Age-related changes in circadian rhythm of serotonin synthesis in ring

doves: Effects of increased tryptophan ingestion

Celia Garau, Sara Aparicio, Ruben V. Rial, Marıa C. Nicolau, Susana Esteban *

Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Ciences de la Salut, Universitat de les Illes Balears, E-07122 Palma de Mallorca, Spain

Received 26 July 2005; received in revised form 15 September 2005; accepted 27 September 2005

Available online 3 November 2005

Abstract

Alterations in the function of the hypothalamic suprachiasmatic nucleus (SCN) with age have been reported. As serotonin is an important

regulator of the circadian clock located in SCN, this work studied the changes produced in the synthesis of serotonin with age using the

accumulation of 5-HTP after decarboxylase inhibition as a measure of serotonin synthesis in the brain in vivo, in young and old ring doves at the

onset of lights-on and lights-off. A diurnal cycle in tryptophan hydroxylation was observed in young animals, with an increased daylight synthesis

and metabolism of 5-HT in hippocampus, neostriatum and hypothalamus. A single dose of melatonin (1 mg/kg, i.p., 1 h) at lighttime produced an

inhibitory effect on the synthesis of 5-HT. In contrast, differences in 5-HT synthesis and metabolism between day and night dissappeared in old

animals indicating an absence of a circadian rhythm in 5-HT synthesis and metabolism. The administration of L-tryptophan (240 mg/kg, i.p.)

strongly increased the 5-HT synthesis in young animals only during lights-off time while it increased in old ones irrespective of the administration

time. These results suggest that the supplemental administration of tryptophan might aid to improve the descent in 5-HT that normally occurs, as

animals get old.

q 2005 Elsevier Inc. All rights reserved.

Keywords: Serotonin synthesis; Aging; Tryptophan; Avian rhythms; Melatonin, Ring doves

1. Introduction

Daily rhythms in vertebrate physiology are generated and

maintained by the biological clock located in the hypothalamic

suprachiasmatic nucleus (SCN) (Moore and Eichler, 1972;

Stephan and Zucker, 1972). Serotonin (5-HT) is an important

regulator of the circadian clock located in the SCN. This clock

is synchronized by photic and non-photic signals. Light, the

principal synchronizer, is received by the retinal ganglion cells

and transmitted directly to the SCN through the retinohy-

pothalamic tract (Moore and Eichler, 1972), and indirectly

through the geniculohypothalamic tract (Card and Moore,

1989). Non-photic signals arrive to the SCN by a direct

serotonergic pathway from mesencephalic raphe nuclei mainly

originated from the median raphe nucleus (MRN) (Azmitia and

0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2005.09.010

* Corresponding author. Address: Valdes, Laboratori de Fisiologia,

Departament de Biologia Fonamental i Ciences de la Salut, Universitat de

les Illes Balears, Ctra. Valldemossa Km 7,5 E-07122 Palma de Mallorca, Spain.

Tel.: C34 971 173145; fax: C34 971 173184.

E-mail address: susana.esteban@uib.es (S. Esteban).

Segal, 1978; Hay-Schmidt et al., 2003) and secondly from the

dorsal raphe nucleus (DRN) (Kawano et al., 1996), also they

arrive indirectly by serotonergic projections from the dorsal

raphe nucleus to the thalamic intergeniculate leaflets which

project to the SCN (Azmitia and Segal, 1978).

Many aspects of circadian function change with age,

including changes in phase relationship of rhythms to the

environmental time signals, reduced sensitivity of the circadian

pacemaker to time cues, decreased amplitude of the circadian

rhythms and increased sleep fragmentation (Czeisler et al.,

1992; Myers and Badia, 1995; Penev et al., 1995; Scarbrough

et al., 1997; Turek et al., 1995; Van Reeth et al., 1992; Zee

et al., 1992). The neural mechanisms causing the age-related

changes in circadian timing system are far from being

elucidated. Some alterations in the function of the SCN have

been identified (Krajnak et al., 1998; Moore and Eichler, 1972).

An interesting candidate to explain some of the age-related

changes in the SCN is 5-HT. It is known that 5-HT takes part in

the regulation of some parameters of circadian timing system

which change with age as in the circadian rhythm of wheel

running or in the modulation of phase shifts (Cutrera et al.,

1994; Penev et al., 1995, 1997). In addition, age-related

changes in 5-HT receptors have been observed in the DRN

Experimental Gerontology 41 (2006) 40–48

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C. Garau et al. / Experimental Gerontology 41 (2006) 40–48 41

(Duncan et al., 1999) and the SCN (Duncan et al., 2000).

Numerous relationships have been demonstrated between the

5-HT system and the SCN. For instance, electrical stimulation

of the DRN and MRN in hamsters evoked 5-HT release in the

SCN, an effect which was blocked by systemic injection of 5-

HT antagonists (Glass et al., 2003). The release of 5-HT in the

SCN was rhythmic and correlated with circadian variations in

the levels of the limiting enzyme in 5-HT synthesis, tryptophan

hydroxylase (Barassin et al., 2002). However, in vivo changes

in the circadian 5-HT synthesis during aging have not been

well studied in spite of plenty of evidence that 5-HT acts as an

inhibitory transmitter that modulates the responses of the

circadian clock to light.

The aim of this work is to study the circadian pattern of 5-HT

synthesis measuring the activity of tryptophan hydroxylase, the

most commonly assay to monitor 5-HT synthesis in vivo

(Carlsson and Lindqvist, 1973). The study has been performed in

three different brain areas of ring doves (Streptopelia risoria):

hypothalamus which contains the homologue of the mammalian

SCN, hippocampus and striatum, two brain regions which

receive a high amount of 5-HT innervations. The effects of the

administration of L-tryptophan, the amino acid precursor of

5-HT synthesis, in young and old ring doves have been studied

too, to analyze possible improvement of the age related changes

that could occur in 5-HT synthesis and metabolism. The ring

dove was chosen as experimental animal because their circadian

characteristics, diurnal and monocyclic, are similar to those of

human beings and also because of their rather well known age

related changes in the melatonin secretion rhythm (Terron et al.,

2002).

2. Material and methods

2.1. Animals

Young (y6 months, 130 g, nZ20) and old (O8 years,

170 g, nZ22) Streptopelia risoria ring doves, have been used.

The animals were individually housed under controlled

environmental conditions (22 8C; 70% humidity), kept under

a 12/12 h light/dark cycle (lights on at 08.00 h daily), with

standard bird seed food and tap water ad libidum.

2.2. Drug treatments of animals

Ring doves received a single intraperitoneally (i.p.)

administration of saline (1 ml/kg) or L-tryptophan

(240 mg/kg, 1 ml/kg) either at the beginning of lights period

(08.00 h) or dark period (20.00 h). After a lapse of 15 min, the

animals received 3-hydroxybenzylhydrazine HCl (NSD 1015,

100 mg/kg, 1 ml/kg, i.p.) (see below) and after a new lapse of

45 min they were sacrificed. In another experiment, ring doves

received a single dose of melatonin (1 mg/kg, 1 ml/kg, i.p.) at

lights on to analyze the effect of melatonin on 5-HT synthesis.

Unless otherwise stated, all used drugs and reagents were

obtained from Sigma chemical, St Louis Mo. The study

followed the ‘principles of laboratory animal care’ (NIH

publication No. 85-23, revised 1996) and was performed

according to the guidelines of the ethical committee of the

Universitat de les Illes Balears.

2.3. Synthesis of serotonin: tryptophan hydroxylase activity

To test the changes in the synthesis of 5-HT after the vehicle

or tryptophan administration, the in vivo activity of tryptophan

hydroxylase (tryptophan-5-monoxygenase; EC 1.14.16.4), was

assessed. The tryptophan hydroxylase is rate-limiting enzyme

for the synthesis of 5-HT and was determined by measuring the

accumulation of 5-hydroxytryptophan (5-HTP) within 45 min

after inhibition of the aromatic L-amino acid decarboxylase

(EC 4.1.1.28) by a maximally effective dose of NSD 1015

(100 mg/kg, i.p.) (Carlsson and Lindqvist, 1973). The 5-HTP-

accumulation method is the most commonly used assay system

to monitor the in vivo rate of tryptophan hydroxylation in the

brain. The synthesis of 5-HTP was measured in three brain

regions: hippocampus, striatum and hypothalamus.

2.4. Brain samples and chromatographic analyses

The animals were killed by decapitation and their brain was

quickly removed and dissected on an ice-cold plate into

hippocampus, hypothalamus and neostriatum (striatum),

following the anatomical guidelines of the Avian Nomencla-

ture Forum (2004), and then were stored at K80 8C for further

analysis. The brain regions were weighed, placed individually

into cold tubes containing 1 ml of 0.4 M HClO4, 0.01%

K2EDTA and 0.1% Na2S2O5 and homogenized with an Ultra-

Turrax homogenizer (type Tp 18/10). The homogenate was

centrifuged at 40,000 g for 15 min at 4 8C. The resulting

supernatant was filtered through 0.45 mm syringe filters

(Spartan-3, Aldrich Chemical, Milwaukee, Wis., USA) and

aliquots (20–40 ml) were injected into the HPLC system for

determination of 5-HTP (the precursor in 5-HT synthesis),

5-HT and its metabolite, the 5-hydroxyindoleacetic acid

(5-HIAA) as described previously (see Fig. 1 for representative

chromatograms). A Spherisorb S3 ODS1 C18 reversed-phase

column (3 mm particle size range, 4.6 mm!10 cm) coupled to

a Tracer ODS2 C18 (2–5 mm particle size range) pre-column

(Teknokroma) were used. The mobile phase consisted of 0.1 M

KH2PO4; 2.1 mM octane sulphonic acid; 0.1 mM K2EDTA,

2 mM NaCl and 12% (vol/vol) methanol (pH 2.7–2.8, adjusted

with 85% H3PO4) and was pumped at a flow rate of 0.8 ml/min

with a Waters M-510 solvent delivery system. The compounds

were detected electrochemically by means of a cell with a

glassy carbon-working electrode with an applied potential of

C0.75 V against an in situ Ag/AgCl reference electrode

(ISAAC; Waters Concorde Electrochemical detector). The

current produced was monitored using an interphase Waters

bus SAT/IN Module connected to a computer. The concen-

trations of 5-HTP, 5-HIAA and 5-HT in a given sample were

calculated by interpolating the corresponding peak height into

a parallel standard curve using the software Millennium32

(Waters).

Fig. 1. Representative chromatographic (HPLC-ED) analyses of precursor

amino acids, monoamines and metabolites in striatum from a saline-treated

young ring dove (A) and a tryptophan (240 mg/kg, i.p., 1 h)-treated ring doves

(B), at nighttime. The retention times for the various compounds were (in min):

2.92 (NA noradrenaline), 3.82 (dopa), 6.27 (dopamine DA), 7.74 (5-HIAA 5-

hydroxyindoleacetic acid), 9.72 (5-HTP) and 15.21 (5-HT). The big peak

between 5-HTP and 5-HT (retention time between 10 and 12 min) is the

NSD1015. (A) Values in ng/g tissue: 2498 (5-HIAA), 235 (5-HTP) and 1792

(5-HT). (B) Values in ng/g tissue: 1379 (5-HIAA), 1669 (5-HTP) and 3099

(5-HT). Note that the administration of tryptophan at nighttime increased

markedly the levels of 5-HTP and 5-HT but did not alter significantly the levels

of 5-HIAA. See text for further details.

C. Garau et al. / Experimental Gerontology 41 (2006) 40–4842

2.5. Drugs and reagents

The following drugs and reagents were used: 3-hydro-

xybenzylhydrazine HCl (NSD 1015; Sigma chemical

company, St Louis MO, USA); melatonin (N-acetyl-5-

methoxytryptamine; Sigma chemical); L-tryptophan-methyl

ester (Sigma chemical). Other reagents were from Sigma or

Amersham.

2.5.1. Statistics

Results are expressed as meansGSEM of the number of

determinations. One-way ANOVA followed by Scheffe’s test

was used for the statistical evaluations. The significance level

chosen was P!0.05.

3. Results

3.1. Diurnal and nocturnal synthesis of 5-HT in young and old

ring doves

Fig 2 shows the 5-HTP accumulated during 45 min after

the inhibition of decarboxylase enzyme with NSD as well as

the 5-HT and 5-HIAA levels in the hippocampus, the striatum

and the hypothalamus of young and old ring doves at different

time of day. In young animals, a diurnal variation in

tryptophan hydroxylation was clearly observed, with greater

accumulation of 5-HTP at daytime than at nighttime (3.1-, 4.8-

and 2.3-fold higher during the light period verses dark period

for hippocampus, striatum and hypothalamus respectively).

The 5-HT content followed a similar pattern reaching the

maximum during the day (2.4-, 4.9- and 2.6-fold higher during

light, in the same brain regions respectively). Also the

metabolite 5-HIAA showed the maximum levels at daytime

(1.5-, 2.4- and 1.9-fold higher during the day than night in the

same regions, respectively). In marked contrast, old animals

did not show differences between diurnal and nocturnal levels

of 5-HTP, 5-HT and 5-HIAA in the three studied regions. In

general, the old animals maintained a quite low level of 5-HT

synthesis and metabolism, similar to that of young ring doves

at the dark period, independently of the environmental light

(Fig. 2).

3.2. Effect of melatonin on diurnal synthesis and turnover

of 5-HT in ring doves

In order to assess the possible mediation of melatonin on

the observed circadian rhythm of 5-HT synthesis in ring

doves, the effect of a single dose of melatonin (1 mg/kg, i.p.)

in young and old animals on diurnal synthesis of 5-HT was

studied. Fig 3 shows the 5-HTP accumulated after the

inhibition of decarboxylation as well as the 5-HT and

5-HIAA amounts in hippocampus, striatum and hypothalamus

of ring doves. In young ring doves, the accumulation of

5-HTP was inhibited by melatonin (37, 67 and 41% in

hippocampus, striatum and hypothalamus respectively). In

these animals, melatonin did not modify significantly the

5-HT content in any of the studied brain regions but marked

reduced the 5-HIAA levels (73, 86 and 35% in the same brain

regions), suggesting an inhibitory effect of melatonin on the

synthesis and release of 5-HT in the brain of young ring doves.

In old ring doves, treatment with the same dose of melatonin

did not alter significantly the accumulation of 5-HTP, the

content of 5-HT and 5-HIAA levels in any of the three brain

regions (Fig. 3).

3.3. Effects of tryptophan on diurnal and nocturnal synthesis

and metabolism of 5-HT in young and old ring doves

In young animals, the administration of L-tryptophan

increased strongly the accumulation of 5-HTP when it was

administered at nighttime in hippocampus (373%), striatum

(620%) and hypothalamus (217%) (see the representative

Fig. 2. Diurnal and nocturnal synthesis and metabolism of 5-HT in young (light bars) and old (dark bars) animals, at the onset of daytime (8.0 h) or nighttime (20.0 h)

in the hippocampus, striatum and hypothalamus. Animals received NSD1015 45 min before the sacrifice. Bars represent meansGSEM derived from 4 to 5 animals

of 5-HTP accumulated along 45 min, 5-HT and the metabolite 5-HIAA, in ng/g of wet tissue. One way ANOVA followed by Scheffe’s test was used for statistical

evaluation *P!0.05; **P!0.01; ***P!0.001 when compared with the diurnal young group.

C. Garau et al. / Experimental Gerontology 41 (2006) 40–48 43

chromatogram corresponding to striatum in Fig. 1), but no

significantly changes were observed after the lighttime

administration of tryptophan (24, 10 and 26% in the same

brain regions) (Fig. 4). These results indicate that, under control

conditions, the limiting enzyme of 5-HT synthesis—tryptophan

hydroxylase—was saturated by its substrate, at day but not at

Fig. 3. Acute effect (1 h) of melatonin (1 mg/kg, i.p.) (dark bars) on the accumulation of 5-HTP, 5-HT and 5-HIAA of young and old ring doves at daytime (8.0 h), in

hippocampus, striatum and hypothalamus. Ring doves received saline (light bars) or melatonin (dark bars) 15 before NSD1015 and were sacrificed after another

45 min. Bars represent meansGSEM derived from four animals of 5-HTP accumulated along 45 min, 5-HT and the metabolite 5-HIAA, in ng/g of wet tissue. One

way ANOVA followed by Scheffe’s test was used for statistical evaluation *P!0.05, **P!0.01, ***P!0.001 when compared with the corresponding saline

control group.

C. Garau et al. / Experimental Gerontology 41 (2006) 40–4844

C. Garau et al. / Experimental Gerontology 41 (2006) 40–48 45

night. In the same way, L-tryptophan significantly elevated the

content of 5-HT in all brain regions only when it was

administered at nighttime (190, 412 and 126% in hippo-

campus, striatum and hypothalamus, respectively), but did not

modify 5-HT content after lighttime administration (Fig. 4).

Interestingly, the metabolism of 5-HT, reflected by the

5-HIAA levels, was not modified after the administration of

L-tryptophan neither at day nor at night in any brain region

(Fig. 4), suggesting that the increased 5-HT synthesis

observed after nighttime L-tryptophan administration could

have been used for melatonin synthesis. In marked contrast

with the young animals, the administration of L-tryptophan to

old ones increased the 5-HTP accumulation, both at day (158,

196 and 680% in hippocampus, striatum and hypothalamus,

respectively) and at night (402, 218 and 408%, respectively),

indicating that, in old animals under control conditions, the

tryptophan hydroxylase enzyme was not saturated by its

substrate either at day and at night (Fig. 4). L-tryptophan also

significantly increased the contents of 5-HT in all brain

regions when it was administered at day (148, 181 and 379%,

respectively) and at night (231, 164 and 368%, respectively)

(Fig. 3). The 5-HIAA levels only were consistently increased

after diurnal administrarion of L-tryptophan in the hypothala-

mus (439%) (Fig. 4).

4. Discussion

5-HT is a neurotransmitter involved in many functions

throughout the brain, as the synchronisation of the circadian

clock located in the suprachiasmatic nucleus (SCN) and its

fundamental role as precursor in the melatonin synthesis.

Ageing brings many changes in the function of the circadian

timing system, so the present work found changes in the

rhythmic 5-HT synthesis with age, and studied the effects of

tryptophan supply on the circadian 5-HT synthesis rhythm.

A diurnal cycle in tryptophan hydroxylation was clearly

observed in young animals, with a major daytime synthesis and

metabolism of 5-HT. In line with the present results, a

circadian dependency of 5-HT release, with higher levels

during light phase was also observed in hippocampal (Monnet,

2002) and hypothalamic slices of rats (Blier et al., 1989). The

tryptophan hydroxylase protein levels detected by immunoau-

toradiography also showed circadian changes in rats (Malek

et al., 2004). Variation of 5-HT responses throughout the day

has been also described (Klein and Moore, 1979; Martin,

1991). In addition, abundant evidences indicate that melatonin

synchronizes various circadian neural and hypothalamic

endocrine processes (Dubocovich, 1995). However, the

participation of melatonin in the circadian rhythm of 5-HT

release has been questioned from in vitro studies in the

hypothalamus of rats (Cardinali et al., 1975). In fact, melatonin

Fig. 4. Acute effects (1 h) of tryptophan (240 mg/kg, i.p.) (black bars) on the accumul

of daytime (8.0 h) and nighttime (20.0 h), in hippocampus, striatum and hypothalam

before NSD1015 and were sacrificed after another 45 min. Tryptophan-treated an

derived from 4 to 5 animals of 5-HTP accumulated along 45 min, 5-HT and the met

test was used for statistical evaluation *P!0.05, **P!0.01 when compared with

reduced the spontaneous 5-HT release in slices of rat

hippocampus (Monnet, 2002) during the dark phase. Trying

to asses the possible mediation of melatonin on the observed

circadian rhythm of 5-HT synthesis in young ring doves, the

effect of a single dose of melatonin on diurnal synthesis of

5-HT was studied in the present work revealing an inhibitory

effect of melatonin on the synthesis and release of brain 5-HT.

Moreover, it was found that melatonin reduced the synthesis

and metabolism of 5-HT down to nocturnal levels, suggesting a

physiological role of melatonin in the 5-HT circadian synthesis

and release. Also agreeing with our results, a marked reduction

of 5-HT content in the rat hypothalamus was observed after the

administration of pharmacological doses of melatonin during

the light period (Lin and Chuang, 2002). Different experimen-

tal approaches in vitro (Monnet, 2002; Cardinali et al., 1975)

and in vivo (Miguez et al., 1994) showed an inhibitory effect of

melatonin on 5-HT release during the dark but not during the

light phase. Pinealectomy in rats (Miguez et al., 1995) and in

birds (Cassone et al., 1983) also modified the release of 5-HT

confirming the modulatory role of melatonin. In this context,

the present study demonstrated that the circadian rhythm of

5-HT synthesis in vivo in different brain regions of ring doves

was dependent on the presence of melatonin. It is well known

that the secretion of this hormone by the pineal gland during

the night synchronizes several processes (Dubocovich, 1995;

Monnet, 2002) including the 5-HT synthesis in ring doves,

as observed in the present work. Although the mechanism

mediating the action of melatonin on 5-HT synthesis remains

unknown, it has been reported that some effects of melatonin

were antagonized by the 5-HT1A antagonist pindolol (Lin

and Chuang, 2002). In this sense, it is well known that

the activation of 5-HT1A receptors inhibits 5-HT synthesis and

release (Adell et al., 1993; Esteban et al., 1999).

In marked contrast to young animals, the absence of a

circadian rhythm in 5-HT synthesis and metabolism was

observed in the old animals. A deficient effect of melatonin on

diurnal synthesis and metabolism of 5-HT in old ring doves in

comparison to young ones was also observed. These results are

consistent with the deteriorated melatonin rhythm during aging

(Reiter, 1992) that likewise weakens and become desynchro-

nized other circadian rhythms (Reiter, 1995). In ring doves of

the same age groups that the studied in the present work, the

circadian rhythm of serum melatonin showed a clear biphasic

cycle in young ring doves but absence of circadian rhythm was

observed in old animals (Terron et al., 2002). In addition, it is

known that the number and the sensitivity of melatonin

receptors decline with age (Gauer et al., 1998; Zhadanova,

2005). It has been observed numerous age dependent

physiological changes in brain 5-HT. For instance, age related

reductions in the binding of 5-HT receptors have been found in

the brain of humans (Rosier et al., 1996; Wang et al., 1995) and

ation of 5-HTP, 5-HT and 5-HIAA of young and old ring doves both at the onset

us. Control young (light bars) and old (dark bars) ring doves received saline 15

imals received tryptophan 15 min before NSD. Bars represent meansGSEM

abolite 5-HIAA, in ng/g of wet tissue. One way ANOVA followed by Scheffe’s

the corresponding saline control group.

"

C. Garau et al. / Experimental Gerontology 41 (2006) 40–4846

C. Garau et al. / Experimental Gerontology 41 (2006) 40–48 47

importantly in the SCN of rodents (Duncan et al., 2000) where in

addition the spontaneous firing of neurons showed a day/night

rhythm in young but not in old mice (Nygard et al., 2005).

Another important trait of the aging is the reduction in the

amplitude of the diurnal rhythms. In this context, our results

indicate that the diurnal 5-HT synthesis and metabolism were

strongly reduced in old animals compared with the observed in

young ring doves. Studies in humans showed a decrease of

serum/plasma tryptophan concentration related with aging and

associated with an enhanced indoleamine (2,3)-dioxygenase

(IDO) activity, which degrades tryptophan to form kynurenine

derivatives (Frick et al., 2004). In agreement, old ring doves

showed a significant decrease in the plasma melatonin levels

compared with young animals (Terrron et al., 2002) a result

which was already reported in other animal species (Turek

et al., 1999).

On the other hand, the rate of pineal melatonin synthesis is

dependent on the 5-HT levels. It also has been reported that

both tryptophan administration and a high plasma ratio of

tryptophan/neutral amino acids increases the availability of

brain tryptophan and consequently the 5-HT levels (Esteban

et al., 2004; Fernstrom and Wurtman, 1971). At night, when the

synthesis of melatonin is activated, the increased 5-HT

stimulated melatonin production (Esteban et al., 2004; Hajak

et al., 1991). On the contrary, the blocker of 5-HT synthesis

parachlorophenylalanine (PCPA), also inhibits the melatonin

release (Miguez et al., 1997). In order to examine the role of

substrate supply for the rhythmic synthesis of 5-HT, tryptophan

was administered at the beginning of either light or dark phases

to the different age groups of ring doves. The administration of

L-tryptophan to young animals, strongly increased the synthesis

of 5-HT only at nighttime, probably approaching the substrate

saturation of tryptophan hydroxylase and indicating that, under

control conditions, the limiting enzyme of 5-HT synthesis,

tryptophan hydroxylase, is saturated by its tryptophan substrate

at day but not at night. As the hydroxylation of tryptophan is

the rate-limiting step in the synthesis of 5-HT, tryptophan

hydroxylase determines the physiological concentration of

5-HT in vivo. Interestingly, the metabolism of 5-HT reflected

on the 5-HIAA levels was not modified after L-tryptophan

injection neither at day nor at night, suggesting that the

increased synthesis of 5-HT at night could have been used to

the synthesis of melatonin, which is known that fundamentally

occurs during night in mammals (Borjigin et al., 1995) and

birds (Bernard et al., 1997).

In marked contrast with young animals, the administration

of L-tryptophan to old animals increased 5-HT synthesis,

irrespective of the time of administration. This suggests that,

under control conditions, tryptophan hydroxylase was always

far from being saturated by its tryptophan substrate. Moreover,

the 5-HIAA levels were strongly increased in hypothalamus

after L-tryptophan ingestion, but only during lighttime. This

indicates an increase in 5-HT metabolims at daytime but not at

nightime and suggests again an increased use of the 5-HT for

melatonin synthesis.

Taken together, the results confirm that the synthesis of

5-HT and probably melatonin can be modulated by tryptophan

ingestion. The available 5-HT and melatonin results to be

dependent, first on an adequate dietary supply of tryptophan,

and second on the balance between 5-HT’s use as a

neurotransmitter and its availability as precursor for melatonin

synthesis, which is deeply dependent on the environmental

light. If the decrease in 5-HT and melatonin which normally

occurs as animals age could be prevented, perhaps some

complaints of aging could also be delayed. In this aspect, the

supplemental administration of tryptophan might aid to

improve some age-related degenerative conditions.

Acknowledgements

This investigation was supported by DGICYT Grant BFI

2002-04583-C02-029. The authors wish to thank the gently

technical assistance of David Moranta in this work. The two

first authors contributed equally to this work.

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