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Age-related changes in circadian rhythm of serotonin synthesis in ring doves: Effects of increased tryptophan ingestion
Experimental Gerontology 41 (2006) 40–48 www.elsevier.com/locate/expgero
Age-related changes in circadian rhythm of serotonin synthesis in ring doves: Effects of increased tryptophan ingestion Celia Garau, Sara Aparicio, Rube´n V. Rial, Marı´a C. Nicolau, Susana Esteban * Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Cie`nces 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 retinohypothalamic 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 * Corresponding author. Address: Valde´s, Laboratori de Fisiologia, Departament de Biologia Fonamental i Cie`nces 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: [email protected] (S. Esteban).
0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.09.010
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
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(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 5HT 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
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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-HTPaccumulation 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 Nomenclature 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 UltraTurrax 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 concentrations 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).
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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-, 4.8and 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 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 5hydroxyindoleacetic 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.
2.5. Drugs and reagents The following drugs and reagents were used: 3-hydroxybenzylhydrazine HCl (NSD 1015; Sigma chemical company, St Louis MO, USA); melatonin (N-acetyl-5methoxytryptamine; 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.
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
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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.
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
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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.
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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 hippocampus, 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 hypothalamus (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 immunoautoradiography 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
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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 experimental 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 desynchronized 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 "
Fig. 4. Acute effects (1 h) of tryptophan (240 mg/kg, i.p.) (black bars) on the accumulation of 5-HTP, 5-HT and 5-HIAA of young and old ring doves both at the onset of daytime (8.0 h) and nighttime (20.0 h), in hippocampus, striatum and hypothalamus. Control young (light bars) and old (dark bars) ring doves received saline 15 before NSD1015 and were sacrificed after another 45 min. Tryptophan-treated animals received tryptophan 15 min before NSD. 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 when compared with the corresponding saline control group.
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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 (Terrro´n 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
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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. References Adell, A., Carceller, A., Artigas, F., 1993. In vivo brain dialysis study of the somatodendritic release of serotonin in the Raphe nuclei of the rat: effects of 8-hydroxy-2-(di-n-propylamino)tetralin. J. Neurochem. 60, 1673–1681. Azmitia, E.C., Segal, M., 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in, the rat. J. Comp. Neurol. 79, 641–667. Barassin, S., Raison, S., Saboureau, M., Bienvenu, C., Maitre, M., Malan, A., Pevet, P., 2002. Circadian tryptophan hydroxylase levels and serotonin release in the suprachiasmatic nucleus of the rat. Eur. J. Neurosci. 15, 833–840. Bernard, M., Iuvone, P.M., Cassone, V.M., Roseboom, P.H., Coon, S.L., Klein, D.C., 1997. Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina. J. Neurochem. 68, 213–224. Blier, P., Galzin, A.M., Langer, S.Z., 1989. Diurnal variation in the function of serotonin terminals in the rat hypothalamus. J. Neurochem. 52, 453–459. Borjigin, J., Wang, M.M., Snyder, S.H., 1995. Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland. Nature 378, 783– 785. Card, J.P., Moore, R.Y., 1989. Organization of lateral geniculate-hypothalamic connections in the rat. J. Comp. Neurol. 284, 135–147. Cardinali, D.P., Nagle, C.A., Freire, F., Rosner, J.M., 1975. Effects of melatonin on neurotransmitter uptake and release by synaptosome-rich homogenates of the rat hypothalamus. Neuroendocrinology 18, 72–85. Carlsson, A., Lindqvist, M., 1973. In-vivo measurements of tryptophan and tyrosine hydroxylase activities in mouse brain. J. Neural. Transm. 34, 79–91. Cassone, V.M., Lane, R.F., Menaker, M., 1983. Daily rhythms of serotonin metabolism in the medial hypothalamus of the chicken: effects of pinealectomy and exogenous melatonin. Brain Res. 289, 129–134. Cutrera, R.A., Ouarour, A., Pevet, P., 1994. Effects of the 5-HT1a receptor agonist 8-OH-DPAT and other non-photic stimuli on the circadian rhythm of wheel-running activity in hamsters under different constant conditions. Neurosci. Lett. 172, 27–30. Czeisler, C.A., Dumont, M., Duffy, J.F., Steinberg, J.D., Richardson, G.S., Brown, E.N., Sanchez, R., Rios, C.D., Ronda, J.M., 1992. Association of sleep-wake habits in older people with changes in output of circadian pacemaker. Lancet 340, 933–936. Dubocovich, M.L., 1995. Melatonin receptors: are there multiple subtypes? Trends Pharmacol. Sci. 16, 50–56. Duncan, M.J., Short, J., Wheeler, D.L., 1999. Comparison of the effects of aging on 5-HT7 and 5-HT1A receptors in discrete regions of the circadian timing system in hamsters. Brain Res. 829, 39–45.
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Age-related changes in circadian rhythm of serotonin synthesis in ring doves: Effects of increased tryptophan ingestion Celia Garau, Sara Aparicio, Rube´n V. Rial, Marı´a C. Nicolau, Susana Esteban * Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Cie`nces 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 retinohypothalamic 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 * Corresponding author. Address: Valde´s, Laboratori de Fisiologia, Departament de Biologia Fonamental i Cie`nces 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: [email protected] (S. Esteban).
0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.09.010
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
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(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 5HT 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
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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-HTPaccumulation 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 Nomenclature 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 UltraTurrax 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 concentrations 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).
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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-, 4.8and 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 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 5hydroxyindoleacetic 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.
2.5. Drugs and reagents The following drugs and reagents were used: 3-hydroxybenzylhydrazine HCl (NSD 1015; Sigma chemical company, St Louis MO, USA); melatonin (N-acetyl-5methoxytryptamine; 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.
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
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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.
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
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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.
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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 hippocampus, 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 hypothalamus (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 immunoautoradiography 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
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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 experimental 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 desynchronized 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 "
Fig. 4. Acute effects (1 h) of tryptophan (240 mg/kg, i.p.) (black bars) on the accumulation of 5-HTP, 5-HT and 5-HIAA of young and old ring doves both at the onset of daytime (8.0 h) and nighttime (20.0 h), in hippocampus, striatum and hypothalamus. Control young (light bars) and old (dark bars) ring doves received saline 15 before NSD1015 and were sacrificed after another 45 min. Tryptophan-treated animals received tryptophan 15 min before NSD. 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 when compared with the corresponding saline control group.
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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 (Terrro´n 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
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