The aim of this study was to determine the effect of pharmacological doses of melatonin (MEL) and L-tryptophan (L-TRP) on depression-like behavior in female rats submitted to the forced swimming test (FST) after 2, 4, 6 or 8 weeks of treatment. This will allow exploring the different mechanisms of L-TRP actions particularly that due to its conversion into MEL. For this purpose, four groups of 24 rats each were constituted; (Group 1: Control): received saline solution NaCl (0.9%), (Group 2: MEL4): received 4 mg/Kg of MEL, (Group 3: L-TRP4): received 4 mg/Kg of L-TRP and (Group 4: L-TRP20): received 20 mg/Kg of L-TRP. Animals of each group were distributed on 4 subgroups of 6 rats submitted to different time treatments. The duration of immobility (TIM) and struggling period (TST) of rats in FST were measured after 2, 4, 6 and 8 weeks of drug treatment and the effects of MEL and L-TRP were compared. Chronical administration of different doses of MEL or L-TRP failed to induce any anti-depressant activity in rats subjected to FST after 2 weeks of treatment. However, after 4 weeks, daily administration of MEL at 4 mg/Kg significantly reduced the immobility period and enhanced struggling time. After 6 weeks, MEL at 4 mg/Kg and L-TRP at 20 mg/Kg were both effective in reducing immobility and increasing struggling movement, their effects being statistically comparable. All treatments were able to significantly reduce immobility time and increase struggling duration after 8 weeks, but L-TRP at 4 mg/Kg was less potent than MEL and L-TRP at 20 g/Kg. The antidepressant-like activity of L-TRP was dose and time dependent, and that of MEL was time dependent. In conclusion, the study showed that at pharmacological doses, MEL and L-TRP have anti-depressant action, and such effect is dependent on time treatment; MEL is more effective than L-TRP. In conclusion, L-TRP, through MEL, 5-HT or by itself could modulate aminergic neurotransmission in the different brain areas to ensure its behavioral effects.
Melatonin (MEL) is a methoxyindole secreted mainly, but not exclusively by the pineal gland since pinealectomy abolishes its synthesis and its rhythmic pattern release. The initial step of MEL biosynthesis involves the uptake of L-tryptophan (L-TRP) from the circulation into the pinealocytes, followed by its conversion into 5-hydroxytryptophan by TRP-hydroxylase. Further decarboxylation by L-aromatic aminoacid decarboxylase leads to serotonin (5-HT) formation. 5-HT is first acetylated by N-acetyltransferase (NAT), which is probably the ratelimiting step, and then methylated by hydroxyindole orthomethyltransferase (HIOMT) to MEL. Characteristically, pineal MEL is secreted in a circadian manner with high levels occurring in all studied species at night. In mammals, the MEL rhythm is generated by an endogenous circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, which is entrained by the light/dark cycle to the 24 h [
Among these functions, MEL is involved in the control of circadian and seasonal rhythms [3,4], sleep regulation [
The relationship between MEL levels, pineal function, and mood or psychiatric disorders is strengthened by some evidences based on clinical observations in Humans and experimental administration of MEL in animals. Thus, in Humans, nocturnal MEL levels are low in subjects with major depressive and panic disorder [11,13]. Some healthy individuals with a dysthymic disposition (mild or episodic depression) also have lower-than-normal nocturnal MEL levels [
As it’s mentioned above, MEL is a derivative of serotonin, which in turn is derived from the essential amino acid L-TRP. Unlike MEL, little is known about the effect of chronic administration of L-TRP. This aminoacid plays an instrumental role in balancing mood and sleep patterns and may be of some benefit in the treatment of some psychiatric disorders since changes in TRP metabolism under stress are assumed to be a risk factor for major depression [
and reverses antidepressant-induced remission [
The involvement of MEL in mediating of TRP action is strongly suggested in many reports as in hypnotic function and affective and sedative action of TRP [23,27,28]. In support of this, TRP administration or a high plasma ratio between TRP and large neutral amino acids enhances brain TRP levels, and accelerates 5-HT and MEL synthesis. Indeed, administration of TRP increases the availability of 5-HT in the brain of different species [
However, the literature influence of chronic effects of L-TRP, extending over several weeks on affective behavior, is not sufficiently explored, studies are generally interested in acute administration, experimental depletion or oral supplementation of L-TRP. In the present study, we 1) examine the effect of chronic administration of MEL (4 mg/kg b.w.) and L-TRP (4 and 20 mg/kg b.w.) on depression-related behavior in female rats and 2) analyze the time dependence by comparing these effects during 2, 4, 6 and 8 weeks of treatment. This will allow us to explore the different mechanisms of L-TRP actions particularly that due to its conversion into MEL.
This experimental study was performed on female Wistar rats initially weighing (100 ± 20) g. Animals were housed by six in cage (36 cm long, 20 cm wide and 15 cm high). All rats were maintained under LD 12/12 (12 h Light/12h Darkness) and at a standard temperature of (21 ± 1) ˚C. Water and food were provided ad libitum. At the beginning of experiments, the colony room was transferred in long photoperiod LD: 16/8 (16 h Light/8h Darkness).
Ninety sex animals used in the present experiment were divided into 4 groups of 24 animals as following: (group 1: control): rats injected subcutaneously, once daily with saline solution NaCl (0.9%) as vehicle containing 5% ethanol, (group 2: MEL4): rats injected with 4 mg/Kg of MEL (group 3: L-TRP4): rats injected with 4 mg/Kg of L-TRP and (group 4: L-TRP20): rats injected with 20 mg/Kg of L-TRP. MEL (Sigma Lot No. 112K0998 France)
and L-TRP (Sharlau, ServiChim, Casablanca, morocco) were dissolved in 5% ethanol. All injections were made approximately at 4:00 pm (2 h before dark phase). Each group was subdivided on 4 experimental subgroups of 6 animals designed to compare emotional behavior of rats after 2, 4, 6 and 8 weeks of treatment. At the end of each period of treatment, the rats were subjected to the Porsolt forced swimming-induced behavioral despair test (FST) to study the effect of drugs on duration of immobility (TIM) and struggling period (TST).
The forced swimming test (FST) is a maze usually used to assess the depressive-like behavior [33,34]. Swimming sessions were conducted by placing the rat in individual glass cylinders (height: 50 cm; diameter: 30 cm) containing 30 cm of water at (23˚C ± 2˚C). During the session, rats were forced to swim for 5 min and the duration of immobility was measured. The latency to the first bout of immobility was also recorded starting immediately after placing the rats in the cylinder. A rat was judged immobile when it ceased all active behaviors (i.e. struggling, swimming and jumping) and remained passively floating or making minimal movements necessary to maintain the nostrils above water. High percent time floating is interpreted as an increased depressive-like response [33,35].
The animals of control groups showed a relative variation in their behavior activity over the time in FST. To eliminate this variation, the activity of control group for each time was considered as the reference level expressed as 100% of activity. To evaluate the effect of various treatments, the individual response of animals of treated groups were calculated relative to the mean control group activity, i.e. 100%. Thus, the intensity of the response of animals of treated groups was established as decreased or increased individual activities relative to the basal level for each time.
All data are expressed as the means ± standard error of the means (S.E.M.). To determine the differences between experimental groups statistical analysis was performed by analysis of variance (ANOVA) 1st/2nd order followed by a post-hoc tests (Fisher LSD) or Student test “t”. Differences were considered significant when p < 0.05, very significant when p < 0.01 and highly significant when p < 0.001.
After 2 weeks of treatment immobility time (TIM) was not affected by MEL (NaCl/MEL4: (p = 0.19 > 0.05) or L-TRP [NaCl/L-TRP4: (p = 0.10 > 0.05) and NaCl/LTRP20 (p = 0.56 > 0.05)]. After 4 weeks, MEL, but not different doses of L-TRP, significantly decreased TIM with comparison to control group (NaCl/MEL4: (p < 0.05). TIM of L-TRP20 was lower than control group even if the difference was not significant (NaCl/L-TRP20: p > 0.05). No significant difference was observed between L-TRP4 and L-TRP20 (L-TRP4/L-TRP20: p > 0.05).
After 6 weeks, both MEL and L-TRP at 20 mg/Kg significantly and similarly reduced TIM [(NaCl/MEL: p = 0.00005 < 0.001); (NaCl/L-TRP20: p = 0.0001 < 0.001)]. In contrast, L-TRP at 4 mg/Kg didn’t affect this parameter (NaCl/L-TRP4: p = 0.21 > 0.05). No significant difference was noted between L-TRP4 and L-TRP20: (LTRP4/L-TRP20: p > 0.05). After 8 weeks, all treated groups showed a TIM significantly lower than control group [(NaCl/MEL4: p = 0.00004 < 0.001); (NaCl/LTRP4: (p = 0.002 < 0.01); (NaCl/L-TRP20 (p = 0.0001 < 0.001)]; the effects of MEL and TRP at 20 mg/kg being significantly comparable.
Similarly, after 2 weeks, no significant difference was noted between all groups with regard to struggling time (TST): [(NaCl/MEL4: (p = 0.36 > 0.05); (NaCl/L-TRP4: p = 0.88 > 0.05); (NaCl/L-TRP20: p = 0.36 > 0.05)] respectively. After 4 weeks, MEL, but not different doses of L-TRP, significantly increased TST with comparison to control group [(NaCl/MEL4: p = 0.01 < 0.05); (NaCl/ L-TRP4: p = 0.89 > 0.05); (NaCl/L-TRP20: p = 0.18 > 0.05)]. After 6 weeks, both MEL and L-TRP at 20 mg/Kg, but not L-TRP at 4 mg/Kg, significantly enhanced TST [(NaCl/L-TRP4: p = 0.02 < 0.05); (NaCl/L-TRP4: (p = 0.46 > 0.05); (NaCl/L-TRP20: p = 0.004 < 0.01)]. After 8 weeks, different treatement were all able to increase significantly TST: [(NaCl/MEL4: p = 0.007 < 0.01); (NaCl/L-TRP4: p = 0.018 < 0.05); (NaCl/L-TRP20: p = 0.0006 < 0.001)]; the effects of MEL and TRP being significantly comparable.
For the control group, no significant difference was observed in immobility and struggling behavior over the time; the values of TIM and TST remaining comparable after 2, 4, 6 and 8 weeks of treatment.
MEL decreased TIM, since the TIM values obtained after 4, 6 and 8 weeks of treatment with MEL at 4 mg/kg were significantly lower than after 2 weeks [(2w/4w: p < 0.001); (2w/6w: p < 0.001); (2w/8w: p < 0.001)]. In addition, the TIM observed after 6 and 8 weeks of treatment were significantly lower than after 4 weeks [(4w/ 6w: p < 0.05); (4w/8w: p < 0.001)] and this TIM after 8 weeks of treatment was significantly lower than after 6 weeks (6w/8w: p < 0.001). Inversely, MEL enhanced TST, and its effects increased proportionally with number of weeks [(2w/6w: p < 0.001); (2w/8w: p < 0.001); [(4w/6w: p < 0.01); (4w/8w: p < 0.001) (6w/8w: p < 0.001)].
The decrease of TIM under L-TRP treatment at 4 mg/Kg depended on the duration of treatment. The difference was more significant and the effect greater when the number of weeks between the compared groups is important [(2w/4w: p < 0.05); (2w/6w: p < 0.001); (2w/8w: p < 0.001)]. In addition, the TIM after 8 weeks of treatment was significantly lower than after 4 weeks (4w/8w: p < 0.05). With regard to TST, the effect obtained at 8
weeks was significantly higher compared to other times [(2w/8w: p < 0.001); (4w/8w: p < 0.001) (6w/8w: p < 0.001)].
The effects of L-TRP at 20 mg/Kg on TIM were very similar to those of MEL: [(2w/4w: p < 0.001); (2w/6w: p < 0.001); (2w/8w: p < 0.001); (4w/6w: p < 0.001); (4w/ 8w: p < 0.001)]. The same observation was made for the evolution of TST with time: [(2w/6w: p < 0.001); (2w/8w: p < 0.001); [(4w/6w: p < 0.001); (4w/8w: p < 0.001)]except the difference between 6 and 8 weeks is not significant.
The aim of this study was to investigate the influence of pineal indole MEL and its precursor L-TRP administration on 1) depressive-like behaviors in FST and 2) to analyze the evolution of their effects over the time. The comparison of L-TRP and MEL effects will allow us to explore the different action ways of L-TRP.
We found that in FST, rats injected by MEL or L-TRP
showed a low level of depressive-like behavior since the immobility time of treated animals was lower and struggling movements were higher and more frequent than control. These findings suggest that depression measurement is more sensitive to drug treatment since MEL and L-TRP administration interacted with depression provoking test situations. Consequently, MEL and L-TRP may exert antidepressive-like effects in this paradigm. In addition, the antidepressant-like action of both MEL and L-TRP was time-dependent and dose-dependent for L-TRP.
These results are in accordance with many reports on rodents in the literature and with our previous report in male and female rats [17,19]. This report showed that MEL administrated chronically in rats produced a real antidepressant power that is expressed in FST since it decreased TIM and enhanced TST. Similar result in both sexes of rats, showing that MEL decreased swimming in the FST, was previously observed by Brotto et al. [
Moreover, we reported that like MEL, chronic administration of L-TRP was able to reduce immobility and to enhance struggling of animals in FST, L-TRP being less potent than MEL at the same dose. Indeed, after 4 weeks, 20 mg/Kg of L-TRP were necessary to obtain a comparable behavioral effect to MEL at 4 mg/Kg, whereas LTRP at 4 mg/Kg required at least 6 and 8 weeks for inducing depressive-like effect.
Unfortunately, in the literature, chronically effects of L-TRP have not been well investigated, studies are generally interested in acute TRP administration or depletion or oral supplementation. Our study has the advantage of following the TRP effect over several weeks. It is consistent with the report of Wong and Ong [
To ensure its behavioral functions, L-TRP may cross the blood brain barrier, attain different areas of the brain and act by itself or by its conversion into MEL and/or 5-HT. The cerebral L-TRP amount not only depends on the absolute concentration of blood TRP but also the relative concentrations of these neutral that compete with TRP in the blood. Then the aminoacid will be picked up by pinealocytes but also serotonergic neurons. Under physiological conditions, fluctuations in brain L-TRP may sound on the metabolism of 5-HT and MEL in the brain. Indeed, the limiting enzyme, TRP-hydroxylase, is not saturated by its substrate. An increase in brain L-TRP therefore leads automatically, and to a certain extent, an increase in the rate of biosynthesis of 5-HT. The studies suggest that charge of L-TRP TRP hydroxylase is only saturated at 50% [50,51]. For this reason, it has been suggested that the administration of L-TRP could, to some extent, stimulate the brain serotonergic melatoninergic activity and reduce some psycho-biological disorders associated with dysfunction of serotonergic and melatoninergic activities [
In this study, it’s possible that L-TRP administration raises circulating MEL levels, which in turn could act on the central nervous system to regulate affective response. In support of this hypothesis: 1) MEL and L-TRP have comparable antidepressant-like properties; 2) TRP is the precursor of MEL and 3) MEL concentrations are affected by the amount of TRP available in the blood and brain [
Moreover, several studies have shown that in humans, administration of L-TRP induced an increase in brain levels of TRP but also of 5-HIAA (50%) in the cerebrospinal fluid, which suggests an increase in the synthesis of serotonin [50,60]. Increasing levels of TRP in humans, serotonin synthesis can be doubled, since the enzyme is only half saturated under normal conditions [
As the pathophysiology of depression could be a consequence of alteration of 5-HT activity in many brain structures [62,63], behavioral actions of TRP, obtained in this study, could be mediated by 5-HT. In support of this, report of Wong and Ong [
Otherwise, there is evidence in the literature that the depression is characterized by decreased function in the noradrenergic locus coeruleus, serotoninergic dorsal and median raphe, and dopaminergic ventral tegmental area systems [
Given the considerations mentioned above, without neglecting other ways of action, it can be assumed that TRP induced antidepressant activity obtained in this study would be the logical consequence of its conversion into MEL which is known to exert the same actions [17,19]. In this sense, TRP through MEL, 5-HT or by itself could regulate noradrenergic, serotoninergic and dopaminergic systems in the different parts of the brain to ensure its behavioral effects.
This work was supported by the project PROTARS (D14/ 03) between the University Ibn Toufail Kenitra and CNRST (Morocco), by GDRI Neurosciences (France, Morocco) and the European Neuromed project. Thanks to Dr P. Pévet, of the Institute of Cellular and Integrative Neurosciences, Department of Neurobiology of Rhythms, UPR-3212 CNRS, University of Strasbourg, France, for the gift of the MEL and to Dr D. Boussaoud of UMR INSERM 1106, Aix-Marseille Université, Faculté de Médecine, Marseille, France for support and encouragement.