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Neurochemistry International 61 (2012) 1144–1150

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Neurochemistry International

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Effects of the putative alstonine on glutamate uptake in acute hippocampal slices ⇑ Ana P. Herrmann a,b, , Paula Lunardi b, Luísa Klaus Pilz a, Ana C. Tramontina b, Viviane M. Linck a,b, Christopher O. Okunji c, Carlos A. Gonçalves b, Elaine Elisabetsky a,b a Laboratório de Etnofarmacologia, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, 90050-170 Porto Alegre, RS, Brazil b Programa de Pós-graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600, 90035-000 Porto Alegre, RS, Brazil c International Centre for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Enugu State, Nigeria article info abstract

Article history: A dysfunctional glutamatergic system is thought to be central to the negative symptoms and cognitive Received 8 March 2012 deficits recognized as determinant to the poor quality of life of people with schizophrenia. Modulating Received in revised form 13 August 2012 glutamate uptake has, thus, been suggested as a novel target for . Alstonine is an Accepted 15 August 2012 sharing with atypical antipsychotics the profile in animal models relevant to schizophrenia, Available online 25 August 2012 though divergent in its mechanism of action. The aim of this study was to evaluate the effects of alstonine on glutamate uptake. Additionally, the effects on glutathione content and extracellular S100B levels were Keywords: assessed. Acute hippocampal slices were incubated with (10 lM), (10 and 100 lM) Alstonine or alstonine (1–100 M), alone or in combination with (100 M), and 5-HT receptor antag- Antipsychotics l l 2 Schizophrenia onists (0.01 lM and 0.1 lM SB 242084). A reduction in glutamate uptake was observed with Astrocytes alstonine and clozapine, but not haloperidol. Apomorphine abolished the effect of clozapine, whereas 5- Glutamate uptake HT2A and 5-HT2C antagonists abolished the effects of alstonine. Increased levels of glutathione were S100B observed only with alstonine, also the only compound that failed to decrease the release of S100B. This study shows that alstonine decreases glutamate uptake, which may be beneficial to the glutamatergic deficit observed in schizophrenia. Noteworthily, the decrease in glutamate uptake is compatible with the reversal of MK-801-induced social interaction and working memory deficits. An additional potential benefit of alstonine as an antipsychotic is its ability to increase glutathione, a key cellular antioxidant reported to be decreased in the brain of patients with schizophrenia. Adding to the characterization of the novel mechanism of action of alstonine, the lack of effect of apomorphine in alstonine-induced

changes in glutamate uptake reinforces that D2 receptors are not primarily implicated. Though clearly

mediated by 5-HT2A and 5-HT2C receptors, the precise mechanisms that result in the effects of alstonine on glutamate uptake warrant elucidation. Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction prompted the idea of a glutamatergic dysfunction in the patho- physiology of schizophrenia. PCP, as well as the related drugs With lifetime prevalence commonly estimated in 1% of the pop- ketamine and MK-801, induce in normal volunteers psychosis-like ulation, schizophrenia is a severe psychiatric disorder marked by and neurocognitive disturbances similar to those of schizophrenia; profound retreat from reality and incapacitating cognitive deficits. unlike dopaminergic , these drugs mimic all the symptoms It belongs to a group of complex multifactorial disorders, for which dimensions of the disease (Javitt, 2010). It is now widely accepted the etiology is understood as the intricate interaction between ge- that the dopaminergic alterations classically associated to schizo- netic and environmental factors (Gogos and Gerber, 2006; van Os phrenia may, in fact, be secondary to an underlying deficit in gluta- et al., 2010). matergic transmission (Carlsson, 2006; Marek et al., 2010). Of note, The observations of the behavioral effects induced by phencyc- Stone et al. (2010) reported in prodromic subjects with mild psy- lidine (PCP), an antagonist of glutamate NMDA receptors, chotic symptoms a negative relationship between hippocampal glutamate levels and striatal [18F]-DOPA uptake, reinforcing the idea of a coupling between glutamatergic transmission and striatal ⇑ Corresponding author at: Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, sala 202, 90050-170 Porto dopaminergic activity. Alegre, RS, Brazil. Tel./fax: +55 51 33083121. Glutamate is released into the synapse and rapidly removed by E-mail address: [email protected] (A.P. Herrmann). a family of excitatory amino acid transporters (EAATs) localized in

0197-0186 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.neuint.2012.08.006 A.P. Herrmann et al. / Neurochemistry International 61 (2012) 1144–1150 1145 neurons and glial cells. Five members of EAATs have been identi- controlled environmental conditions (12-h light/dark cycle at a fied and termed EAAT1–5 to human homologs, or GLAST, GLT-1 constant temperature of 22 ± 1 °C) with free access to food and and EAAC-1 representing the rodent homologs of EAATs1–3, water. The study was approved by the University Ethics Committee respectively (Kanai and Hediger, 1992; Pines et al., 1992; Storck (approval #18237), and followed institutional policies on experi- et al., 1992; Fairman et al., 1995; Arriza et al., 1997). EAAT2/ mental animal handling. All efforts were made to minimize animal GLT-1 is predominantly expressed in astrocytes and provides more suffering, to reduce the number of animals used, and to utilize than 90% of total glutamate uptake in the central nervous system alternatives to in vivo techniques. (Tanaka et al., 1997). Given that astrocytes are fundamental in the control of gluta- 2.2. Drugs and reagents mate homeostasis, a significant role has recently been assigned to glial–neuronal interactions in the pathophysiology of schizo- Alstonine hydrochloride was isolated from Picralima nitida Th. & phrenia. There is increasing evidence that structural and functional H. Dur. (Apocynaceae) by Christopher O. Okunji, as earlier de- alterations in glial cells are present in the brain of people with scribed (Okunji et al., 2005). Clozapine, altanserin and SB 242084 schizophrenia (Cotter et al., 2001; Kondziella et al., 2006; Bernstein were purchased from Sigma Chemical Co. (St. Louis, MO, USA), et al., 2009; Katsel et al., 2011). Additionally, a DNA array study apomorphine was purchased from Research Biochemicals Interna- showed that the genes most frequently altered in the disorder tional (Natick, MA, USA), DL-threo-b-benzyloxyaspartic acid are related to glial function (Sugai et al., 2004). Signaling mole- (DL-TBOA) was acquired from Tocris Bioscience (Bristol, UK) and cules, such as S100B and GFAP, have also been examined as mark- haloperidol was used as commercial HaldolÒ from Janssen-Cilag ers of astrocytic function. Several studies reported increased S100B Farmacêutica Ltd. (São Paulo, SP, Brazil). Monoclonal anti-S100B levels in the serum or cerebrospinal fluid (CSF) of patients with antibody (SHB1), 4-(2-hydroxyethyl)-1-ethanesulfonic schizophrenia (Wiesmann et al., 1999; Lara et al., 2001; Rother- acid (Hepes), o-phenylenediamine (OPD), 3-(4,5-dimethylthiazol- mundt et al., 2001, 2004a,b, 2007; Schroeter et al., 2003; Schmitt yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and L-glutamate et al., 2005); however, the effects of antipsychotic drugs in this were purchased from Sigma (St. Louis, MO, USA), polyclonal rabbit parameter remain unclear (Steiner et al., 2010). anti-S100 from Dako (Glostrup, Denmark), and horseradish perox- It is undisputable that over the past decades major advances 3 idase-conjugated anti-rabbit IgG and L-[2,3- H]-glutamate from have been accomplished in the understanding of the neurobiologi- Amersham International (Buckinghamshire, UK). All other chemi- cal processes involved in schizophrenia. The acquired knowledge, cals were purchased from local commercial suppliers. however, has not translated into better treatment options, since all currently available antipsychotics share their mechanism of ac- tion with drugs discovered almost 60 years ago. Moreover, the 2.3. Hippocampal slices modest and controversial benefits of antipsychotics on cognitive deficits and negative symptoms, combined with the unwanted side Rats were killed by decapitation. Hippocampi were removed effects, result in high rates of treatment discontinuation (Lieber- and sliced in transverse sections of 0.3 mm using a McIlwain tissue man et al., 2005). The development of drugs with innovative mech- chopper. Slices were transferred to 24-well plates containing anisms of action is, thus, required (Gründer et al., 2009). 0.3 mL of Hepes-buffered saline solution (120 mM NaCl, 2 mM We have previously reported the properties of alstonine, a puta- KCl, 1 mM CaCl2, 1 mM MgSO4, 1 mM KH2PO4, 10 mM glucose tive antipsychotic which has consistently shown and and 25 mM Hepes, pH 7.4) per well. The medium was changed antipsychotic-like effects in several mouse models (Costa-Campos every 15 min for 2 h at room temperature. After this stabilization et al., 1998, 2004a; de Moura Linck et al., 2008). Alstonine is an in- period, the drugs were added and the slices incubated for 1 h at dole alkaloid present in plant species traditionally used in Nigeria 30 °C. Treatments consisted of haloperidol (10 lM), clozapine (10 to treat mentally ill patients (Costa-Campos et al., 1998). Although and 100 lM), alstonine (1, 10 and 100 lM), apomorphine (100 lM), altanserin (0.01 lM) and SB 242084 (0.1 lM). Drug con- its mechanism of action remains unclear, D2 receptors do not seem to be directly involved in its antipsychotic-like effects, and block- centrations were chosen based on pilot experiments and/or previ- ous reports (Steiner et al., 2010; Nardin et al., 2011). All ade of 5-HT2A/C receptors by abolished its anxiolytic ac- tion (Costa-Campos et al., 2004a). Recent data showed that the experiments were performed in triplicate (three slices from the ability of alstonine to counteract MK-801-induced working mem- same animal were used for each treatment condition). ory deficit, social withdrawal and hyperlocomotion was also blocked by pretreatment with ritanserin (Linck et al., 2012). 2.4. Glutamate uptake Considering the growing role attributed to astrocytes in brain signaling and the recognized association between glutamatergic After the incubation period, the medium was replaced by dysfunction and schizophrenia, the aim of this study was to evalu- Hank’s balanced salt solution (HBSS) containing 137 mM NaCl, ate the effects of alstonine on glutamate uptake and other astro- 0.63 mM Na2HPO4, 4.17 mM NaOHCO3, 5.36 mM KCl, 0.44 mM cytic parameters. The effects of alstonine were compared to KH2PO4, 1.26 mM CaCl2, 0.41 mM MgSO4, 0.49 mM MgCl2 and typical and atypical antipsychotics (haloperidol and clozapine, 5.55 mM glucose, pH 7.4. The slices were maintained at 35 °C, respectively). For this purpose, we used acute hippocampal slices, and the assay was started by adding 0.1 mM L-glutamate and 3 elected for preserving the neuronal circuitry and the interactions 0.66 lCi/mL L-[2,3- H]-glutamate; this high unlabeled glutamate between neurons and glia. concentration renders the assay insensitive to changes in gluta- mate release. Incubation was stopped after 5 min by removing the medium and rinsing the slices three times with ice-cold HBSS; 2. Materials and methods it has been shown that up to 5 min the glutamate uptake is linear in this slice preparation (Thomazi et al., 2004). Slices were then 2.1. Animals lysed in a solution containing 0.5 N NaOH. Sodium-independent (nonspecific) uptake was determined using a solution with N- Experiments were performed with 30-day-old male Wistar rats methyl-D-glutamine instead of NaCl. Sodium-dependent glutamate obtained from our breeding colony (Department of Biochemistry, uptake was obtained by subtracting nonspecific uptake. Radioac- UFRGS, Porto Alegre, Brazil). Animals were maintained under tivity was measured with a scintillation counter (2800TR TriCarb 1146 A.P. Herrmann et al. / Neurochemistry International 61 (2012) 1144–1150

Liquid Scintillation Analyzer, Perkin-Elmer, Waltham, MA, USA). Fi- 3. Results nal glutamate uptake was expressed as nmol/mg protein/min. As an indication that most of the sodium-dependent uptake in In order to verify if cell viability (Fig. 1A) and integrity (Fig. 1B) this slice preparation is glutamate transporter-dependent, 75% of were not compromised by drug treatments, two assays were per- the uptake was inhibited by the transporter blocker DL-TBOA formed in parallel; no significant differences were observed for

(300 lM), in comparison to 85% inhibition in sodium-free condi- MTT reduction (F6, 53 = 2.15, p > 0.05) or LDH activity (F6, 56 = 0.25, tions (basal: 6463 ± 699, sodium-free: 940 ± 185, 300 lM DL- p > 0.05), indicating that slices remained viable and integrate. TBOA: 1613 ± 161, in cpm, data not shown). These same parameters were also evaluated for apomorphine, altanserin and SB 242084, with no significant alterations (data not shown). 2.5. Glutathione content Glutamate uptake in hippocampal slices was significantly

(F6, 55 = 8.63, p < 0.0001) altered by treatments (Fig. 2A). Post hoc Total reduced glutathione content was determined as previ- analysis revealed that 10 and 100 lM clozapine and alstonine sig- ously described (Browne and Armstrong, 1998). Briefly, slices nificantly decreased glutamate uptake (49% and 59%, respectively, were homogenized in sodium phosphate buffer (0.1 M, pH 8.0) at highest concentrations). The co-incubation of the drugs with containing 5 mM EDTA and protein was precipitated with 1.7% 100 lM apomorphine also affected glutamate uptake (F7, 50 = 9.83, meta-phosphoric acid. Supernatant was assayed with o-phthaldial- p < 0.0001), as shown in Fig. 2B. Post hoc analysis indicated that dehyde (1 mg/ml of methanol) at room temperature for 15 min. apomorphine had no effects per se, and did not interfere with alst- Fluorescence was measured using excitation and emission wave- onine-induced decrease in glutamate uptake; interestingly, how- lengths of 350 and 420 nm, respectively. A calibration curve was ever, the decrease in glutamate uptake induced by clozapine at performed with standard glutathione solutions (0–500 lM). Final 100 lM was abolished in the presence of apomorphine. concentrations were calculated and expressed as nmol/mg protein. The role of 5-HT2A and 5-HT2C receptors on alstonine-induced reduction of glutamate uptake was investigated. Two-way ANOVA

revealed a main effect of 100 lM alstonine (F1, 26 = 11.79, p < 0.01), 2.6. Extracellular S100B content and of the 5-HT2A antagonist altanserin (F1, 26 = 4.23, p < 0.05), as well as interaction between alstonine and altanserin

Immediately after the incubation period, 10 lL of medium were (F1, 26 = 14.25, p < 0.001) (Fig. 3A). Two-way ANOVA also revealed collected and kept in À20 °C until S100B levels were determined by a main effect of 100 lM alstonine (F1, 26 = 71.89, p < 0.01) and of ELISA, as previously described (Leite et al., 2008). Briefly, 50 lLof the 5-HT2C antagonist SB 242084 (F1, 26 = 7.46, p < 0.05), as well Tris buffer were added to the samples and incubated for 2 h in a as interaction between alstonine and SB 242084 (F1, 26 = 40.46, microtiter plate previously coated with monoclonal anti-S100B. p < 0.001) (Fig. 3B). These results show that the effect of alstonine

Polyclonal anti-S100 was incubated for 30 min; peroxidase- on glutamate uptake involve both 5-HT2A and 5-HT2C receptors. conjugated anti-rabbit antibody was added for additional 30-min incubation. The color reaction with o-phenylenediamine was mea- sured at 492 nm. The standard S100B curve ranged from 0.002 to 1 ng/mL. Results were expressed as a percentage of the control.

2.7. Cell viability and integrity

Cell viability was assayed by the colorimetric MTT reduction method (Hansen et al., 1989). Briefly, slices were incubated with 0.5 mg/mL of MTT at 30 °C for 30 min. The formazan product gen- erated during the incubation was solubilized in dimethyl sulfoxide (DMSO). Absorbance values were measured at 560 and 630 nm. Cell integrity was indicated by lactate dehydrogenase (LDH) activ- ity in the incubation medium. Determination was carried out by a colorimetric commercial kit (Doles Reagentes e Equipamentos para Laboratórios Ltd., Brazil) according to the manufacturer’s instruc- tions. Results were expressed as percentage of the control.

2.8. Protein content

Total protein concentrations were determined by the modified method of Lowry (Peterson, 1977), using bovine serum albumin as standard.

2.9. Statistical analysis

Data were analyzed by one-way ANOVA followed by Newman– Keuls post hoc test. Two-way ANOVA was employed when analyz- Fig. 1. Effects of haloperidol (Hal), clozapine (Clz) and alstonine (Alst) on cell viability and integrity. (A) MTT reduction to formazan. (B) Activity of lactate ing the effects of two independent variables. GraphPad Prism 5 for dehydrogenase released into the medium. Hippocampal slices were incubated for Windows was used for the statistical analysis; p < 0.05 was set as 1 h with the various drugs at the indicated concentrations. Data represent statistically significant. Results represent mean + SEM. mean + SEM and are expressed as percentage of control. One-way ANOVA. A.P. Herrmann et al. / Neurochemistry International 61 (2012) 1144–1150 1147

Fig. 4. Effects of haloperidol (Hal), clozapine (Clz) and alstonine (Alst) on glutathione content. Hippocampal slices were incubated for 1 h with the drugs and total glutathione content was measured. Data represent mean + SEM and are expressed as nmol/mg protein. Ã = p < 0.05 compared to control. One-way ANOVA/ Newman–Keuls.

Fig. 2. Effects of haloperidol (Hal), clozapine (Clz), alstonine (Alst) and apomor- phine (Apo) on glutamate uptake in hippocampal slices. (A) Glutamate uptake after incubation with the drugs per se. (B) Glutamate uptake after co-incubation of drugs and apomorphine. Data represent mean + SEM and are expressed as nmol/mg protein/min. Ã = p < 0.05, ÃÃ = p < 0.01, ÃÃÃ = p < 0.001 compared to control. One- way ANOVA/Newman–Keuls. Fig. 5. Effects of haloperidol, clozapine and alstonine on S100B secretion. Hippo- campal slices were incubated for 1 h with the drugs and S100B levels in the medium were measured. Data represent mean + SEM and are expressed as percentage of control. Ã=p < 0.05 compared to control. One-way ANOVA/New- man–Keuls.

alstonine in its highest concentration (100 lM) was able to in- crease (70%) glutathione levels in comparison to control. As shown in Fig. 5, treatment with antipsychotic drugs signifi-

cantly altered S100B secretion (F6, 133 = 3.15, p < 0.01). Post hoc analysis revealed that haloperidol at 10 lM and clozapine at 100, but not 10 lM, reduced (29% and 25%, respectively) the secretion of this protein.

4. Discussion

In the present study, we show the effects of the alkaloid alsto- nine and known antipsychotics in biochemical parameters relevant to the pathophysiological basis of schizophrenia. One significant finding is the observation that alstonine and clozapine, but not hal- operidol, reduced glutamate uptake in acute hippocampal slices. Though the mechanism by which glutamate uptake is reduced re- quires further clarification, a generalized effect on sodium gradi- ents seems unlikely given the absence of evidence that + + Fig. 3. Effects of 5-HT2A and 5-HT2C receptor antagonists on alstonine-induced antipsychotics induce changes in Na ,K-ATPase activity or mem- decrease in glutamate uptake. Glutamate uptake after co-incubation of 100 lM branes sodium gradients (Seibt et al., 2012; López Ordieres et al., alstonine with 0.01 lM altanserin (A) and 0.1 lM SB 242084 (B). Data represent 2005), as well as the reversal seen with the serotonin antagonists. mean + SEM and are expressed as nmol/mg protein/min. Ã = p < 0.01 compared to control. Two-way ANOVA/Newman–Keuls. Specifically for alstonine, no changes in or serotonin lev- els were seen in the striatum from mice acutely treated with alst- Total glutathione content was significantly altered in hippo- onine in behaviorally effective doses (Linck et al., 2011), again campal slices submitted to drug treatments (F6, 50 = 3.76, arguing against a generalized effect that would also alter the so- p < 0.01), as shown in Fig. 4. Post hoc analysis indicated that only dium-dependent monoamine transporters. The nearly complete 1148 A.P. Herrmann et al. / Neurochemistry International 61 (2012) 1144–1150 inhibition of glutamate uptake in the presence of the transport altanserin and SB 242084, consistent with the ability of ritanserin inhibitor TBOA advances the idea that it is the high-affinity so- to prevent the behavioral effects of alstonine (Costa-Campos et al., dium-dependent glutamate transport that was evaluated in this 2004a; Linck et al., 2012). The proposal that serotonin is associated study. with alstonine mechanism of action is also supported by the in- It is known that it is the glutamate transport by astrocyte that creased levels of serotonin and 5-HIAA in frontal cortex and stria- determines the availability of released glutamate to pre- and tum from alstonine treated mice (Linck et al., 2011). Though it is post-synaptic receptors, as well as its spillover to extra-synaptic clear that alstonine modulates serotonin pathways, it remains to sites (Diamond, 2001; Huang and Bergles, 2004; Huang et al., be clarified whether alstonine activates receptors, increases seroto-

2004; Dunlop et al., 2006). Several studies have documented al- nin release, or both. Likewise, since 5-HT2A/C receptors are ex- tered expression and function of astrocytic glutamate transporters pressed in both neurons and astrocytes, data from cultured in schizophrenia (Ohnuma et al., 1998, 2000; Smith et al., 2001; astrocytes controlled for the expression of EAAT1/EAAT2 would Matute et al., 2005), and it has been suggested that an increased better clarify if glutamate transport is affected in neurons, astro- glutamate uptake by astrocytes may considerably contribute to cytes or both. Most importantly, the congruence from in vitro its purported glutamatergic hypofunction (Nanitsos et al., 2005). and in vivo data shows that whatever the specific transport af- Reducing glutamate uptake can, thus, be regarded as a viable strat- fected, glutamatergic transmission is significantly modulated by egy to improve glutamatergic function, and is compatible with the alstonine. ability of alstonine and clozapine to counteract MK-801-induced Glutathione is a tripeptide consisting of glycine, cysteine and behavioral effects. In agreement with our findings, it has been pre- glutamate, which participates in essential aspects of cellular viously reported that clozapine reduced both the expression of the homeostasis. The cellular ability to synthesize glutathione is an GLT-1 glutamate transporter and the glutamate uptake in astrocyte important factor in the management of oxidative stress-induced cultures (Vallejo-Illarramendi et al., 2005), as well as the glutamate neurotoxicity. Schizophrenia has been consistently associated with transport in rat prefrontal cortex (Melone et al., 2003). Moreover, reduced levels of glutathione, leading to potential NMDA receptors the decreased the gene expres- dysfunction (Do et al., 2000; Steullet et al., 2006; Yao et al., 2006; sion of glial and neuronal glutamate transporters in cortical and Gysin et al., 2007; Matsuzawa et al., 2008; Raffa et al., 2009; hippocampal regions, suggesting a transcriptional mechanism to Gawryluk et al., 2011; Micó et al., 2011). Our results show that, strengthen glutamatergic transmission (Segnitz et al., 2009). On among the antipsychotics studied, only alstonine was able to in- the contrary, selective D2 receptor antagonists, such as haloperidol, crease the intracellular levels of glutathione in hippocampal slices; were shown to be less effective than atypical antipsychotics to this may represent an additional valuable feature of alstonine. The attenuate the effects induced by NMDA receptor antagonists alstonine-induced increment in glutathione content could result (Meltzer et al., 2011), in agreement with the lack of effects of from increased synthesis, regeneration (NADPH-dependent), haloperidol on glutamate uptake. It remains to be clarified which sparing or decreased exportation by astrocytes (for a review see subtypes of transporters are altered by alstonine, as well as if the Hirrlinger and Dringen, 2010). Additional experiments, including mechanism is common to the atypical agents above mentioned. those with astrocyte cultures, are required to elucidate the precise Although reducing glutamate uptake can be advantageous to mechanism(s) by which alstonine increases glutathione. minimize glutamatergic hypofunction and is suggested as a novel S100B is a calcium-binding protein secreted by astrocytes into target for antipsychotics (Nanitsos et al., 2005), it is well estab- the synapse, where it is thought to participate in synaptic plasticity lished that excessive glutamate availability may overactivate (Nishiyama et al., 2002), being neurotrophic at nanomolar and NMDA and AMPA receptors leading to excitotoxicity. In the case apoptotic at micromolar levels (Ahlemeyer et al., 2000; Kögel of alstonine, the glutamatergic modulation through reduced up- et al., 2004). Increased serum and CSF S100B levels in patients with take does not seem to reach excitotoxic levels, since unlike cloza- schizophrenia have been interpreted as a marker of structural pine alstonine lacks proconvulsant properties in a kindling damage or, alternatively, as a sign of increased astrocyte activation paradigm (Costa-Campos et al., 2004b). Congruent with the (Wiesmann et al., 1999). It has been suggested that antipsychotic evidence that alstonine lacks affinity for dopamine receptors drugs could normalize these elevated levels (Ling et al., 2007; (Costa-Campos et al., 1998), the co-incubation with the dopamine Steiner et al., 2009, 2010; Nardin et al., 2011). Consistent with apomorphine did not alter the alstonine-induced decrease results obtained by Steiner et al. (2010) in C6 and OLN-93 cell cul- in glutamate uptake, but abolished that of clozapine. tures, we here show that both the haloperidol Save for dopamine and glutamate, it has been shown that some and the atypical clozapine decreased the release of S100B in hippo- atypical antipsychotics act as 5-HT2A/C receptor antagonists and/or campal slices. In contrast, alstonine did not alter S100B levels, inverse agonists (Meltzer et al., 2011). This modula- again reinforcing differences in receptor targets between alstonine tion is thought to be decisive for the alleged advantages of these and known antipsychotics. Considering that the mechanism of over classical agents (Gründer et al., 2009). However, S100B secretion remains unclear, it is of interest to evaluate the agonists of these receptors may also present a desirable antipsy- effects of alstonine in S100B levels under pharmacological condi- chotic-profile, since activation of 5-HT2A receptors enhances dopa- tions of dopaminergic activation before the meaning of this result mine release, whereas 5-HT2C activation inhibits it (Bortolozzi can be properly addressed. et al., 2005; Di Giovanni et al., 2006; Pehek et al., 2006; Huang et al., 2011). Several groups have demonstrated that astrocytes ex- press various 5-HT receptor subtypes, including 5-HT2A and 5-HT2C 5. Conclusions (Deecher et al., 1993; Merzak et al., 1996; Hirst et al., 1998; Cohen et al., 1999). One known effect of serotonin and selective serotonin The key finding of this study is the decrease in glutamate uptake inhibitors at these receptors is the elevation of intracellu- induced by alstonine, and its mediation by 5-HT2A and 5-HT2C lar calcium levels in glial cells (Hagberg et al., 1998; Schipke et al., receptors. A growing body of evidence suggests that particularly 2011), which was shown to trigger the efflux of glutamate (Meller the cognitive and negative symptoms of schizophrenia result from et al., 2002). It is conceivable that changes in astrocytic glutamate a dysfunctional glutamatergic system. Consequently, decreasing uptake may also result from changes in calcium levels when these glutamate uptake may represent an important feature for develop- serotonin receptors are modulated. We here show that alstonine- ing newer antipsychotic , especially effective in the induced decrease in glutamate uptake is prevented by both symptoms that are currently undertreated. A.P. Herrmann et al. / Neurochemistry International 61 (2012) 1144–1150 1149

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