Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 39, Num. 2, 2007, pp. 75-79

Indian Journal of Pharmacology, Vol. 39, No. 2, March-April, 2007, pp. 75-79

Research Paper

Role of 5-HT _1A receptors in antidepressant-like effect of dichloromethane fraction of Kielmeyera coriacea in rats subjected to the forced swim test

Otobone FJ, Sela VR, Obici S, Moreira LY, Cortez DAG, Audi EA

Department of Pharmacy and Pharmacology, University of Maringá, PR
Correspondence Address:Department of Pharmacy and Pharmacology, University of Maringá, PR eaaudi@uem.br

Date of Submission: 20-Sep-2006
Date of Decision: 09-Apr-2007
Date of Acceptance: 11-Apr-2007

Code Number: ph07020

Abstract

Keywords: Depression, dorsal raphe nucleus, Kielmeyera coriacea, 5-HT _1A receptor

Kielmeyera coriacea Mart. (Clusiacea) is a Brazilian plant commonly known as pau santo . A decoction of the stems is used to treat several tropical diseases including schistosomasis, leishmaniasis, malaria and fungal and bacterial infections. ^[1] High performance liquid chromatography (HPLC) analysis of the hydroethanolic extract (HE) and the dichloromethane (DcM) semipurified fraction of K. coriacea stems detected the presence of xanthones, triterpenes and their biphenyl derivatives. ^[2],[3] Previous studies conducted in our laboratory showed that chronic administration of the HE and the DcM semipurified fraction of K. coriacea stems by gavage reduced the immobility time in rats subjected to the forced swimming test (FST) suggesting an antidepressant-like effect. ^[3],[4]

Drugs acting on the serotonin (5-HT, 5-hydroxytryptamine) system have been shown to be effective in the treatment of depressive disorders. ^[5] Somatodendritic 5-HT _1A autoreceptors located in the dorsal raphe nucleus (DRN) in the midbrain, play a particularly important role in this antidepressant activity. These receptors are considered to be the principal inhibitory regulators of 5-HT neuronal activity, playing an important role in the physiological control of the ascending 5-HT pathway. Stimulation of these 5-HT _1A receptors in the DRN reduces the firing of 5-HT neurons and produces an anti-5-HT effect through the reduction in 5-HT release. ^[6],[7],[8]

A delay of usually 2-4 weeks in the onset of antidepressant activity ^[5],[6] is attributed to activation of the inhibitory somatodendritic 5-HT _1A autoreceptors located in the DRN. This DRN somatodendritic 5-HT _1A autoreceptor-mediated inhibition of antidepressant activity can be induced by exogenously applied 5-HT, 5-HT _1A agonists or by acute administration of 5-HT re-uptake inhibitors. All these factors inhibit neuronal firing and consequently, decrease 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. ^[8]

Current strategies aim to improve the efficacy of antidepressant drugs including the co-administration of 5-HT _1A and / or 5-HT _1A/1B autoreceptor antagonists. These 5-HT antagonists can facilitate the antiimmobility effect of subeffective doses of antidepressant drugs in rats and mice in the FST ^[9] and thus, accelerate the effects of these drugs in patients with major depression. ^[10]

The FST is one of the most frequently used screening tests for antidepressant drugs in rodents. It shows great sensitivity to different antidepressant classes and permits determination of the neurobiological mechanisms underlying stress and antidepressant responses. Different classes of antidepressant drugs are effective in decreasing immobility time in the FST. ^[11]

In this study, we examined the involvement of 5-HT neurotransmission, particularly that mediated by somatodendritic 5-HT _1A autoreceptors in the antidepressant-like effect of the DcM semipurified fraction of a K. coriacea stem extract. We assessed the effects of chronic administration of the test substance (DcM fraction) alone or in combination with intraDRN microinjections of 5-HT1A/1B agonists or antagonists in rats subjected to the FST or open field test (OFT). An effective dose of the DcM fraction was given along with an intraDRN microinjection of the 5HT _1A agonist, (+) 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT) while a subactive dose of the DcM fraction was given in combination with either the selective 5-HT _1A antagonist, WAY ₁₀₀₆₃₅ or the 5HT _1A/1B/ β adrenergic antagonist, (-)-pindolol.

Materials and Methods

Animals

Male Wistar rats, each weighing 180-200 g (Central Biotιrio, University of Maringα-UEM) 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 freely available food and water. All experiments were carried out between 8:00 and 12:00 h. The experimental procedures adopted were approved by the UEM Ethics Committee (# 084-02/COBEA) and followed the norms recommended as international guiding principles for Biomedical Research Involving Animals (CIMS), Geneva, 1985.

Plant material, extract and DcM semipurified fraction

K. coriacea was collected near Mogi-Guaηu, Sγo Paulo, Brazil in July 1999. A voucher specimen (# SP 298-463) was deposited with the Herbarium of the State Botanical Institute, Sγo Paulo, Brazil. The species was identified by Dr. Maria Claudia Young of the same institution. Dried and crushed stems (1.0 kg) of K. coriacea were exhaustively extracted by maceration with 38 L of ethanol/water (9:1) at room temperature for seven days, yielding 167.3 g of extract after evaporation of the solvents and lyophilization. After lyophilization, the crude HE extract from K. coriacea stems (167.3 g) was stored in a freezer at -15°C. In order to assess the stability of the DcM fraction, comparative chromatographic analyses were conducted before chronic treatment of the animals. The HE extract (110.6 g) was submitted to vacuum column chromatography (208.3 g silica gel) and eluted with hexane (1000 mL), dichloromethane (1000 mL), ethyl acetate (1000 mL), acetone (1000 mL) and methanol/water (1000 mL), yielding five fractions (F1: 1.8 g), (F2: 28.6 g), (F3: 21.6 g), (F4: 11.1 g) and (F5: 29.5 g) respectively. [Patent application # 001342 with the National Patents Institute (INPI) on October 9, 2002].

Stereotaxic operations

Rats anaesthetized with sodium thiopental (Thiopentax ^® , 45 mg/kg i.p, Cristαlia) and local anesthetic agents (2% xylocaine + adrenaline) were positioned in the stereotaxic frame and cannulated using a guide cannula stereotaxically placed at the DRN. The stereotaxic coordinates were: AP = -7.1 mm, L = 4.0 mm, DV = 5.3 mm, from the Paxinos and Watson atlas ^[12] with reference to bregma for the tip of the 15 mm-long stainless-steel cannula (0.6 mm external diameter) positioned 0.2 mm above the DRN. 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 cannulas were fixed to the skull with stainless-steel screws and self-polymerising acrylate resin and were closed with a stainless-steel wire to prevent obstruction. The animals were allowed to recover for five or six days before the experiments.

Microinjections

The rats were given microinjections while they were awake. A 17-mm needle (0.3 mm outer diameter) was inserted into the guide cannula so that it extended 0.2 mm beyond the cannula tip. The microinjection needle was connected to a 10 µL microsyringe (Hamilton 71-RN, USA) through a filled polyethylene tube (PE 10). The polyethylene tube was filled either with the drugs or with the control substance by aspiration. Solutions were injected with a microinfusion pump (Insight Equipments, Brazil) at a flow rate of 0.2 µl/30 s. After each infusion, the needle was left in place for 30 s before being removed to allow the drug to be absorbed.

Treatments

The DcM fraction from K. coriacea stems was dissolved in a vehicle (water + 5% dimethylsulfoxide, DMSO). Solutions of (+)-8-OH-DPAT hydrobromide (Sigma-RBI), (-)-pindolol (Sigma-RBI) and WAY ₁₀₀₆₃₅ (Sigma-RBI) were all dissolved in saline. To investigate the effects of 5-HT _1A autoreceptors located intraDRN on immobility time in FST or on the number of crossings in the OFT, rats were chronically treated by gavage (45 days) with the vehicle or DcM or received an intraDRN microinjection of the 5-HT _1A ligands (WAY ₁₀₀₆₃₅ , (-)-pindolol or (+)-8-OH-DPAT) or saline. The doses of the DcM were based on previous studies conduced in our laboratory. ^[3] (+) 8-OH-DPAT, WAY ₁₀₀₆₃₅ and (-) pindolol doses used in our study were based in another studies. ^{[13],[14],[15],[16]}

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 water of 45 cm depth at 25 ± 1°C. The test employed is essentially similar to that described by Porsolt et al . ^[11] except for the water level. The water level has been increased (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 procedural modification is consistent with the practice of other authors ^[17] and should be considered the present standard method. Animals were exposed to a pretest for 15 min, 24 h prior to the 5 min swim test. Each animal was considered immobile when it ceased to struggle and swim and remained floating in the water, only moving 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 x 40 x 40 cm) square field, which was divided into 25 identical squares. The rats were observed to evaluate locomotor activity measured by the total number of crossings in the squares. After 30 s of 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 number of crossings (all four feet being moved from one square to be placed in another constituted one crossing) by a trained observer. Two fluorescent lights provided diffuse overhead illumination (200 lux at the level of the arena).

Histology

Histological verification of the injection sites was carried out at the end of the behavioral experiments. The animals were anaesthetized by the intraperitoneal route (i.p.) with sodium thiopental and their brains were perfused through the heart with saline followed by 10% formaldehyde solution before being removed for histological analysis. After fixation, the brains were sectioned into 50 µm-thick slices in the coronal plane on a freezing microtome. The sites of the injections in the DRN were determined by comparing the sections with the Paxinos and Watson atlas. ^[12] If the injection site was located outside the DRN (20.0%), the animal was discarded.

Data analysis

Results are expressed as the mean ± SE for each group. One-way or two-way analysis of variance (ANOVA) followed by an unequal Tukey's test for multiple comparisons, were used for the dose-effect or associated-treatments studies, respectively. Effects were considered statistically significant at P ≤ 0.05.

Results

[Table - 1] shows the effects of intraNDR microinjections of saline, (+) 8-OH-DPAT (0.10; 0.20 or 0.33 µg), (-) -pindolol (0.10, 0.20 or 0.40 µg) or WAY ₁₀₀₆₃₅ (0.11; 0.22 or 0.43 µg) on immobility time (s) in the FST and the crossings number in the OFT in rats chronically treated with vehicle (water + 5% DMSO). The 5-HT _1A agonist, (+) 8-OHDPAT (0.33 µg) significantly ( P < 0.05) increased immobility time in the FST. and signficantly decreased ( P < 0.05) the number of crossings in the OFT (F _(3.34) =4.7; P = 0.007). However, different doses of the 5-HT _1A antagonists, (-)-pindolol and WAY ₁₀₀₆₃₅ did not affect the immobility time in the FST (F _(3.38) = 1.7, P = 0.18) or (F _(3.28) = 0.6, P = 0.59) or the number of crossings in the OFT (F _(3.38) =0.9, P = 0.42) or (F _(3.28) =0.5, P = 0.67) respectively.

[Table - 2] shows the results of the treatment with saline or (+)-8-OH-DPAT (0.20 or 0.33 µg) in rats chronically treated with vehicle or the DcM fraction (5.0 mg/kg) in the FST. The two-way ANOVA showed an effect of (+)-8-OH-DPAT treatment (F _(2.52) = 25.0, P = 0.00001), of DcM treatment (F _(1.52) = 6.5, P = 0.01) and of (+)-8-OH-DPAT X DcM interaction (F _(2.52) = 6.76, P = 0.002) on immobility time. Post hoc Tukey's test detected that immobility time was significantly decreased by the DcM (5.0 mg/kg) and increased by (+)-8-OH-DPAT (0.33 µg) compared with the control group. The addition of each dose of (+)-8-OH-DPAT reversed the antiimmobility effect produced by the DcM.

[Table - 3] shows the results of the treatment with saline, (-)-pindolol (0.10 µg) or WAY ₁₀₀₆₃₅ (0.43 µg) in rats chronically treated with vehicle or a subactive dose of DcM (4.0 mg/kg) in the FST. The two-way ANOVA showed an effect of (-)-pindolol treatment (F _(1.30) = 8.6, P = 0.006) as well as a significant (-)-pindolol (0.10 µg) X DcM (4.0 mg/kg) interaction, (F _(1.30) = 6.3, P = 0.01), but not of DcM treatment (F _(1.30) = 0.4, P = 0.53) on immobility time. In post hoc comparisons, Tukey's test detected a significant reduction in immobility time produced by the combination of (-)-pindolol and a subactive dose of DcM when compared to just the subactive dose of DcM ( P < 0.05). The two way ANOVA detected a main effect of WAY ₁₀₀₆₃₅ treatment (F(1,30)=14.6, P =0.0006), the subactive dose of DcM (4.0 mg/kg), (F(1,30)=16.1, P =0.0004) and of the combination of WAY ₁₀₀₆₃₅ and DcM (F(1,30)=13, 8, P =0.0008) on immobility time in the FST on rats treated with a subactive dose of DcM (4.0 mg/kg). Post hoc Tukey`s comparisons showed a significant antiimmobility effect in rats exposed to the combination of WAY ₁₀₀₆₃₅ with DcM (4.0 mg/kg) as compared to the control, DcM- or WAY ₁₀₀₆₃₅ -treated groups.

[Table - 4] shows the response of the number of crossings in the OFT to treatment with saline or (+)-8-OH-DPAT (0.20 or 0.33 µg) in rats chronically treated with vehicle or DcM (5.0 mg/kg) in the OFT. The two-way ANOVA shows an effect of (+)-8-OH-DPAT treatment (F _(2.52) = 4.9, P = 0.01), but not of DcM treatment (F _(1,52) = 0.03, P = 0.87) or of (+)-8-OH-DPAT X DcM interaction (F _(2.52) = 2.6, P = 0.09) on the number of crossings. Post hoc Tukey`s comparisons indicated significant differences ( P < 0.05) between the (+)-8-OH-DPAT (0.20 µg)-treated group and (+)-8-OH-DPAT (0.30 µg)-treated group, showing a (+)-8-OH-DPAT effect on the number of crossings in the OFT.

[Table - 5] shows the effect of treatment of (-)-pindolol (0.10 µg) or WAY ₁₀₀₆₃₅ (0.43 µg) on the number of crossings in rats chronically treated with a subactive dose of DcM (4.0 mg/kg) and subjected to the OFT. The two-way ANOVA shows no significant effect of (-)-pindolol (F _(1.30) = 2.3, P = 0.14), of DcM treatment (F _(1.30) = 0.004, P = 0.95) or of the association of (-)-pindolol X DcM (F _(1.30) = 0.17, P = 0.68). The two-way ANOVA indicated no effect of WAY ₁₀₀₆₃₅ (F _(1.30) = 3,0, P = 0,09), of DcM (F _(1.30) = 0.04, P = 0.85) or of the association of WAY ₁₀₀₆₃₅ X DcM (F _(1.30) = 0.02, P = 0.89).

Discussion

This study demonstrates that the DcM semipurified fraction from K. coriacea extract stems was active in the FST model of depression in rats. This result confirms previous dose-response curves obtained in our laboratory. These dose-response curves established the lowest active dose for chronic treatment effects and determined whether chronic administration was effective at doses that were ineffective after acute or subacute treatment with the DcM fraction from K. coriacea.^[3] Thus, the DcM fraction was active in the FST when administered for 45 days at a dose of 5.0 mg/kg but not at a dose of 4.0 mg/kg. This antiimmobility effect of the DcM fraction does not reflect a general increase in motor activity because the same dose which induced an antidepressant-like effect did not increase locomotion in the OFT. The antiimmobility effect detected after chronic but not after acute or subacute treatment of DcM in the FST is consistent with other findings. ^[18]

Acute administration of selective 5-HT (serotonin) re-uptake inhibitors (SSRIs) and other antidepressant drugs induces an increase in the concentration of 5-HT in the vicinity of 5-HT cell bodies in midbrain raphe nuclei. It also triggers activation of 5-HT _1A somatodendritic autoreceptors, thus inhibiting the firing activity of the 5-HT neurons and reducing the release of 5-HT in the forebrain. ^[6],[7],[8] Studies have shown that intraraphe or systemic administration of 5-HT _1A antagonists, WAY ₁₀₀₆₃₅ or pindolol, markedly potentiates the activity of SSRIs. This potentiation is due to the blockade of activation of 5-HT _1A receptors by SSRIs, reducing negative feedback and resulting in increased 5-HT neuronal cell firing as well as inducing an increase of extracellular 5-HT levels in forebrain areas. ^{[19],[20],[21]} The combined administration of SSRI and pindolol has been found to accelerate the onset of antidepressant treatment in preclinical and clinical studies and / or to improve antidepressant therapy in resistant patients. ^[9],[10]

In the present study, significant potentiation of the antiimmobility effect of a subactive dose of the DcM fraction by pretreatment with the 5-HT _1A/1B/ βadrenergic antagonist (-)-pindolol or with the 5-HT _1A antagonist WAY ₁₀₀₆₃₅ in the FST clearly highlights the involvement of 5-HT _1A receptors in the antidepressant-like effect of the DcM fraction of K. coriacea. Pindolol is also known to block β-adrenoceptors but considering the reports of increased incidence of depressive disorders due to β-adrenergic blockers, it is unlikely that β-adrenoceptor inhibition enhances the efficacy of antidepressant drugs. ^[22] Studies show that agents capable of blocking 5-HT _1A autoreceptors including the mixed 5-HT _1A/1B/β-adrenergic receptor blockers and SSRIs, produce an enhanced antidepressant effect whereas β-adrenoceptor antagonists lacking 5-HT _1A receptor affinity do not enhance the antidepressant effect of SSRIs. ^[23] Despite differences in specificity between (-)-pindolol and WAY ₁₀₀₆₃₅ for 5-HT _1A receptors, the facilitation of an antidepressant-like effect of DcM by both compounds is probably due to the antagonist activity of the DcM fraction on 5-HT _1A receptors.

Our results are consistent with other reports ^[9],[10] where (-)-pindolol or WAY ₁₀₀₆₃₅ produced no alterations in immobility times in the FST or in the number of crossings in the OFT. Thus, our data showed that both (-)-pindolol and WAY ₁₀₀₆₃₅ exerted antagonist-like properties on 5-HT _1A receptors. Alone they produced no effect but when combined with a subactive dose of DcM, they facilitated its antidepressant-like effect in the FST through inhibition of 5-HT _1A somatodendritic autoreceptors in the DRN and enhanced release of 5-HT.

In our study, an intraDRN microinjection of (+)-8-OH-DPAT (0.33 µg) increased immobility time in the FST and reduced locomotion in the OFT. Both effects could be related to a reduced serotonergic function mediated by the activation of 5-HT _1A somatodendritic autoreceptors in the DRN. An intraDRN microinjection of (+)-8-OH-DPAT inhibits neuronal firing and reduces the release of 5-HT in innervated structures in the forebrain. ^[13] Behavioral studies demonstrate that activation of 5-HT _1A receptors by intraDRN 5-HT _1A agonists triggers the inhibition of motility whereas 5-HT _1A antagonists have the opposite effect. This suggests that both effects are mediated by 5-HT _1A receptors located in the DRN. ^[24]

The involvement of 5-HT _1A receptors in the antidepressant-like effect of the DcM fraction of K. coriacea , as well as in the increase in the immobility time produced by the (+) 8-OH-DPAT is confirmed by the annulation of the effects produced in the FST by each one in the combined treatment. However, the influence of 5-HT _1A agonists on the release of several neurotransmitters in the limbic regions of the brain has been shown in different studies and may be an alternative explanation for the results observed in our study. ^[8]

Despite the possible involvement of different neurotransmitters / receptors in the antidepressant-like effect of the combination of the active dose of the DcM fraction and (+)-8-OH-DPAT in the FST, (+)-8-OH-DPAT, the synergism observed with the subactive dose of the DcM fraction and (-)-pindolol or WAY ₁₀₀₆₃₅ , suggests the involvement of 5-HT _1A receptors. In conclusion, the DcM semipurified fraction of K. coriacea stem extract interacts with 5-HT _1A ligands and produces an antidepressant-like effect in the FST. Thus, the active principle(s) in the DcM semipurified fraction show promise for their use in the treatment of mood disorders such as depression.

Acknowledgements

References

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