Developmental changes in brain indoles, serum tryptophan and other serum neutral amino acids in the rat

Developmental Brain Research, 1 (1981) 551-564

551

Elsevier/North-HollandBiomedicalPress

DEVELOPMENTAL CHANGES IN BRAIN INDOLES, SERUM TRYPTOPHAN AND OTHER SERUM NEUTRAL AMINO ACIDS IN THE RAT

STEVEN H. ZEISEL, CHARLOTTE MAURON, CAROL J. WATKINS and RICHARD J. WURTMAN Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.)

(Accepted December31st, 1980) Key words: tryptophan -- serotonin-- 5-HIAA -- development-- amino acids

SUMMARY The rates at which brain neurons synthesize and release serotonin depend in part on brain tryptophan concentrations; these, in turn, vary directly with serum (or plasma) tryptophan, and inversely with the serum concentrations of other large neutral amino acids (LNAA). Concentrations of serum tryptophan, LNAA and brain indoles were examined in samples drawn at noontime from rats aged 0-59 days. Developmental changes in serum tryptophan largely paralleled those in the tryptophan/LNAA ratio, and brain tryptophan concentrations. Brain serotonin and 5hydroxyindole acetic acid (5-HIAA) levels also increased postnatally; the changes in 5HIAA tended to parallel those in brain tryptophan while those in serotonin did not.

INTRODUCTION The synthesis rates and levels of serotonin in brains of adult rats vary in parallel with brain tryptophan concentrations17-19, 45. Consumption of food generates daily rhythms in plasma and serum concentrations of tryptophan and other large neutral amino acids (LNAA)16,20,50; these, in turn, cause changes in brain tryptophan levels which can be predicted from the plasma tryptophan/LNAA ratio14,15,19. Dependence of brain tryptophan on plasma amino acid pattern is a property of the carrier mechanism which transports tryptophan and other LNAA across the blood-brain barrier: at normal plasma amino acid concentrations the carrier's kinetics are such that the amino acids are competitive for uptake sitesaS,at. Consumption of a carbohydrate meal, or administration of tryptophan, increases serum tryptophan concentrations relative to those of its competitors (chiefly tyrosine, phenylalanine, leucine, 0165-3806/81/0000-0000/$02.50© Elsevier/North-HollandBiomedicalPress

552 isoleucine and valine): hence, brain tryptophan concentrations rise 8,15,1s. In contrast, consumption of a high-protein meal or administration of other LNAA reduces serum tryptophan relative to its competitors, thus lowering brain tryptophan concentrations 14,15,19. The enzyme that limits the rate at which brain neurons synthesize serotonin, tryptophan hydroxylase (tryptophan 5-monooxygenase, EC. 1.12.16.4), has relatively low affinity for tryptophan (i.e. its K~n is on the order of tissue tryptophan concentrationsZ5). Hence, treatments that increase or decrease brain tryptophan levels cause parallel changes in the synthesis of serotonin 8,15,17-19 and ultimately, in its release as estimated by brain 5-hydroxyindole acetic acid (5-H1AA) concentrations15,39. Although there is evidence that, in neonatal rats, brain serotonin synthesis also depends on plasma amino acid content4, 24, few data are available on correlations between this indoleamine and the amino acids during development. Serotonincontaining cell bodies are detectable histochemically within the rat's raphe nuclei by the end of the second trimester of pregnancy za,34. At birth, whole-brain serotonin concentrations are 25-50 % of those measured in adult rats 2-4,24,29,34,46. During the perinatal period, the intraperitoneal injection of tryptophan does increase serum tryptophan, brain tryptophan, brain serotonin and 5-HIAA concentrations 4,24. This report examines some of the changes in tryptophan and serotonin that occur in developing rats. It shows that parallel changes occur in serum tryptophan, the serum tryptophan/LNAA ratio, brain tryptophan and brain 5-HIAA concentrations. Brain serotonin levels also increase, but not parallel to these other indices. Our experiments used rats of various ages that suckled or consumed rat chow, and that were killed at a single time of day; hence, they provide no information about the effects that experimental manipulations of tryptophan availability have on brain indoles, nor about the age at which brain indole levels of rats begin to exhibit characteristic variations which depend on diet composition. MATERIALS AND METHODS Sprague-Dawley rats (300-400 g; Charles River Breeding Laboratories, Wilmington, Mass.) were maintained in individual tubs. Dams were placed in the tubs 2 weeks before giving birth to the litters used in this study. The ambient temperature was 24 °C; light (Vita-Lite; 300 #W/sq.cm; Duro-Test, North Bergen, N.J.) was provided between 08.00 and 20.00 h; food (Charles River Rat, Mouse and Hamster Original Chow; 23% protein, 72% CHO, and 4.5% fat) and water were offered ad libitum (infants were offered chow beginning on day 7; they were not observed eating chow until after day 15). On the day of parturition the dams were observed, deliveries timed, and selected newborns decapitated at birth (day 1, 0 h) or 6 h later. All remaining newborns were randomly placed in 13 litters of 10 rats each. At midday of days 2, 3, 6, 8, 10, 13, 16, 20, 30, 41 and 59, groups of 13 rats were decapitated. Infants were randomly chosen from among all litters, and the number of litters was progressively decreased, maintaining litter sizes of 8-10 rats.

553 To determine whether fasting affected brain serotonin synthesis in 3-day-old rats, an experiment was performed on two groups of 10 animals each, selected as described above. One group (fed) remained continuously with a lactating dam, while the other group (fasted) was placed with a nonlactating female (which had delivered a litter 2 months previously) for 7 h. Both groups were weighed at the beginning and end of this period; all fed rats gained weight while all fasted rats barely maintained or even lost weight. At the end of the 7 h period, the stomach contents of the neonates were examined; stomachs of all fed animals contained milk, while those of the fasted rats had little or no milk. At the time of decapitation, blood and brains were collected. Blood was placed on ice, allowed to clot, and then centrifuged at 16,000 × g for 10 min. Serums were aspirated and stored at --80 °C until assayed. Brains were immediately frozen on dry-ice, and stored at --80 °C until assayed. Neutral amino acids were measured in aliquots of serum (deproteinized with 5sulfosalicylic acid) using a Beckman amino acid analyzer 119C (Beckman Instruments, Palo Alto, Calif)20. Serum tryptophan concentrations were measured fluorimetrically after condensation of the tryptophan with formaldehyde12, 30. The tryptophan in brain homogenates was extracted and then measured using the same fluorimetric method12, 30. Tissues were homogenized in 3-6 ml of 0.1 M trichloroacetic acid (TCA) using a polytron homogenizer (Kinematica GMBH, Luzern, Switzerland), and 0.14).4 #l aliquots of whole homogenates were diluted to 4 ml using 7.5 ~ TCA. Samples were centrifuged for 10 min at 22,000 × g, and the supernatant fluids assayed as described above. Brain serotonin and 5-HIAA were isolated using ion exchange chromatography21; serotonin was measured using high pressure liquid chromatography (HPLC)21; 5-HIAA was measured using a fluorimetric method1,1°. Weighed brains (0.2-1.8 g) were disrupted with a sonicator in an ice bath for 1 min (Heat Systems/Ultrasonics W225R Cell Disruptor, Plainview, N.Y.) in 1-3 ml of 0.05 N HC1 containing 50 #g/ml of ascorbic acid and 50 ng/ml of epinine (deoxyepinephrine; Sigma Chemicals, St. Louis, Mo.). Epinine was used as an internal standard because it is eluted from the column near, but distinct from, the catecholamines and serotonin. Recoveries of serotonin and 5-HIAA were also determined in brain tissues to which 100 ng of serotonin and 5-HIAA had been added. Aliquots of the sonicates, after addition of perchloric acid, were mixed and centrifuged at 16,000 x g for 10 min. The supernatant fluids were adjusted to pH 4.0 with potassium hydroxide and centrifuged at 1000 × g for 10 min. The supernatant fluids were then applied to a 4.5 cm × 6 mm Amberlite column (CG-50, 200-400 mesh; Mallinkrodt, Paris, Ky.) which had previously been washed with 1 M sodium acetate (pH 6) and water. The sample effluent and a 1 ml water wash were collected for analysis of 5-HIAA. The column was then washed with 2 ml of 1.2 N HC1 containing 250 ng of ascorbic acid, eluting monoamines including serotonin. These monoamine fractions were stored at --20 °C until assayed. The 5-HIAA effluents were adjusted to pH 1.5-2.0 by addition of 2 M HC1, and

554 applied to 3 cm × 6 mm Sephadex columns (GI0; Bio-Rad, Richmond, Calif.) which had previously been washed with 0.1 M HC1 containing 0.1 o/ascorbic acid, and then /O with water. The sample effluents and 2 ml washes (0.1 M HCI) were discarded; 5H I A A was collected in 2.5 ml of 0.02 M ammonium hydroxide. Aliquots of I ml were assayed for 5-HIAA after condensation with ortho-pthaldialdehyde (OPT) in the presence of cysteine and 10 M HCI 1,1°. Internal standards (5-HIAA) were recovered with efficiencies of 39 and 41% from two samples of brain, and sample values were corrected to reflect this. Serotonin was measured using H P L C zl. We used a Bioanalytical Systems LC-54 apparatus (Bioanalytical Systems, West Lafayette, Ind.), equipped with a waxy carbon working electrode ( + 0 . 8 V), and a/zBondapak C18-column (Waters Associates, Milford, Mass.). Our buffer was composed of 11% methanol in 0.07 M sodium phosphate (pH 5.0) containing 0.1 mM sodium E D T A and 0.25 mM sodium octyl sulfate. Injections (50-100/A) were made with electrode sensitivity of 2-10 nA/V, and a flow rate of 2 ml/min (elution of serotonin took approximately 18 min; Fig. 1). Serotonin recoveries from two samples of brain were 74 and 81%, and sample values were corrected to reflect this.

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Fig. 1. HPLC separation of serotonin. Extracts of brain, prepared as described in Materials and Methods section, were subjected to HPLC on a/~Bondpak CI 8 column, using a buffer composed of 11% methanol in 0.07 M sodium phosphate (pH 5) containing 0.1 mM EDTA and 0.25 mM sodium octyl sulfate; the flow rate was 2 ml/min. Serotonin was detected at approximately 18 min with a waxy carbon working electrode (+ 0.8 V, 5 nA/V electrode sensitivity). A, norepinephrine; B, dopamine; C, epinine; D, serotonin.

555

Data were analyzed statistically using one-way analysis of variance and Scheffe's test a. Comparison of data from fed versus fasted 3-day-old rats was made using the ttest< RESULTS During development, almost parallel changes occurred in serum and brain tryptophan concentrations, and serum tryptophan/LNAA ratios. Brain serotonin concentrations increased between birth and 59 days of age; however, these changes were not parallel to those in tryptophan. 5-HIAA levels, an indirect measure of neuronal serotonin release ag, did tend to parallel the variations in brain tryptophan concentration (Figs. 2 and 3).

Serum tryptophan Serum total (free plus albumin-bound) tryptophan concentration (Fig. 2) was higher at birth (34.7 #g/ml :k 1.5 S.E.M.) than at 6 h of age (22.5 #g/ml i 2.0 S.E.M.; P < 0.01). It declined still further by 3 days of age (9.2 #g/ml ± 0.75 S.E.M.; P < 0.001 different from birth or 6 h), after which it increased, reaching maximal concentrations at 13 days of age (34.2 #g/ml ± 1.0 S.E.M.; P < 0.001 different from day 3). By day 16, serum tryptophan declined to adult levels (23.5 #g/ml q- 1.0 S.E.M.; P < 0.001 different from day 13). Tryptophan/LNAA ratio The tryptophan/LNAA ratio (calculated by dividing serum tryptophan concentration by the sum of the concentrations of serum tyrosine, phenylalanine, valine, leucine and isoleucine; Fig. 2) was higher at 6 h of age (0.15 ± 0.02 S.E.M.) than it was at its nadir between 3 and 6 days of age (0.05 ~ 0.01 S.E.M.; P < 0.01 different from 6 h). The ratio increased after 6 days, to reach a maximal value at 20 days of age (0.20 40.01 S.E.M.; P < 0.001 different from day 3); thereafter, it fell, but not significantly. The serum concentrations of valine, isoleucine and leucine declined between birth and 6 h, but by 3 days had risen to values similar to those observed in adults. Serum phenylalanine concentrations declined between birth and 6 h, remaining at adult-like values after this age. Tyrosine concentrations declined for several days after birth, and then increased, returning to neonatal levels by day 8-13. Brain tryptophan The brain tryptophan concentration (Fig. 2) was highest at 6 h after birth (12.9 /~g/g wet weight 4- 2.1 S.E.M.), after which it declined, reaching a nadir at 3 days (2.7 /zg/g ± 0.3 S.E.M.; P < 0.001 lower than 6 h). Between 3 and 13 days of age there was a small (though non-significant) increase in brain tryptophan concentration (rising to 4.9 #g/g zk 0.4 S.E.M.), followed by a small decrease by 59 days (3.1 #g/g -q- 0.4 S.E.M.). Brain tryptophan levels (concentration x brain weight) also were higher at birth than at 3 days of age, and subsequently rose to adult values (Fig. 3).

556

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Fig. 2. Developmental changes in serum tryptophan, tryptophan/LNAA ratio, brain tryptophan, serotonin and 5-HIAA concentrations. Rat pups were randomly selected from 13 litters, and groups of 13 rats were decapitated on day of birth (day 1 ; 0 and 6 h postpartum) and at noontime on days 2, 3, 6, 8, 10, 13, 16, 20, 30, 41 and 59. Data are expressed as means ~ S.E.M. [ T r y p t . ] / [ T + P + V + I + L ] , serum tryptophan/LNAA ratio; brain [5-HT], brain serotonin concentration; brain [5-HIAA], brain 5-hydroxyindole acetic acid concentration.

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Fig. 3. Developmental changes in brain tryptophan, serotonin and 5-HIAA levels (concentration × brain weight). Rat pups were selected and sacrificed as described in Fig. 2. Levels were calculated per

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Brain serotonin

Brain serotonin concentrations (Fig. 2) were lowest at birth (0.09 Fg/g -t- 0.01 S.E.M.) when they were only 25 ~ of the values measured at 59 days (0.36/~g/g ~ 0.04 S.E.M.; P < 0.001 different from birth). Brain serotonin levels (concentration x brain weight) were 38 times greater in adults than in newborns (Fig. 3). Brain serotonin levels did not decline after birth: during the period in which brain tryptophan concentration was lowest (3-13 days) brain serotonin concentrations remained constant (0.15 /~g/g). After this age, they rose to attain adult values. Brain 5-HIAA

Brain 5-HIAA concentrations (Fig. 2) were low at birth (0.28 /~g/g q- 0.04 S.E.M.), increased rapidly by 6 h of age (0.6/~g/g zk 0.07 S.E.M.; P < 0.05 different from birth) and then declined to a nadir at 3 days (0.32 Fg/g q- 0.04 S.E.M.; P < 0.05 different from 6 h). Thereafter, they increased, peaking at 13 days (0.56 Fg/g -4- 0.03 S.E.M.) and then declined slowly to adult (59 day) values (0.33 #g/g q- 0.02 S.E.M.). Brain 5-HIAA levels (concentration × weight) rose between birth and 6 h, then decreased slightly until 3 days of age, after which they rose rapidly, reaching adult values by 20 days of age (Fig. 3).

558 TABLE 1 Effect of Jasting on serum tryptophan, serum tJTptophan/neutral amino acids ratio, brain tryptophan, brain 5-HIAA and brain seroconin concentrations

Rat pups (3-day-old) were selected at random from 13 litters. Ten pups were left with their lactating dam (fed) and 10 were placed with a nonlactating female for 7 h. At the end of this period, rats were decapitated and brains and serums collected. Data are expressed as mean ± S.E.M. Fed

Serum [tryptophan] (/zg/ml) Serdm [tryptophan]/[LNAA] Brain [tryptophan] (pg/g wet weight) Brain [serotonin] (/zg/gwet weight) Brain [5-HIAA] (/~g/gwet weight) Brain weight (g wet weight)

9.32 ± 0.05 ± 2.70 ± 0.14 ~ 0.32 ± 0.275 ±

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0.72 0.01 0.30 0.02 0.04 0.02

17.31 ± 1.27"* 0.08 i 0.02 5.50 i 0.40** 0.13 ± 0.01 0.40 :~ 0.05* 0.289 i 0.004

* P < 0.05 by t-test ** P < 0.001 by t-test Effect o f fasting

Serum tryptophan concentrations in 3-day-old rats that had been fasted for 7 h were higher (17.50 #g/ml ± 1.25 S.E.M.) than in rats allowed continuous access to a lactating dam (fed group; 9.20 #g/ml ~ 0.75 S.E.M.; P < 0.001). The serum tryptophan/LNAA ratio also tended to be higher in fasted (0.08 i 0.01 S.E.M.) than in fed animals (0.05 ± 0.01 S.E.M.), as were brain tryptophan concentrations (5.50 pg/g ± 0.40 S.E.M. vs 2.70 #g/g ~ 0.30 S.E.M.; P < 0.001). Brain serotonin concentrations were unchanged by fasting (0.13 #g/g :~ 0.01 S.E.M. in fasted rats vs 0.14/zg/g ~ 0.02 S.E.M. in fed rats), but brain 5-HIAA concentrations, like those of brain tryptophan, were elevated by fasting (0.46/~g/g :~ 0.05 S.E.M. fasted vs 0.32 #g/g :~ 0.04 fed; P < 0.05). Brain weight was not significantly altered by a 7 h fast (Table I). DISCUSSION These data show that serum tryptophan concentrations and serum tryptophan/ L N A A ratios exhibit characteristic changes during development in the rat, which tend to parallel the concurrent changes in brain tryptophan. Brain serotonin concentrations are lowest at birth and increase 4-fold as the rat matures; they correlate poorly with changes in brain tryptophan concentration. In contrast, brain 5-HIAA concentrations do correlate with serum tryptophan/LNAA ratios and with brain tryptophan concentrations. In our study of the metabolic responses of 3-day-old rats to variations in nutrient intake, we found that fasting increases serum tryptophan and brain 5-HIAA concentrations over those observed in continuously feeding animals. Our experiments were designed to provide descriptive information about amino acid and indole levels in tissues of the developing rat. They do not, in general, provide information about the mechanisms by which consumption of particular foods influences brain indole synthesis. We did not study the response of rats at each age to different foods, nor responses to injections of pure amino acids. Future studies will address these questions.

559

Serum tryptophan Serum total tryptophan concentration was high at birth. Thereafter, when sampled at midday, it dropped to its lowest value at day 3, peaked at day 13, and then declined slightly, remaining at a relatively constant level between days 16 and 59 (Fig. 2). Our values for serum tryptophan were higher than previously described4, 47, but exhibited a similar age-dependence. Various hypotheses might explain the variations observed in serum tryptophan concentrations during development: the postnatal fall in serum tryptophan could reflect the cessation of its transplacental transport 24, its accelerated metabolism or utilization (for rapid growth and protein synthesis), or its relatively low dietary intake. As the rat matures, tryptophan's metabolism or its utilization might decrease (i.e. as growth rate diminishes), or the dietary intake of tryptophan might increase, resulting in the higher serum tryptophan concentrations observed in adults. The eating habits of the rat influence plasma amino acid patterns 16 and also alter the activities of hepatic enzymes33,40,49 that metabolize tryptophan and other amino acids. Such diet-induced rhythms appear within 48 h of birth 3a, but are opposite in phase to those observed in the adult rat: pups tend to eat during the day, while adults (including mothers) search for and eat food at night. The reversal from the neonatal to the adult cycle occurs around days 21-23 of life 33. We sacrificed all animals (except newborns) at midday; therefore, the above developmental changes in eating pattern might have contributed to the observed variations in amino acid concentrations. Noontime serum tryptophan concentrations (Fig. 2) may have dropped at days 16-20 because rats had begun to eat at night. Serum tryptophan exists in two states, free or bound to albumin 86. In the neonatal rat most serum tryptophan is free 4, probably because albumin concentrations are low and concentrations of free fatty acids (which compete with tryptophan for albumin binding sites9) are high 44. As the rat matures, progressively more tryptophan tends to be bound to albumin 4. Some investigators have postulated that only the free (dialyzable) tryptophan is transported into braing,la; however, in adult rats, brain tryptophan levels are more accurately predicted by the serum tryptophan/LNAA ratio than by serum free tryptophan concentration15,19, 81. In adult rats, the fraction of tryptophan that is free in vitro greatly underestimates the fraction of tryptophan capable of entering the brain in vivo; this is because the blood-brain barrier amino acid transport mechanism is capable of stripping off tryptophan from albumin a6. In vivo, 49-85 ~ of the total tryptophan in adult rat serum is free and available for transport into the brain (depending on albumin and fatty acid concentrations36). We did not distinguish between dialyzable and albumin-bound tryptophan in our developing animals. Tryptophan/LNAA ratio The serum tryptophan/LNAA ratio was lower at birth than at 6 h; thereafter, noontime ratios dropped to lowest values at 3 and 6 days of age, then rose to adult levels by day 16. At birth, the serum concentrations of the other LNAA (phenylalanine, tyrosine, leucine, isoleucine and valine) were relatively high when compared with those in adults. This is in agreement with the findings of Lajtha and Toth 2s.

560

Brain tryptophan Brain tryptophan concentrations were also lower at birth than at 6 h of age (their maximal value). Thereafter, noontime brain tryptophan concentrations declined, reaching a nadir between 3 and 6 days and rising slightly thereafter to adult values. These observations demonstrate a pattern similar to, but more exaggerated than those previously described 4,47. During the first week of life, the pattern of developmental changes in brain tryptophan were similar to that for the tryptophan/LNAA ratios. In adult rats, a constant relationship exists between brain tryptophan concentration and the serum tryptophan/LNAA ratio14,15,1L When animals consume particular meals that cause the ratio to rise or fall, they cause parallel changes in brain tryptophan. Our present studies do not allow us to determine the age at which this relationship develops, as we did not attempt to manipulate serum tryptophan/LNAA ratio by dietary or other means to see if brain tryptophan changed. At the one age at which we fasted animals (3 days), we did note that an increase in the serum tryptophan/LNAA ratio was associated with an increase in brain tryptophan concentration. We were not able to make inter-age predictions about brain tryptophan for any given serum tryptophan/LNAA ratio, as the relationship changed during development (Fig. 4). We observed higher brain tryptophan concentrations in newborns than in adults, despite lower serum tryptophan/LNAA ratios in the neonates. This difference could be caused by changes in the uptake of tryptophan into the brain, in its metabolism within the brain, or its excretion from the brain. Blood-brain barrier transport of tryptophan could be less sensitive postnatally to competition from the LNAA, resulting in more tryptophan entry than expected. In newborn rabbits (less than 24-h-old), the blood-brain barrier carrier for tryptophan exhibits a lower affinity i5-+ 6 hr

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561 (3-fold higher Kin) and higher capacity than in the adult (W.M. Pardridge, personal communication). These changes might make the carrier less susceptible to competition. Alterations in brain tryptophan utilization and metabolism occur during growth and cell division, as tryptophan is incorporated into proteins and proteolipids 5. Changes in the water content of brain (from approximately 85 % at birth, 88 % at 10 days, 80 % after 30 days22) could also artifactually alter brain concentrations. (All calculations were made using wet weights.) Brain serotonin Brain serotonin concentrations increased after birth, as previously reported26, 46. The low concentration of serotonin observed in neonatal brain (25 % of adult values) is consistent with the small number of functional serotonergic neurons probably present at birthZr, 46. The rate-limiting step in serotonin biosynthesis, the hydroxylation of tryptophan, is catalyzed by an enzyme (tryptophan hydroxylase) which has much less activity per g brain weight in neonates than in adults 4t. Some investigators have concluded that this enzyme is saturated with tryptophan in the neonatal brain; however, others have drawn opposite conclusions and have suggested that manipulations which increased neonatal brain tryptophan also accelerated brain serotonin synthesise4. Perhaps it is more useful to consider serotonin content per neuron rather than per whole brain, as the former measurement is not influenced by developmental changes in neuron numbers and might be more likely to reflect changes in serotonin biosynthesis caused by alterations in tryptophan availability. Brain 5-HIAA Brain 5-HIAA concentrations were lowest at birth, rose to maximal values at 6 h, declined by 50 % at 3 days, and then rose until 20 days, after which they slowly fell until they reached concentrations similar to those first observed at 3 days. A previous study in which rats were sampled at 1, 2, 4, 6, 11 and 22 days, reported that brain 5HIAA concentrations were constant between birth and 5 days (0.35/~g/g) and then increased (to 0.45/~g/g47). Tissari46, who sampled rats only on days --5, 1, 8 and 22, noted that brain stem and hemispheric 5-HIAA concentration seemed to increase linearly with age. Our results are of a similar order of magnitude (0.30-0.60/~g/g); probably we were able to observe marked changes in brain 5-HIAA concentrations because we chose small enough time intervals between sampling. We also used a different, more sensitive technique for the measurement of 5-HIAA than did previous authors. The concentration of 5-HIAA in brain depends on the rate of its formation from released serotoninz9 and on the rate of its elimination from brain 46. The efflux of 5-HIAA from brain, mediated by a carrier in the blood-brain barrier, appears to be slower in the newborn than in the adulta, 46. This may contribute to the relatively high 5-HIAA values we observed in the 6-h-old rat. Our observations on 5-HIAA indicate that serotonin release may be lower at 3 days than it is at 6-10 days of age. A major transition in suckling behavior occurs around 10-15 days of age, after which pups no longer rapidly attach to their mother

562 and suckle 4s. Administration of serotonergic agonists inhibits suckling in rats, while serotonergic antagonists reinstate suckling 4s. Perhaps serotonin neurons tend to inhibit suckling and have their output modulated by the availability of tryptophan. Effect o f fasting We noted that fasted animals had higher serum tryptophan, brain tryptophan and 5 - H I A A concentrations than did fed animals. Weaning might result in periods o f fasting for rat pups, with consequent alterations in blood tryptophan and brain serotonin release. Other investigators have noted increased brain serotonin and 5H I A A in fasted adult rats11, 27, though Perez-Cruet et al. 37 did not observe such changes. Malnourished rat pups have been noted to have particularly high brain trypt o p h a n concentrations 4. Periods of fasting, or diets which are not balanced in amino acid content, disaggregate hepatic polyribosomes zs,a2, decrease muscle protein synthesis, and accelerate the catabolism of protein in skeletal muscles of rats 3z,42,43. These metabolic changes cause increases in the half-lives of serum amino acids, probably including tryptophan3L ACKNOWLEDGEMENTS We thank Drs. Candace Gibson and Bruce Glaeser for advice and assistance in assay techniques. These studies were supported in part by grants from the National Institutes of Health (AM-14228) and the National Aeronautics and Space Administration ( N G R 22-009-627). Dr. Zeisel is a John A. and George L. H a r t f o r d Fellow of the John A. H a r t f o r d Foundation.

REFERENCES 1

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Atack, C. and Lindquist, M., Conjoint native and orthopthaldialdehyde-condensate assays for the fluorometric determination of 5-hydroxyindoles in brain, Naunyn-Schmiedeberg's Arch. Pharmak. 279 (1973) 257-284. Baker, P. C. and Quay, W. B., Serotonin metabolism in early embryogenesis, and the development of brain retinal tissues: a review, Brain Res., 12 (1969) 273-295. Bennett, D. S. and G iarman, N. J., Schedule of appearance of serotonin and associated enzymes in developing rat brain, J. Neurochem., 12 (1965) 273-295. Bourgoin, S., Faivre-Bauman, A., Benda, P., Glowinski, J. and Hamon, M., Plasma tryptophan and serotonin metabolism in the CNS of the newborn rat, J. Neurochem., 23 (1974) 319-327. Bruning, J. and Kintz, B., The t-test for a difference between two independent means. In J. Bruning and B. Kintz (Eds.), Computational Handbook of Statistics, Scott, Foresman, Glenview, Ill., 1977, pp. 10-13. Bruning, J. and Kintz, B., Scheffe's test. In J. Bruning and B. Kintz (Eds.), Computational Handbook of Statistics, Scott, Foresman, Glenview, Ill., 1977, pp. 125-128. Clouet, D. H. and Gaitonde, M. K., The changes with age in the protein composition of the rat brain, J. Neurochem., 1 (1955) 125-133. Colmenares, J. L., Wurtman, R. J. and Fernstrom, J. D., Effect of ingesting a carbohydrate-fat meal on the levels and synthesis of 5-hydroxyindoles in various regions of the rat central nervous system, J. Neurochem., 25 (1975) 825-829. Curzon, G., Friedel, J. and Knott, P. J., The effect of fatty acids on the binding of tryptophan to plasma protein, Nature (Loud.), 242 (1973) 198-200.

563 10 Curzon, G. and Green, A. R., Rapid method for the determination of serotonin and 5-HIAA in small regions of rat brain, Brit. J. Pharmacol., 39 (1970) 653-655. 11 Curzon, G., Joseph, M. H. and Knott, P. J., Effects of immobilization and food deprivation on rat brain tryptophan metabolism, J. Neurochem., 19 (1972) 1967-1974. 12 Denkla, W. D. and Dewey, H. K., The determination of tryptophan in plasma, liver and urine, J. Lab. clin. Med., 69 (t967) 160-169. 13 Fernando, J. C. R., Knott, P. J. and Curzon, G., The relevance of both plasma free tryptophan and insulin to rat brain tryptophan concentration, J. Neurochem., 27 (1976) 343-345. 14 Fernstrorn, J. D. and Failer, D. V., Neutral amino acids in the brain: changes in response to food ingestion, J. Neurochem., 30 (1978) 1531-1538. 15 Fernstrom, J. D., Failer, D. V. and Shabshelowitz, H., Acute reduction of brain serotonin and 5-HIAA following food consumption: correlation with the ratio of serum tryptophan to the sum of competing amino acids, J. Neural Transm., 36 (1975) 113-121. 16 Fernstrorn, J. D., Larin, F. and Wurtman, R. J., Daily variations in the concentrations of individual amino acids in rat plasma, Life Sci., 10 (1971) 813-819. 17 Fernstrom, J. D. and Wurtman, R. J., Brain serotonin content: physiologic dependence on plasma tryptophan levels, Science, 173 (1971) 149-152. 18 Fernstrorn, J. D. and Wurtman, R. J., Brain serotonin content: increase following ingestion of a carbohydrate diet Science, 174 (1971) 1023-1025. 19 Fernstrom, J. D. and Wurtman, R. J., Brain serotonin content : physiological regulation by plasma neutral amino acids, Science, 178 (1972) 414-416. 20 Fernstrom, J. D., Wurtman, R. J., Hammarstrom-Wiklund, B., Rand, W. M., Munro, H. N. and Davidson, C. S., Diurnal variations in plasma neutral amino acid concentrations in patients with cirrhosis: effects of dietary protein, Amer. J. clin. Nutr., 32 (1979) 1923-1933. 21 Gibson, C. J., Deikel, S. M., Young, S. N. and Binik, Y. M., Behavioural and biochemical effects of tryptophan, tyrosine and phenylalanine in mice, Psychopharmacology, submitted for publication. 22 Himwich, W., Introduction. In W. Himwich (Ed.), Biochemistry of the Developing Brain, Dekker, New York, 1973, p. 7. 23 Honova, E., Miller, S. A., Ehrenkranz, R. A. and Woo, A., Tyrosine transaminase: development of daily rhythm in liver of neonatal rat, Science, 162 (1968) 999-1001. 24 Howd, R. A., Nelson, M. F. and Lytle, L. D., L-tryptophan and rat fetal brain serotonin, Life Sci., 17 (1975) 803-812. 25 Jequier, E., Robinson, D. S., Lovenberg, W. and Sjoerdsma, A., Further studies on tryptophan hydroxylase in rat brainstem and beef pineal, Biochem. PharmacoL, 18 (1969) 1071-1081. 26 Kato, R., Serotonin content of rat brain in relation to sex and age, J. Neurochem., 5 (1960) 202. 27 Knott, P. J., Joseph, M. H. and Curzon, G., Effects of food deprivation and immobilization on tryptophan and other amino acids in rat brain, J. Neurochem., 20 (1973) 249-251. 28 Lajtha, A. and Toth, J., Perinatal changes in free amino acid pool of the brain in mice, Brain Res., 55 (1973) 238-241. 29 Lauder, J. M. and Bloom, F. E., Ontogeny of mgnoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. I. Cell differentiation, J. comp. NeuroL, 155 (1974) 469-481. 30 Lehmann, J., Light-A source of error in the fluorometric determination of tryptophan, J. Lab. clin. Invest., 28 (1971) 49-55. 31 Madras, B. K., Cohen, E. L., Messing, R., Munro, H. N. and Wurtman, R. J., Relevance of serum free tryptophan to tissue tryptophan concentrations, Metabolism, 23 (1974) 1107-1116. 32 McFarlane, A. S., Plasma protein metabolism in dietary deprivation. In H. N. Munro and J. B. Allison (Eds.), Mammalian Protein Metabolism, 1Iol. 1, Academic Press, New York, 1964, pp. 326-327. 33 Miller, S. A., Protein metabolism during growth and development. In H. N. Munro (Ed.), Mammalian Protein Metabolism, Iiol. 3, Academic Press, New York, 1969, pp. 183-236. 34 Olson, L. and Sieger, A., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histo-chemical observations, Z. Anat. EntwickL Gesch., 137 (1972) 301-316. 35 Pardridge, W. M., Regulation of amino acid availability to brain. In R. J. Wurtman and J. J. Wurtman (Eds.), Nutrition and the Brain, Vol. 1, Raven Press, New York, 1977, pp. 141-204. 36 Pardridge, W. M., Tryptophan transport through the blood-brain barrier: in vivo measurement of free and albumin bound amino acids, Life Sci., 25 (1979) 1519-1528. 37 Perez-Cruet, J., Tagliamonte, A., Tagliamonte, P. and Gessa, G. L., Changes in brain serotonin metabolism associated with fasting and satiation in rats, Life Sci., 11 (1972) 31-39.

564 38 Proncznk, A. W., Baliga, B. S., Triant, J. W. and Munro, H. N., Comparison of the effect of amino acid supply on hepatic polyribosome profiles in vivo and in vitro, Biochim. hiophys. Acta (Amst.) 157 (1968) 204-206. 39 Reinhard, J. F. and Wurtman, R. J., Relation between brain 5-HIAA levels and the release of serotonin into brain synapses, Life Sci., 21 (1977) 1741-1746. 40 Ross, D. G., Fernstrom, J. D. and Wurtman, R. J., The role of dietary protein in generating daily rhythms in rat liver tryptophan pyrrolase and tyrosine transaminase, Metabolism, 22 (1973) 1175-1184. 41 Schmidt, M. J. and Sanders-Buch, E., Tryptophan hydroxylase activity in the developing rat brain, J. Neurochem., 18 (1971) 2549-2551. 42 Sidransky, H., Sarma, D. S. R., Bongiorno, M. and Verney, E., Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice, J. biol. Chem., 243 (1968) 1123-1132. 43 Sidransky, H. and Verney, E., Decreased protein synthesis in the skeletal muscle of rats force-fed a threonine devoid diet, Biochim. biophys. Acta (Amst.), 138 (1967) 426-429. 44 Snell, K. and Walker, D. G., Glucose metabolism in the newborn rat. Temporal studies in vivo, Biochem. J., 132 (1973) 739-752. 45 Tagliamonte, A., Tagliamonte, P., Perez-Cruet, J., Stern, S. and Gessa, G. L., Effect of psychotropic drugs on tryptophan concentration in the rat brain, J. Pharmacol. exp. Ther., 177 (1971) 475-480. 46 Tissari, A. H., Serotoninergic mechanisms in ontogenesis. In L. Boreus (Ed.), FetalPharmacology, Raven Press, New York, 1973, pp. 237-253. 47 Tyce, G. M., Flock, E. V. and Owen, C. A. Jr., Tryptophan metabolism in the brain of the developing rat. In W. Himwich (Ed.), The Developing Brain, Elsevier, Amsterdam, 1964, pp. 198-203. 48 Williams, C. L., Rosenblatt, J. S. and Hall, W. G., Inhibition of suckling in weaning age rats: a possible serotonergic mechanism, J. comp. physioL Psychol., 93 (1979) 414-429. 49 Wurtman, R. J., Daily rhythms in tyrosine transaminase and other hepatic enzymes that metabolize amino acids: mechanisms and possible consequences, Life Sci., 15 (1974) 827-847. 50 Wurtman, R. J., Rose, C. M., Chou, C. and Larin, F. F., Daily rhythms in the concentration of various amino acids in human plasma, New Eng. J. Med., 279 (1968) 171-175.