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DARPP-32 expression in rat brain after electroconvulsive stimulation
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
DARPP-32 expression in rat brain after electroconvulsive stimulation Daniela V.F. Rosaa,b , Renan P. Souzaa,b , Bruno R. Souzaa,b , Bernardo S. Mottaa,b , Fernando Caetanoa,b , Luciano K. Jornadac , Gustavo Feierc , Marcus V. Gomezb , João Quevedoc , Marco A. Romano-Silvaa,b,⁎ a
Grupo de Pesquisa em Neuropsiquiatria Clínica e Molecular, Universidade Federal de Minas Gerais, Av Antonio Carlos 6627, Belo Horizonte 31270-901 Minas Gerais, Brazil b Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos 6627, Belo Horizonte 31270-901 Minas Gerais, Brazil c Laboratorio de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Although electroconvulsive therapy (ECT) has been used as a treatment for mental disorder
Accepted 17 August 2007
since 1930s, little progress has been made in the mechanisms underlying its therapeutic or
Available online 25 August 2007
adverse effects. The aim of this work was to analyze the expression of DARPP-32 (a protein with a central role in dopaminergic signaling) in striatum, cortex, hippocampus and
Keywords:
cerebellum of Wistar rats subjected to acute or chronic electroconvulsive stimulation (ECS).
Electroconvulsive therapy
Rats were submitted to a single stimulation (acute) or to a series of eight stimulations,
DARPP-32
applied one every 48 h (chronic). Animals were killed for collection of tissue samples at time
Electroconvulsive stimulation
zero, 0.5, 3, 12, 24 and 48 h after stimulation in the acute model and at the same time intervals
Depression
after the last stimulation in the chronic model. Our results indicated that acute ECS produces
Schizophrenia
smaller changes in the expression of DARPP-32 but, interestingly, chronic ECS increased transient expression of DARPP-32 in several time frames, in striatum and hippocampus, after the last stimulation. Results on the expression of proteins involved in signaling pathways are relevant for neuropsychiatric disorders and treatment, in particular ECT, and can contribute to shed light on the mechanisms related to therapeutic and adverse effects. © 2007 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Departamento de Farmacologia – ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos, 6627, 31270-901 Belo Horizonte-MG, Brazil. Fax: +5531 3499 2983. E-mail address: [email protected] (M.A. Romano-Silva). Abbreviations: BDNF, brain derived neurotrophic factor; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element binding protein; DA, dopamine; DARPP-32, dopamine and cAMP regulated phosphoprotein of Mr 32 kDa; D1R, dopamine receptor type 1; D2R, dopamine receptor type 2; ECS, electroconvulsive stimulation; ECT, electroconvulsive therapy; PI3K, phosphatidylinositide 3-kinase; PKA, cAMP-dependent protein kinase; PP1, protein phosphatase-1; Ser, serine; Thr, threonine; TrkB, tyrosine kinase receptor B; VEGF, vascular endothelial growth factor; 5-HTT, serotonin receptor; 5-HT1A, serotonin receptor type 1A; 5-HT2A, serotonin receptor type 2A 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.08.043
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1.
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Introduction
Electroconvulsive therapy (ECT) has been used as a treatment for mental disorders since 1930s. It is defined as a medical procedure in which a brief electrical stimulus is used to induce a cerebral seizure under controlled conditions (Rosen et al., 2003). Studies about ECT have progressed rapidly over the last 20 years, providing new insights into the mechanism of action, improving both its acute and long-term efficacy and decreasing cognitive problems associated with this treatment. Despite a large number of hypothesis on the mechanism of action of ECT have been proposed, it remains not well established. The main indications for ECT include depression, mania, catatonia and schizophrenia (Abrams, 1998). Particularly in the treatment of severe major depression, evidences for the effectiveness of ECT are clear and convincing (The UK ECT Review Group, 2003; Barichello et al., 2004). Evidence for dopaminergic system dysregulation in depression is supported by a variety of reports, ranging from studies of dopamine (DA) and DA-metabolite levels, to neuroimaging, histopathological and neuroendocrine studies. Specifically, a number of reports suggest not only that depression may be linked to abnormally low dopamine levels, but also that the severity of depression is inversely correlated to central nervous system DA metabolite levels (Papakostas, 2006). DARPP-32 (dopamine and cAMP regulated phosphoprotein of Mr 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum. When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on Thr34, it is converted into a potent inhibitor of protein phosphatase-1 (PP-1). DARPP-32 Thr34 phosphorylation leads to an increase in the state of phosphorylation of downstream PP1 substrates, including several neurotransmitter receptors and voltage-gated ion channels (Greengard et al., 1999). In addition to Thr34, DARPP-32 is phosphorylated at Thr75 by cyclin-dependent kinase 5 (Cdk-5). DARPP-32 phosphorylated at Thr75 inhibits PKA activity and thereby reduces the efficacy of DA signaling (Bibb et al., 1999). Svenningsson et al. (2006) showed that acute administration of fluoxetine in mice produced changes in phosphorylation of DARPP-32 in prefrontal cortex, hippocampus and striatum, while chronic treatment, in addition to phosphorylation changes, increased levels of DARPP-32 mRNA and protein. It has also been recently shown that the expression of DARPP-32 was downregulated in post-mortem brains of patients with schizophrenia (Albert et al., 2002). Electroconvulsive stimulation (ECS) was shown to have many effects in experimental animals and those findings contributed toward a better understanding of the therapeutic and side effects of ECT (Newman et al., 1998). To our knowledge, no studies have examined the effect of acute or chronic ECS on DARPP-32 expression. To demonstrate changes in the protein expression profile after acute or chronic ECS, we analyzed DARPP-32 levels in the striatum, cortex, hippocampus and cerebellum of Wistar rats. Our results showed that acute ECS induced small changes in the expression of DARPP32 but, interestingly, chronic ECS induced a sustained increase of the expression of DARPP-32 for several time frames after the last stimulation.
2.
Results
2.1.
Effect of ECS on DARPP-32 expression in striatum
DARPP-32 expression in the striatum of rats submitted to acute and chronic ECS was examined. Acute ECS did not show changes in expression levels of DARPP-32 during the time analyzed (Fig. 1A). DARPP-32 expression increased after chronic ECS at 3, 12 and 24 h when compared to sham and time zero groups (p b 0.05). After 48 h, DARPP-32 expression returned to basal (Fig. 1B).
2.2.
Effect of ECS on DARPP-32 expression in cortex
Twenty-four hours after acute ECS, DARPP-32 expression was increased compared to the sham group (p b 0.05) (Fig. 2A). No difference was observed in DARPP-32 levels after chronic ECS (Fig. 2B).
2.3.
Effect of ECS on DARPP-32 expression in hippocampus
DARPP-32 expression was not altered in the hippocampus after acute stimulation (Fig. 3A). The greatest increase of DARPP-32 levels (1888 ± 180%), after chronic stimulation, was at time zero (p b 0.05) when compared to sham group. When compared to time zero, differences were decreased in 30 min (338 ± 18%), 3 h (860 ± 112%), 12 h (676 ± 101%), 24 h (559 ± 2%) and 48 h (1339 ± 29%) (Fig. 3B).
2.4.
Effect of ECS on DARPP-32 expression in cerebellum
Acute or chronic ECS did not show alteration in DARPP-32 expression in cerebellum (Figs. 4A and B).
3.
Discussion
ECS affects several brain regions, particularly hippocampus, frontal cortex, neostriatum, entorhinal cortex, temporalparietal cortex and several monoaminergic nuclei that project into these areas (Fochtmann, 1994). It is known that sine wave ECT can lead to memory deficits and attention/executive functions deterioration (Fujita et al., 2006). DARPP-32 seems to be related to these processes (Heyser et al., 2000). Acute ECS increased DARPP-32 levels in the cerebral cortex 24 h after stimulation. In all other regions examined (striatum, hippocampus and cerebellum), no differences were observed. Chronic ECS induced more significant changes in DARPP-32 expression in striatum and especially in the hippocampus. It has been proposed that the great benefits of ECT are derived from chronic treatment and repeated sessions in the clinical practice (Thienhaus et al., 1990). Guitary and Nestler (1992) demonstrated that regulation of DARPP-32 immunoreactivity was induced by chronic administration of lithium; however, it was not observed in several other examined brain regions. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in DARPP-32 levels in frontal cortex. Increased levels of DARPP-32 could reflect a common
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with ECT, the molecular mechanism seems to be different. In our experimental model, we have not studied the expression of DARPP-32 in the isolated prefrontal cortex, but instead we measured protein expression in the whole cortex. This might be the reason why we only observed, except a transient increase after acute ECS, negligible changes in DARPP-32 expression in the cerebral cortex following ECS.
Fig. 1 – DARPP-32 expression in rat striatum after acute and chronic electroconvulsive stimulation. Western blots and densitometry analyses of DARPP-32 relative levels in extract prepared from rat striatum after acute (A) and chronic ECS (B). Changes were noted in DARPP-32 levels after chronic ECS. For all densitometry analyses, results are presented in arbitrary units normalized by actin. Data represent means±SD. for duplicates of n=5. *Different from sham (control) (p<0.05) and **different from 0 h (p<0.05); analyses of ANOVA.
functional effect on frontal cortex of long-term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs. Although these pharmacological agents share therapeutic application
Fig. 2 – Expression of DARPP-32 in cortex after electroconvulsive stimulation. DARPP-32 levels of rat cortex after acute (A) and chronic ECS (B). In acute ECS, DARPP-32 increased only after 24 h when compared to sham group. Values are expressed as means ± SD (n = 5 for each group). *Different from sham (control) (p < 0.05) analyses of ANOVA.
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Fig. 3 – DARPP-32 expression in rat hippocampus after acute and chronic electroconvulsive stimulation. Western blots and densitometry analyses of DARPP-32 relative levels in extract prepared from rat hippocampus after acute (A) and chronic ECS (B). The greatest increase in this region was in DARPP-32 levels after chronic stimulation. DARPP-32 increased at time 0 when compared to sham group, and decreased at 30 min, 0, 3, 12, 24 and 48 h when compared to time zero. Levels returned to basal at 48 h. For all densitometry analyses, results are presented in arbitrary units normalized by actin. Data represent means ± SD for duplicates of n = 5. ). *Different from sham (control) (p < 0.05) and **different from 0h (p < 0.05); analyses of ANOVA.
Fig. 4 – Expression of DARPP-32 in cerebellum after electroconvulsive stimulation. Levels of rat cerebellum after acute (A) and chronic ECS (B). For all densitometry analysis, results are presented in arbitrary units normalized by actin. Data represent means ± SD. for duplicates of n = 5.
Prefrontal cortex and striatum receive serotonergic and dopaminergic converging afferent fibers. It was previously shown that ECS has effects on the serotonergic system: increased serotonergic transmission in the hypothalamus via desensitization of 5-HT1A autoreceptors; decreased serotonin transporter 5-HTT mRNA expression; and/or increased 5-HT2A receptor by acute
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and/or chronic ECS protocols. These data could partially explain the efficiency of ECT on medication-resistant depression (Dremencov et al., 2002; Gur et al., 2002; Shen et al., 2001). In addition, regulation of DARPP-32 phosphorylation induced by serotonin was also previously shown (Svenningsson et al., 2002), which was mediated primarily via activation of 5-HT4 and 5-HT6, regulating DARPP-32 phosphorylation at Thr34 and Thr75, whereas regulation at Ser137 was mediated primarily by 5-HT2 receptors. These three pathways appeared to inhibit PP1 through synergistic mechanisms. Yoshida et al. (1998, 1997) investigated the effect of repeated ECS on extracellular concentrations of DA in the frontal cortex of rats, using in vivo microdialysis, and concluded that the ECS effect was greater in the striatum than in the frontal cortex. Our results showed transient increase of DARPP-32 expression in striatum after chronic ECS. It has been reported that dopaminergic pathways are able to enhance DARPP-32 expression and Nomikos et al. (1991) showed interstitial striatal DA elevation up to 1310% of baseline when ECS was administered. In cultured embryonic mouse striatal cells, dopamine was shown to regulate the expression of BDNF mRNA and protein through activation of D1 receptor (Kuppers and Beyer, 2001). BDNF belongs to the neurotrophin family and interacts with the high-affinity tyrosine kinase receptor B (TrkB). Previous studies have shown that both acute and chronic ECS enhance the expression of BDNF and TrkB in rat brain (Nibuya et al., 1995; Zetterstrom et al., 1998; Conti et al., 2007; Ploski et al., 2006). Furthermore, functional cAMP-responsive element (CRE) site was very recently identified in the promoter III of human BDNF, which participates in dopamine modulation of BDNF expression induced via D1R (Nibuya et al., 1996; Fang et al., 2003) and it has also been shown that CREB and its phosphorylated state were increased after ECS (Yagasaki et al., 2006). Chronic ECS induces significant alterations in hippocampus. Zis et al. (1992) showed an increase in homovanilic acid, a DA metabolite, in hippocampus. Interestingly, seizure activity induced by the convulsant agent flurothyl did not influence hippocampus dialysate DA concentrations, suggesting that the ECS-induced DA release was related to the passage of current and not to the seizure activity (Zis et al., 1991). Converging evidence point to hippocampal neurogenesis as an important factor in the pathophysiology of depression and as target of antidepressants (Dranovsky and Hen, 2006). ECS has been shown to have robust effects in rat hippocampus trophic factors such as the induction of BDNF/TrkB/MAP kinase pathway, VEGF and other regulators of neurogenesis and angiogenesis (Newton et al., 2003, 2006; Altar et al., 2004). Thus, the ECS-dependent induction of DARPP-32 could be mediated by activation of PI3K via BDNF acting through its tyrosine kinase receptor (TrkB) (Stroppolo et al., 2001). Such regulation of DARPP-32 expression was also found in other normal and neoplastic tissues (El-Rifai et al., 2002; Belkhiri et al., 2005; Garcia-Jimenez et al., 2005; Hansen et al., 2006; Souza et al., 2006a,b), linking the expression and phosphorylation of DARPP-32 to cell survival and proliferation. We observed marked changes in DARPP-32 expression in the hippocampus after chronic ECS, with a high increase just after the last session of ECS. Although the expression levels are kept significantly higher when compared to sham shock,
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the difference between 0 and 30 min was striking. We have ruled out possible artifacts since the data were obtained from five different animals with small variability among them, as shown by the standard deviation. Although there is no supporting data in the literature regarding synthesis and turnover of DARPP-32, we could speculate that application of the last session on a animal that was already primed by the anterior sessions would produce a peak due to the presence of larger amounts of mRNA; however, the turnover machinery would quickly reduce DARPP-32 to levels that are still higher than those observed in sham shock animals. We are performing experiments involving parallel mRNA and protein quantification in order to further clarify that observation. Although cerebellum has been classically identified as a region involved in motor coordination there is increasing evidence that it plays an important role in higher cognitive functions such as memory, learning and attention (Leiner et al., 1989). Our data showed that ECS was not able to produce changes in DARPP-32 protein levels in cerebellum. In conclusion, our results showed that ECS was able to change transient expression of DARPP-32, especially in the hippocampus, which has a strategic role in signaling pathways relevant to the pathophysiology and treatment of several psychiatric disorders, where the use of ECT is evidencebased. The induction of DARPP-32 affects several different downstream pathways such as those involving receptors, ion channels and transcription factors (Greengard, 2001). Further experiments are needed to shed more light on the role of DARPP-32 and other mechanisms linked to signaling pathways related to the biological effects of neuropsychiatric treatments such as ECT.
4.
Experimental procedures
4.1.
Animals and study design
Adult male Wistar rats (250–300 g) were caged in groups of five with food and water ad libitum and were maintained on a 12 h-light-dark cycle (lights on at 7 AM), at a temperature of 23 ± 1 °C. Experiments were performed between 2 and 5 PM. The rats were divided into two groups: acute and chronic treatment. In the acute treatment, animals received a single ECS. In the chronic treatment group, animals received eight ECS every other day. In both groups, they were killed by decapitation at different time points after the last ECS, i.e., time 0, 0.5, 3, 12, 24 and 48 h after stimulation (n = 5 animals per group). The hippocampus, striatum, total cortex and cerebellum were immediately dissected out after decapitation and stored at − 80 °C for posterior analysis. In vivo studies were performed in accordance with the National Institutes of Health guidelines and with the approval of the Ethics Committee from Universidade do Extremo Sul Catarinense.
4.2.
Electroconvulsive stimulation
ECS was applied via bilateral ear clip electrodes (Barichello et al., 2004). The stimulus parameters were 150 V, 60 Hz, sine wave, during 2 s. Each stimulation elicited tonic–clonic seizures. The
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sham groups (acute and chronic) were handled identically to the ECS-treated rats except no current was applied. This protocol closely resembles ECT in clinical practice, either in number of sessions or in stimulation intensity.
4.3.
Preparation of tissue sample
Frozen tissue samples of different brain areas were sonicated in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 0.5 mM sodium orthovanadate, 2 mM okadaic acid, 10% glycerol, 1% Nonidet P40, 2% protease inhibitor) for 90 s (1 s on, 9 s off) and were kept on ice throughout the procedure until centrifugation at 13,000×g for 20 min at 4 °C. Supernatants were transferred to plastic tubes and protein was quantified (Bradford, 1976).
4.4.
Immunoblotting
Equal amounts of protein (100 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE 15%) and transferred to nitrocellulose membranes (Towbin et al., 1979). The membranes were sequentially immunoblotted using antibodies raised against DARPP-32 (H-62, 1:500 dilution, Santa Cruz Biotechnology) and actin (1:2000 dilution, Chemicon International,) for 2 h. Antibody binding was revealed by incubation with a goat anti-rabbit horseradish peroxidase-linked IgG (1:20000 dilution, Molecular Probes,) or a goat anti-mouse horseradish peroxidase-linked IgG (1:7000 dilution, Molecular Probes) for 1 h, and the ECL immunoblotting detection system (Amersham Biosciences). Chemiluminescense was detected by autoradiography using Kodak autoradiography film and apparent bands of DARPP-32 and actin were quantified by densitometry and analyzed using Scion Image version Beta 4.0.2 (Scion Image software Frederick, MO, USA). Samples from the sham group and groups exposed to ECS were run on the same immunoblots and then analyzed together. For each experiment, values obtained for DARPP-32 were corrected using actin values as a reference. Normalized data from multiple experiments were averaged and statistical analysis was carried out as described in the figure legends.
4.5.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA). Values are expressed as mean±SD (n=5 for each group). The sham (control) group was handled identical to the ECS-treated rats except that no current was applied. Differences were considered significant when pb 0.05.
Acknowledgments This research was supported by grants from CNPq, FAPESC, Instituto Cérebro e Mente and UNESC to J. Quevedo; MVG and MAR-S are CNPq research fellows and RPS, BSM and FSC are holders of CNPq studentships and DVFR and BRS are holders of CAPES studentships. Financial support from CNPq Universal grant proc. #471837/2004-0, Programa Institutos do Milênio/ CNPq/FINEP and FAPEMIG # CBB-453/04.
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Souza, B.R., Souza, R.P., Rosa, D.V.F., Guimarães, M.M., Correa, H., Romano-Silva, M.A., 2006a. Dopaminergic intracellular signal integrating proteins: relevance to schizophrenia. Dialogues Clin. Neurosci. 7 (4), 95–100. Souza, R.P., Rosa, D.V., Souza, B.R., Romano-Silva, M.A., 2006b. Darpp32. AfCS-Nature Molecule Pages. doi:10.1038/mp. a000752.01. Stroppolo, A., Guinea, B., Tian, C., Sommer, J., Ehrlich, M.E., 2001. Role of phosphatidylinositide 3-kinase in brain-derived neurotrophic factor-induced DARPP-32 expression in medium size spiny neurons in vitro. J. Neurochem. 79, 1027–1032. Svenningsson, P., Tzavara, E.T., Liu, F., Fienberg, A.A., Nomikos, G.G., Greengard, P., 2002. DARPP-32 mediates serotonergic neurotransmission in the forebrain. Proc. Natl. Acad. Sci. U. S. A. 99, 3188–3193. Svenningsson, P., Chergui, K., Rachleff, I., Flajolet, M., Zhang, X., Yaacoubi, M., Vaugeois, J.M., Nomikos, G., Greengard, P., 2006. Alterations in 5-HT1B receptor function by p11 in depression-like states. Science 311, 77–80. The UK ECT Review Group, 2003. Efficacy and safety of electroconvulsive therapy in depressive disorders: a systematic review and meta-analysis. The Lancet 361, 799–808. Thienhaus, O.J., Margletta, S., Bennet, J.A., 1990. A study of the clinical efficacy of maintenance ECT. J. Clin. Psychiatry 51, 141–144. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354. Yagasaki, Y., Numakawa, T., Kumamaru, E., Hayashi, T., Su, T., Kunugi, H., 2006. Chronic antidepressants potentiate via sigma-1 receptors the brain-derived neurotrophic factor-induced signaling for glutamate release. J. Biol. Chem. 281, 12941–12949. Yoshida, K., Higushi, H., Kamata, M., Yoshimoto, M., Shimizu, T., Hishikawa, Y., 1997. Dopamine releasing response in rat striatum to single and repeated electroconvulsive shock treatment. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 21, 707–715. Yoshida, K., Higushi, H., Kamata, M., Yoshimoto, M., Shimizu, T., Hishikawa, Y., 1998. Single and repeated electroconvulsive shocks activate dopaminergic and 5-hydroytryptaminergic neurotransmission in the frontal cortex of rats. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 22, 435–444. Zetterstrom, T.S., Pei, Q., Grahame-Smith, D.G., 1998. Repeated electroconvulsive shock extends the duration of enhanced gene expression for BDNF in rat brain compared with a single administration. Brain Res. Mol. Brain Res. 57, 106–110. Zis, A.P, Nomikos, G.G., Damsma, G., Fibiger, H.C., 1991. In vivo neurochemical effects of electroconvulsive shock studied by microdialysis in the rat striatum. Psychopharmacology 103, 343–350. Zis, A.P., Nomikos, G.G., Brown, E.E., Damsma, G., Fibiger, H.C., 1992. Neurochemical effects of electrically and chemically induced seizures: an in vivo microdialysis study in the rat hippocampus. Neuropsychopharmacology 7, 189–195.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
DARPP-32 expression in rat brain after electroconvulsive stimulation Daniela V.F. Rosaa,b , Renan P. Souzaa,b , Bruno R. Souzaa,b , Bernardo S. Mottaa,b , Fernando Caetanoa,b , Luciano K. Jornadac , Gustavo Feierc , Marcus V. Gomezb , João Quevedoc , Marco A. Romano-Silvaa,b,⁎ a
Grupo de Pesquisa em Neuropsiquiatria Clínica e Molecular, Universidade Federal de Minas Gerais, Av Antonio Carlos 6627, Belo Horizonte 31270-901 Minas Gerais, Brazil b Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos 6627, Belo Horizonte 31270-901 Minas Gerais, Brazil c Laboratorio de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Although electroconvulsive therapy (ECT) has been used as a treatment for mental disorder
Accepted 17 August 2007
since 1930s, little progress has been made in the mechanisms underlying its therapeutic or
Available online 25 August 2007
adverse effects. The aim of this work was to analyze the expression of DARPP-32 (a protein with a central role in dopaminergic signaling) in striatum, cortex, hippocampus and
Keywords:
cerebellum of Wistar rats subjected to acute or chronic electroconvulsive stimulation (ECS).
Electroconvulsive therapy
Rats were submitted to a single stimulation (acute) or to a series of eight stimulations,
DARPP-32
applied one every 48 h (chronic). Animals were killed for collection of tissue samples at time
Electroconvulsive stimulation
zero, 0.5, 3, 12, 24 and 48 h after stimulation in the acute model and at the same time intervals
Depression
after the last stimulation in the chronic model. Our results indicated that acute ECS produces
Schizophrenia
smaller changes in the expression of DARPP-32 but, interestingly, chronic ECS increased transient expression of DARPP-32 in several time frames, in striatum and hippocampus, after the last stimulation. Results on the expression of proteins involved in signaling pathways are relevant for neuropsychiatric disorders and treatment, in particular ECT, and can contribute to shed light on the mechanisms related to therapeutic and adverse effects. © 2007 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Departamento de Farmacologia – ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos, 6627, 31270-901 Belo Horizonte-MG, Brazil. Fax: +5531 3499 2983. E-mail address: [email protected] (M.A. Romano-Silva). Abbreviations: BDNF, brain derived neurotrophic factor; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element binding protein; DA, dopamine; DARPP-32, dopamine and cAMP regulated phosphoprotein of Mr 32 kDa; D1R, dopamine receptor type 1; D2R, dopamine receptor type 2; ECS, electroconvulsive stimulation; ECT, electroconvulsive therapy; PI3K, phosphatidylinositide 3-kinase; PKA, cAMP-dependent protein kinase; PP1, protein phosphatase-1; Ser, serine; Thr, threonine; TrkB, tyrosine kinase receptor B; VEGF, vascular endothelial growth factor; 5-HTT, serotonin receptor; 5-HT1A, serotonin receptor type 1A; 5-HT2A, serotonin receptor type 2A 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.08.043
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1.
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Introduction
Electroconvulsive therapy (ECT) has been used as a treatment for mental disorders since 1930s. It is defined as a medical procedure in which a brief electrical stimulus is used to induce a cerebral seizure under controlled conditions (Rosen et al., 2003). Studies about ECT have progressed rapidly over the last 20 years, providing new insights into the mechanism of action, improving both its acute and long-term efficacy and decreasing cognitive problems associated with this treatment. Despite a large number of hypothesis on the mechanism of action of ECT have been proposed, it remains not well established. The main indications for ECT include depression, mania, catatonia and schizophrenia (Abrams, 1998). Particularly in the treatment of severe major depression, evidences for the effectiveness of ECT are clear and convincing (The UK ECT Review Group, 2003; Barichello et al., 2004). Evidence for dopaminergic system dysregulation in depression is supported by a variety of reports, ranging from studies of dopamine (DA) and DA-metabolite levels, to neuroimaging, histopathological and neuroendocrine studies. Specifically, a number of reports suggest not only that depression may be linked to abnormally low dopamine levels, but also that the severity of depression is inversely correlated to central nervous system DA metabolite levels (Papakostas, 2006). DARPP-32 (dopamine and cAMP regulated phosphoprotein of Mr 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum. When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on Thr34, it is converted into a potent inhibitor of protein phosphatase-1 (PP-1). DARPP-32 Thr34 phosphorylation leads to an increase in the state of phosphorylation of downstream PP1 substrates, including several neurotransmitter receptors and voltage-gated ion channels (Greengard et al., 1999). In addition to Thr34, DARPP-32 is phosphorylated at Thr75 by cyclin-dependent kinase 5 (Cdk-5). DARPP-32 phosphorylated at Thr75 inhibits PKA activity and thereby reduces the efficacy of DA signaling (Bibb et al., 1999). Svenningsson et al. (2006) showed that acute administration of fluoxetine in mice produced changes in phosphorylation of DARPP-32 in prefrontal cortex, hippocampus and striatum, while chronic treatment, in addition to phosphorylation changes, increased levels of DARPP-32 mRNA and protein. It has also been recently shown that the expression of DARPP-32 was downregulated in post-mortem brains of patients with schizophrenia (Albert et al., 2002). Electroconvulsive stimulation (ECS) was shown to have many effects in experimental animals and those findings contributed toward a better understanding of the therapeutic and side effects of ECT (Newman et al., 1998). To our knowledge, no studies have examined the effect of acute or chronic ECS on DARPP-32 expression. To demonstrate changes in the protein expression profile after acute or chronic ECS, we analyzed DARPP-32 levels in the striatum, cortex, hippocampus and cerebellum of Wistar rats. Our results showed that acute ECS induced small changes in the expression of DARPP32 but, interestingly, chronic ECS induced a sustained increase of the expression of DARPP-32 for several time frames after the last stimulation.
2.
Results
2.1.
Effect of ECS on DARPP-32 expression in striatum
DARPP-32 expression in the striatum of rats submitted to acute and chronic ECS was examined. Acute ECS did not show changes in expression levels of DARPP-32 during the time analyzed (Fig. 1A). DARPP-32 expression increased after chronic ECS at 3, 12 and 24 h when compared to sham and time zero groups (p b 0.05). After 48 h, DARPP-32 expression returned to basal (Fig. 1B).
2.2.
Effect of ECS on DARPP-32 expression in cortex
Twenty-four hours after acute ECS, DARPP-32 expression was increased compared to the sham group (p b 0.05) (Fig. 2A). No difference was observed in DARPP-32 levels after chronic ECS (Fig. 2B).
2.3.
Effect of ECS on DARPP-32 expression in hippocampus
DARPP-32 expression was not altered in the hippocampus after acute stimulation (Fig. 3A). The greatest increase of DARPP-32 levels (1888 ± 180%), after chronic stimulation, was at time zero (p b 0.05) when compared to sham group. When compared to time zero, differences were decreased in 30 min (338 ± 18%), 3 h (860 ± 112%), 12 h (676 ± 101%), 24 h (559 ± 2%) and 48 h (1339 ± 29%) (Fig. 3B).
2.4.
Effect of ECS on DARPP-32 expression in cerebellum
Acute or chronic ECS did not show alteration in DARPP-32 expression in cerebellum (Figs. 4A and B).
3.
Discussion
ECS affects several brain regions, particularly hippocampus, frontal cortex, neostriatum, entorhinal cortex, temporalparietal cortex and several monoaminergic nuclei that project into these areas (Fochtmann, 1994). It is known that sine wave ECT can lead to memory deficits and attention/executive functions deterioration (Fujita et al., 2006). DARPP-32 seems to be related to these processes (Heyser et al., 2000). Acute ECS increased DARPP-32 levels in the cerebral cortex 24 h after stimulation. In all other regions examined (striatum, hippocampus and cerebellum), no differences were observed. Chronic ECS induced more significant changes in DARPP-32 expression in striatum and especially in the hippocampus. It has been proposed that the great benefits of ECT are derived from chronic treatment and repeated sessions in the clinical practice (Thienhaus et al., 1990). Guitary and Nestler (1992) demonstrated that regulation of DARPP-32 immunoreactivity was induced by chronic administration of lithium; however, it was not observed in several other examined brain regions. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in DARPP-32 levels in frontal cortex. Increased levels of DARPP-32 could reflect a common
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with ECT, the molecular mechanism seems to be different. In our experimental model, we have not studied the expression of DARPP-32 in the isolated prefrontal cortex, but instead we measured protein expression in the whole cortex. This might be the reason why we only observed, except a transient increase after acute ECS, negligible changes in DARPP-32 expression in the cerebral cortex following ECS.
Fig. 1 – DARPP-32 expression in rat striatum after acute and chronic electroconvulsive stimulation. Western blots and densitometry analyses of DARPP-32 relative levels in extract prepared from rat striatum after acute (A) and chronic ECS (B). Changes were noted in DARPP-32 levels after chronic ECS. For all densitometry analyses, results are presented in arbitrary units normalized by actin. Data represent means±SD. for duplicates of n=5. *Different from sham (control) (p<0.05) and **different from 0 h (p<0.05); analyses of ANOVA.
functional effect on frontal cortex of long-term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs. Although these pharmacological agents share therapeutic application
Fig. 2 – Expression of DARPP-32 in cortex after electroconvulsive stimulation. DARPP-32 levels of rat cortex after acute (A) and chronic ECS (B). In acute ECS, DARPP-32 increased only after 24 h when compared to sham group. Values are expressed as means ± SD (n = 5 for each group). *Different from sham (control) (p < 0.05) analyses of ANOVA.
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Fig. 3 – DARPP-32 expression in rat hippocampus after acute and chronic electroconvulsive stimulation. Western blots and densitometry analyses of DARPP-32 relative levels in extract prepared from rat hippocampus after acute (A) and chronic ECS (B). The greatest increase in this region was in DARPP-32 levels after chronic stimulation. DARPP-32 increased at time 0 when compared to sham group, and decreased at 30 min, 0, 3, 12, 24 and 48 h when compared to time zero. Levels returned to basal at 48 h. For all densitometry analyses, results are presented in arbitrary units normalized by actin. Data represent means ± SD for duplicates of n = 5. ). *Different from sham (control) (p < 0.05) and **different from 0h (p < 0.05); analyses of ANOVA.
Fig. 4 – Expression of DARPP-32 in cerebellum after electroconvulsive stimulation. Levels of rat cerebellum after acute (A) and chronic ECS (B). For all densitometry analysis, results are presented in arbitrary units normalized by actin. Data represent means ± SD. for duplicates of n = 5.
Prefrontal cortex and striatum receive serotonergic and dopaminergic converging afferent fibers. It was previously shown that ECS has effects on the serotonergic system: increased serotonergic transmission in the hypothalamus via desensitization of 5-HT1A autoreceptors; decreased serotonin transporter 5-HTT mRNA expression; and/or increased 5-HT2A receptor by acute
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and/or chronic ECS protocols. These data could partially explain the efficiency of ECT on medication-resistant depression (Dremencov et al., 2002; Gur et al., 2002; Shen et al., 2001). In addition, regulation of DARPP-32 phosphorylation induced by serotonin was also previously shown (Svenningsson et al., 2002), which was mediated primarily via activation of 5-HT4 and 5-HT6, regulating DARPP-32 phosphorylation at Thr34 and Thr75, whereas regulation at Ser137 was mediated primarily by 5-HT2 receptors. These three pathways appeared to inhibit PP1 through synergistic mechanisms. Yoshida et al. (1998, 1997) investigated the effect of repeated ECS on extracellular concentrations of DA in the frontal cortex of rats, using in vivo microdialysis, and concluded that the ECS effect was greater in the striatum than in the frontal cortex. Our results showed transient increase of DARPP-32 expression in striatum after chronic ECS. It has been reported that dopaminergic pathways are able to enhance DARPP-32 expression and Nomikos et al. (1991) showed interstitial striatal DA elevation up to 1310% of baseline when ECS was administered. In cultured embryonic mouse striatal cells, dopamine was shown to regulate the expression of BDNF mRNA and protein through activation of D1 receptor (Kuppers and Beyer, 2001). BDNF belongs to the neurotrophin family and interacts with the high-affinity tyrosine kinase receptor B (TrkB). Previous studies have shown that both acute and chronic ECS enhance the expression of BDNF and TrkB in rat brain (Nibuya et al., 1995; Zetterstrom et al., 1998; Conti et al., 2007; Ploski et al., 2006). Furthermore, functional cAMP-responsive element (CRE) site was very recently identified in the promoter III of human BDNF, which participates in dopamine modulation of BDNF expression induced via D1R (Nibuya et al., 1996; Fang et al., 2003) and it has also been shown that CREB and its phosphorylated state were increased after ECS (Yagasaki et al., 2006). Chronic ECS induces significant alterations in hippocampus. Zis et al. (1992) showed an increase in homovanilic acid, a DA metabolite, in hippocampus. Interestingly, seizure activity induced by the convulsant agent flurothyl did not influence hippocampus dialysate DA concentrations, suggesting that the ECS-induced DA release was related to the passage of current and not to the seizure activity (Zis et al., 1991). Converging evidence point to hippocampal neurogenesis as an important factor in the pathophysiology of depression and as target of antidepressants (Dranovsky and Hen, 2006). ECS has been shown to have robust effects in rat hippocampus trophic factors such as the induction of BDNF/TrkB/MAP kinase pathway, VEGF and other regulators of neurogenesis and angiogenesis (Newton et al., 2003, 2006; Altar et al., 2004). Thus, the ECS-dependent induction of DARPP-32 could be mediated by activation of PI3K via BDNF acting through its tyrosine kinase receptor (TrkB) (Stroppolo et al., 2001). Such regulation of DARPP-32 expression was also found in other normal and neoplastic tissues (El-Rifai et al., 2002; Belkhiri et al., 2005; Garcia-Jimenez et al., 2005; Hansen et al., 2006; Souza et al., 2006a,b), linking the expression and phosphorylation of DARPP-32 to cell survival and proliferation. We observed marked changes in DARPP-32 expression in the hippocampus after chronic ECS, with a high increase just after the last session of ECS. Although the expression levels are kept significantly higher when compared to sham shock,
39
the difference between 0 and 30 min was striking. We have ruled out possible artifacts since the data were obtained from five different animals with small variability among them, as shown by the standard deviation. Although there is no supporting data in the literature regarding synthesis and turnover of DARPP-32, we could speculate that application of the last session on a animal that was already primed by the anterior sessions would produce a peak due to the presence of larger amounts of mRNA; however, the turnover machinery would quickly reduce DARPP-32 to levels that are still higher than those observed in sham shock animals. We are performing experiments involving parallel mRNA and protein quantification in order to further clarify that observation. Although cerebellum has been classically identified as a region involved in motor coordination there is increasing evidence that it plays an important role in higher cognitive functions such as memory, learning and attention (Leiner et al., 1989). Our data showed that ECS was not able to produce changes in DARPP-32 protein levels in cerebellum. In conclusion, our results showed that ECS was able to change transient expression of DARPP-32, especially in the hippocampus, which has a strategic role in signaling pathways relevant to the pathophysiology and treatment of several psychiatric disorders, where the use of ECT is evidencebased. The induction of DARPP-32 affects several different downstream pathways such as those involving receptors, ion channels and transcription factors (Greengard, 2001). Further experiments are needed to shed more light on the role of DARPP-32 and other mechanisms linked to signaling pathways related to the biological effects of neuropsychiatric treatments such as ECT.
4.
Experimental procedures
4.1.
Animals and study design
Adult male Wistar rats (250–300 g) were caged in groups of five with food and water ad libitum and were maintained on a 12 h-light-dark cycle (lights on at 7 AM), at a temperature of 23 ± 1 °C. Experiments were performed between 2 and 5 PM. The rats were divided into two groups: acute and chronic treatment. In the acute treatment, animals received a single ECS. In the chronic treatment group, animals received eight ECS every other day. In both groups, they were killed by decapitation at different time points after the last ECS, i.e., time 0, 0.5, 3, 12, 24 and 48 h after stimulation (n = 5 animals per group). The hippocampus, striatum, total cortex and cerebellum were immediately dissected out after decapitation and stored at − 80 °C for posterior analysis. In vivo studies were performed in accordance with the National Institutes of Health guidelines and with the approval of the Ethics Committee from Universidade do Extremo Sul Catarinense.
4.2.
Electroconvulsive stimulation
ECS was applied via bilateral ear clip electrodes (Barichello et al., 2004). The stimulus parameters were 150 V, 60 Hz, sine wave, during 2 s. Each stimulation elicited tonic–clonic seizures. The
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sham groups (acute and chronic) were handled identically to the ECS-treated rats except no current was applied. This protocol closely resembles ECT in clinical practice, either in number of sessions or in stimulation intensity.
4.3.
Preparation of tissue sample
Frozen tissue samples of different brain areas were sonicated in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 0.5 mM sodium orthovanadate, 2 mM okadaic acid, 10% glycerol, 1% Nonidet P40, 2% protease inhibitor) for 90 s (1 s on, 9 s off) and were kept on ice throughout the procedure until centrifugation at 13,000×g for 20 min at 4 °C. Supernatants were transferred to plastic tubes and protein was quantified (Bradford, 1976).
4.4.
Immunoblotting
Equal amounts of protein (100 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE 15%) and transferred to nitrocellulose membranes (Towbin et al., 1979). The membranes were sequentially immunoblotted using antibodies raised against DARPP-32 (H-62, 1:500 dilution, Santa Cruz Biotechnology) and actin (1:2000 dilution, Chemicon International,) for 2 h. Antibody binding was revealed by incubation with a goat anti-rabbit horseradish peroxidase-linked IgG (1:20000 dilution, Molecular Probes,) or a goat anti-mouse horseradish peroxidase-linked IgG (1:7000 dilution, Molecular Probes) for 1 h, and the ECL immunoblotting detection system (Amersham Biosciences). Chemiluminescense was detected by autoradiography using Kodak autoradiography film and apparent bands of DARPP-32 and actin were quantified by densitometry and analyzed using Scion Image version Beta 4.0.2 (Scion Image software Frederick, MO, USA). Samples from the sham group and groups exposed to ECS were run on the same immunoblots and then analyzed together. For each experiment, values obtained for DARPP-32 were corrected using actin values as a reference. Normalized data from multiple experiments were averaged and statistical analysis was carried out as described in the figure legends.
4.5.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA). Values are expressed as mean±SD (n=5 for each group). The sham (control) group was handled identical to the ECS-treated rats except that no current was applied. Differences were considered significant when pb 0.05.
Acknowledgments This research was supported by grants from CNPq, FAPESC, Instituto Cérebro e Mente and UNESC to J. Quevedo; MVG and MAR-S are CNPq research fellows and RPS, BSM and FSC are holders of CNPq studentships and DVFR and BRS are holders of CAPES studentships. Financial support from CNPq Universal grant proc. #471837/2004-0, Programa Institutos do Milênio/ CNPq/FINEP and FAPEMIG # CBB-453/04.
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