The main objective of this work was to 1 ) study the influence of endogenous melatonin (Mel) abolishment via pi n ealectomy and 2 ) explore exogenous Mel effect on anxiety-like and depressive-like behavior in male and female rats. Rats were shamoperated (Sh) or pinealectomized (Px) and following subgroups were selected 1 ) Px/NaCl (0.9%) and Sh/NaCl (0.9%) : rats injected subcutaneously, once daily for 8 weeks, with saline solution NaCl (0.9%) as vehicle ; 2 ) Px/Mel (4 mg/Kg) and Sh/Mel (4 mg/Kg): Rats similarly injected with 4 mg/Kg of Mel. All animals were housed under a photoperiod of (LD:16/8). After different treatments animals were tested in the open-field test (OFT), elevated plus maze test (EPM) to determine anxiety-like behavior, and forced swimming test (FST) to evaluate depressive-like level. Our results revealed that level of anxiety-like and depressive-like behavior are significantly higher in Px/NaCl (0.9%) when compared to Sh/NaCl (0.9%) group, suggesting that pinelectomy induced an anxiogenic and depressant effects. The Px effects would be due to the absence of endogenous Mel synthesis and release. Additionally, we clearly demonstrated that the level of anxiety-like and depressive-like behavior are higher in Px/Mel (4 mg/Kg) and Sh/Mel when compared respectively to Px/NaCl (4 mg/Kg) and Sh/NaCl groups suggesting an anxiolytic and antidepressant effects of exogenous Mel. Behavioral responses were sex dependent since the difference between females and males, especially, after melatonin administration, were statistically significant. These experiments provide evidence that pinealectomy and Mel regulated emotionally behavior in male and female rats.
Pineal gland is located in the epithalamia of the brain. It synthesizes several hormones, especially Mel which is shown in all groups from plants [
Rhythmic Mel production in the rat is controlled by a circadian rhythm of the pineal N-acetyltransferase (NAT) activity which synthesizes the Mel precursor N-acetylserotonin from serotonin [
The main experimental protocols used to understand the different Mel actions on the biological functions are surgical removal of the pineal gland, namely called pinealectomy and Mel administration. Mel is involved in the circadian regulation of many physiological functions such as reproduction, immunity, sleep, visual and cerebrovascular, functions [13-15]. In rodents, Mel is known to be implicated in several processes such as sedative and anticonvulsant [
The biological effects of Mel may be mediated via its receptors (MT1, MT2 and MT3) widely distributed in the brain and peripheral organs. In the many mammals, MT1 are shown especially in SCN, pars tuberalis, paraventricular nucleus, cerebellar cortex, hippocampus and cortex. MT2 are characterized in dorsal thalamus, corpus cerebella and MT3 in hypothalamus, thalamus, frontal cortex, kidney, liver and lung [29-31]. MT1 and MT2 inhibit cyclic AMP synthesis [
Given the considerations mentioned above, an experimental research is needed for illuminating the role of pineal gland and Mel treatment in anxiety and depression. The administration of Mel with or without abolishment of Mel hormone via pinealectomy may produce different effects on anxiety and depression behavior. This allows us to understand the influence of endogenous and exogenous Mel on emotionally behavior in the both sex of rats.
This experimental study was performed on male and female Wistar rats initially weighing (80 ± 20) g. Animals were housed by six in cages (36 cm long, 20 cm wide and 15 cm high). All rats were maintained on a 12 h Light/12h dark cycle and at a standard temperature (21˚C ± 1˚C). Water and food were provided ad libitum. At the beginning of treatment, the colony room was maintained under a long photoperiod LD: 16/8 (16 h Light/8h Darkness).
Rats were sham operated (Sh) or pinealectomized (Px) after being anesthetized with chloral hydrate (0.5 ml/100 g, sigma-aldrich, laborchemikalien Gmbh, Germany) according to the classical procedure described in literature. After anesthesia, the intact animals were placed in stereotaxic apparatus. The skin of the skull was incised along the suture lambda and the bone was excised in a circle of about 5 mm of diameter whose center corresponds to the lambda. The venous sinus was exposed and perforated with fine forceps and the pineal gland removed by suction. Dressing coagulant was immediately applied to the venous sinuses to stop the bleeding and close the wound, and then the skin was stitched. In sham operated animals, the bone was excised and then closed and the skin of skull was stitched. A dual test was adopted to ensure the success of pinealectomy 1) after each pinealectomy the presence of the pineal in the bottle of reception after aspiration is carefully checked and the content of bottle was emptied between two operations; 2) in addition, once behavioral study is completed, the animals were killed, brains and blood were collected for possible analysis. Before freezing the brain, the absence of the pineal in all pinealectomized animals was noted. Surgeries were performed in Dr. N. Lakhdar-Ghazal laboratory at the University of Mohammed V of Rabat (Morocco) and rats were transferred to University of Kénitra. The survival rate of animals after pinealectomy was about 60%.
One week after surgery, four subgroups were selected; each subgroup consists of six (6) animals. 1) Px/NaCl (0.9%) and Sh/NaCl (0.9%): rats injected subcutaneously, once daily for 8weeks, with saline solution NaCl (0.9%) as vehicle containing 5% ethanol; 2) Px/Mel (4 mg/Kg) and Sh/Mel (4 mg/Kg): rats similarly injected with 4 mg/Kg of Mel (N-acetyl-5-methoxytryptamine; Sigma Lot No. 112K0998 France), dissolved in 5% ethanol. All injections were made approximately at 4:00 pm.
A first experimental groups: Px/NaCl (0.9%); Sh/NaCl (0.9%) were designed to compare emotional behavior of intact and Px rats and thus to determine the effects of endogenous Mel. The second experimental groups: Px/Mel (4 mg/Kg) and Sh/Mel (4 mg/Kg)] were used to compare emotional behavior of intact and Px rats and thus to determine the effects of exogenous Mel treatment. At the end of treatment, the rats were subjected to different behavioral tests undertaken in following order: OFT, EPM and FST.
The OFT is used to measure the anxiety-like behavior in rodents. The maze adopted is made of wood (100 cm × 100 cm) enclosed with 40 cm high walls and placed under strong illumination (100 W, 2 m above the apparatus) [
The EPM is an ethological model of anxiety in rodents provoked by the novelty and repulsion as a result of elevation and illumination of the maze [
The FST is an excellent maze used to assess the depressive-like behavior [
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.
4.1.1.1. Time Spent in the Central Area (TCA) (
The sex factor (F(1.32) = 74.72, p < 0.001), and the treatment factor (F(1.32) = 37.76, p < 0.001) significantly affected the TCA. An interaction was found between treatment and sex F(1.32) = 24.64, p < 0.001). Indeed, the females of the groups Sh/Mel and Px/Mel showed a TCA significantly and respectively higher compared to males of similar groups (p < 0.001 and p < 0.0001).
A comparison between the groups showed that females of Px/NaCl group spend significantly less time in the central area compared to the animals of Sh/NaCl group (p = 0.04 < 0.05). Also, females of Px/Mel group spend significantly less time in the central area compared to the animals of Sh/Mel group (p = 0.000004 < 0.001). Similar or more pronounced results were obtained in males (p = 0.0001 < 0.001). Females of Px/NaCl group spend significantly less time in the central area compared to the animals of Sh/Mel (p = 0.00001 < 0.001). In contrast, no effect was obtained in males. Females of Px/Mel group spend significantly more time in the central area compared to the animals of Px/NaCl group (p = 0.02 < 0.05). Similarly, Females of Sh/Mel group spend significantly more time in the central area compared to the animals of Sh/NaCl group (p = 0.000002 < 0.001). However, no effect in males was observed. Males of Sh/NaCl group spend significantly more time in the central area compared to the animals of Px/Mel group (p < 0.001). No significant difference was observed between these groups in females.
4.1.1.2. Number of Returns to the Center (NRC) (
The sex factor (F(1.32) = 49.19, p < 0.001), and the treatment factor (F(1.32) = 16.91, p < 0.001) significantly affected the NRC. An interaction was found between treatment and sex (F(1.32) = 9.11, p < 0.001). Indeed, the females of the groups Sh/Mel and Px/Mel showed a NRC significantly and respectively higher compared to males of similar groups (p = 0.0001 < 0.001 and p < 0.0001).
The results of
4.1.1.3. Locomotors Activity (NTS) (
Locomotors activity was unaffected by any treatment (F(1.32) = 34.00, p > 0.05), and no effect of sex (F(1.32) = 1.49, p = 0.23 > 0.05) was noted. The interaction between sex and other treatments did not reach significance F(1.32) =1.63, p = 0.20 > 0.05). The values of all groups were comparable.
4.1.2.1. Entry to Open Arms (EOA) (
Statistical analysis showed that EOA was significantly affected by sex factor (F(1.20) = 15.39, p = 0.001 < 0.01)), and the melatonin treatment (F(1.20) = 7.47, p = 0.001 < 0.01). An interaction was found between treatment and sex (F(1.20) = 18.31, p = 0.0005 < 0.001). The females of Px group treated with Mel (Px/Mel) visited open arms significantly more than males of similar group (p = 0.01 < 0.05). Females and males of Px/NaCl groups visited open arms significantly less than animals of both sex of Sh/NaCl groups (p < 0.05). Additionally, females of Sh/Mel visited open arms significantly more than animals of Sh/NaCl (p < 0.05).
4.1.2.2. Time Spent in Open Arms (TOA) (
This parameter was affected by melatonin treatment (F(1.32) = 4.29, p = 0.01), but not by sex factor (F(1.32) = 3.65, p > 0.05). No interaction was found between treat-
ment and sex F(1.32) = 2.47, p > 0.05). A comparison between the groups showed that females of Sh/Mel group spend significantly more time in the open arms compared to the animals of sh/NaCl (p = 0.01), Px/NaCl (p < 0.01)
and those Px/Mel (p < 0.05). Conversely, no significant effect was found in males.
4.1.2.3. Total Entries in Arms (TEA) (
Locomotors activity was unaffected by sex (F(1.32) = 6.64, p > 0.05) or by melatonin treatment (F(1.32) =
0.75, p > 0.05). No interaction was found between treatment and sex F(1.32) = 0.32, p = 0.8 > 0.05). There was no significant difference between all groups.
4.1.3.1. Immobility Time (TIM) (
Pinealectomy significantly increased immobility time in animals of both sex although the effects were more evident in females. Indeed, females of Px/NaCl group showed a TIM significantly higher than those of Sh/NaCl (p < 0.05) and higher than those of all other groups. A similar result was observed in males. Also, animals of Px/NaCl and Px/Mel group showed a TIM significantly higher than those of Sh/Mel (p < 0.01 and p < 0.05 respectively). In contrast, Mel decreased immobility time especially in females. Thus, animals of Px/Mel and Sh/Mel groups showed a TIM significantly and respectively lower than those of Px/NaCl (p < 0.05) and Sh/NaCl (p < 0.05).
4.1.3.2. Struggling Time (TST) (
Px rats exhibited a lower struggling behavior when compared to other groups in animals of both sex although the effects were more pronounced in females. Indeed, animals of Px/NaCl group showed a TST significantly lower than animals of Sh/NaCl (p < 0.001) as well as of all other groups. Also, animals of Px/Mel group showed a low struggling behavior than those of Sh/Mel (p < 0.001).
Conversely, animals treated by Mel exhibited vigorous struggling behavior in males as well as in females. Thus, animals of Px/Mel and Sh/Mel groups showed a TST significantly and respectively higher than those of Px/NaCl (p < 0.05) and Sh/NaCl. Similarly, in males, a significant difference was noted between Px/NaCl and Px/Mel (p < 0.05).
The present study examines two important aspects: effect of pinealectomy and Mel administration on 1) anxietylike behavior in OFT and EPM and 2) depressive-like behavior in FST in male and female rats.
In OFT, Px rats showed a high anxiety-like behavior since the time passed at the center of the OFT and number of returns to the center was lower than sham operated animals. In this maze, the number of the entries to the edge of the OFT increased and the total distance travelled decreased if the anxiety of the animal is high [39,40].
In the EPM, Px rats visited open arms less frequently and spent less time in these arms than sham operated animals. In this test, the number of the entries to closed arms is increasing and the total distance travelled is decreasing when the anxiety level of the animal is high [
treatment interacted with anxiety provoking test situations. An additional argument of the role of pineal gland was given by comparison of behavioral response of Sh/Mel and Px/Mel treated animals. Thus, in OFT, Px/Mel showed a high level of anxiety-like with comparison to Sh/Mel group. Such result was also observed in the EPM confirming that Px/Mel treated rats visited open arms less frequently and spent less time in these arms than those of sham operated treated by the pineal hormone.
Taking the observations mentioned above, our results showed that the anxiety-like behavior was significantly affected by the pinealectomy since the control subjects were more mobile in the anxiogenic environmental of the OFT and EPM than the Px subjects. On the other hand, Mel secretion in rats follows a circadian pattern which is high throughout the darkness [
The assumption made above was verified by comparing Sh/Mel and Px/Mel groups which showed a lower anxiety-like behavior than their respective control subjects i.e. Sh/NaCl and Px/NaCl groups in the two anxiety-tests. This suggests that Mel is efficient in reducing the anxiety since the time passed at the center of the OFT and at the open arms of EPM was longer especially in the Mel treated animals. Mel was even capable to cancel or reverse the anxiogenic effect of pinealectomy since anxiety-like level was higher in Px/Mel vs Px/NaCl, such effect was approximately similar to than of intact animals (Sh/NaCl). The finding that Mel induced anxiolytic effect supported our previous observation [
The second finding concerns the influence of pinealectomy on depressive-like behavior measured in the FST. We demonstrated that Px rats showed a high level of depressive-like behavior since the immobility time and struggling behavior were lower than Sh animals, suggesting that the depressive-like measurement is more sensitive to the removal of pineal gland. The despair of rat is high when it ceased all active behaviors (struggling, swimming, and jumping) and remained passively floating. High percent time floating is interpreted as an increased depressive-like response [
The third finding was the pinealectomy and Mel effects on anxiety-like and depressive-like behaviors were depending on the sex. Indeed, whether in anxiety or depressive measurement tests, females showed a positive and coherent response to different treatments, while the males were generally less active and showed partially significant effects. This confirmed our previous observation concerning the existence of a sexual dimorphism in the behavioral response [
Otherwise, there is evidence in the literature that the depression and anxiety are characterized by decreased function in the noradrenergic locus coeruleus, serotoninergic dorsal and median raphe, and dopaminergic ventral tegmental area systems [
Finally, Melatonin is one hormone from a set of compounds synthesized by the pineal gland as 5-methoxytryptophol (5-ML), 5-methoxytryptamine (5-MT) and 5-methoxyindole acetic acid (5-MIAA) from a common substrate i.e. serotonin (5-HT). If the influence of melatonin on the emotional behavior is widely accepted, the role of other hormones of the pineal gland is not excluded [
The authors thank Paul Pévet from University of Strasbourg (France) for support and generously providing the Mel, Nouria Lakhdar-Ghazal and Bouhaddou from University Mohammed V of Rabat (Morocco) for technical assistance for pinealectomy realization and Samira Boulbaroud for helpful discussions and comments on an earlier version of this manuscript. This research was supported by PROTARS (D14/03, CNRST Morocco), GDRINeurosciences (France-Morocco) and Neuromed project.