- Email: [email protected]
Increased neurotrophin-3 in drug-free subjects with bipolar disorder during manic and depressive episodes
Journal of Psychiatric Research 44 (2010) 561–565
Contents lists available at ScienceDirect
Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires
Increased neurotrophin-3 in drug-free subjects with bipolar disorder during manic and depressive episodes Brisa Simões Fernandes a,b,c,d, Clarissa Severino Gama a,b,c,d, Julio C. Walz a,b,d, Keila Maria Ceresér a,b,d, Gabriel Rodrigo Fries b,d,e, Gabriela Colpo b, Bianca Aguiar b, Bianca Pfaffenseller b, Marcia Kauer-Sant’Anna a,b,d, Flavio Kapczinski a,b,c,d,* a
Bipolar Disorder Program, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Laboratory of Molecular Psychiatry, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Schizophrenia Program, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil d INCT for Translational Medicine, Brazil e Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil b c
a r t i c l e
i n f o
Article history: Received 1 November 2009 Received in revised form 21 November 2009 Accepted 26 November 2009
Keywords: Bipolar disorder NT-3 Biological marker Depression Mania Mood stabilizers
a b s t r a c t Bipolar disorder (BD) has been increasingly associated with abnormalities in neuroplasticity. Previous studies demonstrated that neurotrophin-3 (NT-3) plays a role in the pathophysiology of mood disorders. The influence of medication in these studies has been considered a limitation. Thus, studies with drugfree vs. medicated patients are necessary to evaluate the role of medication in serum NT-3 levels. About 10 manic and 10 depressive drug-free, and 10 manic and 10 depressive medicated patients with BD type I were matched with 20 controls for sex and age. Patients were assessed using SCID-I, YMRS and HDRS. Serum NT-3 levels in drug-free and medicated patients is increased when compared with controls (2.51 ± 0.59, 2.56 ± 0.44 and 1.97 ± 0.33, respectively, p < 0.001 for drug-free/medicated vs. control). Serum NT-3 levels do not differ between medicated and drug-free patients. When analyzing patients according to mood states, serum NT-3 levels are increased in both manic and depressive episodes, as compared with controls (2.47 ± 0.43, 2.60 ± 0.59 and 1.97 ± 0.33, respectively, p < 0.001 for manic/depressive patients vs. controls). There is no difference in serum BDNF between manic and depressive patients. Results suggest that increased serum NT-3 levels in BD are likely to be associated with the pathophysiology of manic and depressive symptoms. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Bipolar disorder (BD) is a highly disabling illness, characterized by the presence of manic and depressive symptoms (Yatham et al., 2009). Although a growing body of evidence strongly suggests that a neurobiological basis may underlie the pathophysiology of BD, its etiology is poorly understood. In recent years, many psychiatric disorders, particularly mood disorders such as BD, have been recognized as conditions that affect neurotrophins (Cunha et al., 2006; Gama et al., 2007; Walz et al., 2007; Kapczinski et al., 2008a,b; de Oliveira et al., 2009; Fernandes et al., 2009a; Kapczinski et al., 2009a,b). An emerging body of evidence links the expression of these neurotrophic factors to mood disorders. Brain-derived neurotrophic factor (BDNF) is so far the best studied member of this family, being put forward as a predictor of response to treat* Corresponding author. Address: Hospital de Clínicas de Porto Alegre, Av. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil. Tel.: +55 51 21018845. E-mail address: [email protected] (F. Kapczinski). 0022-3956/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2009.11.020
ment (Kapczinski et al., 2008a; Fernandes et al., 2009b; Kapczinski et al., 2009a; Tramontina et al., 2009) and as a possible biomarker of diagnosis to discriminate unipolar from bipolar depression (Fernandes et al., 2009a). Neurotrophin-3 (NT-3), an important member of the neurotrophin family, couples to the same signal transduction pathways as BDNF through their respective receptors. Decreased expression of these factors could lead to alterations in the structure and function of subpopulations of hippocampal neurons (Duman and Monteggia, 2006). In addition, it has been reported that NT-3 modulates basal synaptic transmission and long-term potentiation in rat hippocampus (Kato et al., 2003). There is a paucity of studies about NT-3 in peripheral serum. To our knowledge, there is only one report of serum NT-3 levels in BD, which shows that NT-3 is increased during both manic and depressive episodes, but not in euthymia (Walz et al., 2007). A question that frequently emerges in studies about serum neurotrophins in BD is the influence of medication use. This is considered to be an important limitation in most of the studies concerning neurotrophins. Little is known about whether the use
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B.S. Fernandes et al. / Journal of Psychiatric Research 44 (2010) 561–565
of medication in these studies alters their final results. Recently, we have shown that serum BDNF levels are similar in BD regarding subjects on and off medication (de Oliveira et al., 2009). As aforementioned, serum NT-3 data are scarce in the BD field; whether the use of mood stabilizers and antipsychotics plays or not a role in serum NT-3 levels in mood states of BD remains unclear. Therefore, there is a need for studies that compare serum NT-3 in patients on and off medication. The aim of this report is to investigate whether drug-free patients have differential levels of circulating serum NT-3 levels, when compared to medicated patients with BD. For this purpose, we evaluated serum NT-3 concentrations in medicated and drugfree BD type I during manic and depressive episodes.
2. Methods and materials
severity of manic and depressive episodes was evaluated using the Young Mania Rating Scale (YMRS) (Young et al., 1978) and Hamilton Depression Rating Scale (HDRS) (Hamilton, 1960), respectively. Twenty drug-free patients (10 manic and 10 depressed) were included in this study. This group did not receive any psychotropic medication for at least two weeks (five weeks if fluoxetine or depot medication was used) before blood sampling. The 20 medicated patients (10 manic and 10 depressed) and 20 controls were matched for age and gender to drug-free bipolar subjects. Controls were recruited from the Hospital de Clinicas de Porto Alegre catchment area. This group was screened with the non-patient version of the SCID to exclude current psychiatric morbidity. Those that had first-degree relatives with BD, schizophrenia or other psychiatric disorders were excluded. Controls were not on medication. All participants gave a written informed consent before entering the study, which was approved by the local ethics committee.
2.1. Subjects and measurement 2.2. Biochemical measurement Manic and depressive drug-free and medicated patients with BD type I were recruited from the Bipolar Disorders Program and Psychiatry Inpatient Unit – Hospital de Clinicas de Porto Alegre and Pronto Atendimento da Vila Cruzeiro do Sul, Porto Alegre, Brazil, between 2006 and 2008. To be included in this study, patients had to present with a manic or depressive episode according to the Diagnostic and Statistical Manual of Mental Disorders – Fourth Edition (DSM-IV) and Structured Clinical Interview for DSM-IV-Axis I Disorders (SCID-I) criteria. The diagnosis of manic and depressive episodes was established according to SCID-I (APA, 2000). The
Five milliliters of blood were withdrawn from each subject at afternoon by venipuncture into a free-anticoagulant vacuum tube. Blood was immediately centrifuged at 4000g for 10 min, and serum was kept frozen at 80 °C until assayed. Serum NT-3 levels were assessed with an ELISA-sandwich from a commercial kit which was handled according to the manufacturer’s instructions (Chemicon, USA). Briefly, microtiter plates (96-well flat-bottom) were coated for 12 h with the samples diluted 1:3 in sample diluent and the standard curve ranged from 7.8 to 500 pg of NT-3. Then,
Table 1 Characteristics of drug-free and medicated bipolar patients and controls. Group* Characteristics
Drug-free patients
Drug-treated patients
Controls
p value
Male sex – no. (%)a All patients Manic patients Depressive patients
5/20 (25.0) 4/10 (40.0) 1/10 (10.0)
5/20 (25.0) 4/10 (40.0) 1/10 (10.0)
5/20 (25.0) 5/20 (25.0) 5/20 (25.0)
1.000 1.000 1.000
Age – yearsb All patients Manic patients Depressive patients
38.85 ± 12.36 (20) 44.00 ± 12.08 (10) 32.83 ± 10.53 (10)
39.11 ± 7.46 (20) 44.67 ± 9.63 (10) 36.33 ± 7.81 (10)
39.43 ± 10.27 (20) 39.43 ± 10.27 (20) 39.43 ± 10.27 (20)
0.955 0.920 0.528
Length of illness – yearsc All patients Manic patients Depressive patients
14.06 ± 8.81 (20) 16.00 ± 8.19 (10) 12.12 ± 9.52 (10)
13.87 ± 9.26 (20) 11.75 ± 9.18 (10) 16.00 ± 8.37 (10)
– – –
0.860 0.290 0.330
Presence of psychosis All patients Manic patients Depressive patients
15/20 (75.0) 9/10 (90.0) 5/10 (50.0)
12/20 (60.0) 8/10 (80.0) 3/10 (30.0)
– – –
0.370 0.490 0.870
31.26 ± 10.67 (10) 4.00 ± 3.32 (10)
28.00 ± 16.37 (10) 2.83 ± 2.77 (10)
– –
0.607 0.511
6.43 ± 8.22 (10) 23.86 ± 8.61 (10)
10.33 ± 13.79 (10) 23.00 ± 7.01 (10)
– –
0.580 0.849
2.51 ± 0.59 (20) 2.38 ± 0.38 (10) 2.63 ± 0.73 (10)
2.56 ± 0.44 (20) 2.57 ± 0.48 (10) 2.56 ± 0.44 (10)
1.97 ± 0.33 (20) 1.97 ± 0.33 (20) 1.97 ± 0.33 (20)
0.001 0.003 0.002
YMRS scorec,d Manic patients Depressive patients HDRS scorec,d Manic patients Depressive patients NT-3 in pg/mg proteinb,e All patients Manic patients Depressive patients
Abbreviations: YMRS (Young Mania Rating Scale); HDRS (Hamilton Depression Rating Scale); and NT-3 (neurotrophin-3). * Columns show mean ± standard deviation (SD) for all categories except male sex and presence of psychosis. The number of participants in each group is shown in parenthesis. a Chi-square test. b One-way ANOVA test with Tukey post-test. c Unpaired t test. d YMRS and HDRS in drug-free patients = drug-treated groups for manic and depressive patients. e NT-3 in controls < drug-free/drug-treated groups for all patients, and for manic and depressive patients separately.
B.S. Fernandes et al. / Journal of Psychiatric Research 44 (2010) 561–565
the plates were washed four times with wash solution and incubated for 2 h at room temperature with rabbit monoclonal antiNT-3 antibody diluted 1:1000 in sample diluent. After washing, a second incubation with peroxidase conjugated anti-rabbit antibody diluted 1:1000 for 1 h at room temperature was carried out. After addition of streptavidin-enzyme, substrate, and stop solution, the amount of NT-3 was determined (absorbance was set at 450 nm). The standard curve demonstrates a direct relationship between optical density (OD) and NT-3 concentration. We accepted intra- and inter-assay variation of ±4.0% and ±9.4%, respectively, as indicated by the manufacturer. Total protein was measured by Lowry’s method using bovine serum albumin as a standard. 2.3. Statistical analysis Statistical analysis was performed using SPSS 17.0 for Windows. All values are presented as mean ± standard deviation (SD) or percentage (%), as appropriate. v2 tests were used to evaluate associations between categorical variables. For the comparisons of continuous variables among the groups we employed one-way analysis of variance (ANOVA) with individual differences assessed using Tukey’s correction for the multiple comparisons if the ANOVA was statistically significant. Independent t test was also used. Pearson’s correlation coefficient was used to analyze the correlation between NT-3 and age in years, and the YMRS and HDRS scores. Two-tailed p values < 0.05 were considered to be statistically significant. 3. Results A total of 40 patients and 20 controls were included. The characteristics of drug-free and medicated patients with BD and controls are summarized in Table 1. Medicated and drug-free patients and controls are similar in terms of gender and age. Medicated and drug-free patients are similar regarding gender, age, presence of psychosis, age at first mood episode, length of illness, and YMRS and HDRS scores. Of the 20 medicated patients, 18 (90%) were on mood stabilizers and 14 (70%) on antipsychotics; 6 (30%) were also on antidepressants. Serum NT-3 levels in the drug-free and medicated patients with BD are increased when compared to controls. NT-3 levels do not differ between medicated and drug-free patients (Table 1, Fig. 1).
Fig. 1. Serum neurotrophin-3 (NT-3) levels in pg/mg protein in patients with bipolar disorder (BD) during manic or depressive episodes, according to the use or not of medication, and in healthy controls. Data are expressed as mean and 95% confidence interval (95% CI). *p < 0.001 for drug-free/medicated patients vs. control (one-way ANOVA with Tukey post-test).
563
When analyzing the patients according to mood state, there are no differences among manic (n = 20) and depressive (n = 20) patients and controls (n = 20) regarding gender and age, and no difference between manic and depressive patients regarding length of illness (data not shown). As expected, manic patients had significantly higher rates on YMRS than depressive patients (28.50 ± 12.47 vs. 3.21 ± 3.04, respectively, p < 0.001), and depressive patients had significantly higher rates on HDRS than manic patients (21.79 ± 9.62 vs. 7.60 ± 9.53, respectively, p < 0.001). Serum NT-3 levels are increased in those with BD during both manic (p < 0.004) and depressive (p < 0.001) episodes, as compared with healthy subjects (2.47 ± 0.43, 2.60 ± 0.59 and 1.97 ± 0.33, respectively, p < 0.001 for manic/depressive patients vs. controls) (Fig. 2). A significant moderate positive correlation between the severity of manic symptoms (YMRS scores) and serum NT-3 levels is observed in manic medicated and drug-free patients (r = 0.41, p = 0.032). In addition, serum NT-3 levels are positively correlated with depressive symptoms (HDRS scores) in depressive drug-free and medicated patients (r = 0.42, p = 0.016). There is no correlation between serum NT-3 levels and age (r = 0.60, p = 0.82). 4. Discussion As far as we are aware, this is the first study to report serum NT3 during manic and depressive episodes in patients on and off medication. In this study, we were able to show that serum NT-3 levels are increased in subjects with BD either on or off medication during manic and depressive episodes. In addition, drug-free and medicated subjects with BD had similar serum NT-3 levels. Our group has showed an increase in serum NT-3 levels in medicated patients with BD during manic and depressive states (Walz et al., 2007). Moreover we found an increase in serum NT-3 levels not only in medicated patients during manic and depressive episodes, but also in medication-free patients during mood episodes. In addition, we found no difference between patients with BD on and off medication during mood states. It may be possible that the mood state is more relevant to the increase in serum NT-3 levels than medication itself. Neurotrophic signaling systems are key regulatory factors in cellular plasticity and survival. BDNF and NT-3 are important pieces in the pathophysiology of bipolar disorder (Kapczinski et al., 2008a). It has been reported that NT-3 is temporarily and
Fig. 2. Serum neurotrophin-3 (NT-3) levels in pg/mg protein in drug-free and in medicated patients with bipolar disorder (BD) during manic or depressive episodes, and in healthy controls. Data are expressed as mean and 95% confidence interval (95% CI). *p < 0.001 for drug-free/medicated patients vs. control (one-way ANOVA with Tukey post-test).
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spatially expressed in response to the loss of BDNF in some neuronal populations (Agerman and Ernfors, 2003). Furthermore, NT-3 infusion increases BDNF mRNA expression in the cerebral cortex (Schutte et al., 2000) and induces BDNF-like effects inducing TrKB phosphorylation (Giehl et al., 2001). We are tempted to speculate that an increase in serum NT-3 levels occurs in tandem with a decrease in serum BDNF levels (Cunha et al., 2006; de Oliveira et al., 2009). This possible negative correlation between both neurotrophins may represent a compensatory response to the mood episodes. We previously demonstrated that both lithium and valproate modulate serum and hippocampal NT-3 levels in an animal model of mania (Walz et al., 2008). The extent to which these findings in an animal model could be translated to the clinical setting remains to be determined. Nevertheless, this finding supports the notion that the regulation of neurotrophic signaling systems might be associated with the therapeutic effects of mood stabilizers. It has been demonstrated that NT-3 crosses the brain–blood barrier and there is a strong positive correlation (r = 0.80) between serum and brain NT-3 levels. This positive correlation suggests that peripheral changes may correspond, at least in part, to central NT-3 changes (Pan et al., 1998). In addition, a biomarker in the periphery would be useful as a predictor of response to treatment, or as a possible tool to support clinical diagnosis in the field of mood disorders (Kapczinski et al., 2009b; Fernandes et al., 2009a). Some limitations of the present study relate to the cross-sectional design as well as the fact that all subjects were assessed in a tertiary referral center including mostly severely ill patients. Thus, the present study may not be representative of the whole spectrum of BD. It should also be mentioned that drug-free patients had slightly higher serum NT-3 levels, when compared to the medicated group, particularly the depressive ones (not statistically significant). Therefore, it is possible that a larger sample would identify differences between medicated and unmedicated patients. In conclusion, these findings confirm the results of a previous study of our group reporting that serum NT-3 levels are increased in medicated patients with BD during manic and depressive episodes. They add to the notion that serum NT-3 levels are increased not only in patients on medication but on patients off medication as well. Moreover, our study converges with other research findings of serum NT-3 in subjects with BD on and off mood stabilizers and antipsychotics. This raises the possibility that serum NT-3 levels could be considered a state-related biomarker in BD.
Author contributions BSF designed the study, wrote the protocol, participated in data acquisition and interpretation, and was responsible for the analysis and interpretation of data, drafting the article and final approval of this version. CSG, JCW, GC, KMC, GRF, BA and BP participated in study design, data acquisition and interpretation, drafting the article and final approval of this version. MK was responsible for drafting the article and final approval of this version. FK was responsible for study design and interpretation of data, drafting the article and final approval of this version.
Role of funding sources This study was supported by Stanley Medical Research Institute, NARSAD, INCT for Translational Medicine, CNPq, CAPES and FIPE– HCPA. These agencies had no role in study design, acquisition and interpretation of data or writing the report.
Financial disclosures Flavio Kapczinski has received research grants from FIPE–HCPA, CNPq, CAPES, SMRI, NARSAD, Lilly, AstraZeneca, and Janssen. Marcia Kauer-Sant’Anna has received research grants from AstraZeneca, FIPE–HCPA, CNPq, CAPES, SMRI, NARSAD, and Lilly. Clarissa S. Gama has received Grant/Research Support from CNPq, FIPE– HCPA, and Endeavour Award. She has been a paid speaker for AstraZeneca. Brisa Simões Fernandes, Keila Ceresér, Bianca Aguiar, Bianca Pfaffenseller, Laura Stertz, Gabriela Colpo and Gabriel Fries have declared no conflict of interest. These agencies had no role in study design, acquisition and interpretation of data or writing the report.
Acknowledgements This study was supported by FIPE–HCPA and CNPq. Brisa Simões Fernandes is supported by a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
References Agerman K, Ernfors P. Differential influence of BDNF and NT3 on the expression of calcium binding proteins and neuropeptide Y in vivo. Neuroreport 2003;14(17):2183–7. APA. Diagnostic and statistical manual of mental disorders. Washington, DC; 2000. Cunha AB, Frey BN, Andreazza AC, Goi JD, Rosa AR, Goncalves CA, et al. Serum brainderived neurotrophic factor is decreased in bipolar disorder during depressive and manic episodes. Neuroscience Letters 2006;398(3):215–9. de Oliveira GS, Cereser KM, Fernandes BS, Kauer-Sant’Anna M, Fries GR, Stertz L, et al. Decreased brain-derived neurotrophic factor in medicated and drug-free bipolar patients. Journal of Psychiatric Research 2009;43(14):1171–4. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biological Psychiatry 2006;59(12):1116–27. Fernandes BS, Gama CS, Kauer-Sant’Anna M, Lobato MI, Belmonte-de-Abreu P, Kapczinski F. Serum brain-derived neurotrophic factor in bipolar and unipolar depression: a potential adjunctive tool for differential diagnosis. Journal of Psychiatric Research 2009a;43(15):1200–4. Fernandes B, Gama CS, Massuda R, Torres M, Camargo D, Kunz M, et al. Serum brainderived neurotrophic factor (BDNF) is not associated with response to electroconvulsive therapy (ECT): a pilot study in drug resistant depressed patients. Neuroscience Letters 2009b;453(3):195–8. Gama CS, Andreazza AC, Kunz M, Berk M, Belmonte-de-Abreu PS, Kapczinski F. Serum levels of brain-derived neurotrophic factor in patients with schizophrenia and bipolar disorder. Neuroscience Letters 2007;420(1):45–8. Giehl KM, Rohrig S, Bonatz H, Gutjahr M, Leiner B, Bartke I, et al. Endogenous brainderived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2001;21(10):3492–502. Hamilton M. A rating scale for depression. Journal of Neurology, Neurosurgery, and Psychiatry 1960;23:56–62. Kapczinski F, Fernandes BS, Kauer-Sant’Anna M, Gama CS, Yatham L, Berk M. The concept of staging in bipolar disorder: the role of BDNF and TNF-alpha as biomarkers. Acta Neuropsychiatrica 2009a;21(6):272–4. Kapczinski F, Teixeira AL, Magalhaes PVS, Kauer-Sant’Anna M, Klamt F, Pasquali MA, et al. A systemic toxicity index developed to assess peripheral changes in mood episodes. Molecular Psychiatry 2009. doi:10.1038/mp.2009.11. Kapczinski F, Frey BN, Kauer-Sant’Anna M, Grassi-Oliveira R, Berk M. Brain-derived neurotrophic factor and neuroplasticity in bipolar disorder. Expert Review of Neurotherapeutics 2008a;8(7):1101–13. Kapczinski F, Vieta E, Andreazza AC, Frey BN, Gomes FA, Tramontina J, et al. Allostatic load in bipolar disorder: implications for pathophysiology and treatment. Neuroscience and Biobehavioral Reviews 2008b;32(4):675–92. Kato A, Fukazawa Y, Ozawa F, Inokuchi K, Sugiyama H. Activation of ERK cascade promotes accumulation of Vesl-1S/Homer-1a immunoreactivity at synapses. Brain Research. Molecular Brain Research 2003;118(1-2):33–44. Pan W, Banks WA, Kastin AJ. Permeability of the blood–brain barrier to neurotrophins. Brain Research 1998;788(1–2):87–94. Schutte A, Yan Q, Mestres P, Giehl KM. The endogenous survival promotion of axotomized rat corticospinal neurons by brain-derived neurotrophic factor is mediated via paracrine, rather than autocrine mechanisms. Neuroscience Letters 2000;290(3):185–8. Tramontina JF, Andreazza AC, Kauer-Sant’anna M, Stertz L, Goi J, Chiarani F, et al. Brain-derived neurotrophic factor serum levels before and after treatment for acute mania. Neuroscience Letters 2009;452(2):111–3.
B.S. Fernandes et al. / Journal of Psychiatric Research 44 (2010) 561–565 Walz JC, Andreazza AC, Frey BN, Cacilhas AA, Cereser KM, Cunha AB, et al. Serum neurotrophin-3 is increased during manic and depressive episodes in bipolar disorder. Neuroscience Letters 2007;415(1):87–9. Walz JC, Frey BN, Andreazza AC, Cereser KM, Cacilhas AA, Valvassori SS, et al. Effects of lithium and valproate on serum and hippocampal neurotrophin-3 levels in an animal model of mania. Journal of Psychiatric Research 2008;42(5):416–21. Yatham LN, Kennedy SH, Schaffer A, Parikh SV, Beaulieu S, O’Donovan C, et al. Canadian Network for Mood and Anxiety Treatments (CANMAT) and
565
International Society for Bipolar Disorders (ISBD) collaborative update of CANMAT guidelines for the management of patients with bipolar disorder: update 2009. Bipolar Disorders 2009;11(3):225–55. Young RC, Biggs JT, Ziegler VE, Meyer DA. A rating scale for mania: reliability, validity and sensitivity. The British Journal of Psychiatry: The Journal of Mental Science 1978;133:429–35.
Contents lists available at ScienceDirect
Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires
Increased neurotrophin-3 in drug-free subjects with bipolar disorder during manic and depressive episodes Brisa Simões Fernandes a,b,c,d, Clarissa Severino Gama a,b,c,d, Julio C. Walz a,b,d, Keila Maria Ceresér a,b,d, Gabriel Rodrigo Fries b,d,e, Gabriela Colpo b, Bianca Aguiar b, Bianca Pfaffenseller b, Marcia Kauer-Sant’Anna a,b,d, Flavio Kapczinski a,b,c,d,* a
Bipolar Disorder Program, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Laboratory of Molecular Psychiatry, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Schizophrenia Program, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil d INCT for Translational Medicine, Brazil e Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil b c
a r t i c l e
i n f o
Article history: Received 1 November 2009 Received in revised form 21 November 2009 Accepted 26 November 2009
Keywords: Bipolar disorder NT-3 Biological marker Depression Mania Mood stabilizers
a b s t r a c t Bipolar disorder (BD) has been increasingly associated with abnormalities in neuroplasticity. Previous studies demonstrated that neurotrophin-3 (NT-3) plays a role in the pathophysiology of mood disorders. The influence of medication in these studies has been considered a limitation. Thus, studies with drugfree vs. medicated patients are necessary to evaluate the role of medication in serum NT-3 levels. About 10 manic and 10 depressive drug-free, and 10 manic and 10 depressive medicated patients with BD type I were matched with 20 controls for sex and age. Patients were assessed using SCID-I, YMRS and HDRS. Serum NT-3 levels in drug-free and medicated patients is increased when compared with controls (2.51 ± 0.59, 2.56 ± 0.44 and 1.97 ± 0.33, respectively, p < 0.001 for drug-free/medicated vs. control). Serum NT-3 levels do not differ between medicated and drug-free patients. When analyzing patients according to mood states, serum NT-3 levels are increased in both manic and depressive episodes, as compared with controls (2.47 ± 0.43, 2.60 ± 0.59 and 1.97 ± 0.33, respectively, p < 0.001 for manic/depressive patients vs. controls). There is no difference in serum BDNF between manic and depressive patients. Results suggest that increased serum NT-3 levels in BD are likely to be associated with the pathophysiology of manic and depressive symptoms. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Bipolar disorder (BD) is a highly disabling illness, characterized by the presence of manic and depressive symptoms (Yatham et al., 2009). Although a growing body of evidence strongly suggests that a neurobiological basis may underlie the pathophysiology of BD, its etiology is poorly understood. In recent years, many psychiatric disorders, particularly mood disorders such as BD, have been recognized as conditions that affect neurotrophins (Cunha et al., 2006; Gama et al., 2007; Walz et al., 2007; Kapczinski et al., 2008a,b; de Oliveira et al., 2009; Fernandes et al., 2009a; Kapczinski et al., 2009a,b). An emerging body of evidence links the expression of these neurotrophic factors to mood disorders. Brain-derived neurotrophic factor (BDNF) is so far the best studied member of this family, being put forward as a predictor of response to treat* Corresponding author. Address: Hospital de Clínicas de Porto Alegre, Av. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil. Tel.: +55 51 21018845. E-mail address: [email protected] (F. Kapczinski). 0022-3956/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2009.11.020
ment (Kapczinski et al., 2008a; Fernandes et al., 2009b; Kapczinski et al., 2009a; Tramontina et al., 2009) and as a possible biomarker of diagnosis to discriminate unipolar from bipolar depression (Fernandes et al., 2009a). Neurotrophin-3 (NT-3), an important member of the neurotrophin family, couples to the same signal transduction pathways as BDNF through their respective receptors. Decreased expression of these factors could lead to alterations in the structure and function of subpopulations of hippocampal neurons (Duman and Monteggia, 2006). In addition, it has been reported that NT-3 modulates basal synaptic transmission and long-term potentiation in rat hippocampus (Kato et al., 2003). There is a paucity of studies about NT-3 in peripheral serum. To our knowledge, there is only one report of serum NT-3 levels in BD, which shows that NT-3 is increased during both manic and depressive episodes, but not in euthymia (Walz et al., 2007). A question that frequently emerges in studies about serum neurotrophins in BD is the influence of medication use. This is considered to be an important limitation in most of the studies concerning neurotrophins. Little is known about whether the use
562
B.S. Fernandes et al. / Journal of Psychiatric Research 44 (2010) 561–565
of medication in these studies alters their final results. Recently, we have shown that serum BDNF levels are similar in BD regarding subjects on and off medication (de Oliveira et al., 2009). As aforementioned, serum NT-3 data are scarce in the BD field; whether the use of mood stabilizers and antipsychotics plays or not a role in serum NT-3 levels in mood states of BD remains unclear. Therefore, there is a need for studies that compare serum NT-3 in patients on and off medication. The aim of this report is to investigate whether drug-free patients have differential levels of circulating serum NT-3 levels, when compared to medicated patients with BD. For this purpose, we evaluated serum NT-3 concentrations in medicated and drugfree BD type I during manic and depressive episodes.
2. Methods and materials
severity of manic and depressive episodes was evaluated using the Young Mania Rating Scale (YMRS) (Young et al., 1978) and Hamilton Depression Rating Scale (HDRS) (Hamilton, 1960), respectively. Twenty drug-free patients (10 manic and 10 depressed) were included in this study. This group did not receive any psychotropic medication for at least two weeks (five weeks if fluoxetine or depot medication was used) before blood sampling. The 20 medicated patients (10 manic and 10 depressed) and 20 controls were matched for age and gender to drug-free bipolar subjects. Controls were recruited from the Hospital de Clinicas de Porto Alegre catchment area. This group was screened with the non-patient version of the SCID to exclude current psychiatric morbidity. Those that had first-degree relatives with BD, schizophrenia or other psychiatric disorders were excluded. Controls were not on medication. All participants gave a written informed consent before entering the study, which was approved by the local ethics committee.
2.1. Subjects and measurement 2.2. Biochemical measurement Manic and depressive drug-free and medicated patients with BD type I were recruited from the Bipolar Disorders Program and Psychiatry Inpatient Unit – Hospital de Clinicas de Porto Alegre and Pronto Atendimento da Vila Cruzeiro do Sul, Porto Alegre, Brazil, between 2006 and 2008. To be included in this study, patients had to present with a manic or depressive episode according to the Diagnostic and Statistical Manual of Mental Disorders – Fourth Edition (DSM-IV) and Structured Clinical Interview for DSM-IV-Axis I Disorders (SCID-I) criteria. The diagnosis of manic and depressive episodes was established according to SCID-I (APA, 2000). The
Five milliliters of blood were withdrawn from each subject at afternoon by venipuncture into a free-anticoagulant vacuum tube. Blood was immediately centrifuged at 4000g for 10 min, and serum was kept frozen at 80 °C until assayed. Serum NT-3 levels were assessed with an ELISA-sandwich from a commercial kit which was handled according to the manufacturer’s instructions (Chemicon, USA). Briefly, microtiter plates (96-well flat-bottom) were coated for 12 h with the samples diluted 1:3 in sample diluent and the standard curve ranged from 7.8 to 500 pg of NT-3. Then,
Table 1 Characteristics of drug-free and medicated bipolar patients and controls. Group* Characteristics
Drug-free patients
Drug-treated patients
Controls
p value
Male sex – no. (%)a All patients Manic patients Depressive patients
5/20 (25.0) 4/10 (40.0) 1/10 (10.0)
5/20 (25.0) 4/10 (40.0) 1/10 (10.0)
5/20 (25.0) 5/20 (25.0) 5/20 (25.0)
1.000 1.000 1.000
Age – yearsb All patients Manic patients Depressive patients
38.85 ± 12.36 (20) 44.00 ± 12.08 (10) 32.83 ± 10.53 (10)
39.11 ± 7.46 (20) 44.67 ± 9.63 (10) 36.33 ± 7.81 (10)
39.43 ± 10.27 (20) 39.43 ± 10.27 (20) 39.43 ± 10.27 (20)
0.955 0.920 0.528
Length of illness – yearsc All patients Manic patients Depressive patients
14.06 ± 8.81 (20) 16.00 ± 8.19 (10) 12.12 ± 9.52 (10)
13.87 ± 9.26 (20) 11.75 ± 9.18 (10) 16.00 ± 8.37 (10)
– – –
0.860 0.290 0.330
Presence of psychosis All patients Manic patients Depressive patients
15/20 (75.0) 9/10 (90.0) 5/10 (50.0)
12/20 (60.0) 8/10 (80.0) 3/10 (30.0)
– – –
0.370 0.490 0.870
31.26 ± 10.67 (10) 4.00 ± 3.32 (10)
28.00 ± 16.37 (10) 2.83 ± 2.77 (10)
– –
0.607 0.511
6.43 ± 8.22 (10) 23.86 ± 8.61 (10)
10.33 ± 13.79 (10) 23.00 ± 7.01 (10)
– –
0.580 0.849
2.51 ± 0.59 (20) 2.38 ± 0.38 (10) 2.63 ± 0.73 (10)
2.56 ± 0.44 (20) 2.57 ± 0.48 (10) 2.56 ± 0.44 (10)
1.97 ± 0.33 (20) 1.97 ± 0.33 (20) 1.97 ± 0.33 (20)
0.001 0.003 0.002
YMRS scorec,d Manic patients Depressive patients HDRS scorec,d Manic patients Depressive patients NT-3 in pg/mg proteinb,e All patients Manic patients Depressive patients
Abbreviations: YMRS (Young Mania Rating Scale); HDRS (Hamilton Depression Rating Scale); and NT-3 (neurotrophin-3). * Columns show mean ± standard deviation (SD) for all categories except male sex and presence of psychosis. The number of participants in each group is shown in parenthesis. a Chi-square test. b One-way ANOVA test with Tukey post-test. c Unpaired t test. d YMRS and HDRS in drug-free patients = drug-treated groups for manic and depressive patients. e NT-3 in controls < drug-free/drug-treated groups for all patients, and for manic and depressive patients separately.
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the plates were washed four times with wash solution and incubated for 2 h at room temperature with rabbit monoclonal antiNT-3 antibody diluted 1:1000 in sample diluent. After washing, a second incubation with peroxidase conjugated anti-rabbit antibody diluted 1:1000 for 1 h at room temperature was carried out. After addition of streptavidin-enzyme, substrate, and stop solution, the amount of NT-3 was determined (absorbance was set at 450 nm). The standard curve demonstrates a direct relationship between optical density (OD) and NT-3 concentration. We accepted intra- and inter-assay variation of ±4.0% and ±9.4%, respectively, as indicated by the manufacturer. Total protein was measured by Lowry’s method using bovine serum albumin as a standard. 2.3. Statistical analysis Statistical analysis was performed using SPSS 17.0 for Windows. All values are presented as mean ± standard deviation (SD) or percentage (%), as appropriate. v2 tests were used to evaluate associations between categorical variables. For the comparisons of continuous variables among the groups we employed one-way analysis of variance (ANOVA) with individual differences assessed using Tukey’s correction for the multiple comparisons if the ANOVA was statistically significant. Independent t test was also used. Pearson’s correlation coefficient was used to analyze the correlation between NT-3 and age in years, and the YMRS and HDRS scores. Two-tailed p values < 0.05 were considered to be statistically significant. 3. Results A total of 40 patients and 20 controls were included. The characteristics of drug-free and medicated patients with BD and controls are summarized in Table 1. Medicated and drug-free patients and controls are similar in terms of gender and age. Medicated and drug-free patients are similar regarding gender, age, presence of psychosis, age at first mood episode, length of illness, and YMRS and HDRS scores. Of the 20 medicated patients, 18 (90%) were on mood stabilizers and 14 (70%) on antipsychotics; 6 (30%) were also on antidepressants. Serum NT-3 levels in the drug-free and medicated patients with BD are increased when compared to controls. NT-3 levels do not differ between medicated and drug-free patients (Table 1, Fig. 1).
Fig. 1. Serum neurotrophin-3 (NT-3) levels in pg/mg protein in patients with bipolar disorder (BD) during manic or depressive episodes, according to the use or not of medication, and in healthy controls. Data are expressed as mean and 95% confidence interval (95% CI). *p < 0.001 for drug-free/medicated patients vs. control (one-way ANOVA with Tukey post-test).
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When analyzing the patients according to mood state, there are no differences among manic (n = 20) and depressive (n = 20) patients and controls (n = 20) regarding gender and age, and no difference between manic and depressive patients regarding length of illness (data not shown). As expected, manic patients had significantly higher rates on YMRS than depressive patients (28.50 ± 12.47 vs. 3.21 ± 3.04, respectively, p < 0.001), and depressive patients had significantly higher rates on HDRS than manic patients (21.79 ± 9.62 vs. 7.60 ± 9.53, respectively, p < 0.001). Serum NT-3 levels are increased in those with BD during both manic (p < 0.004) and depressive (p < 0.001) episodes, as compared with healthy subjects (2.47 ± 0.43, 2.60 ± 0.59 and 1.97 ± 0.33, respectively, p < 0.001 for manic/depressive patients vs. controls) (Fig. 2). A significant moderate positive correlation between the severity of manic symptoms (YMRS scores) and serum NT-3 levels is observed in manic medicated and drug-free patients (r = 0.41, p = 0.032). In addition, serum NT-3 levels are positively correlated with depressive symptoms (HDRS scores) in depressive drug-free and medicated patients (r = 0.42, p = 0.016). There is no correlation between serum NT-3 levels and age (r = 0.60, p = 0.82). 4. Discussion As far as we are aware, this is the first study to report serum NT3 during manic and depressive episodes in patients on and off medication. In this study, we were able to show that serum NT-3 levels are increased in subjects with BD either on or off medication during manic and depressive episodes. In addition, drug-free and medicated subjects with BD had similar serum NT-3 levels. Our group has showed an increase in serum NT-3 levels in medicated patients with BD during manic and depressive states (Walz et al., 2007). Moreover we found an increase in serum NT-3 levels not only in medicated patients during manic and depressive episodes, but also in medication-free patients during mood episodes. In addition, we found no difference between patients with BD on and off medication during mood states. It may be possible that the mood state is more relevant to the increase in serum NT-3 levels than medication itself. Neurotrophic signaling systems are key regulatory factors in cellular plasticity and survival. BDNF and NT-3 are important pieces in the pathophysiology of bipolar disorder (Kapczinski et al., 2008a). It has been reported that NT-3 is temporarily and
Fig. 2. Serum neurotrophin-3 (NT-3) levels in pg/mg protein in drug-free and in medicated patients with bipolar disorder (BD) during manic or depressive episodes, and in healthy controls. Data are expressed as mean and 95% confidence interval (95% CI). *p < 0.001 for drug-free/medicated patients vs. control (one-way ANOVA with Tukey post-test).
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spatially expressed in response to the loss of BDNF in some neuronal populations (Agerman and Ernfors, 2003). Furthermore, NT-3 infusion increases BDNF mRNA expression in the cerebral cortex (Schutte et al., 2000) and induces BDNF-like effects inducing TrKB phosphorylation (Giehl et al., 2001). We are tempted to speculate that an increase in serum NT-3 levels occurs in tandem with a decrease in serum BDNF levels (Cunha et al., 2006; de Oliveira et al., 2009). This possible negative correlation between both neurotrophins may represent a compensatory response to the mood episodes. We previously demonstrated that both lithium and valproate modulate serum and hippocampal NT-3 levels in an animal model of mania (Walz et al., 2008). The extent to which these findings in an animal model could be translated to the clinical setting remains to be determined. Nevertheless, this finding supports the notion that the regulation of neurotrophic signaling systems might be associated with the therapeutic effects of mood stabilizers. It has been demonstrated that NT-3 crosses the brain–blood barrier and there is a strong positive correlation (r = 0.80) between serum and brain NT-3 levels. This positive correlation suggests that peripheral changes may correspond, at least in part, to central NT-3 changes (Pan et al., 1998). In addition, a biomarker in the periphery would be useful as a predictor of response to treatment, or as a possible tool to support clinical diagnosis in the field of mood disorders (Kapczinski et al., 2009b; Fernandes et al., 2009a). Some limitations of the present study relate to the cross-sectional design as well as the fact that all subjects were assessed in a tertiary referral center including mostly severely ill patients. Thus, the present study may not be representative of the whole spectrum of BD. It should also be mentioned that drug-free patients had slightly higher serum NT-3 levels, when compared to the medicated group, particularly the depressive ones (not statistically significant). Therefore, it is possible that a larger sample would identify differences between medicated and unmedicated patients. In conclusion, these findings confirm the results of a previous study of our group reporting that serum NT-3 levels are increased in medicated patients with BD during manic and depressive episodes. They add to the notion that serum NT-3 levels are increased not only in patients on medication but on patients off medication as well. Moreover, our study converges with other research findings of serum NT-3 in subjects with BD on and off mood stabilizers and antipsychotics. This raises the possibility that serum NT-3 levels could be considered a state-related biomarker in BD.
Author contributions BSF designed the study, wrote the protocol, participated in data acquisition and interpretation, and was responsible for the analysis and interpretation of data, drafting the article and final approval of this version. CSG, JCW, GC, KMC, GRF, BA and BP participated in study design, data acquisition and interpretation, drafting the article and final approval of this version. MK was responsible for drafting the article and final approval of this version. FK was responsible for study design and interpretation of data, drafting the article and final approval of this version.
Role of funding sources This study was supported by Stanley Medical Research Institute, NARSAD, INCT for Translational Medicine, CNPq, CAPES and FIPE– HCPA. These agencies had no role in study design, acquisition and interpretation of data or writing the report.
Financial disclosures Flavio Kapczinski has received research grants from FIPE–HCPA, CNPq, CAPES, SMRI, NARSAD, Lilly, AstraZeneca, and Janssen. Marcia Kauer-Sant’Anna has received research grants from AstraZeneca, FIPE–HCPA, CNPq, CAPES, SMRI, NARSAD, and Lilly. Clarissa S. Gama has received Grant/Research Support from CNPq, FIPE– HCPA, and Endeavour Award. She has been a paid speaker for AstraZeneca. Brisa Simões Fernandes, Keila Ceresér, Bianca Aguiar, Bianca Pfaffenseller, Laura Stertz, Gabriela Colpo and Gabriel Fries have declared no conflict of interest. These agencies had no role in study design, acquisition and interpretation of data or writing the report.
Acknowledgements This study was supported by FIPE–HCPA and CNPq. Brisa Simões Fernandes is supported by a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
References Agerman K, Ernfors P. Differential influence of BDNF and NT3 on the expression of calcium binding proteins and neuropeptide Y in vivo. Neuroreport 2003;14(17):2183–7. APA. Diagnostic and statistical manual of mental disorders. Washington, DC; 2000. Cunha AB, Frey BN, Andreazza AC, Goi JD, Rosa AR, Goncalves CA, et al. Serum brainderived neurotrophic factor is decreased in bipolar disorder during depressive and manic episodes. Neuroscience Letters 2006;398(3):215–9. de Oliveira GS, Cereser KM, Fernandes BS, Kauer-Sant’Anna M, Fries GR, Stertz L, et al. Decreased brain-derived neurotrophic factor in medicated and drug-free bipolar patients. Journal of Psychiatric Research 2009;43(14):1171–4. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biological Psychiatry 2006;59(12):1116–27. Fernandes BS, Gama CS, Kauer-Sant’Anna M, Lobato MI, Belmonte-de-Abreu P, Kapczinski F. Serum brain-derived neurotrophic factor in bipolar and unipolar depression: a potential adjunctive tool for differential diagnosis. Journal of Psychiatric Research 2009a;43(15):1200–4. Fernandes B, Gama CS, Massuda R, Torres M, Camargo D, Kunz M, et al. Serum brainderived neurotrophic factor (BDNF) is not associated with response to electroconvulsive therapy (ECT): a pilot study in drug resistant depressed patients. Neuroscience Letters 2009b;453(3):195–8. Gama CS, Andreazza AC, Kunz M, Berk M, Belmonte-de-Abreu PS, Kapczinski F. Serum levels of brain-derived neurotrophic factor in patients with schizophrenia and bipolar disorder. Neuroscience Letters 2007;420(1):45–8. Giehl KM, Rohrig S, Bonatz H, Gutjahr M, Leiner B, Bartke I, et al. Endogenous brainderived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2001;21(10):3492–502. Hamilton M. A rating scale for depression. Journal of Neurology, Neurosurgery, and Psychiatry 1960;23:56–62. Kapczinski F, Fernandes BS, Kauer-Sant’Anna M, Gama CS, Yatham L, Berk M. The concept of staging in bipolar disorder: the role of BDNF and TNF-alpha as biomarkers. Acta Neuropsychiatrica 2009a;21(6):272–4. Kapczinski F, Teixeira AL, Magalhaes PVS, Kauer-Sant’Anna M, Klamt F, Pasquali MA, et al. A systemic toxicity index developed to assess peripheral changes in mood episodes. Molecular Psychiatry 2009. doi:10.1038/mp.2009.11. Kapczinski F, Frey BN, Kauer-Sant’Anna M, Grassi-Oliveira R, Berk M. Brain-derived neurotrophic factor and neuroplasticity in bipolar disorder. Expert Review of Neurotherapeutics 2008a;8(7):1101–13. Kapczinski F, Vieta E, Andreazza AC, Frey BN, Gomes FA, Tramontina J, et al. Allostatic load in bipolar disorder: implications for pathophysiology and treatment. Neuroscience and Biobehavioral Reviews 2008b;32(4):675–92. Kato A, Fukazawa Y, Ozawa F, Inokuchi K, Sugiyama H. Activation of ERK cascade promotes accumulation of Vesl-1S/Homer-1a immunoreactivity at synapses. Brain Research. Molecular Brain Research 2003;118(1-2):33–44. Pan W, Banks WA, Kastin AJ. Permeability of the blood–brain barrier to neurotrophins. Brain Research 1998;788(1–2):87–94. Schutte A, Yan Q, Mestres P, Giehl KM. The endogenous survival promotion of axotomized rat corticospinal neurons by brain-derived neurotrophic factor is mediated via paracrine, rather than autocrine mechanisms. Neuroscience Letters 2000;290(3):185–8. Tramontina JF, Andreazza AC, Kauer-Sant’anna M, Stertz L, Goi J, Chiarani F, et al. Brain-derived neurotrophic factor serum levels before and after treatment for acute mania. Neuroscience Letters 2009;452(2):111–3.
B.S. Fernandes et al. / Journal of Psychiatric Research 44 (2010) 561–565 Walz JC, Andreazza AC, Frey BN, Cacilhas AA, Cereser KM, Cunha AB, et al. Serum neurotrophin-3 is increased during manic and depressive episodes in bipolar disorder. Neuroscience Letters 2007;415(1):87–9. Walz JC, Frey BN, Andreazza AC, Cereser KM, Cacilhas AA, Valvassori SS, et al. Effects of lithium and valproate on serum and hippocampal neurotrophin-3 levels in an animal model of mania. Journal of Psychiatric Research 2008;42(5):416–21. Yatham LN, Kennedy SH, Schaffer A, Parikh SV, Beaulieu S, O’Donovan C, et al. Canadian Network for Mood and Anxiety Treatments (CANMAT) and
565
International Society for Bipolar Disorders (ISBD) collaborative update of CANMAT guidelines for the management of patients with bipolar disorder: update 2009. Bipolar Disorders 2009;11(3):225–55. Young RC, Biggs JT, Ziegler VE, Meyer DA. A rating scale for mania: reliability, validity and sensitivity. The British Journal of Psychiatry: The Journal of Mental Science 1978;133:429–35.