Serotonergic Neurons of Dorsal Raphe Nucleus on the Effect of a Xanthone from Kielmeyera coriacea Stems in Behavioral Tests

Abstract

This study investigated the influence of serotonergic neurons of the dorsal raphe nucleus (DRN) on the effect of 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone, obtained from a hydroethanolic extract (HE) from Kielmeyera coriacea Mart. (Clusiaceae) stems. Intra-DRN microinjection (0.25 μ l/30 s) of xanthone or 5-HT_1A ligands and its associations were performed in rats submitted to the forced swimming (FST) and to the open field (OFT) tests. Xanthone (0.3, 0.6 or 0.9 pmol), WAY₁₀₀₆₃₅ (5-HT_1A antagonist; 0.2, 0.4 or 0.8 nmol) or (+)pindolol (5-HT_{1A/1B/ßadrenergic} antagonist; 0.2, 0.4 or 0.8 nmol) did not alter immobility time in the FST. The 5-HT_1A agonist, (+)8-OH-DPAT (0.6, 0.8, or 1.0 nmol) increases the immobility time in higher dose. Associated treatment of WAY₁₀₀₆₃₅ (0.8 nmol) or (−) pindolol (0.4 nmol) and xanthone (0.3 pmol), produced an anti-immobility effect, showing a synergic effect. Xanthone (0.3 pmol) abolished the increase on immobility time produced by (+)8-OH-DPAT (1.0 nmol). WAY₁₀₀₆₃₅ (0.8 nmol) blocked the increase in immobility time produced by (+)8-OH-DPAT (1.0 nmol). Crossings number in the OFT was not altered by any tested compound or associated treatment. These results suggest that the serotonergic neurons of the DRN, through the 5-HT_1A somatodendritic autoreceptors, are involved in the xanthone effects on FST.

Keywords::

• Dorsal raphe nucleus
• forced swimming test
• serotonin
• Kielmeyera coriacea
• xanthone
• 5-HT_1A receptor

Introduction

Kielmeyera coriacea Mart. (Clusiaceae), popularly known as the “pau santo”, is a tree indigenous to the central Brazilian plateau (Alves et al., 2000). Hypericum perforatum L., a plant from the same family as the Kielmeyera coriacea, is known as an effective alternative treatment for mild to moderate depression (Butterweck et al., 1998; Linde et al., 1996). Previous studies showed that a hydroethanolic (HE) extract of Kielmeyera coriacea Mart. stems and its semi-pure dichloromethane constituent reduce immobility time when chronically administered in rats submitted to the forced swimming test (FST) (Martins et al., 2004, 2006). Tested in vitro, the HE extract of Kielmeyera coriacea stems inhibits, in a concentration-dependent manner, the synaptossomal uptake of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) (Goulart et al., 2007). Another study conducted in our laboratory showed that the extract of Kielmeyera coriacea leaves did not reduce immobility time in the FST, but increased the open-arm entries in the plus maze test (PMT), suggesting an anxiolytic-like effect (Audi et al., 2002).

Analysis by high performance liquid chromatography (HPLC), detected the presence of 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone in the HE and semi-pure dichloromethane fraction of Kielmeyera coriacea stems extract, but not in the dichloromethane leaves extract. This result suggests that this xanthone is related to the anti-immobility effect produced by HE and semi-pure dichloromethane fraction of Kielmeyera coriacea stems extract. The 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone is structurally related to the tricyclic antidepressant compound (Cortez et al., 1998).

The central 5-HT system plays an important role in the etiology and treatment of anxiety and mood disorders. Ascending 5-HT pathways originating from dorsal or median (DRN or MRN) raphe nuclei have received attention so far for controlling aversion, modulating through different sub-types of receptors, neural substrates of anxiety, panic, and depression (Deakin & Graeff, 1991; Graeff, 1993; Handley, 1995).

Somatodendritic 5-HT_1A autoreceptors, located in the raphe nuclei, are considered to be the principal inhibitory regulator of 5-HT neuronal activity playing an important role in the physiological control of ascending 5-HT pathway. The stimulation of these 5-HT_1A receptors reduces the firing of 5-HT neurons and produces an anti 5-HT effect, through reduction in the 5-HT release (Barnes & Sharp, 1999; Beck et al., 2004).

The effectiveness of the different antidepressant drugs in treating depressive disorders has been observed only after chronic administration (Hjorth et al., 2000; Pineyro & Blier, 1999). The delay in the onset of antidepressant activity, which requires a period of two to four weeks before this activity can be observed (Ballenger, 1993; Barnes & Sharp, 1999), is attributed to activation of the inhibitory somatodendritic 5-HT_1A autoreceptors, located in the DRN and MRN. Exogenously applied 5-HT, 5-HT_1A agonist ligands (Romaniuk et al., 2001) or acute administration of 5-HT uptake inhibitors produce inhibition of neuronal firing, decrease in the 5-HT release in different areas of the forebrain, and may hinder the therapeutic efficacy of an antidepressant. After continued stimulation, these feedback mechanisms become desensitized and the enhanced 5-HT availability is able to enhance 5-HT neurotransmission (Barnes & Sharp, 1999).

Current strategies aim at improving the efficacy of antidepressant drugs, including the co-administration of 5-HT_1A and/or 5-HT_1A/1B autoreceptor antagonists. These antagonists can facilitate the anti-immobility effect of sub-effective doses of antidepressant drugs in the FST in rats and mice (Tatarczynska et al., 2002) and accelerate the effects of these drugs in patients with major depression (Artigas et al., 1996).

This work investigated the involvement of serotonergic neurotransmission, particularly mediated by somatodendritic 5-HT_1A autoreceptors locate in DRN, on the effect of xanthone, obtained from Kielmeyera coriacea stems, in rats submitted to the FST and the OFT. We examined if xanthone, alone or in combination with 5-HT_1A ligands, could affect the behaviour of rats in these tests.

Materials and Methods

Animals

Male Wistar rats aged 55–63 days, weighing 240–270 g (Central Biotério, University of Maringá) were used. The rats were housed in groups of four per cage and maintained on a 12 h light:dark cycle (lights on at 7:00 h) under controlled temperature (22° ± 1°C), with food and water freely available. The animals were maintained in this situation for three days before the surgery for acclimatization, and during the post-surgery period. All experiments were carried out between 8:00 and 12:00 h. The experimental procedures adopted were approved by the UEM Ethics Committee (no. 084-02/COBEA), and follow the norms recommended as international guiding principles for Biomedical Research Involving Animals (CIMS), Geneva, 1985.

Plant material and extract

Kielmeyera coriacea was collected near Mogi Guaçu (São Paulo, Brazil) in July 1999 for analysis and identification and the species identification was performed by Dr. Maria Claudia Young of the Instituto Botânico de São Paulo. A voucher specimen (no. SP 298463) was deposited with the Herbarium of the State Botanical Institute, São Paulo, Brazil. Dried and crushed stems (1.0 kg) of Kielmeyera coriacea were exhaustively extracted with 38 L of ethanol/water (9:1) at room temperature for 7 days, yielding 167.3 g of extract after evaporation of the solvents and lyophilization as described in Goulart et al. (2007).

Isolation of the constituents

The lyophilized extract EH (10 g) obtained from Kielmeyera coriacea stems was submitted to vacuum-column chromatography to yield six fractions after the organic solvent was removed under vacuum at 40°C. The fractions were analyzed by HPLC-UV, and the dichloromethane fraction was purified by gel filtration columns on Sephadex LH-20 with isocratic organic solvent. The resulting fraction (42 g) was rechromatographed in silica gel (230–400 mesh) mesh successively, and the semi-pure compound (300 mg) was submitted to gel filtration or Sephadex LH-20 (CHCl₃–MeOH (50:50) and MeOH) to yield 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (146 mg) (Cortez et al., 1998).

HPLC analysis

The HPLC-UV analyses were performed on a Shimadzu SCL-10A system controller with a Shimadzu SIL-10A auto-injector with an injection volume of 20 μ l, a Shimadzu SPD-M10AVP spectrophotometer diode-array detector at 254 nm and two Shimadzu LC-10AD pumps with a flow of 1 ml/min. Data processing was carried out by a Class-LC10 version 1.41. The column was a MetaSil ODS (5 μ m, 150 × 4.6 mm); step gradient of acetonitrile-water (containing 0.05% TFA): 10% → 100% CH₃CN over 40 min.

Stereotaxic operations

Rats anaesthetized with sodium pentobarbital (Thiopental, 45 mg/kg intraperitoneally, Cristália) associated with local anesthesia (2% xylocaine with adrenaline) were positioned in the stereotaxic frame and cannulated using guide cannulas stereotaxically placed at the DRN. The stereotaxic coordinates from the Paxinos and Watson Atlas (1998) with reference to bregma for the tip of the 15 mm long stainless-steel cannula (0.6 mm external diameter) rested 0.2 mm above the DRN were: AP = −7.1 mm, L = 4.0 mm, V = 5.3 mm. The cannula was inserted into the DRN at an angle of 34° to the horizontal plane, to avoid penetration of the midline sinus and the brain aqueducts. The stainless-steel guide cannula was fixed to the skull with stainless-steel screws and self-polymerizing acrylate resin, and was closed with a stainless-steel wire to prevent obstruction. The animals were allowed to recover for 5–6 days before the experiments.

Treatments

The xanthone and (−)pindolol (RBI) were dissolved in a vehicle containing saline (0.9% NaCl) plus 2% Tween 80. (+)8-OH-DPAT hydrobromide (RBI) and WAY₁₀₀₆₃₅ (RBI), were dissolved in saline. The 5-HT_1A ligands, saline or vehicle were microinjected intra-DRN 10 min before the xanthone or vehicle. The animals were subjected to the behaviour tests 10 min after the last treatment. Drug doses were based on previous studies conducted in our laboratory.

Microinfusions

Microinjections were made in awake rats while they were gently handled by the experimenter. A 17-mm needle (0.3 mm outer diameter) was inserted into the guide cannula, so that it extended 0.2 mm beyond its tip. The microinjection needle was connected to a 10 μ L micro-syringe (Hamilton 71-RN) by a filled polyethylene tube. The polyethylene tube was filled either with the drugs or with the control substance by aspiration. Solutions were injected with a microinfusion pump (Insight) at a flow rate of 0.25 μ l/30 s. After each infusion the needle was left in place for 30 s before being removed, to allow the drug to be absorbed.

Forced swimming test (FST) and open field test (OFT)

Rats were individually forced to swim in an open cylindrical container (diameter 30 cm, height 60 cm), containing 45 cm of water at 25° ± 1°C. The test employed is essentially similar to that described by Porsolt et al. (1978), except for the water level. The water level is increased (to 45 cm) in order to increase the sensitivity of the test. The rats lack a sense of the water's depth, and their tails do not touch the bottom of the cylinder. This modification in the procedure is consistent with the practice of other authors (Detke & Lucki, 1996), and should be considered the present standard method. Animals were exposed to a pre-test for 15 min, 24 h prior to the 5 min swim test. Each animal was considered immobile when it ceased struggling and swimming, and remained floating in the water, moving only as necessary to keep its head above water. After the test, the animals were removed from the water, dried by the experimenter, and placed in cages. After 24 h, the same animals were individually placed in the OFT (40 × 40 × 40 cm), divided into 25 identical squares, for evaluation of locomotor activity measured by the total crossings number. After 30 s for habituation, the 5 min swim session in the FST or the 5 min locomotion session in the OFT was videotaped for subsequent measurement of the time of immobility or the crossings number (four feet placed in the same square) by a trained observer was recorded. Two fluorescent lights provided diffuse overhead illumination (200 lux at the level of the arena).

Data analysis

Results are expressed as the mean ± SEM for each group. The immobility time in the FST and the motor activity in the OFT were measured in seconds and in crossings number, respectively. Data were evaluated by one-way or by two-way ANOVA for dose-effect curves or associated treatments, respectively, followed by unequal Tukey's test. Differences between groups were considered significant if p ≤ 0.05.

Results

Table 1 shows the effect of the (−)pindolol (0.2, 0.4, or 0.8 nmol), WAY₁₀₀₆₃₅ (0.2, 0.4, or 0.8 nmol) and (+)8-OH-DPAT (0.6, 0.8 or 1.0 nmol) on immobility time in the FST and on the crossings number in the OFT compared to the respective control treated groups. The different doses of (−)pindolol, (F_(3.32) = 3.43, p = 0.030) or WAY₁₀₀₆₃₅ (F_(3.38) = 0.11, p = 0.95) did not alter, whereas (+)8-OH-DPAT (1.0 nmol) significantly increased (p < 0.05) immobility time (F_(3.33) = 4.71, p = 0.008). The crossings number was not significantly altered by any treatment.

Table 1.  Effect of the 5-HT_1A ligands or control microinjected intra DRN of the rats.

Compound (nmol)	Immobility time (s) in FST	Number of crossings in OFT
(−)Pindolol
C	198.9 ± 15.31	110.8 ± 7.06
0.2	222.2 ± 9.734	131.0 ± 8.61
0.4	178.0 ± 13.50	117.1 ± 19.49
0.8	202.1 ± 14.70	125.5 ± 4.80
WAY100635
C	204.8 ± 10.54	116.4 ± 8.82
0.2	209.9 ± 6.12	1364 ± 11.76
0.4	210.9 ± 9.15	144.5 ± 12.24
0.8	205.2 ± 11.72	137.2 ± 10.08
(+)8-OH-DPAT
C	204.8 ± 10.54	116.4 ± 8.82
0.6	205.0 ± 5.73	108.5 ± 13.59
0.8	215.4 ± 9.68	107.1 ± 15.07
1.0	242.5 ± 4.31^a	96.00 ± 9.19

Data are means ± SEM of immobility time (s) and number of crossings in the OFT for each group (n = 7–16) in rats microinjected (0.25 μ l/30 s) intra DRN with 5-HT_1A ligands or saline 10 min before vehicle. Significant differences calculated by Tukey's test shows ^ap ≤ 0.05. C, Control group (saline + vehicle).

Table 2 shows that intra-DRN microinjections (0.25 μ l/30 s) of xanthone (0.3, 0.6, 0.9 pmol) did not induce significant changes on immobility time (F_(3.38) = 0.298, p = 0.83) in the FST or on crossings number in the OFT (F_(3.38) = 0.87; p = 0.47) compared to the treated control group (saline + vehicle).

Table 2.  Effect of the xanthone or control microinjected intra DRN of the rats.

Xanthone (pmol)	Immobility time (s) in FST	Number of crossings in OFT
C	198.9 ± 15.31	111.0 ± 8.00
0.3	200.4 ± 10.87	111.4 ± 6.81
0.6	188.0 ± 15.87	96.67 ± 9.71
0.9	183.6 ± 20.78	121.1 ± 14.61

Data are means ± SEM of immobility time (s) and number of crossings in the OFT for each group (n = 7–16) in rats microinjected intra DRN (0.25 μ l/30 s) with saline 10 min before vehicle or xanthone (0.3, 0.6, or 0.9 pmol). C, Control group (saline + vehicle). Tukey's test shows p > 0.05.

Table 3 illustrates the effect observed with associated treatment of WAY₁₀₀₆₃₅ (0.8 nmol) and (+)8-OH-DPAT (1.0 nmol) microinjected intra-DRN on immobility time in the FST. The two-way ANOVA showed no effect of (+)8-OH-DPAT (F_(1,34) = 0.02, p = 0.89), but a significant effect of WAY₁₀₀₆₃₅ (F_(1,34) = 10.69, p = 0.002) and WAY₁₀₀₆₃₅ X (+) 8-OH-DPAT interaction (F_(1,34) = 10.93, p = 0.002) on immobility time. Post hoc comparisons showed that WAY₁₀₀₆₃₅ blocked the increased immobility produced by (+)8-OH-DPAT (p < 0.05).

Table 3.  Effects of association of 8-OH-DPAT pretreated with WAY₁₀₀₆₃₅ both microinjected intra-DRN of rats.

	Saline	(+)8-OH-DPAT (1 nmol)
Vehicle	204.8 ± 12.13	242.5 ± 12.87
WAY100635 (0.8 nmol)	205.2 ± 11.51	164.3 ± 10.97^a

Data are means ± SEM of immobility time in the FST for each group (n = 7–16) in rats microinjected (0.25 μ l/30 s) intra-DRN with 8-OH-DPAT (1.0 nmol) or saline treated 10 min previously with vehicle or WAY₁₀₀₆₃₅ (0.8 nmol). C, Control group (saline + vehicle). ^ap ≤ 0.05 versus corresponding control group, according to Tukey's test.

Table 4 illustrates the effect observed with associated treatments of (−)pindolol (0.4 nmol), WAY₁₀₀₆₃₅ (0.4 and 0.8 nmol) or 8-OH-DPAT (0.6, 0.8 and 1.0 nmol) and xanthone (0.3 pmol) microinjected intra-DRN on immobility time in the FST. The two-way ANOVA showed no effect of (−)pindolol (F_(1,39) = 3.84, p = 0.057) treatment, but significant effect of xanthone (F_(1,39) = 11.89, p = 0.001) treatment, as well as a significant (−)pindolol X xanthone interaction (F_(3,39) = 12.62, p = 0.001) on immobility time in the FST. Post hoc comparisons detected that immobility time was significantly decreased by the association of (−)pindolol X xanthone (p < 0.05). In the associated treatment of WAY₁₀₀₆₃₅ and xanthone microinjected intra-DRN, the two-way ANOVA showed a significant effect of WAY₁₀₀₆₃₅ (F_(1,61) = 5.83, p = 0.004), significant effect of xanthone (F_(1.61) = 23.6, p = 0.00009) and significant WAY₁₀₀₆₃₅ X xanthone interaction (F_(1,61) = 7.56, p = 0.001). Post hoc comparisons showed a significant decrease on immobility time produced by WAY₁₀₀₆₃₅ (0.4 and 0.8 nmol) X xanthone (p < 0.05). In the associated treatment of (+)8-OH-DPAT (1.0 nmol) and xanthone (0.3 pmol), the two-way ANOVA showed a significant (+)8-OH-DPAT X xanthone interaction (F_(1,73) = 4.07, p = 0.010), but no significant effect of (+)8-OH-DPAT (F_(1,73) = 1.61, p = 0.19) or xanthone (F_(1,73) = 1.80, p = 0.183) on immobility time in the FST. Post hoc comparisons showed that xanthone blocked the increased immobility produced by higher dose of (+)8-OH-DPAT (p > 0.05) compared to (+)8-OH-DPAT (1.0 nmol) (p < 0.05). The two-way ANOVA did not shows significant effects on the immobility time with (+)8-OH-DPAT (0.6 or 0.8 nmol), (F_(1,34) = 1.68, p = 0.2) or (F_(1,38) = 1.77, p = 0.19) associated to the xanthone (0.3 pmol) in the FST (Table 4).

Table 4.  Effects of the association of the xanthone pretreated with 5-HT_1A ligands both microinjected intra-DRN of rats.

	Vehicle	Xanthone (0.3 pmol)
Saline	204.8 ± 11.70	205.9 ± 9.65
(−)Pindolol (0.4 nmol)	222.2 ± 12.45	145.6 ± 11.82^a
WAY100635 (0.4 nmol)	210.9 ± 12.24	120.0 ± 12.24^a,^b,^c
WAY100635 (0.8 nmol)	205.2 ± 12.24	154.8 ± 10.73^b,^c
8-OH-DPAT (0.6 nmol)	205.0 ± 10.79	225.4 ± 10.80
8-OH-DPAT (0.8 nmol)	199.1 ± 9.76	191.4 ± 9.76
8-OH-DPAT (1.0 nmol)	240.4 ± 10.79^a,^e	187.1 ± 11.45

Data are means ± SEM of immobility time in the FST for each group (n = 7–16) in rats microinjected (0.25 μ l/30 s) with vehicle or xanthone (0.3 pmol) treated 10 min previously with saline or (−)pindolol (0.4 nmol), WAY₁₀₀₆₃₅ (0.4 or 0.8 nmol) or 8-OH-DPAT (0.6, 0.8, or 1.0 nmol). C, Control group (saline + vehicle). Tukey's test shows: ^ap ≤ 0.05 versus corresponding on control group; ^bp ≤ 0.05 versus corresponding value on xantone; ^cp ≤ 0.05 versus corresponding value on WAY₁₀₀₆₃₅ (0.4 nmol); ^dp ≤ 0.05 versus corresponding value on WAY₁₀₀₆₃₅ (0.8 nmol); ^ep ≤ 0.05 versus corresponding value on associated treatment.

The two-way ANOVA detected no alterations in the crossings number evaluated in the OFT for all treatments (results not shown).

Discussion

Stressor models that involve inescapable foot shocks, restraint-induced stress, or the forced swimming test (FST) are able to modify the activity of serotonin (5-HT) and other neurotransmitter systems (Chaouloff et al., 1999). The FST consists in a primary rodent screening test for antidepressant drugs. It shows great sensitivity to different antidepressant classes and permits determination of the neurobiological mechanism underlying stress and antidepressant responses (Porsolt et al., 1977, 1978).

In our study, intra-DRN microinjection of different doses of 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone, 5-HT_1A/1B antagonist, (−)pindolol, or 5-HT_1A antagonist WAY₁₀₀₆₃₅ produced no alterations on immobility time or on crossings number, whereas the 5-HT_1A agonist (+)8-OH-DPAT, increased immobility time in the FST, without altering crossings number in the OFT.

The results on immobility time produced by WAY₁₀₀₆₃₅ and (−)pindolol agree with previously reported evidence using systemic administration, showing that WAY₁₀₀₆₃₅ (Tatarczynska et al., 2002) or (±)pindolol did not shorten immobility time in the FST, whereas 8-OH-DPAT reduced the immobility time in the FST (Redrobe et al., 1996), showing that this behavioral test has been relatively insensitive to the antagonist of 5-HT_1A receptors.

The 5-HT_1A selective agonist, (+)8-OH-DPAT, induces an anti-immobility effect in the FST, and other behavioral and physiological changes in rodents, including a characteristic 5-HT syndrome (Tricklebank et al., 1984), when systemically administered, acting through postsynaptic 5-HT_1A receptors (Luscombe et al., 1993).

In our experiments, the increase in the immobility time observed with intra-DRN microinjection of (+)8-OH-DPAT, suggest a reduction of the central serotoninergic function mediated by activation of the somatodendritic 5-HT_1A auto receptor, producing reduction of 5-HT release in innervated structures in the forebrain.

In fact, our results demonstrated that WAY₁₀₀₆₃₅, was effective in blocking the increase in immobility time produced by (+)8-OH-DPAT in the FST, and confirms the suggestion that the activation of the 5-HT_1A receptors by intra-DRN microinjection of (+)8-OH-DPAT reduced the endogenous 5-HT level in structures in the forebrain.

The association of the microinjected intra-DRN 5-HT_1A antagonists, (−)pindolol or WAY₁₀₀₆₃₅ and xanthone, significantly decreased immobility time in the FST.

5-HT_1A antagonists show ability to block and disinhibit 5-HT_1A somatodendritic receptors in the DRN. In vivo microdialysis studies have shown that intra-DRN or systemic administration of WAY₁₀₀₆₃₅, markedly potentiates the selective serotonin reuptake inhibitors (SSRIs), inducing an increase of extracellular 5-HT levels in forebrain areas (Hjorth et al., 1996; Romero et al., 1996a). Systemic administration of (−)pindolol attenuate, in dose-dependent fashion, the reduction of striatal extracellular 5-HT induced by DRN infusion of citalopram, and normalized the reduction in firing activity of DRN neurons produced by systemic administration of paroxetine (Romero et al., 1996b). Combined administration of (−)pindolol or WAY₁₀₀₆₃₅ can enhance the effect of antidepressant drugs in different animal models of depression (Detke et al., 1995; Dawson et al., 2000; Redrobe et al., 1996).

Our study showed that (−)pindolol and WAY₁₀₀₆₃₅ exerted antagonist-like properties on the 5-HT_1A receptors. By itself it produced no effect, but in association with xanthone it facilitated the anti-immobility effect on FST. This result suggests the level of the endogenous 5-HT is increased to a detectable level in the FST by the associated treatment with xanthone. In corroboration of this hypothesis, in our experiments xanthone reverted the increased immobility time in the FST produced by the 5-HT_1A receptor agonist (+)8-OH-DPAT.

The amplitude of the anti-immobility effect observed with the associations of (−)pindolol or WAY₁₀₀₆₃₅ and xanthone in the FST were similar to those obtained in our laboratory with 15-days intraperitoneal administration of the reference drugs, fluoxetine or nortriptiline (results not shown). The reduction in the immobility time was not influenced by an increase in stimulant effect of the drugs, because the anti-immobility effect on FST was observed at doses which do not affect the crossing number in the OFT.

Behavioral studies demonstrate that activation of 5-HT_1A receptors by intra-DRN 5-HT_1A agonists triggers inhibition of motility; whereas 5-HT_1A antagonists have the opposite effect, suggesting that both effects are mediated by 5-HT_1A receptors located in the DRN (Karakamus et al., 2004). However, in our experiments, no alteration in the crossings number or rearing, both indicative parameters of locomotor activity was detected in the OFT.

In conclusion, these results are a direct evidence that the activation/blockage of the 5-HT_1A receptors by intra-DRN microinjection of 5-HT_1A ligands could be detected using the FST and support the hypothesis that 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone interact with 5-HT_1A ligands in stress-related events observed in the FST. Xanthone could be a prototype drug to be used in the treatment of mood disorders such as depression. This pure constituent of HE from Kielmeyera coriacea stems, 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone, proved to be a prototype drug useful in mood disorders such as depression and anxiety.

Acknowledgements

The authors are thankful to Dra. Rosangela Santana of the Universidade Estadual de Maringá for the statistical support and to Marcos Alberto Trombeli for technical support. This study was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.