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Decreased levels of plasma glutamate in patients with first-episode schizophrenia and bipolar disorder
Schizophrenia Research 95 (2007) 174 – 178 www.elsevier.com/locate/schres
Decreased levels of plasma glutamate in patients with first-episode schizophrenia and bipolar disorder Aitor Palomino a , Ana González-Pinto b , Ana Aldama b , Cristina González-Gómez b , Fernando Mosquera b , Gixane González-García b , Carlos Matute a,⁎ a
Neurotek UPV-EHU, Parque Tecnológico de Bizkaia, E-48170 Zamudio, Vizcaya, Spain b Stanley Research Center, 03-RC-003, Vitoria, Spain
Received 30 November 2006; received in revised form 12 June 2007; accepted 14 June 2007 Available online 13 July 2007
Abstract A variety of studies have suggested that glutamatergic neurotransmission is altered in schizophrenia and bipolar disorder. Here, we tested if plasma glutamate levels are altered in 56 patients diagnosed with schizophrenia, bipolar disorder or non-specified psychosis at the first psychotic episode and at various stages during one-year follow-up. A decrease in the levels of plasma glutamate was observed in all groups of patients at the first psychotic episode. Furthermore, plasma glutamate levels were restored after treatment in all instances. Decreased plasma glutamate levels at first psychotic episodes may reflect impaired glutamate signaling during the initial stages of schizophrenia and bipolar disorder. © 2007 Elsevier B.V. All rights reserved. Keywords: Glutamate; First psychotic episode; Schizophrenia; Bipolar disorder
1. Introduction Glutamate is the major excitatory neurotransmitter in the mammalian brain and its effects are mediated by glutamate-gated ion channels and seven transmembrane, G-protein coupled glutamate receptors. In turn, glutamate homeostasis is driven by glutamate transporters which regulate the extracellular levels of glutamate (Danbolt, 2001). The first evidence of decreased glutamate levels in schizophrenia was reported over 25 years ago (Kim et al., 1980). Since then, mounting evidence has accumulated indicating that the dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia and bipolar disorder (Tsai and Coyle, 2002; Schiffer, ⁎ Corresponding author. 0920-9964/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2007.06.012
2002). In particular, the hypofunction of N-methyl-Daspartate (NMDA) type glutamate receptors is considered to be a key factor in schizophrenia (Olney and Farber, 1995). Thus, the psychotomimetic drug phencyclidine blocks the NMDA receptor channel (Javitt and Zukin, 1991); mice expressing low levels of the NMDAR1 subunit display behaviors which are related to schizophrenia and are ameliorated by antipsychotic treatment (Mohn et al., 1999); and treatment with positive modulators of NMDA receptors induces therapeutic benefit (Konradi and Heckers, 2003). In addition, glutamate transporter expression and function is altered in areas of the brain which are vulnerable to schizophrenia and bipolar disease (McCullumsmith and Meador-Woodruff, 2002; Matute et al., 2005). Overall, these findings suggest that glutamate signaling is impaired in schizophrenia.
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2. Methods
of the sample were female. Total PANSS scores (mean ± S.D.) were 80.98 ± 16.39 at baseline, 57.51 ± 19.57 at one month, 53.33 ± 19.04 at six months, and 51.76 ±16.69 at twelve months. GAF was 30.72 at baseline, 51.28 at one month, 56.19 ± 15.26 at six months, and 56.60 ± 14.47 at twelve months. Patients were diagnosed after 12 months of inclusion using the Structured Clinical Interview for DSM IV, SCID-I. 24 patients were diagnosed with schizophrenia, 17 with bipolar disorder, and the remaining 15 with non-specified psychotic disorders. Patients with schizophrenia had the following demographic characteristics: mean age (± S.E.M), 23.21 ± 1.33 years; % female, 16.7%; % with at least primary level education (Neleven years of schooling), 90% and % with medium or higher socioeconomic status, 50%. Patients with bipolar disorder had the following demographic characteristics: mean age (± S.E.M), 25.29 ± 1.71 years; % female, 35.3%; % with at least primary level education (Neleven years of schooling), 93.3% and % with medium or higher socioeconomic status, 76.9%. Patients with other psychotic disorders had the following demographic characteristics: mean age (± S.E.M), 23.36 ± 2.59 years; % female, 40%; % with at least primary level education (Neleven years of schooling), 92.3% and % with medium or higher socioeconomic status, 69.2%. Each patient was paired by age and sex with a control subject. Patients were treated after the first episode with atypical antipsychotics (62–68%), with lithium or other mood stabilizers together with atypical antipsychotics (23– 26%), with typical antipsychotics (7–11%), or received no treatment (2–4%). Ranges in each treatment group indicate changes in the drugs administered initially at the onset of symptoms and during the first year of illness.
2.1. Patients
2.2. Control subjects
In this study, we examined 56 patients (mean age ± S.E.M., 23.3 ± 0.94 years) from the health catchment area of Vitoria (300.000 inhabitants) (Álava, Spain), who experienced a first psychotic episode during the period 2002–2005. First psychotic episode was defined as the first time a patient displayed positive psychotic symptoms of delusions or hallucinations. This sample of patients consisting of all first-episode patients who required hospitalization and gave informed consent to participate in the study, represented 75% of the total population of patients who had been admitted to the psychiatric emergency care unit. Dropout from the study was around 40% after and occurred principally towards the end of the year of treatment. No significant differences in age, gender, or clinical symptoms were detected between included and excluded patients. 28.4%
In addition, 50 healthy volunteers were selected explicitly for the research and matched pairwise for sex and age (25.19 ± 0.95 years, mean age ± S.E.M.). Twenty one of these controls were used for comparisons with patients with schizophrenia, 15 for comparisons with bipolar disorder and 14 for comparisons with non-specified psychoses. All subjects (case and controls) were recruited from the same community and included after informed consent to participate in the study was obtained. Subjects with mental retardation, organic brain disorders, or drug abuse as a primary diagnosis were excluded. As for the patients, 44% were smokers, 52% consumed alcohol, 13% abused alcohol and 34% abused cannabis. We did not observe differences in the plasma glutamate concentration among these groups, or in comparison to the remaining patients. The exclusion criteria for controls consisted of the
The levels of glutamate in blood plasma may reflect the extracellular concentration of glutamate within the CNS, since brain-to-blood efflux of this amino acid occurs through the blood–brain-barrier (Berl et al., 1961; O'Kane et al., 1999), but the entry from blood into the brain is minimal (Hawkins et al., 2006). Thus, although blood cells and peripheral organs may constitute the primary source of plasma glutamate, exit of glutamate from the CNS also contributes substantially to the overall concentration of glutamate in plasma, as shown in certain pathological conditions (Mallolas et al., 2006). Furthermore, atypical antipsychotic drugs increase extracellular concentrations of glutamate by reducing the expression and function of glutamate transporters (Vallejo-Illarramendi et al., 2005), and as a consequence these drugs may increase plasma glutamate levels. Indeed, several studies have reported an increased concentration of glutamate in the plasma/ serum of patients with schizophrenia who had been treated with antipsychotics (Evins et al., 1997; Tortorella et al., 2001; Goff et al., 2002; van der Heijden et al., 2004). In addition, plasma glutamate levels are higher in treated bipolar disorder patients than in healthy controls (Hoekstra et al., 2006). In the present study, we set out to characterize the levels of plasma glutamate in patients with schizophrenia or bipolar disorder upon the first psychotic episode and at various intervals thereafter. We observed that the concentration of glutamate in plasma is reduced at the onset of disease in both instances but is restored to near normal levels following 1 year treatment.
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A. Palomino et al. / Schizophrenia Research 95 (2007) 174–178
absence of any Axis I disorder, as well as the exclusion criteria applied to the patient group.
Table 2 Glutamate in plasma from patients with schizophrenia, bipolar disorder and non-specified psychosis
2.3. Experimental procedures Blood samples were collected upon arrival at the hospital emergency room and at 1, 6 and 12 months later, using glass whole-blood tubes containing K3-EDTA. Plasma was isolated by centrifugation at 300 ×g for 10 min and frozen at −80 °C until the time of analysis. Plasma levels of glutamate were measured by enzymatic assay. The enzyme glutamate dehydrogenase, in the presence of NADP+, oxidizes glutamate to α-ketoglutarate and generates NADPH. NADPH fluorescence was excited at 340 nm and emitted at 420 nm. The reaction buffer consisted of NaCl (14 mM), KCl (5 mM), MgCl2 (2 mM), pH 7.7 and was supplemented with 1 mM NADP+ and glutamate dehydrogenase (15 U/ml). 40 μl of plasma was diluted with the reaction buffer to a final volume of 300 μl per assay and the fluorescence values were measured after 10 min using a microplate reader (Synergy; Biotek). Glutamate concentration was determined using standard curves constructed with serial dilutions of glutamate. These curves were linear between 5 μM and 100 μM of the transmitter and plasma levels in both patients and controls were within those values. This fluorimetric method is highly reliable under the conditions employed (Bezzi et al., 2001). Repeated measures of the same sample resulted in variations within a range of around ±2%. In turn, we initially monitored inter-individual changes over time in plasma glutamate levels of controls and observed that they were non-significant. Consequently, we did not carry out a follow-up of controls. Finally, plasma levels of tumor necrosis factor-α (TNFα) were measured by ELISA using a commercial kit (KHC3011C; Biosource International). 2.4. Statistical analysis Comparisons of plasma glutamate levels between the total group of first episode patients and controls were Table 1 Glutamate in plasma from patients at the first-psychotic episode and during a 1-year follow First-psychotic episode patients Control Basal 1 month 6 months 12 months
54.22 ± 16.06 41.67 ± 15.84⁎⁎ 42.31 ± 14.17⁎⁎ 47.94 ± 15.07 49.11 ± 12.58
Values (μM) are given as mean ± S.D. ⁎⁎p b 0.01 for comparisons vs. control.
Control Basal 1 month 6 months 12 months
Schizophrenia
Bipolar disorder
Non-specified psychosis
53.39 ± 14.25 45 ± 14.71⁎ 43.55 ± 16.61⁎ 48.83 ± 16.83 50.76 ± 12.24a
54.39 ± 16.77 40.29 ± 17.32⁎⁎ 42.82 ± 13.51⁎ 49.02 ± 13.24 46.74 ± 10.07
53.4 ± 18.73 38.12 ± 14.16⁎ 34.6 ± 8.57⁎ 46.85 ± 17.41 49.85 ± 17.7
Values (μM) are given as mean ± S.D. ⁎p b 0.05, ⁎⁎p b 0.01 for comparisons vs. control, ap b 0.05, 12 months vs. 1 month.
made using one-way ANOVA and the Dunnett post test. Comparisons of plasma glutamate levels within groups and between each patient group and controls were made between pairs matched for age and sex using paired twotailed Student t-tests. Values are given as mean ± S.D., unless otherwise stated, and differences were considered significant when p b 0.05. 3. Results In the total sample of patients, glutamate concentrations were found to be reduced at the onset of psychosis and after the first month of treatment relative to controls (Table 1). At the first psychotic episode, a significant decrease (15–25%) in plasma glutamate levels was found in patients with schizophrenia, bipolar disorder and non-specified psychosis relative to healthy controls matched for sex and age (Table 2). In each of these groups, a similar reduction in the concentration of glutamate was also observed at 1 month after the first psychotic episode. Glutamate levels progressively increased towards control values during the 1-year follow up after the first episode. Furthermore, TNFα plasma levels were unaltered in all the patient groups and time points studied (data not shown), which is in line with previous findings (Baker et al., 1996; Palomino et al., 2006). 4. Discussion The results of this study show that lower plasma levels of glutamate are associated with the onset of psychosis, and in particular with schizophrenia and bipolar disorder. These findings are consistent with the reported alterations in glutamate signaling in these diseases (Tsai and Coyle, 2002; Schiffer, 2002). The fact that this study examined drug-naïve patients suggests that aberrant glutamate signaling occurs early in schizophrenia and bipolar disease. Glutamate is involved in brain development and synaptic plasticity, two
A. Palomino et al. / Schizophrenia Research 95 (2007) 174–178
processes which are altered in schizophrenia (Angelucci et al., 2005) and in bipolar disorder (Einat and Manji, 2006). Recently, we also found decreased levels of plasma brain derived neurotrophic factor (BDNF) to be associated with these disorders (Palomino et al., 2006). BDNF, which is capable of crossing the blood–brain-barrier (Pan et al., 1998), regulates neuronal development and survival and controls the activity of many neurotransmitter systems, including the serotoninergic and glutamatergic systems (Cotman and Berchtold, 2002). Notably, BDNF is released by NMDA receptor activation and plays an important role in increasing synaptic sensitivity by regulating glutamate release during plasticity (Carter, 2006). Remarkably, the decrease in plasma glutamate levels at the first psychotic episode observed in the current study correlates with that reported for BDNF in a similar cohort of patients (Palomino et al., 2006). Additionally, the time course of the medication-induced restoration of plasma glutamate levels towards control values after the first psychotic episode observed in the current study also parallels that of BDNF (Palomino et al., 2006). Overall, these findings suggests that BDNF up-regulation, which is known to be able to drive glutamate release (Matsumoto et al., 2006), may be the cause of the restoration of plasma glutamate levels. The progressive increase in plasma glutamate levels during the 1 year follow-up after the first psychotic episode supports the idea that treatment could affect excitatory neurotransmission by restoring glutamate levels towards control values. Indeed, atypical antipsychotics, which were preferentially used in the patients included in the current study (González-Pinto et al., 2002), are known to enhance plasma glutamate levels in schizophrenia patients (Evins et al., 1997; Goff et al., 2002). The mechanisms by which atypical antipsychotics regulate the levels of glutamate in plasma are not known, but it may be related to their capacity to reduce the expression and function of glutamate transporters (Vallejo-Illarramendi et al., 2005). In turn, the increase in plasma glutamate observed in bipolar patients is consistent with the stimulatory effect of lithium on glutamate release (Hokin et al., 1996). However, the effects of typical antipsychotics on plasma glutamate levels do not appear to have been characterized yet. In our study, the number of patients treated with these drugs was low (7– 11%) and their plasma glutamate levels were comparable to those in the group of patients treated with atypical antipsychotics. Finally, the similar profile of plasma glutamate levels at the first psychotic episode and during the first year of treatment in schizophrenia and bipolar disorder, together with the fact that both pyschoses have multiple
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clinical manifestations in common (González-Pinto et al., 1998, 2004, 2007) supports the hypothesis that similar alterations in molecular signaling may underlie both of these disorders (Gonzalez-Pinto et al., 2003; Craddock et al., 2005), In summary, decreased plasma levels of glutamate may be directly involved in the pathophysiology of psychosis. Future studies focusing on the association between altered levels of glutamate and clinical variables in schizophrenia and bipolar disorder may provide useful markers to predict the outcome of these diseases. Role of the Funding Source Funding for this study was provided by the Stanley Research Foundation (03-RC-003), Fondo de Investigaciones Sanitarias (PI021297, G03/032, CM04/00101), and the Gobierno Vasco (2004/ 11016 and Saiotek program). These institutions had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Contributors Ana González-Pinto and Carlos Matute designed the study and wrote the protocol. Aitor Palomino conducted the experiments and Carlos Matute supervised them. Aitor Palomino, Ana González-Pinto and Carlos Matute supervised the data, managed the literature searches and analysis, and undertook the statistical analysis. Ana González-Pinto, Ana Aldama, Cristina González-Gómez, Fernando Mosquera and Gixane González-García recruited patients, and built and controlled data bases. Aitor Palomino and Carlos Matute wrote the first draft and final manuscript. All authors contributed to and have approved the final manuscript. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgments We thank Dr. D.J. Fogarty for reviewing the English of this manuscript. This study was supported by the Stanley Research Foundation (03-RC-003), the Fondo de Investigaciones Sanitarias (PI021297, G03/032, CM04/00101), the Spanish Ministry of Health, Instituto de Salud Carlos III, the Red de Enfermedades Mentales (REM-TAP Network) and the Gobierno Vasco (2004/11016 and Saiotek program).
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Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., Volterra, A., 2001. CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity. Nat. Neurosci. 4, 702–710. Carter, C.J., 2006. Schizophrenia susceptibility genes converge on interlinked pathways related to glutamatergic transmission and long-term potentiation, oxidative stress and oligodendrocyte viability. Schizophr. Res. 86, 1–14. Cotman, C.W., Berchtold, N.C., 2002. Exercise: a behavioral intervention to enhance brain health and plasticity. Trends Neurosci. 25, 295–301. Craddock, N., O'Donovan, M.C., Owen, M.J., 2005. The genetics of schizophrenia and bipolar disorder: dissecting psychosis. J. Med. Genet. 42, 193–204. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Einat, H., Manji, H.K., 2006. Cellular plasticity cascades: genes-tobehavior pathways in animal models of bipolar disorder. Biol. Psychiatry 59, 1160–1171. Evins, A.E., Amico, E.T., Shih, V., Goff, D.C., 1997. Clozapine treatment increases serum glutamate and aspartate compared to conventional neuroleptics. J. Neural Transm. 104, 761–766. Goff, D.C., Hennen, J., Lyoo, I.K., Tsai, G., Wald, L.L., Evins, A.E., Yurgelun-Todd, D.A., Renshaw, P.F., 2002. Modulation of brain and serum glutamatergic concentrations following a switch from conventional neuroleptics to olanzapine. Biol. Psychiatry 51, 493–497. González-Pinto, A., Gutiérrez, M., Mosquera, F., Ballesteros, J., Lopez, P., Ezcurra, J., Figuerido, J.L., de Leon, J., 1998. First episode in bipolar disorder: misdiagnosis and psychotic symptoms. J. Affect. Disord. 50, 41–44. González-Pinto, A., Tohen, M., Lalaguna, B., Pérez de Heredia, J.L., Fernández de Corres, B., Gutiérrez, M., Micó, J.A., 2002. Treatment of bipolar I rapid cycling patients during dysphoric mania with olanzapine. J. Clin. Psychopharmacol. 22, 450–454. Gonzalez-Pinto, A., van Os, J., Perez de Heredia, J.L., Mosquera, F., Aldama, A., Lalaguna, B., Gutierrez, M., Mico, J.A., 2003. Agedependence of Schneiderian psychotic symptoms in bipolar patients. Schizophr. Res. 61, 157–162. Gonzalez-Pinto, A., van Os, J., Peralta, V., Perez de Heredia, J.L., Mosquera, F., Aldama, A., Gonzalez, C., Gutierrez, M., 2004. The role of age in the development of Schneiderian symptoms in patients with incident psychosis. Acta Psychiatr. Scand. 109, 1–5. González-Pinto, A., Aldama, A., González, C., Mosquera, F., Arrasate, M., Vieta, E., 2007. Predictors of suicide in first affective and nonaffective psychotic in-patients: five-year follow-up of a cohort of 112 first episode patients from a catchment area in Vitoria, Spain. J. Clin. Psychiatry 68, 242–247. Hawkins, R.A., O'Kane, R.L., Simpson, I.A., Vina, J.R., 2006. Structure of the blood–brain barrier and its role in the transport of amino acids. J. Nutr. 136, 218S–226S. Hoekstra, R., Fekkes, D., Loonen, A.J., Pepplinkhuizen, L., Tuinier, S., Verhoeven, W.M., 2006. Bipolar mania and plasma amino acids: increased levels of glycine. Eur. Neuropsychopharmacol. 16, 71–77. Hokin, L.E., Dixon, J.F., Los, G.V., 1996. A novel action of lithium: stimulation of glutamate release and inositol 1,4,5 trisphosphate accumulation via activation of the N-methyl D-aspartate receptor in monkey and mouse cerebral cortex slices. Adv. Enzyme Regul. 36, 229–244.
Javitt, D.C., Zukin, S.R., 1991. Recent advances in the phencyclidine model of schizophrenia. Am. J. Psychiatry 148, 1301–1308. Kim, J.S., Kornhuber, H.H., Schmid-Burgk, W., Holzmuller, B., 1980. Low cerebrospinal fluid glutamate in schizophrenic patients and a new hypothesis on schizophrenia. Neurosci. Lett. 20, 379–382. Konradi, C., Heckers, S., 2003. Molecular aspects of glutamate deregulation: implications for schizophrenia and its treatment. Pharmacol. Ther. 97, 153–179. Mallolas, J., Hurtado, O., Castellanos, M., Blanco, M., Sobrino, T., Serena, J., Vivancos, J., Castillo, J., Lizasoain, I., Moro, M.A., Davalos, A., 2006. A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke. J. Exp. Med. 203, 711–717. Matsumoto, T., Numakawa, T., Yokomaku, D., Adachi, N., Yamagishi, S., Numakawa, Y., Kunugi, H., Taguchi, T., 2006. Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: different dependency on signaling pathways and neuronal activity. Mol. Cell. Neurosci. 31, 70–84. Matute, C., Melone, M., Vallejo-Illarramendi, A., Conti, F., 2005. Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics. Glia 49, 451–455. McCullumsmith, R.E., Meador-Woodruff, J.H., 2002. Striatal excitatory amino acid transporter transcript expression in schizophrenia, bipolar disorder, and major depressive disorder. Neuropsychopharmacology 26, 368–375. Mohn, A.R., Gainetdinov, R.R., Caron, M.G., Koller, B.H., 1999. Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell 98, 427–436. O'Kane, R.L., Martinez-Lopez, I., DeJoseph, M.R., Vina, J.R., Hawkins, R.A., 1999. Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood–brain barrier. A mechanism for glutamate removal. J. Biol. Chem. 274, 31891–31895. Olney, J.W., Farber, N.B., 1995. Glutamate receptor dysfunction and schizophrenia. Arch. Gen. Psychiatry 52, 998–1007. Palomino, A., Vallejo-Illarramendi, A., Gonzalez-Pinto, A., Aldama, A., Gonzalez-Gomez, C., Mosquera, F., Gonzalez-Garcia, G., Matute, C., 2006. Decreased levels of plasma BDNF in firstepisode schizophrenia and bipolar disorder patients. Schizophr. Res. 86, 321–322. Pan, W., Banks, W.A., Fasold, M.B., Bluth, J., Kastin, A.J., 1998. Transport of brain-derived neurotrophic factor across the blood– brain barrier. Neuropharmacology 37, 1553–1561. Schiffer, H.H., 2002. Glutamate receptor genes: susceptibility factors in schizophrenia and depressive disorders? Mol. Neurobiol. 25, 191–212. Tortorella, A., Monteleone, P., Fabrazzo, M., Viggiano, A., De, L.L., Maj, M., 2001. Plasma concentrations of amino acids in chronic schizophrenics treated with clozapine. Neuropsychobiology 44, 167–171. Tsai, G., Coyle, J.T., 2002. Glutamatergic mechanisms in schizophrenia. Annu. Rev. Pharmacol. Toxicol. 42, 165–179. Vallejo-Illarramendi, A., Torres-Ramos, M., Melone, M., Conti, F., Matute, C., 2005. Clozapine reduces GLT-1 expression and glutamate uptake in astrocyte cultures. Glia 50, 276–279. van der Heijden, F.M., Tuinier, S., Fekkes, D., Sijben, A.E., Kahn, R.S., Verhoeven, W.M., 2004. Atypical antipsychotics and the relevance of glutamate and serotonin. Eur. Neuropsychopharmacol. 14, 259–265.
Decreased levels of plasma glutamate in patients with first-episode schizophrenia and bipolar disorder Aitor Palomino a , Ana González-Pinto b , Ana Aldama b , Cristina González-Gómez b , Fernando Mosquera b , Gixane González-García b , Carlos Matute a,⁎ a
Neurotek UPV-EHU, Parque Tecnológico de Bizkaia, E-48170 Zamudio, Vizcaya, Spain b Stanley Research Center, 03-RC-003, Vitoria, Spain
Received 30 November 2006; received in revised form 12 June 2007; accepted 14 June 2007 Available online 13 July 2007
Abstract A variety of studies have suggested that glutamatergic neurotransmission is altered in schizophrenia and bipolar disorder. Here, we tested if plasma glutamate levels are altered in 56 patients diagnosed with schizophrenia, bipolar disorder or non-specified psychosis at the first psychotic episode and at various stages during one-year follow-up. A decrease in the levels of plasma glutamate was observed in all groups of patients at the first psychotic episode. Furthermore, plasma glutamate levels were restored after treatment in all instances. Decreased plasma glutamate levels at first psychotic episodes may reflect impaired glutamate signaling during the initial stages of schizophrenia and bipolar disorder. © 2007 Elsevier B.V. All rights reserved. Keywords: Glutamate; First psychotic episode; Schizophrenia; Bipolar disorder
1. Introduction Glutamate is the major excitatory neurotransmitter in the mammalian brain and its effects are mediated by glutamate-gated ion channels and seven transmembrane, G-protein coupled glutamate receptors. In turn, glutamate homeostasis is driven by glutamate transporters which regulate the extracellular levels of glutamate (Danbolt, 2001). The first evidence of decreased glutamate levels in schizophrenia was reported over 25 years ago (Kim et al., 1980). Since then, mounting evidence has accumulated indicating that the dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia and bipolar disorder (Tsai and Coyle, 2002; Schiffer, ⁎ Corresponding author. 0920-9964/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2007.06.012
2002). In particular, the hypofunction of N-methyl-Daspartate (NMDA) type glutamate receptors is considered to be a key factor in schizophrenia (Olney and Farber, 1995). Thus, the psychotomimetic drug phencyclidine blocks the NMDA receptor channel (Javitt and Zukin, 1991); mice expressing low levels of the NMDAR1 subunit display behaviors which are related to schizophrenia and are ameliorated by antipsychotic treatment (Mohn et al., 1999); and treatment with positive modulators of NMDA receptors induces therapeutic benefit (Konradi and Heckers, 2003). In addition, glutamate transporter expression and function is altered in areas of the brain which are vulnerable to schizophrenia and bipolar disease (McCullumsmith and Meador-Woodruff, 2002; Matute et al., 2005). Overall, these findings suggest that glutamate signaling is impaired in schizophrenia.
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of the sample were female. Total PANSS scores (mean ± S.D.) were 80.98 ± 16.39 at baseline, 57.51 ± 19.57 at one month, 53.33 ± 19.04 at six months, and 51.76 ±16.69 at twelve months. GAF was 30.72 at baseline, 51.28 at one month, 56.19 ± 15.26 at six months, and 56.60 ± 14.47 at twelve months. Patients were diagnosed after 12 months of inclusion using the Structured Clinical Interview for DSM IV, SCID-I. 24 patients were diagnosed with schizophrenia, 17 with bipolar disorder, and the remaining 15 with non-specified psychotic disorders. Patients with schizophrenia had the following demographic characteristics: mean age (± S.E.M), 23.21 ± 1.33 years; % female, 16.7%; % with at least primary level education (Neleven years of schooling), 90% and % with medium or higher socioeconomic status, 50%. Patients with bipolar disorder had the following demographic characteristics: mean age (± S.E.M), 25.29 ± 1.71 years; % female, 35.3%; % with at least primary level education (Neleven years of schooling), 93.3% and % with medium or higher socioeconomic status, 76.9%. Patients with other psychotic disorders had the following demographic characteristics: mean age (± S.E.M), 23.36 ± 2.59 years; % female, 40%; % with at least primary level education (Neleven years of schooling), 92.3% and % with medium or higher socioeconomic status, 69.2%. Each patient was paired by age and sex with a control subject. Patients were treated after the first episode with atypical antipsychotics (62–68%), with lithium or other mood stabilizers together with atypical antipsychotics (23– 26%), with typical antipsychotics (7–11%), or received no treatment (2–4%). Ranges in each treatment group indicate changes in the drugs administered initially at the onset of symptoms and during the first year of illness.
2.1. Patients
2.2. Control subjects
In this study, we examined 56 patients (mean age ± S.E.M., 23.3 ± 0.94 years) from the health catchment area of Vitoria (300.000 inhabitants) (Álava, Spain), who experienced a first psychotic episode during the period 2002–2005. First psychotic episode was defined as the first time a patient displayed positive psychotic symptoms of delusions or hallucinations. This sample of patients consisting of all first-episode patients who required hospitalization and gave informed consent to participate in the study, represented 75% of the total population of patients who had been admitted to the psychiatric emergency care unit. Dropout from the study was around 40% after and occurred principally towards the end of the year of treatment. No significant differences in age, gender, or clinical symptoms were detected between included and excluded patients. 28.4%
In addition, 50 healthy volunteers were selected explicitly for the research and matched pairwise for sex and age (25.19 ± 0.95 years, mean age ± S.E.M.). Twenty one of these controls were used for comparisons with patients with schizophrenia, 15 for comparisons with bipolar disorder and 14 for comparisons with non-specified psychoses. All subjects (case and controls) were recruited from the same community and included after informed consent to participate in the study was obtained. Subjects with mental retardation, organic brain disorders, or drug abuse as a primary diagnosis were excluded. As for the patients, 44% were smokers, 52% consumed alcohol, 13% abused alcohol and 34% abused cannabis. We did not observe differences in the plasma glutamate concentration among these groups, or in comparison to the remaining patients. The exclusion criteria for controls consisted of the
The levels of glutamate in blood plasma may reflect the extracellular concentration of glutamate within the CNS, since brain-to-blood efflux of this amino acid occurs through the blood–brain-barrier (Berl et al., 1961; O'Kane et al., 1999), but the entry from blood into the brain is minimal (Hawkins et al., 2006). Thus, although blood cells and peripheral organs may constitute the primary source of plasma glutamate, exit of glutamate from the CNS also contributes substantially to the overall concentration of glutamate in plasma, as shown in certain pathological conditions (Mallolas et al., 2006). Furthermore, atypical antipsychotic drugs increase extracellular concentrations of glutamate by reducing the expression and function of glutamate transporters (Vallejo-Illarramendi et al., 2005), and as a consequence these drugs may increase plasma glutamate levels. Indeed, several studies have reported an increased concentration of glutamate in the plasma/ serum of patients with schizophrenia who had been treated with antipsychotics (Evins et al., 1997; Tortorella et al., 2001; Goff et al., 2002; van der Heijden et al., 2004). In addition, plasma glutamate levels are higher in treated bipolar disorder patients than in healthy controls (Hoekstra et al., 2006). In the present study, we set out to characterize the levels of plasma glutamate in patients with schizophrenia or bipolar disorder upon the first psychotic episode and at various intervals thereafter. We observed that the concentration of glutamate in plasma is reduced at the onset of disease in both instances but is restored to near normal levels following 1 year treatment.
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absence of any Axis I disorder, as well as the exclusion criteria applied to the patient group.
Table 2 Glutamate in plasma from patients with schizophrenia, bipolar disorder and non-specified psychosis
2.3. Experimental procedures Blood samples were collected upon arrival at the hospital emergency room and at 1, 6 and 12 months later, using glass whole-blood tubes containing K3-EDTA. Plasma was isolated by centrifugation at 300 ×g for 10 min and frozen at −80 °C until the time of analysis. Plasma levels of glutamate were measured by enzymatic assay. The enzyme glutamate dehydrogenase, in the presence of NADP+, oxidizes glutamate to α-ketoglutarate and generates NADPH. NADPH fluorescence was excited at 340 nm and emitted at 420 nm. The reaction buffer consisted of NaCl (14 mM), KCl (5 mM), MgCl2 (2 mM), pH 7.7 and was supplemented with 1 mM NADP+ and glutamate dehydrogenase (15 U/ml). 40 μl of plasma was diluted with the reaction buffer to a final volume of 300 μl per assay and the fluorescence values were measured after 10 min using a microplate reader (Synergy; Biotek). Glutamate concentration was determined using standard curves constructed with serial dilutions of glutamate. These curves were linear between 5 μM and 100 μM of the transmitter and plasma levels in both patients and controls were within those values. This fluorimetric method is highly reliable under the conditions employed (Bezzi et al., 2001). Repeated measures of the same sample resulted in variations within a range of around ±2%. In turn, we initially monitored inter-individual changes over time in plasma glutamate levels of controls and observed that they were non-significant. Consequently, we did not carry out a follow-up of controls. Finally, plasma levels of tumor necrosis factor-α (TNFα) were measured by ELISA using a commercial kit (KHC3011C; Biosource International). 2.4. Statistical analysis Comparisons of plasma glutamate levels between the total group of first episode patients and controls were Table 1 Glutamate in plasma from patients at the first-psychotic episode and during a 1-year follow First-psychotic episode patients Control Basal 1 month 6 months 12 months
54.22 ± 16.06 41.67 ± 15.84⁎⁎ 42.31 ± 14.17⁎⁎ 47.94 ± 15.07 49.11 ± 12.58
Values (μM) are given as mean ± S.D. ⁎⁎p b 0.01 for comparisons vs. control.
Control Basal 1 month 6 months 12 months
Schizophrenia
Bipolar disorder
Non-specified psychosis
53.39 ± 14.25 45 ± 14.71⁎ 43.55 ± 16.61⁎ 48.83 ± 16.83 50.76 ± 12.24a
54.39 ± 16.77 40.29 ± 17.32⁎⁎ 42.82 ± 13.51⁎ 49.02 ± 13.24 46.74 ± 10.07
53.4 ± 18.73 38.12 ± 14.16⁎ 34.6 ± 8.57⁎ 46.85 ± 17.41 49.85 ± 17.7
Values (μM) are given as mean ± S.D. ⁎p b 0.05, ⁎⁎p b 0.01 for comparisons vs. control, ap b 0.05, 12 months vs. 1 month.
made using one-way ANOVA and the Dunnett post test. Comparisons of plasma glutamate levels within groups and between each patient group and controls were made between pairs matched for age and sex using paired twotailed Student t-tests. Values are given as mean ± S.D., unless otherwise stated, and differences were considered significant when p b 0.05. 3. Results In the total sample of patients, glutamate concentrations were found to be reduced at the onset of psychosis and after the first month of treatment relative to controls (Table 1). At the first psychotic episode, a significant decrease (15–25%) in plasma glutamate levels was found in patients with schizophrenia, bipolar disorder and non-specified psychosis relative to healthy controls matched for sex and age (Table 2). In each of these groups, a similar reduction in the concentration of glutamate was also observed at 1 month after the first psychotic episode. Glutamate levels progressively increased towards control values during the 1-year follow up after the first episode. Furthermore, TNFα plasma levels were unaltered in all the patient groups and time points studied (data not shown), which is in line with previous findings (Baker et al., 1996; Palomino et al., 2006). 4. Discussion The results of this study show that lower plasma levels of glutamate are associated with the onset of psychosis, and in particular with schizophrenia and bipolar disorder. These findings are consistent with the reported alterations in glutamate signaling in these diseases (Tsai and Coyle, 2002; Schiffer, 2002). The fact that this study examined drug-naïve patients suggests that aberrant glutamate signaling occurs early in schizophrenia and bipolar disease. Glutamate is involved in brain development and synaptic plasticity, two
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processes which are altered in schizophrenia (Angelucci et al., 2005) and in bipolar disorder (Einat and Manji, 2006). Recently, we also found decreased levels of plasma brain derived neurotrophic factor (BDNF) to be associated with these disorders (Palomino et al., 2006). BDNF, which is capable of crossing the blood–brain-barrier (Pan et al., 1998), regulates neuronal development and survival and controls the activity of many neurotransmitter systems, including the serotoninergic and glutamatergic systems (Cotman and Berchtold, 2002). Notably, BDNF is released by NMDA receptor activation and plays an important role in increasing synaptic sensitivity by regulating glutamate release during plasticity (Carter, 2006). Remarkably, the decrease in plasma glutamate levels at the first psychotic episode observed in the current study correlates with that reported for BDNF in a similar cohort of patients (Palomino et al., 2006). Additionally, the time course of the medication-induced restoration of plasma glutamate levels towards control values after the first psychotic episode observed in the current study also parallels that of BDNF (Palomino et al., 2006). Overall, these findings suggests that BDNF up-regulation, which is known to be able to drive glutamate release (Matsumoto et al., 2006), may be the cause of the restoration of plasma glutamate levels. The progressive increase in plasma glutamate levels during the 1 year follow-up after the first psychotic episode supports the idea that treatment could affect excitatory neurotransmission by restoring glutamate levels towards control values. Indeed, atypical antipsychotics, which were preferentially used in the patients included in the current study (González-Pinto et al., 2002), are known to enhance plasma glutamate levels in schizophrenia patients (Evins et al., 1997; Goff et al., 2002). The mechanisms by which atypical antipsychotics regulate the levels of glutamate in plasma are not known, but it may be related to their capacity to reduce the expression and function of glutamate transporters (Vallejo-Illarramendi et al., 2005). In turn, the increase in plasma glutamate observed in bipolar patients is consistent with the stimulatory effect of lithium on glutamate release (Hokin et al., 1996). However, the effects of typical antipsychotics on plasma glutamate levels do not appear to have been characterized yet. In our study, the number of patients treated with these drugs was low (7– 11%) and their plasma glutamate levels were comparable to those in the group of patients treated with atypical antipsychotics. Finally, the similar profile of plasma glutamate levels at the first psychotic episode and during the first year of treatment in schizophrenia and bipolar disorder, together with the fact that both pyschoses have multiple
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clinical manifestations in common (González-Pinto et al., 1998, 2004, 2007) supports the hypothesis that similar alterations in molecular signaling may underlie both of these disorders (Gonzalez-Pinto et al., 2003; Craddock et al., 2005), In summary, decreased plasma levels of glutamate may be directly involved in the pathophysiology of psychosis. Future studies focusing on the association between altered levels of glutamate and clinical variables in schizophrenia and bipolar disorder may provide useful markers to predict the outcome of these diseases. Role of the Funding Source Funding for this study was provided by the Stanley Research Foundation (03-RC-003), Fondo de Investigaciones Sanitarias (PI021297, G03/032, CM04/00101), and the Gobierno Vasco (2004/ 11016 and Saiotek program). These institutions had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Contributors Ana González-Pinto and Carlos Matute designed the study and wrote the protocol. Aitor Palomino conducted the experiments and Carlos Matute supervised them. Aitor Palomino, Ana González-Pinto and Carlos Matute supervised the data, managed the literature searches and analysis, and undertook the statistical analysis. Ana González-Pinto, Ana Aldama, Cristina González-Gómez, Fernando Mosquera and Gixane González-García recruited patients, and built and controlled data bases. Aitor Palomino and Carlos Matute wrote the first draft and final manuscript. All authors contributed to and have approved the final manuscript. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgments We thank Dr. D.J. Fogarty for reviewing the English of this manuscript. This study was supported by the Stanley Research Foundation (03-RC-003), the Fondo de Investigaciones Sanitarias (PI021297, G03/032, CM04/00101), the Spanish Ministry of Health, Instituto de Salud Carlos III, the Red de Enfermedades Mentales (REM-TAP Network) and the Gobierno Vasco (2004/11016 and Saiotek program).
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