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Golf protein levels in rat striatum are increased by chronic antidepressant administration and decreased by olfactory bulbectomy
Life Sciences 79 (2006) 462 – 468 www.elsevier.com/locate/lifescie
Golf protein levels in rat striatum are increased by chronic antidepressant administration and decreased by olfactory bulbectomy Hideki Taoka a , Takashi Hamamura a,b,⁎, Shiro Endo c , Masahito Miki d , Youmei Lee e , Shinji Miyata a , Kishio Toma f , Takeshi Ishihara a , Hidenori Sagara g , Yutaka Gomita g , Shigetoshi Kuroda a a
Department of Neuropsychiatry, Okayama Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Hamamura Clinic, 5-1-17 Ogawa, Kojima, Kurashiki 711-0911, Japan c Fujisawa Hospital, 383 Kozuka, Fujisawa, Kanagawa 251-8530, Japan d Takaoka Hospital, 5-3-8 Nishiimajuku, Himeji 670-0061, Japan e Mannari Hospital, Tanimannari, Okayama 700-0071, Japan f Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan g Hospital Pharmacy, Okayama University Hospital, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 4 October 2005; accepted 18 January 2006
Abstract There are many studies of the mechanisms of antidepressants; however, most of these studies were conducted on the hippocampus or frontal cortex. In the present study, we hypothesized that the nucleus accumbens and caudate/putamen might be major targets for antidepressant effects. Thus, we focused on Golf protein, a stimulant α-subunit of G protein that is coupled with the dopamine D1 receptor and specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) in the rat brain. We examined the effects of chronic administration of imipramine, fluvoxamine, maprotiline and, as a negative control, cocaine on the level of Golf protein in the rat striatum. We also examined the effect of olfactory bulbectomy. Chronic imipramine treatment (10 mg/kg for 2 or 4 weeks) significantly increased the level of Golf in the striatum (by 17% or 18%, respectively), although this increase was not apparent after only 1 week of treatment. The time course of these changes corresponded well to that of the clinical efficacy of imipramine. Chronic fluvoxamine and maprotiline treatment (20 mg/kg for 2 weeks) also significantly increased the level of Golf (by 9% and 25%, respectively), but cocaine did not alter it significantly. Bulbectomy decreased the Golf protein level by 9%. The increases in Golf protein after chronic administration of these three different classes of antidepressants and the decrease after bulbectomy suggest that Golf protein may play an important role in the antidepressant effect. © 2006 Elsevier Inc. All rights reserved. Keywords: G protein; Dopamine; Imipramine; SSRI; NRI; Bulbectomy; Depression; Motivation; Anhedonia; Nucleus accumbens
Introduction There have been many studies of the mechanisms of antidepressants with respect to individual neurotransmitters, particularly noradrenalin and serotonin [5-HT], and their ⁎ Corresponding author. Department of Neuropsychiatry, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 7008558, Japan. Tel.: +81 86 235 7242; fax: +81 86 235 7246. E-mail address: [email protected] (T. Hamamura). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.01.033
specific receptor subtypes, including noradrenalin β1, 5-HT2 and 5-HT1A (Vetulani and Sulser, 1975; Sulser et al., 1978; Bergstrom and Kellar, 1979; Blackshear and Sanders-Bush, 1982; Kendall and Nahorski, 1985; Blier and de Montigny, 1994). However, no consistent basis for their antidepressant effects has emerged. Recently, investigations of the mechanism of antidepressants have focused on the intracellular signaling pathways modulated by chronic antidepressant administration (Manji et al., 2001). Several lines of evidence suggest that longterm antidepressant treatments activate the G protein-related
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enzyme adenylyl cyclase, followed by activation of cAMP response element-binding protein (CREB), leading subsequently to an increase in brain-derived neurotrophic factor (BDNF). This increased BDNF results in neurogenesis in the rat hippocampus, and these modifications are thought to be involved in the mechanism of the antidepressive effect (Nibuya et al., 1996). Although the hippocampus is undoubtedly involved in depression, the nucleus accumbens is thought to be associated with depression as well (Eisch et al., 2003). The dopaminergic systems in the nucleus accumbens are believed to be involved in hedonia and reward (Salamone et al., 1997; Yadid et al., 2001), and anhedonia and loss of motivation are the core symptoms of depression (Willner, 1983; Naranjo et al., 2001). However, the involvement of the nucleus accumbens in mood regulation and depression is not well studied, and a systematic examination of the role of the nucleus accumbens in mood regulation is required (Nestler et al., 2002). Previously, by studying c-fos expression, we showed the nucleus accumbens and the caudate/putamen were a possible target region for the mood stabilizers lithium and carbamazepine (Lee et al., 1999, 2000). Recently, we also demonstrated that the shell of the nucleus accumbens was the only region in which both a selective serotonin reuptake inhibitor (SSRI) (fluoxetine) and a selective noradrenaline reuptake inhibitor (NRI) (reboxetine) increased Fos-immunoreactivity expression (Miyata et al., 2005); therefore, we considered that this region might be a major target for the antidepressant effects. Thus, we focused on intracellular transduction systems that are localized in the nucleus accumbens and the caudate/putamen. Golf is the stimulant α-subunit of G protein and is coupled with the dopamine D1 receptor (Herve et al., 1993; Zhuang et al., 2000; Corvol et al., 2001). Golf is specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) and olfactory systems in the rat brain and is about 10 times more abundant than Gs in the rat striatum (Herve et al., 1993). Recently, we showed that chronic lithium treatment increased Golf protein levels (Miki et al., 2001). We proposed that this protein might be involved in the modulation of mood, in particular in the mechanism of an antidepressive effect. To test this hypothesis, in the present study we examined the effects on the level of Golf protein in the rat striatum. First, we examined the effect of chronic administration of imipramine, a prototype of the classic tricyclic antidepressant. Next, we examined the effects of two different classes of antidepressants, a selective serotonin reuptake inhibitor, fluvoxamine, and a selective noradrenalin reuptake inhibitor, maprotiline, and as a negative control, cocaine, which is not an antidepressant but does inhibit monoamine reuptake. Last, we examined the effect of olfactory bulbectomy, an animal model of depression, on the level of Golf protein in the rat striatum.
Guidelines for Animal Experimentation of Okayama University Medical School. Male Wistar rats (Clea, Japan; initial weight 210–280 g) were used. They were housed two per cage and maintained under a controlled 12/12-h light/dark cycle (lights on at 0700 h) at a constant temperature (28 °C), with access to water and food ad libitum.
Materials and methods
The animals were killed by decapitation, and their brains were rapidly removed, frozen in powdered dry ice and stored at − 80 °C. The striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) was dissected out at − 20 °C. The striatum was homogenized in a sonicator in ice-cold radioimmunoprecipitation (RIPA) buffer [phosphate-buffered saline
Subjects All efforts were made to minimize animal suffering and the number of animals used in accordance with the
Imipramine administration Ten animals each were treated with either 3 mg/kg or 10 mg/ kg imipramine hydrochloride intraperitoneally (i.p.) once a day for 2 weeks. Imipramine hydrochloride (Sigma, USA) was dissolved in saline (0.9% NaCl) at the concentration of 3 mg/ml or 10 mg/ml. As a control group, 10 animals were injected with 1 ml/kg saline i.p. once a day for 2 weeks. Next, to test the time course of imipramine treatment, 10 animals each were treated with imipramine hydrochloride 10 mg/kg i.p. once a day for 1 or 4 weeks. As a control group, 10 animals each were treated with 1 ml/kg i.p. saline once a day for 1 or 4 weeks. SSRI, NRI and cocaine administration Ten animals each were injected with 20 mg/kg fluvoxamine (provided by Meiji Seika Kaisha Ltd.) i.p., 20 mg/kg maprotiline (Sigma) i.p. or 15 mg/kg cocaine i.p. once a day for 2 weeks. Fluvoxamine and maprotiline were dissolved in saline (0.9% NaCl) at 20 mg/ml, and cocaine was dissolved in saline at 15 mg/ml. As a control group, 10 animals were injected with saline 1 ml/kg i.p. once a day for 2 weeks. All animals were killed on the day following the final treatment with drugs or saline. Olfactory bulbectomy Twenty animals were bilaterally olfactory bulbectomized. As a control group, 20 animals were sham-operated. The olfactory bulbectomy followed the method of Gomita et al. (1984), and the method is described briefly. Animals were anaesthetized using pentobarbital (40 mg/kg i.p.), and two burr holes (approximately 2 mm in diameter, 8 mm anterior to the bregma) were made in the skull just above the bulbs. The olfactory bulbs were bilaterally aspirated by suction through the holes by means of a blunt hypodermic needle attached to a water pump, taking care not to damage the frontal cortex. Blood loss from the burr holes was prevented by filling them with a haemostatic sponge. The wound was sewn closed. Sham-operated rats were treated similarly, except that the olfactory bulbs were not removed. All animals were killed 14 days after operation. Western blotting
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(PBS), 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing 1% phenylmethylsulphonyl fluoride (Sigma, 10 mg/ml in isopropanol), 3% aprotinin (Sigma), 1% sodium orthovanadate (Sigma). We used 3 ml of RIPA buffer per gram of tissue. Homogenates were centrifuged at 15,000 rpm in a refrigerated microcentrifuge for 20 min at 4 °C, and the supernatant fluid was used for samples. Protein samples were mixed with an equal volume of Laemmli sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol) and boiled for 90 s. Samples were subjected to polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels (Bio-Rad, USA). Samples (5 μl) were applied to the wells. SDS-PAGE molecular weight standards (Bio-Rad), including ovalbumin, were also run on each gel as a standard. Electrophoresis was performed at 100 V for 90 min using SDS-PAGE transfer buffer (25 mM Tris, 0.1% SDS, 192 mM glycine). Electrophoretic transfer of proteins onto nitrocellulose membranes (Bio-Rad, USA) was carried out at 650 mA for 60 min in an Amersham wet blotting tank using a transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol). Membranes were blocked in Trisbuffered saline (TBS: 20 mM Tris, 500 mM sodium chloride, pH 7.5) containing 5% dried skim milk for 90 min at room temperature. Membranes were rinsed twice in Tween Trisbuffered saline (TTBS: 20 mM Tris, 500 mM sodium chloride, 0.05% Tween 20, pH 7.5) for 5 min each, and then incubated overnight at room temperature with the primary antibody (Santa Cruz Biotechnology, USA; rabbit anti-Golf, cat. no. sc-385, diluted to 1:3000). They were then rinsed in TTBS buffer twice for 5 min each, and further incubated with a second antibody (Amersham, UK; anti-rabbit IgG, diluted to 1:5000) for 90 min
Fig. 2. Western blotting of Golf protein after imipramine hydrochloride treatment. Golf was detected as two bands. The Golf bands in the group treated with 10 mg/kg/day imipramine hydrochloride for 2 weeks were denser than those of the control group.
at room temperature. Membranes were rinsed once in TTBS buffer for 15 min and four times for 5 min each, incubated with ECL reagent (Amersham) for 60 s, and band images were acquired by Chemi Doc XRS (Bio-Rad). The bands detected by ECL were analyzed quantitatively using Quantity One (BioRad). To examine the specificity of each detected band for Western blotting, a 10-fold excess of blocking peptide was added to the primary antibody. Golf was detected in two bands (Figs. 1 and 2). Each band of Golf was completely abolished by the blocking peptide (Fig. 1), so we combined the two bands for statistical analysis. We adjusted the Golf protein level on different gels using the ratio relative to ovalbumin (45 kDa). The protein level of Golf was quantified as a percentage of the control. Statistical analysis The non-parametric Mann–Whitney U-test was performed on the densities of the groups treated with antidepressants or bulbectomized and the controls. The significance level was set at P < 0.05. To evaluate the dose effect of imipramine hydrochloride, the non-parametric Kruskal–Wallis test was performed on the density of each dose group (3 mg/kg for 2 weeks and 10 mg/kg for 2 weeks) and the control group, and followed by Mann–Whitney U-test to compare each group. The significance level was set at P < 0.05. All data are presented here as the mean ± S.E.M. Results Effects of imipramine hydrochloride treatment on Golf protein levels
Fig. 1. Western blotting of Golf protein and molecular weight marker. A: primary antibody. B: primary antibody with the addition of a 10-fold excess of blocking peptide. Each band of Golf was completely abolished by the blocking peptide. The molecular weight of the Golf protein is 42 kDa (Herve et al., 1993). The 45 kDa-band molecular weight marker is ovalbumin, which was used as a standard to compare the density on different gels.
The level of Golf protein in the group treated with 10 mg/kg/ day imipramine hydrochloride for 2 weeks increased Golf protein levels by 17% compared with levels in the control group (P < 0.05) (Fig. 2). On the other hand, the level of Golf protein in the group treated with 3 mg/kg/day of imipramine did not differ significantly from that of the control group. The protein levels in the imipramine hydrochloride-treated groups, expressed as a
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percentage of the control ± S.E.M., were 98.9 ± 4.0% for treatment with 3 mg/kg/day for 2 weeks, and 117 ± 5.8% for 10 mg/kg/day for 2 weeks. Time course The level of Golf protein in the group treated with imipramine hydrochloride for 1 week (10 mg/kg/day) did not differ significantly from that of the control group. In contrast, 2 weeks of treatment increased Golf protein, and the increased levels of Golf protein were sustained in animals treated for 4 weeks (by 18% compared with those in the control group; P < 0.05). The protein levels in the imipramine hydrochloridetreated groups, expressed as a percentage of the control ± S.E.M., were 89.3 ± 4.2% at 1 week, 117 ± 5.8% at 2 weeks and 118 ± 6.0% at 4 weeks (Fig. 3). Effects of SSRI, NRI or cocaine administration on Golf protein levels All antidepressants increased the level of Golf protein significantly. Two weeks' treatment with 20 mg/kg/day fluvoxamine increased the Golf protein level by 8.9% compared with the level in the control group (P < 0.05), and 20 mg/kg/day maprotiline increased it by 25% (P < 0.01) (Fig. 4). Cocaine administration did not affect the Golf protein level significantly compared with that of the control group (Fig. 4). The Golf protein levels expressed as a percentage of the control ± S.E.M. were 109 ± 3.8% for fluvoxamine, 125 ± 5.2% for maprotiline and 97.5 ± 3.3% for cocaine. Effects of olfactory bulbectomy on Golf protein levels The level of Golf protein in the striatum of olfactory bulbectomized rats was decreased significantly. Bulbectomy
Fig. 3. Time course of the effect of imipramine hydrochloride treatment on Golf protein levels. The levels of Golf protein in the group treated with imipramine hydrochloride for 1 week did not differ significantly from those in the control group. However, the levels of Golf protein in the group treated for 2 weeks increased by 17% compared with those in the control group (P < 0.05). The increased levels of Golf protein were sustained in the group treated for 4 weeks, having increased by 18% compared with those in the control group (P < 0.05). Significant changes are indicated by an asterisk (P < 0.05).
Fig. 4. The effect of SSRI, NRI and cocaine treatment and bulbectomy on Golf protein levels. The levels of Golf protein in the group treated with fluvoxamine for 2 weeks increased by 9% compared with those in the control group (P < 0.05). The levels of Golf protein treated with maprotiline also increased by 25% (P < 0.01). However, the levels of Golf protein treated with cocaine did not differ significantly. The levels of Golf protein after bulbectomy decreased by 8.7% (P < 0.05). Significant changes are indicated by asterisks (*P < 0.05; **P < 0.01).
decreased the Golf protein level by 9% compared with the level in the control group (P < 0.05) (Fig. 4). The Golf protein levels expressed as a percentage of the control ± S.E.M. were 91.3 ± 2.7%. Discussion In the present study, we clearly demonstrated that chronic imipramine administration (2 or 4 weeks) increased the levels of Golf protein in the striatum (nucleus accumbens, caudate/ putamen and olfactory tubercle). This increase was not observed after 1 week of imipramine treatment. The time course corresponded well to that of the clinical efficacy of imipramine in individuals with depression. Several studies have examined the effect of imipramine treatment on the Gproteins Gs, Gi and Go, but the results have been inconsistent (Duman et al., 1989; Lesch et al., 1991; Lesch and Manji, 1992). However, the effect on Golf had not been examined to date. This is the first report on the effect of imipramine treatment on Golf levels in the rat striatum. We also demonstrated that chronic administration of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), or maprotirine, a selective noradrenalin reuptake inhibitor (NRI), increased the level of Golf protein. The increase of Golf protein in the striatum after chronic administration of these three different classes of antidepressants strongly suggests that Golf might be associated with antidepressive effects. Golf is part of a major intracellular signal transduction system as the stimulant α-subunit of G-protein in the striatum, and is thought to be coupled to the dopamine D1 receptor (Herve et al., 1993). Recently a Golf knock-out mouse was developed (Belluscio et al., 1998). Studies of this mutant mouse have demonstrated that Golf is essential for D1stimulated cAMP production or c-fos expression in the
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striatum and D1 agonist-induced hyperactivity (Corvol et al., 2001; Zhuang et al., 2000). These lines of evidence suggest that Golf is functionally coupled with the D1 receptor in the striatum. In animal studies, chronic imipramine administration induced supersensitivity to dopamine agonists (Maj et al., 1984a,b; Rossetti et al., 1991); however, the effect on dopamine receptors was inconsistent (Nowak et al., 1991; Dziedzicka-Wasylewska et al., 1997a,b; Rogoz and Dziedzicka-Wasylewska, 1999). This discrepancy may be explained by the increase in Golf. That is, chronic imipramine treatment increases Golf protein levels, and this increase can lead to the augmentation of dopaminergic neural transmission. Additionally, chronic treatment with SSRIs or NRIs was reported to enhance the dopaminergic system of the nucleus accumbens. Chronic administration of desipramine, one of the NRIs, or citalopram, one of the SSRIs, enhances behavioural effects of dopamine injected into the nucleus accumbens (Maj et al., 1987; Plaznik and Kostowski, 1987). Hypermotility induced by apomorphine injection into the nucleus accumbens was enhanced by chronic desipramine or fluvoxamine treatment (Durlach-Misteli and Van Ree, 1992). Therefore we assume that chronic antidepressant administration may increase D1related G-protein Golf, and subsequently augment dopaminergic neural transmission in the striatum. Although noradrenalin and serotonin have attracted major attention, dopamine is also believed to be involved in the etiology or pathophysiology of depression (Randrup and Braestrup, 1977; Fibiger and Phillips, 1988; Dailly et al., 2004). The dopaminergic system is thought to have a significant role in the etiology of the core symptoms of depression, such as anhedonia and loss of motivation (Willner, 1983). Several studies have reported that the dopamine receptor agonists bromocryptine and pergolide are effective in treatment of depression (Nordin et al., 1981; Waehrens and Gerlach, 1981; Bouras and Bridges, 1982; Theohar et al., 1982; Sitland-Marken et al., 1990; Bouckoms and Mangini, 1993; Inoue et al., 1996; Izumi et al., 2000). On the other hand, drugs that either reduce dopamine levels, such as reserpine, or block dopamine receptors, such as neuroleptics, can induce either dysphoria or a depressed mood (Jimerson, 1987). In animal studies, selective dopamine D1 receptor agonists exerted an antidepressant-like effect in the behavioural despair animal model of depression, and a selective antagonist of dopamine D1 receptors antagonized the anti-immobility effect of imipramine in the same behavioural model (D'Aquila et al., 1994). Thus, because depression and dopamine have a close relationship, we postulate that the increase in Golf protein levels might result in augmentation of D1-related dopaminergic neural transmission to exert the antidepressant effect of antidepressants. On the other hand, cocaine, which can increase extracellular monoamine levels (Reith et al., 1997) but does not possess an antidepressant effect, did not increase Golf protein in this study. Perrine also reported 1, 3 or 14 days administration of cocaine did not change significantly the protein levels of Golf in the nucleus accumbens (Perrine et al., 2005). However, Crawford reported 6 or 7 days cocaine
treatments decreased Golf levels in the nucleus accumbens (Crawford et al., 2004). The reason of this discrepancy may be based on the difference in a medication period. Anyway, these results suggest that the mere quantitative increase of monoamine does not cause the increase of Golf. However, the mechanism of the increase of Golf in the rats treated with the three different classes of antidepressants remains unknown, and further studies are needed. The olfactory bulbectomized (OB) rat has been proposed as a very reliable animal model of depression (Cryan et al., 2002). In the OB rat model, the behavioral syndrome is reversed after chronic, but not acute, antidepressant treatment (Kelly et al., 1997); therefore, OB represents the pathophysiology of depression well. In the present study, we observed that OB decreased the levels of Golf protein in the striatum. This result supports our hypothesis that Golf is involved in the antidepressive effect. Conclusion Chronic imipramine, maprotiline and fluvoxamine treatment increased the levels of Golf protein in the rat striatum. Moreover, olfactory bulbectomy, an animal model of depression, decreased Golf protein. These lines of evidence strongly suggest that Golf is involved in the antidepressive effect. Acknowledgements This study was supported by research grants from the Mitsubishi Pharma Research Foundation (2002) and the Zikei Institute of Psychiatry. References Belluscio, L., Gold, G.H., Nemes, A., Axel, R., 1998. Mice deficient in Golf are anosmic. Neuron 20 (1), 69–81. Bergstrom, D.A., Kellar, K.J., 1979. Adrenergic and serotonergic receptor binding in rat brain after chronic desmethylimipramine treatment. The Journal of Pharmacology and Experimental Therapeutics 209 (2), 256–261. Blackshear, M.A., Sanders-Bush, E., 1982. Serotonin receptor sensitivity after acute and chronic treatment with mianserin. The Journal of Pharmacology and Experimental Therapeutics 221 (2), 303–308. Blier, P., de Montigny, C., 1994. Current advances and trends in the treatment of depression. Trends in Pharmacological Sciences 15 (7), 220–226. Bouckoms, A., Mangini, L., 1993. Pergolide: an antidepressant adjuvant for mood disorders? Psychopharmacology Bulletin 29 (2), 207–211. Bouras, N., Bridges, P.K., 1982. Bromocriptine in depression. Current Medical Research and Opinion 8 (3), 150–153. Corvol, J.C., Studler, J.M., Schonn, J.S., Girault, J.A., Herve, D., 2001. Gαolf is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum. Journal of Neurochemistry 76 (5), 1585–1588. Crawford, C.A., Choi, F.Y., Kohutek, J.L., Yoshida, S.T., McDougall, S.A., 2004. Changes in PKA activity and Gsα and Golfα levels after amphetamineand cocaine-induced behavioral sensitization. Synapse 51 (4), 241–248. Cryan, J.F., Markou, A., Lucki, I., 2002. Assessing antidepressant activity in rodents: recent developments and future needs. Trends in Pharmacological Sciences 23 (5), 238–245. D'Aquila, P.S., Collu, M., Pani, L., Gessa, G.L., Serra, G., 1994. Antidepressant-like effect of selective dopamine D1 receptor agonists in the behavioural despair animal model of depression. European Journal of Pharmacology 262 (1–2), 107–111.
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Golf protein levels in rat striatum are increased by chronic antidepressant administration and decreased by olfactory bulbectomy Hideki Taoka a , Takashi Hamamura a,b,⁎, Shiro Endo c , Masahito Miki d , Youmei Lee e , Shinji Miyata a , Kishio Toma f , Takeshi Ishihara a , Hidenori Sagara g , Yutaka Gomita g , Shigetoshi Kuroda a a
Department of Neuropsychiatry, Okayama Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Hamamura Clinic, 5-1-17 Ogawa, Kojima, Kurashiki 711-0911, Japan c Fujisawa Hospital, 383 Kozuka, Fujisawa, Kanagawa 251-8530, Japan d Takaoka Hospital, 5-3-8 Nishiimajuku, Himeji 670-0061, Japan e Mannari Hospital, Tanimannari, Okayama 700-0071, Japan f Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan g Hospital Pharmacy, Okayama University Hospital, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 4 October 2005; accepted 18 January 2006
Abstract There are many studies of the mechanisms of antidepressants; however, most of these studies were conducted on the hippocampus or frontal cortex. In the present study, we hypothesized that the nucleus accumbens and caudate/putamen might be major targets for antidepressant effects. Thus, we focused on Golf protein, a stimulant α-subunit of G protein that is coupled with the dopamine D1 receptor and specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) in the rat brain. We examined the effects of chronic administration of imipramine, fluvoxamine, maprotiline and, as a negative control, cocaine on the level of Golf protein in the rat striatum. We also examined the effect of olfactory bulbectomy. Chronic imipramine treatment (10 mg/kg for 2 or 4 weeks) significantly increased the level of Golf in the striatum (by 17% or 18%, respectively), although this increase was not apparent after only 1 week of treatment. The time course of these changes corresponded well to that of the clinical efficacy of imipramine. Chronic fluvoxamine and maprotiline treatment (20 mg/kg for 2 weeks) also significantly increased the level of Golf (by 9% and 25%, respectively), but cocaine did not alter it significantly. Bulbectomy decreased the Golf protein level by 9%. The increases in Golf protein after chronic administration of these three different classes of antidepressants and the decrease after bulbectomy suggest that Golf protein may play an important role in the antidepressant effect. © 2006 Elsevier Inc. All rights reserved. Keywords: G protein; Dopamine; Imipramine; SSRI; NRI; Bulbectomy; Depression; Motivation; Anhedonia; Nucleus accumbens
Introduction There have been many studies of the mechanisms of antidepressants with respect to individual neurotransmitters, particularly noradrenalin and serotonin [5-HT], and their ⁎ Corresponding author. Department of Neuropsychiatry, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 7008558, Japan. Tel.: +81 86 235 7242; fax: +81 86 235 7246. E-mail address: [email protected] (T. Hamamura). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.01.033
specific receptor subtypes, including noradrenalin β1, 5-HT2 and 5-HT1A (Vetulani and Sulser, 1975; Sulser et al., 1978; Bergstrom and Kellar, 1979; Blackshear and Sanders-Bush, 1982; Kendall and Nahorski, 1985; Blier and de Montigny, 1994). However, no consistent basis for their antidepressant effects has emerged. Recently, investigations of the mechanism of antidepressants have focused on the intracellular signaling pathways modulated by chronic antidepressant administration (Manji et al., 2001). Several lines of evidence suggest that longterm antidepressant treatments activate the G protein-related
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enzyme adenylyl cyclase, followed by activation of cAMP response element-binding protein (CREB), leading subsequently to an increase in brain-derived neurotrophic factor (BDNF). This increased BDNF results in neurogenesis in the rat hippocampus, and these modifications are thought to be involved in the mechanism of the antidepressive effect (Nibuya et al., 1996). Although the hippocampus is undoubtedly involved in depression, the nucleus accumbens is thought to be associated with depression as well (Eisch et al., 2003). The dopaminergic systems in the nucleus accumbens are believed to be involved in hedonia and reward (Salamone et al., 1997; Yadid et al., 2001), and anhedonia and loss of motivation are the core symptoms of depression (Willner, 1983; Naranjo et al., 2001). However, the involvement of the nucleus accumbens in mood regulation and depression is not well studied, and a systematic examination of the role of the nucleus accumbens in mood regulation is required (Nestler et al., 2002). Previously, by studying c-fos expression, we showed the nucleus accumbens and the caudate/putamen were a possible target region for the mood stabilizers lithium and carbamazepine (Lee et al., 1999, 2000). Recently, we also demonstrated that the shell of the nucleus accumbens was the only region in which both a selective serotonin reuptake inhibitor (SSRI) (fluoxetine) and a selective noradrenaline reuptake inhibitor (NRI) (reboxetine) increased Fos-immunoreactivity expression (Miyata et al., 2005); therefore, we considered that this region might be a major target for the antidepressant effects. Thus, we focused on intracellular transduction systems that are localized in the nucleus accumbens and the caudate/putamen. Golf is the stimulant α-subunit of G protein and is coupled with the dopamine D1 receptor (Herve et al., 1993; Zhuang et al., 2000; Corvol et al., 2001). Golf is specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) and olfactory systems in the rat brain and is about 10 times more abundant than Gs in the rat striatum (Herve et al., 1993). Recently, we showed that chronic lithium treatment increased Golf protein levels (Miki et al., 2001). We proposed that this protein might be involved in the modulation of mood, in particular in the mechanism of an antidepressive effect. To test this hypothesis, in the present study we examined the effects on the level of Golf protein in the rat striatum. First, we examined the effect of chronic administration of imipramine, a prototype of the classic tricyclic antidepressant. Next, we examined the effects of two different classes of antidepressants, a selective serotonin reuptake inhibitor, fluvoxamine, and a selective noradrenalin reuptake inhibitor, maprotiline, and as a negative control, cocaine, which is not an antidepressant but does inhibit monoamine reuptake. Last, we examined the effect of olfactory bulbectomy, an animal model of depression, on the level of Golf protein in the rat striatum.
Guidelines for Animal Experimentation of Okayama University Medical School. Male Wistar rats (Clea, Japan; initial weight 210–280 g) were used. They were housed two per cage and maintained under a controlled 12/12-h light/dark cycle (lights on at 0700 h) at a constant temperature (28 °C), with access to water and food ad libitum.
Materials and methods
The animals were killed by decapitation, and their brains were rapidly removed, frozen in powdered dry ice and stored at − 80 °C. The striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) was dissected out at − 20 °C. The striatum was homogenized in a sonicator in ice-cold radioimmunoprecipitation (RIPA) buffer [phosphate-buffered saline
Subjects All efforts were made to minimize animal suffering and the number of animals used in accordance with the
Imipramine administration Ten animals each were treated with either 3 mg/kg or 10 mg/ kg imipramine hydrochloride intraperitoneally (i.p.) once a day for 2 weeks. Imipramine hydrochloride (Sigma, USA) was dissolved in saline (0.9% NaCl) at the concentration of 3 mg/ml or 10 mg/ml. As a control group, 10 animals were injected with 1 ml/kg saline i.p. once a day for 2 weeks. Next, to test the time course of imipramine treatment, 10 animals each were treated with imipramine hydrochloride 10 mg/kg i.p. once a day for 1 or 4 weeks. As a control group, 10 animals each were treated with 1 ml/kg i.p. saline once a day for 1 or 4 weeks. SSRI, NRI and cocaine administration Ten animals each were injected with 20 mg/kg fluvoxamine (provided by Meiji Seika Kaisha Ltd.) i.p., 20 mg/kg maprotiline (Sigma) i.p. or 15 mg/kg cocaine i.p. once a day for 2 weeks. Fluvoxamine and maprotiline were dissolved in saline (0.9% NaCl) at 20 mg/ml, and cocaine was dissolved in saline at 15 mg/ml. As a control group, 10 animals were injected with saline 1 ml/kg i.p. once a day for 2 weeks. All animals were killed on the day following the final treatment with drugs or saline. Olfactory bulbectomy Twenty animals were bilaterally olfactory bulbectomized. As a control group, 20 animals were sham-operated. The olfactory bulbectomy followed the method of Gomita et al. (1984), and the method is described briefly. Animals were anaesthetized using pentobarbital (40 mg/kg i.p.), and two burr holes (approximately 2 mm in diameter, 8 mm anterior to the bregma) were made in the skull just above the bulbs. The olfactory bulbs were bilaterally aspirated by suction through the holes by means of a blunt hypodermic needle attached to a water pump, taking care not to damage the frontal cortex. Blood loss from the burr holes was prevented by filling them with a haemostatic sponge. The wound was sewn closed. Sham-operated rats were treated similarly, except that the olfactory bulbs were not removed. All animals were killed 14 days after operation. Western blotting
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(PBS), 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing 1% phenylmethylsulphonyl fluoride (Sigma, 10 mg/ml in isopropanol), 3% aprotinin (Sigma), 1% sodium orthovanadate (Sigma). We used 3 ml of RIPA buffer per gram of tissue. Homogenates were centrifuged at 15,000 rpm in a refrigerated microcentrifuge for 20 min at 4 °C, and the supernatant fluid was used for samples. Protein samples were mixed with an equal volume of Laemmli sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol) and boiled for 90 s. Samples were subjected to polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels (Bio-Rad, USA). Samples (5 μl) were applied to the wells. SDS-PAGE molecular weight standards (Bio-Rad), including ovalbumin, were also run on each gel as a standard. Electrophoresis was performed at 100 V for 90 min using SDS-PAGE transfer buffer (25 mM Tris, 0.1% SDS, 192 mM glycine). Electrophoretic transfer of proteins onto nitrocellulose membranes (Bio-Rad, USA) was carried out at 650 mA for 60 min in an Amersham wet blotting tank using a transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol). Membranes were blocked in Trisbuffered saline (TBS: 20 mM Tris, 500 mM sodium chloride, pH 7.5) containing 5% dried skim milk for 90 min at room temperature. Membranes were rinsed twice in Tween Trisbuffered saline (TTBS: 20 mM Tris, 500 mM sodium chloride, 0.05% Tween 20, pH 7.5) for 5 min each, and then incubated overnight at room temperature with the primary antibody (Santa Cruz Biotechnology, USA; rabbit anti-Golf, cat. no. sc-385, diluted to 1:3000). They were then rinsed in TTBS buffer twice for 5 min each, and further incubated with a second antibody (Amersham, UK; anti-rabbit IgG, diluted to 1:5000) for 90 min
Fig. 2. Western blotting of Golf protein after imipramine hydrochloride treatment. Golf was detected as two bands. The Golf bands in the group treated with 10 mg/kg/day imipramine hydrochloride for 2 weeks were denser than those of the control group.
at room temperature. Membranes were rinsed once in TTBS buffer for 15 min and four times for 5 min each, incubated with ECL reagent (Amersham) for 60 s, and band images were acquired by Chemi Doc XRS (Bio-Rad). The bands detected by ECL were analyzed quantitatively using Quantity One (BioRad). To examine the specificity of each detected band for Western blotting, a 10-fold excess of blocking peptide was added to the primary antibody. Golf was detected in two bands (Figs. 1 and 2). Each band of Golf was completely abolished by the blocking peptide (Fig. 1), so we combined the two bands for statistical analysis. We adjusted the Golf protein level on different gels using the ratio relative to ovalbumin (45 kDa). The protein level of Golf was quantified as a percentage of the control. Statistical analysis The non-parametric Mann–Whitney U-test was performed on the densities of the groups treated with antidepressants or bulbectomized and the controls. The significance level was set at P < 0.05. To evaluate the dose effect of imipramine hydrochloride, the non-parametric Kruskal–Wallis test was performed on the density of each dose group (3 mg/kg for 2 weeks and 10 mg/kg for 2 weeks) and the control group, and followed by Mann–Whitney U-test to compare each group. The significance level was set at P < 0.05. All data are presented here as the mean ± S.E.M. Results Effects of imipramine hydrochloride treatment on Golf protein levels
Fig. 1. Western blotting of Golf protein and molecular weight marker. A: primary antibody. B: primary antibody with the addition of a 10-fold excess of blocking peptide. Each band of Golf was completely abolished by the blocking peptide. The molecular weight of the Golf protein is 42 kDa (Herve et al., 1993). The 45 kDa-band molecular weight marker is ovalbumin, which was used as a standard to compare the density on different gels.
The level of Golf protein in the group treated with 10 mg/kg/ day imipramine hydrochloride for 2 weeks increased Golf protein levels by 17% compared with levels in the control group (P < 0.05) (Fig. 2). On the other hand, the level of Golf protein in the group treated with 3 mg/kg/day of imipramine did not differ significantly from that of the control group. The protein levels in the imipramine hydrochloride-treated groups, expressed as a
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percentage of the control ± S.E.M., were 98.9 ± 4.0% for treatment with 3 mg/kg/day for 2 weeks, and 117 ± 5.8% for 10 mg/kg/day for 2 weeks. Time course The level of Golf protein in the group treated with imipramine hydrochloride for 1 week (10 mg/kg/day) did not differ significantly from that of the control group. In contrast, 2 weeks of treatment increased Golf protein, and the increased levels of Golf protein were sustained in animals treated for 4 weeks (by 18% compared with those in the control group; P < 0.05). The protein levels in the imipramine hydrochloridetreated groups, expressed as a percentage of the control ± S.E.M., were 89.3 ± 4.2% at 1 week, 117 ± 5.8% at 2 weeks and 118 ± 6.0% at 4 weeks (Fig. 3). Effects of SSRI, NRI or cocaine administration on Golf protein levels All antidepressants increased the level of Golf protein significantly. Two weeks' treatment with 20 mg/kg/day fluvoxamine increased the Golf protein level by 8.9% compared with the level in the control group (P < 0.05), and 20 mg/kg/day maprotiline increased it by 25% (P < 0.01) (Fig. 4). Cocaine administration did not affect the Golf protein level significantly compared with that of the control group (Fig. 4). The Golf protein levels expressed as a percentage of the control ± S.E.M. were 109 ± 3.8% for fluvoxamine, 125 ± 5.2% for maprotiline and 97.5 ± 3.3% for cocaine. Effects of olfactory bulbectomy on Golf protein levels The level of Golf protein in the striatum of olfactory bulbectomized rats was decreased significantly. Bulbectomy
Fig. 3. Time course of the effect of imipramine hydrochloride treatment on Golf protein levels. The levels of Golf protein in the group treated with imipramine hydrochloride for 1 week did not differ significantly from those in the control group. However, the levels of Golf protein in the group treated for 2 weeks increased by 17% compared with those in the control group (P < 0.05). The increased levels of Golf protein were sustained in the group treated for 4 weeks, having increased by 18% compared with those in the control group (P < 0.05). Significant changes are indicated by an asterisk (P < 0.05).
Fig. 4. The effect of SSRI, NRI and cocaine treatment and bulbectomy on Golf protein levels. The levels of Golf protein in the group treated with fluvoxamine for 2 weeks increased by 9% compared with those in the control group (P < 0.05). The levels of Golf protein treated with maprotiline also increased by 25% (P < 0.01). However, the levels of Golf protein treated with cocaine did not differ significantly. The levels of Golf protein after bulbectomy decreased by 8.7% (P < 0.05). Significant changes are indicated by asterisks (*P < 0.05; **P < 0.01).
decreased the Golf protein level by 9% compared with the level in the control group (P < 0.05) (Fig. 4). The Golf protein levels expressed as a percentage of the control ± S.E.M. were 91.3 ± 2.7%. Discussion In the present study, we clearly demonstrated that chronic imipramine administration (2 or 4 weeks) increased the levels of Golf protein in the striatum (nucleus accumbens, caudate/ putamen and olfactory tubercle). This increase was not observed after 1 week of imipramine treatment. The time course corresponded well to that of the clinical efficacy of imipramine in individuals with depression. Several studies have examined the effect of imipramine treatment on the Gproteins Gs, Gi and Go, but the results have been inconsistent (Duman et al., 1989; Lesch et al., 1991; Lesch and Manji, 1992). However, the effect on Golf had not been examined to date. This is the first report on the effect of imipramine treatment on Golf levels in the rat striatum. We also demonstrated that chronic administration of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), or maprotirine, a selective noradrenalin reuptake inhibitor (NRI), increased the level of Golf protein. The increase of Golf protein in the striatum after chronic administration of these three different classes of antidepressants strongly suggests that Golf might be associated with antidepressive effects. Golf is part of a major intracellular signal transduction system as the stimulant α-subunit of G-protein in the striatum, and is thought to be coupled to the dopamine D1 receptor (Herve et al., 1993). Recently a Golf knock-out mouse was developed (Belluscio et al., 1998). Studies of this mutant mouse have demonstrated that Golf is essential for D1stimulated cAMP production or c-fos expression in the
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striatum and D1 agonist-induced hyperactivity (Corvol et al., 2001; Zhuang et al., 2000). These lines of evidence suggest that Golf is functionally coupled with the D1 receptor in the striatum. In animal studies, chronic imipramine administration induced supersensitivity to dopamine agonists (Maj et al., 1984a,b; Rossetti et al., 1991); however, the effect on dopamine receptors was inconsistent (Nowak et al., 1991; Dziedzicka-Wasylewska et al., 1997a,b; Rogoz and Dziedzicka-Wasylewska, 1999). This discrepancy may be explained by the increase in Golf. That is, chronic imipramine treatment increases Golf protein levels, and this increase can lead to the augmentation of dopaminergic neural transmission. Additionally, chronic treatment with SSRIs or NRIs was reported to enhance the dopaminergic system of the nucleus accumbens. Chronic administration of desipramine, one of the NRIs, or citalopram, one of the SSRIs, enhances behavioural effects of dopamine injected into the nucleus accumbens (Maj et al., 1987; Plaznik and Kostowski, 1987). Hypermotility induced by apomorphine injection into the nucleus accumbens was enhanced by chronic desipramine or fluvoxamine treatment (Durlach-Misteli and Van Ree, 1992). Therefore we assume that chronic antidepressant administration may increase D1related G-protein Golf, and subsequently augment dopaminergic neural transmission in the striatum. Although noradrenalin and serotonin have attracted major attention, dopamine is also believed to be involved in the etiology or pathophysiology of depression (Randrup and Braestrup, 1977; Fibiger and Phillips, 1988; Dailly et al., 2004). The dopaminergic system is thought to have a significant role in the etiology of the core symptoms of depression, such as anhedonia and loss of motivation (Willner, 1983). Several studies have reported that the dopamine receptor agonists bromocryptine and pergolide are effective in treatment of depression (Nordin et al., 1981; Waehrens and Gerlach, 1981; Bouras and Bridges, 1982; Theohar et al., 1982; Sitland-Marken et al., 1990; Bouckoms and Mangini, 1993; Inoue et al., 1996; Izumi et al., 2000). On the other hand, drugs that either reduce dopamine levels, such as reserpine, or block dopamine receptors, such as neuroleptics, can induce either dysphoria or a depressed mood (Jimerson, 1987). In animal studies, selective dopamine D1 receptor agonists exerted an antidepressant-like effect in the behavioural despair animal model of depression, and a selective antagonist of dopamine D1 receptors antagonized the anti-immobility effect of imipramine in the same behavioural model (D'Aquila et al., 1994). Thus, because depression and dopamine have a close relationship, we postulate that the increase in Golf protein levels might result in augmentation of D1-related dopaminergic neural transmission to exert the antidepressant effect of antidepressants. On the other hand, cocaine, which can increase extracellular monoamine levels (Reith et al., 1997) but does not possess an antidepressant effect, did not increase Golf protein in this study. Perrine also reported 1, 3 or 14 days administration of cocaine did not change significantly the protein levels of Golf in the nucleus accumbens (Perrine et al., 2005). However, Crawford reported 6 or 7 days cocaine
treatments decreased Golf levels in the nucleus accumbens (Crawford et al., 2004). The reason of this discrepancy may be based on the difference in a medication period. Anyway, these results suggest that the mere quantitative increase of monoamine does not cause the increase of Golf. However, the mechanism of the increase of Golf in the rats treated with the three different classes of antidepressants remains unknown, and further studies are needed. The olfactory bulbectomized (OB) rat has been proposed as a very reliable animal model of depression (Cryan et al., 2002). In the OB rat model, the behavioral syndrome is reversed after chronic, but not acute, antidepressant treatment (Kelly et al., 1997); therefore, OB represents the pathophysiology of depression well. In the present study, we observed that OB decreased the levels of Golf protein in the striatum. This result supports our hypothesis that Golf is involved in the antidepressive effect. Conclusion Chronic imipramine, maprotiline and fluvoxamine treatment increased the levels of Golf protein in the rat striatum. Moreover, olfactory bulbectomy, an animal model of depression, decreased Golf protein. These lines of evidence strongly suggest that Golf is involved in the antidepressive effect. Acknowledgements This study was supported by research grants from the Mitsubishi Pharma Research Foundation (2002) and the Zikei Institute of Psychiatry. References Belluscio, L., Gold, G.H., Nemes, A., Axel, R., 1998. Mice deficient in Golf are anosmic. Neuron 20 (1), 69–81. Bergstrom, D.A., Kellar, K.J., 1979. Adrenergic and serotonergic receptor binding in rat brain after chronic desmethylimipramine treatment. The Journal of Pharmacology and Experimental Therapeutics 209 (2), 256–261. Blackshear, M.A., Sanders-Bush, E., 1982. Serotonin receptor sensitivity after acute and chronic treatment with mianserin. The Journal of Pharmacology and Experimental Therapeutics 221 (2), 303–308. Blier, P., de Montigny, C., 1994. Current advances and trends in the treatment of depression. Trends in Pharmacological Sciences 15 (7), 220–226. Bouckoms, A., Mangini, L., 1993. Pergolide: an antidepressant adjuvant for mood disorders? Psychopharmacology Bulletin 29 (2), 207–211. Bouras, N., Bridges, P.K., 1982. Bromocriptine in depression. Current Medical Research and Opinion 8 (3), 150–153. Corvol, J.C., Studler, J.M., Schonn, J.S., Girault, J.A., Herve, D., 2001. Gαolf is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum. Journal of Neurochemistry 76 (5), 1585–1588. Crawford, C.A., Choi, F.Y., Kohutek, J.L., Yoshida, S.T., McDougall, S.A., 2004. Changes in PKA activity and Gsα and Golfα levels after amphetamineand cocaine-induced behavioral sensitization. Synapse 51 (4), 241–248. Cryan, J.F., Markou, A., Lucki, I., 2002. Assessing antidepressant activity in rodents: recent developments and future needs. Trends in Pharmacological Sciences 23 (5), 238–245. D'Aquila, P.S., Collu, M., Pani, L., Gessa, G.L., Serra, G., 1994. Antidepressant-like effect of selective dopamine D1 receptor agonists in the behavioural despair animal model of depression. European Journal of Pharmacology 262 (1–2), 107–111.
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