effects of pinealectomy on anxiety and depressive-like ...

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Oct 3, 2014 - This work was supported by the World Federation of Scientists and Medical Science Council,. Medical University, Pleven, contract No 1/2014.
Доклади на Българската академия на науките Comptes rendus de l’Acad´ emie bulgare des Sciences Tome 67, No 12, 2014

MEDECINE Physiologie

EFFECTS OF PINEALECTOMY ON ANXIETY AND DEPRESSIVE-LIKE BEHAVIOUR IN WISTAR RATS Zlatina Nenchovska, Lidia Kortenska, Miroslava Stefanova, Liana Alova, Milena Atanasova∗, Jana Tchekalarova (Submitted by Academician P. Vassileva on October 3, 2014)

Abstract In the present study, we aimed to investigate the influence of endogenous melatonin abolishment via pinea lectomyone motional behaviour associated with anxiety and depressive responses in male Wistar rats. Sham-operated (sham) or pinealectomised (Pin) rats were tested in the hole-board (HB) test, elevated plus maze (EPM), sucrose preference test (SCT) and forced swimming test (FST) one month (Ist trial) and three months (IInd trial) after surgery. Melatonin deficit caused a significant decrease of the head-dipping at the holes accompanied by increased time of stereotype grooming both during the Ist and the IInd trial. Pinealectomy elevated both the number of entries and the time spent on the open arms in EPM test and this effect was significant a month after the removal of the pineal gland. Sucrose preference was decreased in Pin rats compared to sham rats during the light phase in both the Ist and the IInd trial, respectively. The immobility time tested in FS was significantly increased a month after pinealectomy. The observed depressive behaviour in Pin rats was accompanied by a tendency of decreased 5-HT release from the hippocampus. Taken together, a model of melatonin deficit caused an impulsiveand depressive-like behaviour, which was evident three months after pinealectomy. Our results suggest that endogenous melatonin synthetized in the pineal gland affects these behavioural responses through a regulatory mechanism on the hippocampal 5-HT release. Key words: pinealectomy, anxiety, depression, serotonin This work was supported by the World Federation of Scientists and Medical Science Council, Medical University, Pleven, contract No 1/2014.

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Introduction. The serotonin derivative melatonin (N-acetyl-5-methoxytryptamine) is synthesized and released by the pineal gland during the dark period. This hormone has a regulatory influence on several physiological and behavioural processes, including circadian rhythms, sleep-inducing effect and reproduction [1, 2 ]. In addition to its neuroendocrine role, endogenous and exogenous melatonin has been suggested to produce anxiolytic, sedative, analgesic and anticonvulsant effects [3, 4 ]. The efficacy of melatonin and its derivatives in patients with major depression has been mainly associated with its pivotal role as a resynchronizer of circadian rhythms, including the sleep/wake rhythms [5 ]. The resynchronizing properties of a drug recently marketed in Europe for the treatment of major depression, agomelatine, have been confirmed both in animals and humans [6, 7 ]. Stimulation of melatonin M1 and M2 receptors in the suprachiasmatic nuclei has been suggested to underlie entraining influence of agomelatine upon circadian rhythms [8 ]. There are controversial clinical data and few experimental reports considering the role of endogenous melatonin in depression. The serum melatonin levels during the night have been reported to be lower in patients with depression compared to physiological levels [9 ]. However, other studies demonstrated elevated melatonin concentration levels in patients with depression with disrupted circadian pattern of melatonin synthesis [10 ]. Experimental data revealed antidepressant effect of exogenous melatonin in tail suspension test and the forced swim test (FST) [11 ]. However, studies with a model of pinealectomy to characterize the role of deficit of endogenous melatonin on emotional behaviour, including anxiety and depressive-like behaviour, are few and controversial [12 ]. Furthermore, it is uncertain whether the antidepressant effect of melatonin is mediated by central serotonin pathway, which is known to play a crucial role in anxiety and depression. Therefore, the present experiments were designed further to explore the effect of melatonin deficit on the anxiety and depressive-like behavioural responses of Wistar rats. In particular, the possible involvement of 5-HT neurotransmission in melatonin effects is studied to verify the hypothesis that antidepressant and anxiolytic effect of endogenous melatonin is associated with central serotonin pathway. Materials and methods. The experiments were performed on six-weekold male Wistar rats kept under standardized conditions: temperature 21 ± 2 ◦ C, 50–60% humidity, in a 12 h light/dark cycle with lights on at 8 a.m. Animals were housed by three or four in cages. Water and food were provided ad libitum. The experimental design was approvedby governmental authorities fully in accordance with the European CommunitiesCouncil Directives of 24 November 1986 (86/609/EEC). Two experimentalgroupswere tested: Wis-sham (control group, sham operated, n = 10) and Wis-pin (pinealectomized rats, n = 10). The pinealectomy was performed following the methoddescribed by Hoffmann and Reiter [13 ]. In brief, rats were anesthetized (ketamine 40 mg/kg i.p 1692

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and xylazine 20 mg/kg i.p.) and fixed in a stereotaxic apparatus. The skull’s skin was incised along the suture lambda and using a pointed dental burr, a pieceof bone was removed at the juncture of the lambda and thesagittal suture lines. The venous sinus was exposed and grasped and thenthe pineal was grasped with fineforceps and removed. The bone disk was returned to its first position.The skin flaps pulled together. In sham-operatedrats, the same procedure was used except for thefact that the pineal gland was not removed. Four behavioural tests were performed in rats with pinealectomy and shamoperated: the hole board (HB) test, the elevated plus maze (EPM) test, the sucrose preference test (SPT), and forced swimming test (FST). These tests started a month after the surgery procedure within a time period of two weeks in the same sequence and interval of two days in all rats between each test. Before each behavioural test, the rats were transferred to a room next to a room in which the experiments were performed, so that the respective rat that was subjected to a behavioural test was not affected by the presence of other rats. The experiments were performed between 10 a.m. and 1. p.m. For the HB and EPM tests, the behaviour was recorded using an infrared sensitive CCD camera and a video tracking system (SMART PanLab software, Harvard Apparatus, USA). Hole board test. The hole board apparatus was made of a gray polystyrene plexiglas box (50 × 50 × 50) with 15 holes 3 cm in diameter and 1 cm deep. Each animal was placed in the centre of the board and its behaviour was recorded for 5 min. The calculatedmeasures were: 1) snuffed/dip-head hole number and 2) grooming time. Elevated Plus maze test. The apparatus consisted of two open arms (50 × 10 cm), two enclosed arms (50 × 10 × 50 cm), and a central platform (10 × 10 cm) elevated 50 cm above the floor level. At the beginning of the test, the rat was placed on the central platform facing an open arm. The test lasted 5 min. The calculated standard measures were: 1) number of entrances in open arms; 2) time spent in open arms. Sucrose preference test. On the first day (habituation), each cage was supplied with two identical 100 ml graduated water bottles. On the second day (pretest) and third (test), regular water in one of the bottles was replaced with 1% sucrose. The SPT started at 08:00 a.m. Both bottles were weighed after 12 hand replaced by a second pair of preweighed bottles. Sucrose preferencewas expressed as the percentage of the volume of sucrose solutionof the total volume of fluid (sucrose plus regular water) consumed during a 12-h period (light phase – 8:00–20:00 h and dark phase – 20:00–8:00 h, respectively). After each rat trial the equipment was cleaned with 0.1% acetic solution. Forced swim test. The despair-like behaviour was evaluated by a classic forced swimming test (FST) [14 ]. The test was carried out in a clear and transparent cylinder (50 cm tall, 25 cm diameter) filled to a level of 30 cm from the bottom with 24 ◦ C tap water. The water in the apparatus was changed Compt. rend. Acad. bulg. Sci., 67, No 12, 2014

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from rat to rat. Two swimming sessions were conducted: 15 min on the 1st day and 5 min on the 2nd day. After each test, the rat was dried and kept warm by a heating device for 10 min. behaviour during the 2nd day (test) was recorded by two skilled experimenters unaware of the treatment conditions. The parameter measured was immobility in seconds, which occurred when the rat remained motionless, or made only movements necessary to keep its head above the water. Measurement of 5-HT release in the hippocampus. After decapitation of the animal, the hippocampus was isolated on ice and 0.33 mm sections were prepared by a chopper MCIL. Tissue sections were loaded with [3 H] – 5HT, by incubation at 37 ◦ C in 2 ml of a buffer solution containing 0.1 µM [3 H] – 5HT for 60 min. After incubation, the sections were washed with buffer and transferred to perfusion chambers. The perfusion was performed with the tempered aerated solution with 5% SO2 /95% O2 (speed 1 ml/min). Incubation and perfusion solutions contain (mM): NaCl 118; KCl 4.8; CaCl2 1.25; MgSO4 1.2; NaHCO3 25; KH2 PO4 1.2; glucose 11; ascorbic acid 0.03; EDTA 0.03. After one hour perfusion samples were collected at 3 min intervals. The stimulation for 3 min with KCl is performed for 9 min after the start of the collection of samples. The sections were dissolved in 1 ml of 10% TXO. Then [3 H]-5HT was determined in the sections and in the samples,using a method of liquids cintillation measurement in a Beckman scintillation counter. Student’s t-testwas used for HB, EPM, FST and 5-HT release and two way ANOVA (factors: treatment and phase) followed by a post-hoc Mann–Whitney or Wilcoxon t-test for sucrose preference test. p < 0.05 was accepted as an index of statistically significant differences. Results and discussion. Figure 1 shows the effect of pinealectomy on the number of head-dipping (A) and time of grooming (B) during the first and the third month after pinealectomy, respectively. Pinealectomy caused a significant decrease of the head-dipping at the holes both during the first and the third month after the surgery (t-test, p = 0.002) (Fig. 1A). These results suggest that, melatonin deficit caused a sedative effect by a depression of spontaneous exploratory behaviour. Moreover, this depressant effect in Wis-pin group was confirmed by increased time of stereotype grooming (p < 0.005) (Fig. 1B) suggesting increased anxiety behaviour. The suppressed emotional behaviour in the HB test was sustained and evident three months after the pineal gland removal. Pinealectomy significantly elevated both the number of open arm entries and the time spent on the open arms a month after the removal of pineal gland (p < 0.05) and this tendency was still evident three month later (Fig. 2A, B). The observed sedative effect in HB test and anxiolytic effect in EPM test in melatonin deficit model resemble sedative and anxiolytic effects of effective doses of triazolobenzodiazepines in the same tests [15 ]. Furthermore, recently we have reported that spontaneously hypertensive rats (SHRs), considered to model at1694

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Fig. 1. Effect of pinealectomyon the number of head-dipping (A) and grooming time (B) in hole board test during the first and third month after the surgery (n = 10 − 12). Data arepresented as means ± SE, ∗ p < 0.05 Wis-sham vs. Wis-pin

tention deficit hyperactivity disorder, demonstrate low anxiety level in EPM test and decreased head-dipping in HB test compared to normotensive Wsitar rats [16 ]. Furthermore, epileptic rats (Wistar and SHRs) showed also a correlation between the low frequency of head-dipping in HB and decreased anxiety in EPM test [16 ]. We hypothesize that the observed correlation between the increased exploration Compt. rend. Acad. bulg. Sci., 67, No 12, 2014

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Fig. 2. Effect of pinealectomy on the number of entrances (A) andtime spent (B) in open arms in the elevated plus maze test during the first and third month after the surgery (n = 10 − 12). Data are presented as means ± SE; ∗ p < 0.05 sham-operated rats vs. pinelactomized rats

of the open arms in EPM test and diminished exploration of the holes in the HB test reflects a state of impulsive-like behaviour in SHRs and epileptic rats, which is suggested to be characteristic type of behaviour also for pinealectomized rats. 1696

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However, further studies are necessary before this presumption can be verified with great certainty. The literature data considering the anxiety behaviour of pinealectomized rats is controversial from a lack of effect [4, 12, 17 ] to increased anxiety after pinealectomy in EPM test [18 ]. The divergence in the reported data reveals that the influence of melatonin secretion by the pineal gland in anxiety behaviour is not synonymous and that endogenous melatonin is partially involved in this emotional response. Two-way ANOVA showed a main effect of the Phase [F1, 125 = 4.039, p = 0.047] and Drug × Phase interaction [F1, 70 = 4.972, p = 0.029] for the first month. Sham-operated rats were with a higher affinity to sucrose consumption, which was abolished by rats with pinealectomy during both phases one and three months after the surgery (∗ p < 0.05) (Fig. 3). Post hoc test confirmed the observed depressive-like behaviour in pinealectomized rats both after the first and the third month of surgery (∗ p < 0.05). However, this effect was evident only during the light phase. Recently, we have reported that epileptic Wistar rats are also characterized by anhedonia only during the light phase in the chronic epileptic period [19 ]. In the present study, the plasma melatonin levels in pinealectomized rats are not measured though it is suggested that in a model of melatonin deficit the diurnal rhythm of melatonin synthesis and release from the pineal gland are disrupted [13 ]. Although the exogenous melatonin showed to restore the phasedependent depressive like behaviour in epileptic Wistar rats [19 ] our present results suggest that endogenous melatonin has not a direct influence on phase-dependent anhedonia in pinealectomized rats.

Fig. 3. Effect of pinealectomy on diurnal variations in sucrose preference during the first and third month after the surgery (n = 10 − 12). Data are presented as means ± SE. ∗ p < 0.05 Wis-sham vs. Wis-pin 7

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Fig. 4. Effect of pinealectomy on immobility time in the Forced swimming test during the first and third month after the surgery (n = 10 − 12). Data are presented as means ± SE. ∗ p < 0.05 Wis-sham vs. Wis-pin

Pinealectomy significantly increased the duration of immobility time compared with sham-operated Wistar rats during the first month after surgery (∗ p < 0.05) (Fig. 4). This depressive-like behaviour developed by Wis-pin group is consistent with Mrabet et al. [17 ] data in FST. Furthermore, Mrabet et al. [18 ] reported that depressive behaviour in pinelactomized rats is sex-dependent with higher level of immobility and reduced sensitivity to antidepressants in intact male rats compared to females. Pinealectomy showed a tendency to attenuate KCl-evoked [3 H]-5HT release from the hippocampus compared to sham-operated rats (Table 1). Previous findings showed a lack of effect of exogenous melatonin on daytime spontaneous efflux of [3 H]-5HT both from the hippocampus and the pineal gland [20 ]. However, melatonin increased the evoked release in the hippocampus at the higher concenTable

1

Effect of pinealectomy on KCl-Evoked [3 H]-5HT release from the hippocampus (n = 6). Results are expressed as mean percentage ± SE of KCl-Evoked tritium efflux during a 5-min period of evoke

Wis-sham Wis-pin

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1 min

2 min

3 min

4 min

5 min

Mean ± SEM

18.99

5.25

4.43

24.18



11.29 ± 4.951

8.94

3.66

3.34

3.14

9.96

8.34 ± 1.498

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trations during the light phase [20 ], which suggests that endogenous melatonin has a pivotal regulatory role on [3 H]-5HT release in the hippocampus. In conclusion, deficit of melatonin released by the pineal gland causes an impulsive- and depressive-like behaviour, which is sustained and evident three months after pinealectomy. Our results suggest that endogenous melatonin synthetized in the pineal gland affects these behavioural responses through regulatory mechanism on the hippocampal 5-HT release.

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[19 ] Tchekalarova J., Z. Petkova, D. Pechlivanova, S. Moyanova, L. Kortenska, R. Mitreva, V. Lozanov, D. Atanasova, N. Lazarov, A. Stoynev. Epilepsy Behav., 27, 2013, No 1, 174–187. [20 ] Monnet F. P. J. Neuroendocrinol., 14, 2002, No 3, 194–199. Institute of Neurobiology Acad. G. Bonchev St, Bl. 23 Bulgarian Academy of Sciences 1113 Sofia, Bulgaria e-mail: [email protected]

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Department of Biology Medical Faculty Medical University-Pleven 1, Kl. Ohridski St 5850 Pleven, Bulgaria ∗

Z. Nenchovska, L. Kortenska et al.