICAL AND BEHAVIORAL CORRELATES 1. I n t r o du c t i o n . The purpose of the f i n a l study was to determine i f the carbohydrate induced i nc rease i n b r a i n se ro ton in would y i e l d behav io ra l r e s u l t s com-parab le to the t ryptophan induced e l e v a t i o n . To he lp c l a r i f y whether the l a tency measure r e f l e c t s e xp l o r a t o r y behav ior or locomotor a c t i v i t y , r ea r i ng behav ior on both t r i a l s was t abu la ted as another component of e x p l o r a t i o n . Defecat ion and u r i n a t i o n , behav i o r s o f ten nega t i ve l y a s s o c i -ated w i th se ro ton in (Kameyama et a l . , 1980), were a l s o recorded . F i n a l l y , plasma c o r t i c o s t e r one was assayed as a b iochemica l i n d i c a t o r of inc reased b r a i n se ro ton in s i n ce t h i s neu ro t ransm i t t e r has been shown to regu la te the hypo tha lamus/p i tu i t a ry /ad rena l ax i s ( Y u w i l l e r , 1979). 2. Exper imenta l Procedure. The exper imenta l procedure i s o u t l i n ed i n F igure 8. Seventeen hours p r i o r to drug i n j e c t i o n s or d i e t p r e sen t a t i on , ra t s were randomly ass igned to 5 groups and 80 of 88 animals were depr ived of food but not water . The remaining 8 ra t s were g iven f r ee access to both food and water . Fas t -ing was terminated between 1700 and 1800 and animals were t r ea t ed as f o l l ows : Group 1 - (non- fas ted group) - decap i t a t ed , b lood and b ra ins removed; Group 2 - de cap i t a t ed , b lood and b ra ins removed; Group 3 - i n j e c t e d w i th 0.9% s a l i n e and o f f e r ed the high carbo-hydra te , p r o t e i n - f r e e meal ad l i b i t u m ; - 45 -ANIMALS FASTED 2400 1700 NON-FASTED 1700 FASTED BASAL CONTROL SALINE CARBOHYDRATE SALINE CONTROL TRYPTOPHAN NON-FASTED BASAL 1800* 1800* 1800 1900* 1900" 1900* F igure 8. Tryptophan and carbohydrate induced inc reases i n b r a i n serotonin:_ b iochemica l and behav io ra l c o r r e l a t e s : Exper imental des i gn . Bra ins removed f o r ana l y s i s of b r a i n t ryptophan and se ro ton in and b lood c o l l e c t e d f o r plasma c o r t i c o s t e r one . - 46 -Group 4 - i n j e c t e d w i th 0.9% s a l i n e and o f f e red the con t ro 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 th 50 mg/kg t ryptophan and o f f e red the con t ro l d i e t ad l i b i t u m . At 1 or 2 hours post t reatment , r a t s from groups 3, 4 and 5 were i n d i v i d u a l l y p laced i n the novel chamber. Behaviors recorded i n c l uded : (1) l a tency to step-down on 2 t r i a l s ; (2) number of rears per t r i a l once on the f l o o r ; (3) t o t a l number of boluses and u r i n a t i o n pools a f t e r 2 t r i a l s . Immediately f o l l ow i ng behav io ra l t e s t i n g each r a t was decap i t a t ed . Trunk blood was c o l l e c t e d i n hepa r i n i zed tubes . A f t e r c e n t r i f u g a t i o n at (1000 x G) f o r 20 min, plasma was c o l l e c t e d and s to red a t -20°C. Bra ins were removed, r a p i d l y f rozen in l i q u i d n i t r ogen , and s to red at -70°C. S t a t i s t i c s . The S tudent ' s t - t e s t was used to analyze f a s ted versus non- fas ted basal b r a i n t ryp tophan, s e r o t o n i n , and plasma c o r t i c o s t e r o ne . B ra in t r yp tophan , s e r o t o n i n , plasma c o r t i c o s t e r o n e , l a tency 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 ke , measures taken 1 and 2 h r s . po s t t rea tment , were ana lyzed by a Group (3) x Time (2) ANOVA. 3. Re su l t s . B ra in Tryptophan. B ra in t ryptophan from f a s t ed basal or non- fas ted basal r a t s and the time course and peak concen t ra t i ons of b r a i n t ryptophan i n animals fed a high c a r b ohyd r a t e , ' p r o t e i n - f r e e meal , i n j e c t e d wi th t r y p t o -phan, or fed the con t ro l d i e t are presented i n F igure 9 and Appendix Table V. The S tudent ' s t - t e s t i n d i c a t ed tha t b r a i n t ryptophan was h igher i n f a s ted versus non- fas ted basal animals T(13)=4.12, p < .01 . In a d d i t i o n , the ANOVA revea led a Group x Time i n t e r a c t i o n F(2,64) = 119,26, p < .001. Tests f o r s imple main e f f e c t s i n d i c a t ed a main e f f e c t of group a t 1 hour Q-O I— Q_ >-OH CH CO 18 16 14 12 CD g> 10 6 4 -2 " X Basal One Hour Two Hours • • Non-fasted basal Fasted basa l Cont ro l Carbohydrate Tryptophan F igure 9 . B ra in tryptophan f o l l ow i ng i nges t i on of carbohydrate and tryptophan adm in i s t r a t i on (mean + SEM) - 48 -F(2,63)=319,2, p < . 01 , and 2 hours F(2,65)=9.55, p < . 01 , a f t e r food and drug a d m i n i s t r a t i o n . A main e f f e c t of time F(2,65)=175.5, p < . 01 , was a l s o found. Neuman-Keuls post hoc analyses showed tha t animal i n j e c t e d wi th trypotophan e xh i b i t e d h igher b r a i n t ryptophan concent ra t ions than both con t ro l and carbohydrate t r ea ted ra t s at 1 hour post i n j e c t i o n , p < .01 . By 2 hours , b r a i n t ryptophan f e l l i n the tryptophan i n j e c t e d an ima l s , but concent ra t i ons were s t i l l s i g n i f i c a n t l y more e leva ted than c o n t r o l s , p < . 01 . Post hoc ana lyses a l s o revea led that the carbohydrate fed animals d i sp l ayed an inc rease i n b r a i n t ryptophan over c on t r o l s a t 1 and 2 hours a f t e r food p r e s en t a t i o n , p < .01 . B ra in Se ro t on i n . F igure 10 and Appendix Table VI show f a s t ed and non-f a s t ed basa l concen t ra t i ons of b r a i n se ro ton in and the time course and peak l e v e l s o f se ro ton in i n animals fed the con t ro l or exper imenta l d i e t s or i n -j e c t e d wi th t r yp tophan . B ra in se ro ton in i n f as ted and non- fas ted ra t s was not d i f f e r e n t when analyzed by the S tudent ' s t - t e s t . However, the ANOVA revea led a Group x Time F(2,65)=5.30, p < . 0 1 , i n t e r a c t i o n f o r r a t s assayed 1 and 2 hours a f t e r t ryptophan i n j e c t i o n or food p r e sen t a t i on . A main e f f e c t of group was shown a t both 1,F(2,65)=50.1 , p < . 01 , and 2 ,F(2 ,65)=13.91, p < .01 , hours a f t e r treatment by the t e s t of s imple main e f f e c t s . Neuman-Keuls post hoc analyses revea led tha t animals i n -j e c t e d w i th t ryptophan had a h igher concen t ra t i on of b r a i n s e r o t o n i n , p < .01 , than carbohydrate and con t ro l fed ra t s at 1 hour post i n j e c t i o n . By 2 hours b r a i n se ro ton in f e l l , but concent ra t ions were s t i l l h igher than c o n t r o l s , p < .01 . Carbohydrate fed animals were not shown to d i f f e r from con t r o l s at e i t h e r t imes assayed. Plasma Co r t i c o s t e r one . The t ime course o f plasma co r t i c o s t e r one con-cen t r a t i on s i s i l l u s t r a t e d i n F igure 11 and Appendix Table V I I . The ANOVA revea led a main e f f e c t o f group F(2,64)=5.18, p < . 01 . Neuman-Keuls post F igure 10. Bra in se ro ton in f o l l ow i ng i nges t i on of carbohydrate or t ryptophan adm in i s t r a t i on (mean + SEM). F igure 11. Plasma co r t i c o s t e rone f o l l ow i ng carbohydrate i nge s t i on or t ryptophan adm in i s t r a t i on (mean + SEM). - 51 -hoc ana lyses i n d i c a t ed tha t carbohydrate fed ra t s e xh i b i t e d a s i g n i f i c a n t l y h igher l e v e l of c o r t i c o s t e r one than c o n t r o l s , p < . 01 . Food In take . Table IV i n c l udes the amount of food and carbohydrate consumed by animals e i t h e r i n j e c t e d wi th t ryp tophan, o f f e r ed the high carbohydrate meal , or presented the con t ro l d i e t . ANOVA revea led tha t the animals o f f e r ed the high carbohydrate p r o t e i n - f r e e meal ate l e s s food than those g iven the con t ro l d i e t and i n j e c t e d w i th e i t h e r t r y p t o -phan or s a l i n e F(2,62)=14.57, p < .01 . However, carbohydrate consumption d i d not d i f f e r among groups. Latency To Step-Down/Rearing. Mean r ea r i ng and l a tency to step-down f o r animals i n j e c t e d w i th t ryp tophan , o f f e r ed the high carbohydrate meal , or g iven con t ro l d i e t i s g iven in Table V and F igure 12 and Appendix, Table V I I I , r e s p e c t i v e l y . The ANOVA revea led tha t i n j e c t i o n of t ryptophan or a dm i n i s t r a t i o n of a h igh carbohydrate meal d i d not a f f e c t r e a r i ng or the l a tency to step-down on e i t h e r t r i a l , a t 1 or 2 hours post t reatment . U r i n a t i o n and Bo luses . U r i n a t i on and bolus measures are presented i n Table V I . No s i g n i f i c a n t e f f e c t of group was obta ined when the u r i n a -t i o n and bolus data were ana lyzed by ANOVA. However, a t r end , . p< .09, f o r t ime was suggested f o r u r i n a t i o n . Both the t ryptophan and con t ro l animals tended to u r i na t e l ess 2 hours a f t e r i n j e c t i o n or food consumption. 4. D i s c u s s i on . B ra in Tryptophan. The e l e va t i on s i n b ra i n t ryptophan observed i n f a s ted basa l r a t s over tha t of non- fas ted basal ra t s i n t h i s study are i n accord w i th the f i n d i ng s o f Curzon et a l . (1972). 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 to inc rease the l e v e l o f both plasma f r ee t ryptophan and subsequent ly b r a i n t ryptophan (Curzon e t a l . , 1972). As p r ev i ou s l y d i s cu s sed , f a s t i n g has been shown to inc rease - 52 -TABLE IV TOTAL FOOD AND CARBOHYDRATE INTAKE OF ANIMALS FED CONTROL DIET, CARBO-HYDRATE MEAL, OR INJECTED WITH TRYPTOPHAN Treatment Food Intake (g + SEM) Carbohydrate Intake (g + SEM) Contro l (21) 4.74 + 0.23 Carbohydrate (24) 3.06 + 0.21 Tryptophan (23) 4.71 + 0.31 2.56 + 0.12 2.39 + 0.16 2.54 + 0.17 * number of an imals/group c LATENCY TO STEP-DOWN seconds ro o ro o ro o -s o o c -s • • • * ro N -a c+ o -a Q> 3 o DJ -s cr O =3-<< CL -s o o 13 c+ -s o ro - eg -TABLE V EFFECT OF CARBOHYDRATE INGESTION OR A TRYPTOPHAN INJECTION ON REARING Treatment One Hour Two Hours Rear 1 Rear 2 Rear 1 Rear 2 Contro l (12)* 4.42 + 0 . 60 + 3 . 2 7 + 0 . 7 2 ( 1 2 ) 4 . 8 3 + 0 . 9 6 3 . 4 2 + 1 . 0 0 Carbohydrate (12) 4.50 + 0.86 2.75 + 0.59 (12) 3.17 + 0.58 3.50 + 0.73 Tryptophan (11) 3.64 + 0.65 3.09 + 0.63 (12) 2.67 + 0.56 2.83 + 0.76 * number of 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 U r i na t i on One Hour Two Hours Bolus One Hour Two Hours Cont ro l Carbohydrate Tryptophan 1.75 + 0.50 0.42 + 0.23 1.67 + 0.79 0.25 + 0.25 0.67 + 0.31 1 .08 + 0.31 0.75 +• 0.41 0.08 + 0.83 0.50 + 0.50 0.00 + 0.00 0.50 + 0.29 0.58 + 0.40 * 12 animals/group - 56 -f a t t y a c i d output which in turn e leva tes plasma f r ee t ryptophan by d i s p l a c -ing t ryptophan from albumin b ind ing s i t e s . The e f f e c t of carbohydrate i n ge s t i o n and tryptophan dos ing on b ra i n t ryptophan and i t s t ime course r e p l i c a t e d the f i n d i ng s of the second exper iment . B ra in Se r o t on i n . I n j e c t i o n of t ryptophan induced an inc rease i n b r a i n s e ro ton 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 con t ra s t to the prev ious s tudy , b r a i n se ro ton in d id not r i s e i n r a t s fed the high carbohydrate p r o t e i n - f r e e meal . The e l eva ted plasma c o r t i c o s t e r one concent ra t ions a l so observed i n the carbohydrate fed r a t s , cou ld o f f e r an exp lana t i on f o r these c on t r a d i c t o r y r e s u l t s , i . e . the carbohydrate fed group may have been more s t r e s sed than the con t ro l r a t s . Ce r t a i n s t r e s s f u l procedures have been shown to inc rease the turnover of se ro ton in to 5-HIAA ( Y u w i l l e r , 1979). Thus, a l though inc reased se ro ton in syn thes i s may have occu r red , the s t r e s s of a p r o t e i n - f r e e meal a f t e r f a s t -i n g , p lus exposure to the novel chamber cou ld have been powerful enough to cause an even g rea te r turnover of s e r o t o n i n . Conf i rmat ion cou ld be obta ined by measuring l a b e l l e d 5-hydroxyt ryptophan, an i n d i c a t o r of i n -creased se ro ton in s yn t he s i s . In a d d i t i o n , 5-HIAA cou ld be assayed as a measure of se ro ton in tu rnove r . Plasma Co r t i c o s t e r one . Although ra t s i n j e c t e d w i th t ryptophan e xh i b i t e d an inc rease i n b ra i n s e r o t on i n , plasma co r t i c o s t e r one l e v e l s d i d not d i f f e r from those of. con t ro l or carbohydrate fed an ima ls . These r e s u l t s were s u r p r i s i n g s ince they are hot i n agreement w i th those of Modl inger e t a l . (1979, 1980) who showed tha t C o r t i s o l was i n -creased i n humans admin i s te red an o ra l dose of t ryp tophan. In a d d i t i o n , our data are not i n accord w i th the r a p i d l y growing body of l i t e r a t u r e - 57 -which suggests se ro ton in p lays a r o l e i n the r egu l a t i o n of the hypothalamus/ p i t u i t a r y / a d r e n a l a x i s . However, i t i s noteworthy t h a t when the c o r t i -costerone concent ra t i ons of the t ryptophan versus con t ro l r a t s (1 hour post i n j e c t i o n ) were ana lyzed w i th a t - t e s t , the d i f f e r en ce s were s i g n i f i -can t . Un fo r tuna te l y , the des ign of the present experiment i n v a l i d a t e s the use of t h i s s t a t i s t i c . However, i t i s po s s i b l e t ha t a change i n exper imenta l de s i gn , to i n c l ude on ly the t ryptophan and con t ro 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 ng s i n agreement w i th those i n the l i t e r a t u r e . In con t r a s t to the tryptophan e f f e c t , r a t s fed the high carbohydrate , 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 eva ted concent ra t i ons of plasma c o r t i c o s t e r o n e . From t h i s s tudy , s e r o t on i n ' s r o l e i n mediat ing t h i s response i s not c l e a r , e s p e c i a l l y s i nce b ra i n se ro ton in concent ra t ions were not i n c r ea sed . However, s ince b r a i n t ryptophan was e l e v a t ed , i t i s po s s i b l e tha t se ro ton in turnover and re l ease were i n c r ea sed . Conf i rmat ion of the se ro ton in mechanism must awai t f u r t h e r research when 5-HIAA can be assayed or the c o r t i c o s t e r one response to carbohydrate can be analyzed f o l l ow i ng adm in i s t r a t i o n of a se ro ton in b l o cke r . Po s s i b l e ev idence tha t se ro ton in has no r o l e i n the carbohydrate induced inc rease i n plasma c o r t i c o s t e r one comes from a study i n which a sucrose s o l u t i o n was admin i s te red to r a t s (Hart e t a l . , 1980). A l though e leva ted c o r t i c o i d s were shown, the time course appeared to d i f f e r from tha t normal ly found f o r se ro ton in f o l l ow i ng carbohydrate i n g e s t i o n . Rats g iven a high carbohydra te , p r o t e i n - f r e e meal have been repor ted to ex-h i b i t inc reases i n s e ro ton i n and 5-HIAA f o r 2-3 hours a f t e r food presen-t a t i o n . However, Hart e t a l . (1980) observed t ha t the c o r t i c o i d l e v e l s peaked a t 50 min and f e l l to c on t r o l concent ra t ions by 100 min. Thus, - 58 -some non-se ro tonerg i c mechanism cou ld have mediated the c o r t i c o s t e r one response found i n Hart et a l . ' s (1980) experiment and i n the present study f o l l ow i ng carbohydrate i n g e s t i o n . In a d d i t i o n , a c e t y l c h o l i n e has been imp l i c a t ed i n the r egu l a t i on of the hypo tha l amus/p i tu i t a r y /ad rena l system (Jones e t a l . , 1977). Thus, i t may have mediated the co r t i c o s t e r one response a f t e r sugar consumption. However, to da te , the carbohydrate 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 . Al though the mechanism of the carbohydrate induced inc rease i n c o r t i c o s t e r one i s un c l e a r , i t appears t h a t these animals may have been s t r e s sed more than c o n t r o l s . Co r t i co s t e rone re l ease has been shown to occur as pa r t of the s t r e s s response ( Y u w i l l e r , 1979). However, because of the des ign of t h i s s tudy , 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-hydrate per se cou ld have generated the c o r t i c o i d response. But , t h i s seems u n l i k e l y s i nce the amount of carbohydrate eaten was the same among groups. The more probable s t r e s s o r may have been the lack of d i e t a r y p r o t e i n . Food In take . The data from the present study revea l t ha t f a s t ed ra t s o f f e red a high carbohydra te , p r o t e i n - f r e e meal, consume less food than ra t s g iven the con t ro l d i e t or i n j e c t e d w i th t r yp tophan . Thus, the p r o t e i n - f r e e meal may have g rea te r shor t - te rm s a t i e t y va lue , perhaps through induc ing a more pronounced e l e v a t i o n i n plasma g lucose . Crapo e t a l . (1976) observed t ha t i n humans, consumption of a sucrose s o l u t i o n produced a g rea te r r i s e i n plasma glucose than i nge s t i on of the same sugar w i th added p ro t e i n and f a t . The s i g n i f i c a n c e of e l e -vated plasma g lucose i n s a t i e t y has a l s o been demonstrated. Adm in i s t r a t i on of 2-deoxyg lucose, an i n h i b i t o r o f g lucose metabol ism, was observed to s t imu-- 59 -l a t e food in take (Houpt and Hance, 1971), whereas, duodenal and po r ta l i n -fus i ons of g lucose were found to suppress feed ing i n f a s ted animals (Russek, e t a l . , 1980). In a d d i t i o n , in take of carbohydrate may have been regu la ted by se ro -t o n i n . Severa l researchers have suggested tha t se ro ton in may i n f l u ence carbohydrate i n t a ke . Nance and K i l bey (1973) observed tha t sucrose p re -fe rence was inc reased when PCPA t r ea ted r a t s were dep le ted of se ro ton in and tha t t h i s behav ior was reversed by 5-hydroxytryptophan. S i m i l a r l y , Wurtman and Wurtman ,(1979) showed tha t drugs which e l eva te se ro ton in con-c en t r a t i o n lowered carbohydrate i n take but had no e f f e c t on p ro t e i n i n -g e s t i o n . Both researchers suggested se ro ton in may regu la te carbohydrate i n t a ke . Al though the r e s u l t s o f the present study do not prove the above pro-p o s a l , e l eva ted se ro ton in turnover i n the carbohydrate fed ra t s may have mediated the reduc t i on i n food in take so tha t t o t a l carbohydrate consumed would not exceed amounts ingested by con t ro l r a t s . Latency To Step-Down. Ne i the r t ryptophan or carbohydrate admin-i s t e r e d ra t s e xh i b i t e d d i f f e r en ce s in l a tency to step-down from con t ro l fed an ima ls . That se ro ton in a l s o was not e leva ted i n the carbohydrate fed r a t s cou ld e xp l a i n the lack of behav io ra l r e s u l t s i n t h i s group. However, the i n a b i l i t y to repeat the t ryptophan e f f e c t on the l a tency scores i s more d i f f i c u l t to e x p l a i n . One p o s s i b i l i t y cou ld be the un-avo idab le changes in exper imenta l des i gn . Rats were o f f e red food ad l i b i t u m i n the prev ious exper iment. But , i n t h i s s tudy , animals were fas ted 17 hours and then presented food immediately a f t e r i n j e c t i o n . Bo l l e s (1965) observed inc reased home cage a c t i v i t y i n r a t s f a s t ed 24 hours and then l i m i t e d to 10-12 g food d a i l y f o r 15 days, even on the f i r s t day of t e s t i n g . In a d d i t i o n , the r a t s of t h i s study were observed - 60 -to be h i gh l y e x c i t a b l e when handled and exposed to the novel chamber (when compared to animals of the p r e l im i na r y exper iment ) . Thus, the pos s i b l e e f f e c t s o f food dep r i v a t i on cou ld e xp l a i n the non-s i g n i f i c a n t r e s u l t s obta ined i n t h i s s tudy . - 61 -V I I I . SUMMARY In th is ser ies of studies, behavioral and biochemical correlates of the tryptophan and carbohydrate induced increase in brain serotonin were invest i gated. 1. Brain Tryptophan and Serotonin. Brain tryptophan and serotonin were consistent ly found to peak in tryptophan administered rats at 1 hour post i n jec t i on . Both tryptophan and serotonin dropped to control levels by 2 hours af ter i n j ec t i on . This time course was rep l i ca ted. Carbohydrate was found to induce an increase in brain serotonin; however, when animals were also exposed to a novel chamber, the carbo-hydrate induced serotonin elevat ion was no longer present. 2. Latency to Step-Down. Aminals fed ad l ib i tum and injected with tryptophan exhibited an i n -creased latency to step-down and explore a novel chamber. However, these data were not rep l icated in the f i na l study when animals were fasted pr ior to in jec t ion and food presentation. 3. Plasma Cort icosterone. Since brain serotonin has been implicated in the regulat ion of the hypothalamus/pituitary/adrenal ax i s , plasma cort icosterone was analyzed as a biochemical ind icator of increased serotonin a c t i v i t y . Tryptophan administrat ion did not increase plasma cort icosterone above control con-centrat ions, but ingestion of a high carbohydrate, protein-free meal did produce an elevat ion in plasma cort icosterone. - 62 -IX. CONCLUSIONS The data from the present s tud i e s i n d i c a t e t ha t t ryptophan i n j e c t i o n s r e s u l t i n an inc reased l a tency to step-down and exp lo re a novel chamber, p rov i d i ng the animals are fed ad l i b i t u m . However, a t ryptophan induced c o r t i c o i d response was not c l e a r l y demonstrated. In c on t r a s t , carbo-hydrate i nge s t i on a f t e r f a s t i n g d id not a f f e c t step-down l a tency under the cond i t i ons of t h i s exper iment, i . e . food p resen ta t i on a f t e r a 17 hour f a s t and behav io ra l t e s t i n g dur ing the dark phase, but d i d e l eva te plasma c o r t i c o s t e r o n e . 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New York: 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 con t ro l and carbohydrate d i e t * Ingred ients Contro l Kg/1 Kg Carbohydrate Kg/1 Kg 2 Case i n , v i t am in f r ee .240 -3 Dextrose .185 .265 . 4 Dex t r i n .185 .265 5 Sucrose .165 .247 Corn o i l .150 .150 Agar*7 .035 .035 o Minera l mix .027 .027 9 • Cho l ine Ch l o r i de Premix .007 .007 V i tamin m i x ^ .004 .004 Methionine^ 1 .002 -1.000 Kg 1.000 Kg 1 Mod i f i ed from Fernstrom and F a l l e r , (1978). 2 Supp l ied by Tek lad Test D i e t s , ARS/Sprague-Dawley D i v i s i o n of the Mogul Co rpo ra t i on , Madison, Wiscons in . 3 Supp l ied by Grand I s l and B i o l o g i c a l Co . , Grand I s l a nd , New York. 4 Supp l ied by ICN N u t r i t i o n a l B i o chem ica l s , C l e ve l and , Ohio. 5 Supp l ied by B.C. Sugar Re f i n e r y , Vancouver, B r i t i s h Columbia. ^ Supp l ied by Best Foods D iv . Quebec. , The Canadian Starch Co. L t d . , Montreal 7 Supp l ied by ICN N u t r i t i o n a l B i ochemica l s , C l e ve l and , Ohio. - 73 -Appendix Table 1 ( con t ' d ) Conta ins 16.10 g CaHP0 4; 6.82 g KC1; 1.98 g-MgS0 4; 1.54 g Na 2 HP0 4 ; 0.64 g CaC0 3 ; 0.22 g FeCgH^xh^O (16% Fe) ; 0.15 MnS0 4; 0.02302 g ZnC0 3 ; 0.01256 g CuS0 4 ; 0.00221 g NaF l ; 0.001537 g C r C l 3 6 H 2 0 ; 0.0002188 g NaSe; 0.0001962 g K l ; 13 g sucrose; M inera l s supp l i ed by J . T . Baker Chemical Co . , P h i l l i p s b u r g , New Je r sey . 9 Contains 2 g cho l i ne Ch l o r i d e ; 5 g suc rose . Cho l ine c h l o r i d e supp l i ed by ICN Pharmaceut ia ls I n c . , L i f e Sc iences Group, C l e ve l and , Ohio. ^ Contains 200 mg i n o s i t o l ; 120 mg v i t . E (50% d l - a - t o cophe ra 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 a c i d ; 16 mg d l ca l c ium pantothenate; 16 mg v i t . A a ce ta t e ; 12 mg py r i dox ine HC1; 8 mg th iamine 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 . K ) ; 0.1 mg v i t . B-|2; 0.05 mg v i t . D ( c a l c i f e r o l ) ; 13 g sucrose; Vitamins. ' supp l i ed by ICN Pharmaceut ica ls I n c . , L i f e Sc iences Group, C l eve l and , Ohio. 11 Supp l i ed by ICN Pharmaceutals I n c . , L i f e Sc iences Group, C leve land . Oh io . APPENDIX TABLE I I BRAIN TRYPTOPHAN FOLLOWING INGESTION OF A CONTROL OR CARBOHYDRATE MEAL OR'INJECTION OF TRYPTOPHAN Treatment One Hour Two Hours Three Hours Four Hours Cont ro l (5)* 4.48 + 0 .18 + (6) 4.12 + 0.15 (6) 4.22 + 0.18 (6) 4.37 + 0.22 Carbohydrate (5) 5.60 + 0.17 (6) 5.36 + 0.31 (6) 5.33 + 0.13 (6) 5.16 + 0.20 Tryptophan (4) 1 1 . 0 6 + 1 . 2 0 (6) 4 . 9 8 + 0 . 2 4 (6) 4 . 2 1 + 0 . 1 3 * 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 Two Hours Three Hours Four Hours Cont ro l (5)* .629 + .038 + (6) .616 + .030 (6) .589 + .022 (6) .585 + .030 Carbohydrate (5) .683 + .028 (6) .684 + .020 (6) .672 + .033 (6) .631 + .033 Tryptophan (4) .845 + .061 (6) .675 + .031 (6) .657 + .021 * Number of animals/group t Mean se ro ton in (ug/g + SEM) - 76 -APPENDIX TABLE IV LATENCY TO STEP-DOWN FOLLOWING TRYPTOPHAN OR SALINE INJECTIONS Treatment Mean Latency to Step-Down + SEM T r i a l 1 T r i a l 2 t S a l i n e (24) 18.29 + 2.8 7.88 + 2.4 Tryptophan (23) 25.57 + 3.8 16.25 + 3.6 * number of an imals/group t Mean seconds - 77 -APPENDIX TABLE V BRAIN TRYPTOPHAN FOLLOWING CARBOHYDRATE INTAKE OR TRYPTOPHAN ADMINISTRATION Treatment B ra in Tryptophan Non-fasted (8) basa l Fasted basal (7) 4.38 + 0.15 5.42 + 0.37 Treatment One Hour Two Hours Cont ro l (12) 4.76 + 0.09 (12) 4.69 + 0.10 Carbohydrate (12) 6.29 + 0.21 (12) 6.29 + 0.17 Tryptophan (12) 1 6 . 1 8 + 0 . 8 0 (11) 6 . 7 3 + 0 . 2 5 * Number of an imals/group t Mean tryptophan (ug/g + SEM) - 78 -APPENDIX TABLE VI BRAIN SEROTONIN FOLLOWING INGESTION OF A HIGH CARBOHYDRATE MEAL, OR IN-JECTION OF TRYPTOPHAN Treatment B r a i n S e ™ t o n i n ug/g Non-fasted basal (7)* .702 + . 030 + Fasted-basa l (8) .731 + .019 Treatment One Hour Two Hours Cont ro l (12) .632 + .011 .(12) .653 + .014 Carbohydrate (12) .635 + .011 (12) .688 + 0.15 Tryptophan (12) .849 + .023 (11) .786 + .030 * Number of an imals/group f Mean se ro ton i n (ug/g + SEM) - 79 -APPENDIX TABLE VII EFFECT OF CARBOHYDRATE INTAKE OR TRYPTOPHAN ADMINISTRATION ON PLASMA CORTICOSTERONE Treatment Plasma Co r t i co s t e rone b a s a i a S t e d ( 8 ) * 1 4 - 3 t  2 ' 5 9 + Fasted basal (8) 15.4 + 2.72 Treatment One Hour Two Hours Cont ro l (11) 1 4 . 6 0 + 1 . 8 6 (11) 1 3 . 8 7 + 2 . 2 4 Carbohydrate (12) 26.67 + 4.12 (12) 22.44 + 3.24 Tryptophan (12) 23.48 + 3.95 (12) 15.45 + 2.66 Number of animals/group + Mean c o r t i c o s t e r one (ug/100 ml + SEM) APPENDIX TABLE VII I EFFECT OF A TRYPOTOPHAN INJECTION OR INGESTION OF A CARBOHYDRATE OR CONTROL DIET ON LATENCY TO STEP-DOWN Treatment Latency to Step-Down One Hour 1 Latency to Step-Down Two Hours 1 2 Contro l Carbohydrate Tryptophan (12)" 11.88 + 5 . 21 + 11.08 + 5.40 (12) 17.96 + 6.20 8.67 + 4.75 (12) 1 5 . 4 2 + 5 . 6 0 9 . 9 2 + 6 . 0 0 (12) 15.08 + 5.71 1 0 . 1 7 + 4 . 9 5 (12) 2 0 . 0 5 + 5 . 7 1 1 1 . 2 5 + 5 . 9 5 ( 1 1 ) 1 6 . 0 0 + 5 . 6 7 1 0 . 0 0 + 5 . 0 6 number of. animals/group Mean seconds + SEM