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The influence of aripiprazole and olanzapine on neurotransmitters level in frontal cortex of prenatally stressed rats
Environmental Toxicology and Pharmacology 46 (2016) 122–130
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The influence of aripiprazole and olanzapine on neurotransmitters level in frontal cortex of prenatally stressed rats c ´ P. Ratajczak a , K. Kus a , K. Gołembiowska b , K. Noworyta-Sokołowska b , A. Wozniak , a a,∗ T. Zaprutko , E. Nowakowska (Prof. Dr.) a
Department of Pharmacoeconomics and Social Pharmacy, Poznan University of Medical Sciences, Dabrowskiego 79, 60-529 Poznan, Poland Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Cracow, Poland c Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland b
a r t i c l e
i n f o
Article history: Received 22 February 2016 Received in revised form 14 July 2016 Accepted 16 July 2016 Available online 18 July 2016 Keywords: Animal model of schizophrenia Prenatal stress Aripirazole Olanzapine Microdialysis Neurotransmitters
a b s t r a c t Objectives: The study aims to verify whether alterations in the level of neurotransmitters have occurred in prenatally stressed rats (animal model of schizophrenia), and whether aripiprazole (ARI) and olanzapine (OLA) modify this level. Methods: The effects of ARI (1.5 mg/kg) and OLA (0.5 mg/kg) were studied by means of microdialysis in freely moving rats (observation time 120 min). The level of neurotransmitters (DA, 5-HT, NA) and their metabolites (DOPAC, HVA, 5-HIAA) was analyzed by HPLC with coulochemical detection. Results: Obtained results indicate that after a single administration of ARI and OLA in the prenatally stressed rats the increase of DA, DOPAC, and 5-HT was observed. In turn ARI administration increase the level of HVA and 5-HIAA and also decrease the level of NA. After OLA administration the level of NA and HVA increased and no significant change in 5-HIAA was observed. Conclusion: Alterations observed as a result of ARI and OLA administration may be pivotal in identifying animal models of mental disorders and in the analysis of neuroleptics effectiveness. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe mental disease of complex aetiopathogenesis and it often requires prolonged pharmacotherapy (Konradi and Heckers, 2003). As the prevalence of schizophrenia in the global population is high (about 1%) (Nagai et al., 2010), studies aimed at finding the causes of this disorder are an important aspect of medical development in the 21st century. Animal models of schizophrenia (Ratajczak et al., 2013a) that enable the identification of disease symptoms observed in the clinical presentation are one of the basic tools used in experimental studies. One of the models frequently used in experimental studies is the model of prenatal stress (Kuneninn et al., 2003) based on the probability of cognitive or neurodevelopmental disorders as well as abnormal biochemical parameters in animals. This model is based on the assumption that the exposure of pregnant females to intense stress during the third trimester leads to a series of changes in cerebral cytoarchitecture in the developing foetus, ultimately gen-
∗ Corresponding author. E-mail address: [email protected] (E. Nowakowska). http://dx.doi.org/10.1016/j.etap.2016.07.007 1382-6689/© 2016 Elsevier B.V. All rights reserved.
erating cognitive (e.g. spatial memory disorder, impaired object and social recognition, increase locomotor acitity) and biochemical disorders (e.g. GABAergic and presynaptic protein dysregulation) observed in their offspring (Kuneninn et al., 2003; Koenig et al., 2005). In this context animal model of schizophrenia applies only to offspring (not for pregnant mothers). Neurodevelopmental lesions are primarily related to the increasing level of the stress hormone (corticosterone) observed in the pregnant mother; corticosterone crosses the placental barrier and leads e.g. to neural atrophy, especially in the hippocampal region (Koenig et al., 2005). We have proven in our studies that animals stressed prenatally (during fetal development) present with cognitive function disorders (Ratajczak et al., 2012; Ratajczak et al., 2013b; Nowakowska et al., 2014) as well as increased basal level of corticosterone (Ratajczak et al., 2013b) and decreased level of brain derived neutorhropic factor (BDNF) (Nowakowska et al., 2014). The efficient treatment of schizophrenia includes the use of drugs acting on the Central Nervous System (CNS) neurotransmitters, dopamine (DA) D2 receptors in particular (Barnes, 2011). It is estimated that the level of dopamine in subcortical structures increases in schizophrenia, which is the cause of clinically assessed positive symptoms of schizophrenia. On the other hand, DA transmission in the cerebral cortex is weakened, which determines the
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presence of negative symptoms of schizophrenia (Winterer and Weinberger, 2004). With development of neuropsychiatry and introduction of new neuroleptics, the important role of noradrenaline (NA) and serotonine (5-HT), apart from DA, in the course of schizophrenia has been confirmed (Huang et al., 2008). Olanzapine (OLA) and aripiprazole (ARI) are atypical neuroleptics with a mechanism of action reducing positive and negative schizophrenia symptoms (Jafari et al., 2012; Hirose and Kikuchi, 2005). ARI is thought to be a partial agonist of D2 and D3 dopamine receptors and 5-HT1A serotonin receptors as well as an antagonist of 5-HT2A and 5-HT6 receptors (Hirose and Kikuchi, 2005; Burda et al., 2011). On the other hand, OLA is a typical antagonist of D2 , 5HT2A , 5-HT2B , 5-HT2C receptors as well as H1 histamine receptors and ␣1 adrenergic receptors. Due to the receptor profile of those drugs, their antagonistic effect on D2 receptors in the mesolimbic system causes a reduction of positive schizophrenia symptoms (Hirose and Kikuchi, 2005). Our previous studies indicate that OLA and ARI reduce memory disorders in prenatally stressed animals and modify the levels of corticosterone and BDNF (Ratajczak et al., 2013b; Nowakowska et al., 2014), therefore it was interesting to find out whether neuroleptics also affect the levels of neurotransmitters (DA, NA, 5-HT) and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homo-vanillic acid (HVA) and 5-Hydroxyindoleacetic acid (5-HIAA) tested in the frontal cortex of prenatally stressed animals. 2. Materials and methods
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Fig. 1. Experiment schedule. Table 1 Schedule of stress exposures applied in the prenatal stress paradigm. Gestation day
Morning
Midday
Evening
14 15 16 17 18 19 20 21
metal tube 60 min cold exposure swim 15 min open social stress 12 h metal tube 60 min
swim 15 min
metal tube 60 min fast overnight swim 15 min lights on overnight
swim 15 min
metal tube 60 min swim 15 min swim 15 min cold exposure 6 h metal tube 60 min
metal tube 60 min swim 15 min
2.1. Animals Timed pregnant Wistar female rats (15) were purchased from Poznan University of Medical Sciences, Poznan, Poland (licensed by the Ministry of Agriculture in Warsaw, Poland) and arrived at our animal facility on day 2 of gestation. The pregnant animals were housed individually in a light- (lights on 07.00–19.00 h), temperature- and humidity-controlled animal facility. The dams had free access to rat chow (Labofeed B) and water. The total number of animals in the study was 51 (15 females and 36 offspring males). The male rats (36 animals) were divided into three groups – non stressed control group (NSCG) receiving ARI or OLA – (12 non-prenatally stressed rats, offspring of the 5 non-stressed females), prenatally stressed group (PSG) receiving ARI or OLA (12 prenatally stressed rats, offspring of the 5 prenatally stressed females) and “saline” group receiving sodium chloride NaCl dissolved in 1% carboxymethylcellulose vehicle in the 1:1 ratio (not ARI or OLA) (12 non-prenatally stressed rats, offspring of the 5 non-stressed females). Fig. 1 contain schematic picture of conducted experiments. All procedures related to the use of rats in these experiments were conducted with due respect to ethical principles regarding experiments on animals. The study protocol was approved by the Local Ethics Committee for Research on Animals in Poznan. 2.2. Drugs Pure carboxymethylcellulose was obtained from Koch-Light Laboratories (London, England), ARI – Otsuka Pharmaceutical Europe, Bristol-Myers Squibb (Warsaw, Poland), OLA – Zyprexa Eli Lilly (Warsaw, Poland). The rats were administered ARI (1.5 mg/kg ip), OLA (0.5 mg/kg ip) or the vehicle intraperitoneally (ip). ARI and OLA were suspended in saline solution (0.5% carboxymethylcellulose) (2 ml, ip). The controls were given saline solution according to the same schedule. Separate groups of animals were used for different tests.
ARI and OLA dose which was used in the study were calculated based on the previous studies (Ratajczak et al., 2012; Ratajczak et al., 2013b; Nowakowska et al., 2014; Burda et al., 2011; Czubak et al., 2010). All the chemicals used for high performance liquid chromatography (HPLC) were from Merck (Warszawa, Poland). 2.3. Prenatal stress procedure (Kuneninn et al., 2003) Beginning on day 14 of gestation, the pregnant dams were exposed to a repeated variable stress paradigm until delivery of their pups on gestational day 22 or 23 as previously described. The stresses used in this paradigm consisted in: (1) restraint in metal tube for 1 h, (2) exposure to a cold environment (4 ◦ C) for 6 h, (3) overnight food deprivation, (4) 15 min of swim stress in water of room temperature, (5) lights on for 24 h, and (6) social stress induced by overcrowded housing conditions during the dark phase of the cycle. A typical schedule of the stress applications is presented in prenatal stress table (Table 1). Pregnant control dams remained in the animal room from gestational days 14–21 and were only exposed to normal animal room husbandry procedures. All dams delivered their pups vaginally. At weaning, male offspring from each litter were placed with same sex litter mates per cage with free access to rat chow and water. The animals were exposed to normal animal room procedures from that point onward until experimental use on post-natal day 90. 2.4. Microdialysis Rats were anesthetised with ketamine (75 mg/kg im) and xylazine (10 mg/kg im), placed in a stereotactic apparatus (David Kopf Instruments, Tujunga, CA, USA) and subsequently vertical microdialysis probes were implanted in the rat frontal cortex with coordinates (mm) A + 2.8, L + 0.8, V − 6.0 from the dura. Twenty four hours after implantation, probe inlets were connected to a syringe
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DA (% of basal level)
500
naive NSCG
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min Fig. 2. Effect of olanzapine (0.5 mg/kg) on DA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
450
naive NSCG
NA (% of basal level)
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sal-na ive saline OLANZAPINE 0,5 mg/kg
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^
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-20
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m in Fig. 3. Effect of olanzapine (0.5 mg/kg) on NA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
pump (CMA, Sweden) which delivered an artificial CSF (aCSF) composed of (mM): NaCl 147, KCl 4.0, CaCl2 1.2, MgCl2 1.0 at a flow rate of 2 l/min. Baseline samples were collected every 20 min after the washout period. Appropriate drugs were then administered and dialysate fractions were collected for 120 min. At the end of the experiment, the rats were sacrificed and their brains were histologically examined to validate probe placement. 2.5. Analytical procedure Dopamine (DA), serotonin (5-hydroxytryptamine, 5-HT), noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by HPLC with coulochemical detection. Chromatography was performed using the Ultimate 3000 System (Dionex, USA), coulochemical detector Coulochem III (model 5300, ESA, USA) with a 5020 guard cell, a 5014B microdialysis cell and a Hypersil Gold-C18 analytical column (3 × 100 mm). The mobile phase was composed of 0.05 M potassium phosphate buffer adjusted to pH = 3.6, 0.5 mM EDTA, 16 mg/L 1-octanesulfonic acid sodium salt, and a 2.1% methanol. The flow rate during analysis was 0.7 ml/min. The applied potential of a guard cell was +600 mV, while those of microdialysis cell was E1 = −50 mV, E2 = +300 mV
and a sensitivity was set at 50 nA/V. The chromatographic data were processed by Chromeleon v. 6.80 (Dionex, USA) software run on a PC computer. 2.6. Statistical analysis The statistical significance was calculated using repeatedmeasures ANOVA, followed by Tukey’s post-hoc test. The results were considered statistically significant when p < 0.05 or p < 0.01. Only time points after drug administration was calculated in the result section. 3. Results 3.1. Effect of olanzapine on DA, NA, 5-HT, DOPAC, HVA and 5-HIAA extracellular level in frontal cortex of NSCG and PAG rats Single treatment of olanzapine in dose of 0.5 mg/kg increased DA extracellular level in PSG rats from 20 to 120 min after administration compared to the saline treated rats (PSG vs. saline p < 0.01) (Fig. 2). Moreover DA increase was also observed in PSG rats compared to NSCG rats in 20–120 min of the observation (PSG vs. NSCG
P. Ratajczak et al. / Environmental Toxicology and Pharmacology 46 (2016) 122–130
5-HT (% of basal level)
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naive NSCG
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80 40
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^
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OLANZAPINE 0,5 mg/kg
0 -40
-20
0
20
40
60
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m in Fig. 4. Effect of olanzapine (0.5 mg/kg) on 5-HT extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
DOPAC (% of basal level
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naive NSCG schi PSG
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sal-na salineive
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^
50
OLANZAPINE 0,5 mg/kg
0 -40
-20
0
20
min Fig. 5. Effect of olanzapine (0.5 mg/kg) on DOPAC extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
p < 0.01) (Fig. 2). The study also found significant decline of DA level observed from 80 to 120 min of fraction collection compared to the saline treated rats (NSCG vs. saline p < 0.01) (Fig. 2). Olanzapine in a dose of 0.5 mg/kg increased NA extracellular level in NSCG and PSG rats compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 3). After olanzapine administration (0.5 mg/kg) in the PSG rats it was also observed both decrease (20 min and 80–120 min after administration) and increase (40–60 min after administration) of NA level compared to the NSCG rats (PSG vs. NSCG p < 0.01) (Fig. 3). 5-HT extracellular level was slightly but significantly increased at 20, 40, 80 min and decreased at 100–120 min after olanzapine administration in NSCG rats compared to the saline treated rats (NSCG vs. saline p < 0.01) (Fig. 4). Olanzapine also potently increased extracellular 5-HT level in PSG rats from 20 to 120 min after administration compared to the NSCG rats (PSG vs. NSCG p < 0.01). There was also observed significant increase of 5-HT in PSG rats versus saline rats from 20–120 min after OLA administration (PSG vs. saline p < 0.01) (Fig. 4). Intraneuronal metabolite of DA, DOPAC was increased in both NSCG and PSG rats after olanzapine administration compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 4). There
was also significant increase of DOPAC in PSG rats versus NSCG rats after administration of OLA (PSG vs. NSCG p < 0.01) (Fig. 5). Extraneuronal metabolite of DA, HVA was increased in both, NSCG and PSG rats after administration of olanzapine compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 6). There was also significant increase of HVA in PSG rats versus NSCG after administration of OLA (PSG vs. NSCG p < 0.01) (Fig. 6). Serotonin metabolite, 5-HIAA was not affected by olanzapine administration in both, NSCG and PSG rats (Fig. 7). There was also no significant increase of 5-HIAA in PSG versus NSCG rats after administration of OLA (Fig. 7).
3.2. Effect of aripiprazole on DA, NA, 5-HT, DOPAC, HVA and 5-HIAA extracellular level in frontal cortex of NSCG and PSG rats Administration of aripiprazole (1.5 mg/kg) significantly increased DA extracellular level only in PSG rats from 20 to 120 min of the observation period in comparison to the saline treated rats (PSG vs. saline p < 0.01 or p < 0.05) (Fig. 8). There was also significant increase of DA level in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.01 p < 0.05) (Fig. 8).
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HVA (% of basal level)
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50 0 -40
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m in Fig. 6. Effect of olanzapine (0.5 mg/kg) on HVA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HIAA (% of basal level
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OLANZAPINE 0,5 mg/kg
schi PSG
sal-na salineive 60 -40
-20
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min Fig. 7. Effect of olanzapine (0.5 mg/kg) on 5-HIAA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level.
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ARIPIPRAZOLE 1,5 mg/kg
**^^ **^^ **^^
DA (% of basal level)
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naive NSCG
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-20
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min Fig. 8. Effect of aripiprazole (1.5 mg/kg) on DA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆ P < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
P. Ratajczak et al. / Environmental Toxicology and Pharmacology 46 (2016) 122–130
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m in Fig. 9. Effect of aripiprazole (1.5 mg/kg) on NA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HT (% of basal level)
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ARIPIPRAZOLE 1,5
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m in Fig. 10. Effect of aripiprazole (1.5 mg/kg) on 5-HT extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆˆ P < 0.01 versus saline treated naïve rats, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
Aripiprazole (1.5 mg/kg) increased NA extracellular level in NSCG rats only at 40 min after administration compared to saline treated rats (NSCG vs. saline p < 0.01) (Fig. 9). There was also observed decrease in extracellular NA level in PSG rats in compared to the NSCG rats (PSG vs. NSCG p < 0.05) (Fig. 9). Administration of aripiprazole (1.5 mg/kg) increased 5-HT extracellular level in both NSCG and PSG rats from 20 to 120 min after administration in comparison to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 10). There was also significant increase of the 5-HT level in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.01) (Fig. 10). Aripiprazole administration (1.5 mg/kg) increased DOPAC extracellular level only in PSG rats from 20 to 120 min after administration in comparison to the saline treated rats (PSG vs. saline p < 0.05 or p < 0.01) (Fig. 11). There was also significant increase in DOPAC level in PSG rats versus NSCG rats (20–120 min) after administration of ARI (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 11). HVA extracellular level was significantly increased in both NSCG and PSG rats after treatment of aripiprazole in comparison to the saline treated rats (NSCG/PSG vs. saline p < 0.05 or p < 0.01) (Fig. 12). There was also significant decrease of HVA in PSG versus NSCG rats after administration of ARI from 60 to 120 min (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 12).
5-HIAA extracellular level was increased in PSG rats from 20 to 100 min after aripiprazole administration in comparison to the saline treated rats (PSG vs. saline p < 0.05 or p < 0.01) (Fig. 13). There was also significant increase (20–100 min) and decrease (120 min) of 5-HIAA in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 13). 4. Discussion Biochemical studies indicate that the functioning of neurotransmission systems is disturbed in schizophrenia, primarily the dopaminergic and serotoninergic systems, which was the basis of constructing theories on the genesis of the disease in the context of various neurotransmission systems. The dopamine theory of schizophrenia assumes that the clinical presentation of the disease includes increased DA activity in the CNS that can be reduced by antipsychotic drugs capable of reducing the level of dopaminergic activity in the limbic system’s structures (Podgrodzka and Jarema, 2010). According to this acknowledged idea developed for many years, the negative symptoms of schizophrenia are an effect of dopaminergic insufficiency of the mesocortical pathways and positive psychotic symptoms result from hyperactivity of the mesolimbic dopaminergic pathway (Siwek et al., 2010).
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DOPAC (% of basal level
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ARIPIPRAZOLE 1,5 mg/kg
schi PSG sal-na salineive
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m in Fig. 11. Effect of aripiprazole (1.5 mg/kg) on DOPAC extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
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HVA (% of basal level)
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ARIPIPRAZOLE 1,5 mg/kg
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min Fig. 12. Effect of aripiprazole (1.5 mg/kg) on HVA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HIAA (% of basal level
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ARIPIPRAZOLE 1,5 mg/kg
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min Fig. 13. Effect of aripiprazole (1.5 mg/kg) on 5-HIAA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
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Diversely, the serotonin theory substantiated the efficiency of atypical antipsychotics in schizophrenia which affect dopamine receptors but also show strong antagonistic affinity with serotonin receptors (especially 5-HT2A ) (Krzystanek, 2007). Apart from the mechanism related to the antagonistic effect on D2 receptors as well as 5-HT2A and 5-HT2C serotonin receptors, found in all atypical antipsychotics, attention should be paid to a new neuroleptic, aripiprazole, which also is partially agonistic to D2 , D3 , and 5-HT1A receptors (Krzystanek, 2007). Mainly due to its mechanism of action, ARI has been called a stabiliser of the dopamine-serotonin system, acting antagonistically, weakening dopaminergic transmission, in case of dopamine receptor hyperactivity, and acting as an agonist, enhancing DA transmission, in conditions of insufficient dopaminergic activity (Burris et al., 2002). It should also be mentioned that ARI’s partial agonism of D2 receptors in combination with antagonism of 5-HT2A receptors minimises the risk of dopamine insufficiency in the nigrostriatal and tuberoinfundibular pathways, but also the risk of drug-induced parkinsonism or hyperprolactinaemia (Krzystanek, 2007). Obtained results indicate that the level of DA in non-stressed animals (NSCG) was regulated only by the administration of OLA. Conversely, in prenatally stressed rats (PSG), the administration OLA and ARI resulted in an increase of DA level which suggests an agonist effect on D2 receptors located in the frontal cortex (FC). Other studies indicate that ARI caused an increase of DA level or had no effect on the prefrontal cortex (PFC). In several studies, ARI did not cause a DA level increase (Newma-Tancredi et al., 2005), but Li et al. (2004) observed an increased DA level after administering ARI in the dose of 0.3 mg/kg. DA increase observed after OLA administration (OLA—typical antagonist of the D2 receptors) was observed by Bymaster et al. (1999) but other authors failed to obtain any DA level increase in the PFC (Rollema et al., 2000; Volonte et al., 1997). Literature indicate that OLA and ARI also induce the increase of DA levels in the occipital cortex and in the cerebellum (Devoto and Flore, 2006). Meltzer and McGurk (1999) also observed a DA increase in the medial prefrontal cortex (mPFC). Moreover changes in the level of DA in PFC could be related to the cytoarchitecture changes observed in the prenatally stressed rats. The level of DA directly affects the regulation of the level of its metabolites, DOPAC and HVA. The level of DOPAC is regulated by the level of DA throughout the brain (Devoto and Flore, 2006). Administration of antipsychotics can regulate the level of DA, DOPAC and HVA. Obtained results indicate that DOPAC level in NSCG was regulated only by OLA (increase). However, the administration of both OLA and ARI increased DOPAC level in prenatally stressed rats. Moreover, HVA level observed after the administration of OLA and ARI was higher in both NSCG and PSG. The results obtained by Semba et al. (1995) indicate that the administration of both OLA and ARI in doses of (2, 10, and 40 mg/kg) caused an increase of DOPAC concentration in mPFC and in the striatum. In the case of OLA administration, the DOPAC level increase may depend on the drug doses used (Jordan et al., 2004). Earlier studies indicate a strong antagonistic effect of OLA on presynaptic D2 receptors (Volonte et al., 1997; Li et al., 1998; Westerink et al., 1998). In the case of HVA, the studies by Jordan et al. (2004) indicate that both the administration of OLA and ARI can generate increased HVA level in the mPFC and in the striatum. Moreover, Jordan et al. (2004) demonstrated that HVA level observed after the use of OLA and ARI depended on the dose and the duration of drug administration. Single administration of both ARI and OLA in doses of 1 mg/kg, 10 mg/kg, and 20 mg/kg increased the level of this metabolite. Contrary, in groups of animals receiving drugs for 21 days, increased HVA level was observed only in rats receiving ARI (Jordan et al., 2004), which suggests the possibility of tolerance to OLA. Cognitive functions are regulated by the normal level of DA and NA in the CNS (Yamamoto and Horynkiewicz, 2004). Moreover, NA
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is considered to play a crucial role in defensive reactions related to strong stress which can generate disruptions in the level of NA (Yamamoto and Horynkiewicz, 2004). The level of NA is regulated mainly by DA synthesis, both in noradrenergic and dopaminergic neurons (Devoto and Flore, 2006). DA release increases the level of NA, which in turn can act back increasing DA activity (Pan et al., 1996). NA also has an indirect effect on DA reuptake in PFC (Moron et al., 2002). Obtained results indicate that NA level in NSCG was regulated by the administration of OLA and ARI (increased neurotransmitter level). In the case of PSG, the administration of OLA generated an NA level increase, while ARI decreased it. Comparing the efficiency of aripiprazole and clozapine Zocchi et al. (2005) indicated that increased NA level, coexisting with increased DA level, was observed after the use of clozapine in the dose of 10 mg/kg. No changes of NA level were observed after the use of ARI in doses (0.1 mg/kg, 0.3 mg/kg, 3.0 mg/kg, and 30 mg/kg). Blocking 5-HT2 receptors in combination with effect on other neurotransmission systems is believed to be responsible for the anti-deficit, anti-depressant, and pro-cognitive effect (Rybakowski, 2007). ARI is an antagonist of 5-HT2A and 5-HT2C receptors, and an agonist of 5-HT1A receptors (Lieberman, 2004), while OLA is a typical antagonist of 5-HT2A and 5-HT2C receptors. Patient’s mood, possibly regulated by the level of 5-HT and NA, is believed to ˛ 2012). be at the root of schizophrenia (Markowicz-Narekiewicz, Obtained results indicate that the level of 5-HT in the FC of both NSCG and PSG was regulated by the administration of OLA and ARI. According to Carli et al. (2011), the use of 3 mg/kg of ARI can cause an increase of 5-HT level in the mPFC. However, other investigators did not indicate changes of 5-HT level in the mPFC in mice following the administration of ARI (Volonte et al., 1997) and the hippocampus of rats (Assie et al., 2008). Bortolozzi et al. (2007) observed a decreased 5-HT level in the mPFC in mice due to reduced re-uptake of 5-HT. In turn, Knauer et al. (2008) believe the use of OLA can lead to rapid saturation of 5-HT2A receptors located in the FC, which can reduce 5-HT level. Saturation of 5-HT receptors was also observed following the administration of ARI in the studies of Natesan et al. (2006), but only when large doses of the drug were used (over 20 mg/kg). The level of serotonin directly affects the level of 5-HIAA, and obtained results indicate that ARI in PSG generated an increase in the level of 5-HIAA. The effect of changes in 5-HIAA level was not observed in NSCG. Literature reports state that the use of ARI can cause a decrease of 5-HIAA level in the mPFC and in the striatum. It was also found that 5-HIAA level in these structures was reduced after repeated administration, which can be due to sudden functional desynthetisation of 5-HT1A receptor somatodendrites (Kennett et al., 1987; Fuller and Perry, 1989). Through presynaptic activation of 5-HT1A receptors, ARI can reduce serotonin release and thereby reduce 5-HIAA level in the forebrain as was proposed by Jordan et al. (Jordan et al., 2004). However studies by Li et al. (1998) on the effect of OLA on the level of 5-HIAA indicate that regardless of the dose the drug affected 5-HIAA level in the PFC and the nucleus accumbens.
5. Conclusion Both OLA and ARI increase the level of DA and 5-HT as well as their metabolites (DOPAC, HVA) in the frontal cortex. Increased 5HIAA level was observed only following the administration of ARI, while OLA increased the level of NA. The differences in the observed levels of neurotransmitters and their metabolites were probably due to different mechanisms of action of the used neuroleptics. All the neuroleptics used act mainly on dopaminergic receptors. Differences in clinical profiles are the result of the degree to which they
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block particular subtypes of dopamine receptors. The dopaminergic system is a system of neuromodulation acting in midbrain cells on the mesolimbic region, striatum, and cortical areas (Rzewuska, 2009). The dopaminergic activity in the midbrain-cortex circuit allows regulation of many behaviours, that is cognitive functions, movement control, mood, or attention, so disorders of dopaminergic functions can lead to neurological and mental deficits observed in patients. Considering the complexity and episodic character of schizophrenia, aripiprazole seems to be efficient in reducing positive and negative disorders. Conflict of interest The authors declare that there are no conflicts of interest associated with this manuscript. Acknowledgment Project was funded by the National Science Centre—Cracow, Poland. References Assie, M.B., et al., 2008. The antipsychotics clozapine and olanzapine increase plasma glucose and corticosterone levels in rats; comparison with aripiprazole ziprasidone bifeprunox and F15063. Eur. J. Pharmacol. 592, 160–166. Barnes, T., 2011. Evidence-based guidelines for the pharmacological treatment of schizophrenia:recommendations from the British Association for Psychopharmacology. J. Psychopharmacol. 25 (5), 567–620. Bortolozzi, A., et al., 2007. Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT1A receptors but not 5-HT2A receptors. Int. J. Neuropsychopharmacol. 13, 1299–1314. Burda, K., et al., 2011. Influence of aripiprazole on the antidepressant anxiolytic and cognitive functions of rats. Pharmacol. Rep. 63, 898–907. Burris, K.D., et al., 2002. Aripiprazole a novel antipsychiotic is a high-affinity partial agonist at human dopamine D2 receptors. J. Pharmacol. Exp. Ther. 302 (1), 381–389. Bymaster, F., et al., 1999. Olanzapine: a basic science update. Br. J. Psychiatry Suppl. 37, 36–40. Carli, M., et al., 2011. Effects of aripiprazole olanzapine and haloperidol in a model of cognitive deficit of schizophrenia in rats:relationship with glutamate release in the medial prefrontal cortex. Psychopharmacology 214, 639–652. Czubak, A., et al., 2010. Effect of venlafaxine and nicotine on the level of neurotransmitters and their metabolites in rat brains. J. Physiol. Pharmacol. 61 (3), 339–346. Devoto, P., Flore, G., 2006. On the origin of cortical dopamine:is it a co-transmitter in noradrenergic neurons? Neuropharmacology 4, 115–125. Fuller, R.W., Perry, K.W., 1989. Effects of buspirone and its metabolite 1-(2-pyrimidinyl)piperazine on brain monoamines and their metabolities in rats. J. Pharmacol. Exp. Ther. 248, 50–56. Hirose, T., Kikuchi, T., 2005. Aripiprazole a novel antipsychotic agent:Dopamine D2 receptor partial agonist. J. Med. Invest. 52, 284–290. Huang, M., et al., 2008. Asenapine increases dopamine norepinephrine and acetylcholine efflux in the rat medial prefrontal cortex and hippocampus. Neuropsychopharmacol 33, 2934–2945. Jafari, S., et al., 2012. Novel olanzapine analogues presenting a reduced H1 receptor affinity and retained 5HT2A/D2 binding affinity ratio. BMC Pharmacol. 12, 8, http://dx.doi.org/10.1186/1471-2210-12-8. Jordan, S., et al., 2004. In vivo effects of aripiprazole on cortical and striatal dopaminergic and serotonergic function. Eur. J. Pharmacol. 483, 45–53. Kennett, G.A., et al., 1987. Single administration of 5-HT(1A) presynaptic but not postsynaptic receptor-mediated responses:relationship to antidepressant-like action. Eur. J. Pharmacol. 138, 53–60. Knauer, C.S., et al., 2008. Validation of a rat in vivo [(3)H]M100907 binding assay to determine a translatable measure of 5-HT(2A) receptor occupancy. Eur. J. Pharmacol. 591, 136–141. Koenig, J.I., et al., 2005. Prenatalexposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring:potential relevance to schizophrenia. Behav. Brain Res. 156 (2), 251–261.
Konradi, C., Heckers, S., 2003. Molecular aspects of glutamate dysregulation:implications for schizophrenia and its treatment. Pharmacol. Ther. 97, 153–179. Krzystanek, M., 2007. Schizofrenia zaburzenia typu schizofrenii (schizotypowe) I ˛ ˛ dla studentów pielegniarstwa. urojeniowe Psychiatria. Podrecznik Kuneninn, A.K., et al., 2003. Repeated variable prenatal stress alters pre- and postsynaptic gene expression in the rat frontal pole. J. Neurochem. 86 (3), 736–748. Li, X.M., et al., 1998. Olanzapine increases in vivo dopamine and norepinephrine release in rat prefrontal cortex nucleus accumbens and striatum. Psychopharmacology 136, 153–161. Li, Z., et al., 2004. Aripiprazole a novel antipsychotic drug preferentially increases dopamine release in the prefrontal cortex and hippocampus in rat brain. Eur. J. Pharmacol. 493, 75–83. Lieberman, J.A., 2004. Dopamine partial agonists A new class of antipsychotics. CNS Drugs 18, 251–267. ˛ ˛ ˛ Markowicz-Narekiewicz, A.E., 2012. Zwiazek miedzy wydzielaniem ´ a powstawaniem chorób psychicznych −na szczegółowo neuroprzekazników omówionym przykładzie depresji. Neurokogniwistyka w Patologii I Zdrowiu, 55–59. Meltzer, H.Y., McGurk, S.R., 1999. The effect of clozapine risperidone and olanzapine on cognitive function in schizophrenia. Schizophr. Bull. 25, 233–255. Moron, J.A., et al., 2002. Dopamine uptake through the norepinephrine transporter in brain regions with low levels of the dopamine transporter:evidence from knock-out mouse lines. J. Neurosci. 22, 389–395. Nagai, T., et al., 2010. Dysfunction of dopamine release in the prefrontal cortex of dysbindin deficient sandy mice: an in vivo microdialisis study. Neurosci. Lett. 470 (2), 134–138. Natesan, S., et al., 2006. Dissociation between in vivo occupancy and functional antagonism of dopamine D2 receptors: comparing aripiprazole to other antipsychotics in animal models. Neuropsychopharmacology 31, 1854–1863. Newma-Tancredi, A., et al., 2005. Novel antipsychotics activate recombinant human and native rat serotonin 5-HT(1A) receptors:affinity efficacy and potential implication for treatment of schizophrenia. Int. J. Neuropsychopharmacol. 11, 1–16. Nowakowska, E., et al., 2014. The influence of aripiprazole olanzapine and enrichment environment on depressant-like behavior spatial memory dysfunction and hippocampal level of BDNF in prenatally stressed rats. Pharmacol. Rep. 66 (3), 404–411. Pan, W.H., et al., 1996. Locally application of amphetamine into the ventral tegmental area enhances dopamine release in the nucleus accumbens and the medial prefrontal cortex through noradrenergic neurotransmission. J. Pharmacol. Exp. Ther. 278, 725–731. Podgrodzka, M., Jarema, M., 2010. Practical aspects of the use of olanzapine in the treatment of schizophrenia and bipolar disorders. Psychiatria 7 (5), 180–188. Ratajczak, P., et al., 2012. The effect of ethyl alcohol on the function of spatial memory in rats. Arzneimittelforschung 62 (12), 614–623. Ratajczak, P., et al., 2013a. Animal model of schizophrenia—devopmental preparation in rats. Acta Neurobiol. Exp. 73 (4), 472–484. Ratajczak, P., et al., 2013b. Influence of aripiprazole and olanzapine on behavioral dysfunctions of adolescent rats exposed to stress in perinatal period. Pharmacol. Rep. 65, 30–43. Rollema, H., et al., 2000. 5-HT1A receptor activation contributes to ziprasidone-induced dopamine release in the rat prefrontal cortex. Biol. Psychiatry 48, 229–237. Rybakowski, J., 2007. Atypical antipsychotic drugs in the treatment of psychiatric disorders. Przewodnik Lekarza 2, 169–174. Semba, J., et al., 1995. Behavioural and neurochemical effects of OPC-14597 a novel antipsychotic drug on dopaminergic mechanisms in rat brain. Neuropharmacology 34, 785–791. Siwek, M., et al., 2010. Aripiprazole in acute schizophrenia—case studiem. Wiadomo´sci Psychiatryczne 13 (3), 157–162. Volonte, M., et al., 1997. BIMG 80 a novel potential antipsychotic drug:evidence for multireceptor actions and preferential release of dopamine in prefrontal cortex. J. Neurochem. 69, 182–190. Westerink, B.H., et al., 1998. Antipsychotic drugs induce similar effects on the release of dopamine and noradrenaline in the medical prefrontal cortex of the rat brain. Eur. J. Pharmacol. 361, 27–33. Winterer, G., Weinberger, D., 2004. Genes dopamine and cortical signal-to-noise ratio in schizophrenia. Trends Neurosci. 27, 683–690. Yamamoto, K., Horynkiewicz, O., 2004. Proposal for a noradrenaline hypothesis of schizophrenia. Prog. NeuroPsychopharmacol. 28, 913–922. Zocchi, A., et al., 2005. Aripiprazole increases dopamine but not noradrenaline and serotonin levels in the mouse prefrontal cortex. Neurosci. Lett. 387, 157–161. Rzewuska, M., 2009. Układ dopaminergiczny i leki przeciwpsychotyczne. Psychiatria 2 (3), 115–123.
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The influence of aripiprazole and olanzapine on neurotransmitters level in frontal cortex of prenatally stressed rats c ´ P. Ratajczak a , K. Kus a , K. Gołembiowska b , K. Noworyta-Sokołowska b , A. Wozniak , a a,∗ T. Zaprutko , E. Nowakowska (Prof. Dr.) a
Department of Pharmacoeconomics and Social Pharmacy, Poznan University of Medical Sciences, Dabrowskiego 79, 60-529 Poznan, Poland Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Cracow, Poland c Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland b
a r t i c l e
i n f o
Article history: Received 22 February 2016 Received in revised form 14 July 2016 Accepted 16 July 2016 Available online 18 July 2016 Keywords: Animal model of schizophrenia Prenatal stress Aripirazole Olanzapine Microdialysis Neurotransmitters
a b s t r a c t Objectives: The study aims to verify whether alterations in the level of neurotransmitters have occurred in prenatally stressed rats (animal model of schizophrenia), and whether aripiprazole (ARI) and olanzapine (OLA) modify this level. Methods: The effects of ARI (1.5 mg/kg) and OLA (0.5 mg/kg) were studied by means of microdialysis in freely moving rats (observation time 120 min). The level of neurotransmitters (DA, 5-HT, NA) and their metabolites (DOPAC, HVA, 5-HIAA) was analyzed by HPLC with coulochemical detection. Results: Obtained results indicate that after a single administration of ARI and OLA in the prenatally stressed rats the increase of DA, DOPAC, and 5-HT was observed. In turn ARI administration increase the level of HVA and 5-HIAA and also decrease the level of NA. After OLA administration the level of NA and HVA increased and no significant change in 5-HIAA was observed. Conclusion: Alterations observed as a result of ARI and OLA administration may be pivotal in identifying animal models of mental disorders and in the analysis of neuroleptics effectiveness. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe mental disease of complex aetiopathogenesis and it often requires prolonged pharmacotherapy (Konradi and Heckers, 2003). As the prevalence of schizophrenia in the global population is high (about 1%) (Nagai et al., 2010), studies aimed at finding the causes of this disorder are an important aspect of medical development in the 21st century. Animal models of schizophrenia (Ratajczak et al., 2013a) that enable the identification of disease symptoms observed in the clinical presentation are one of the basic tools used in experimental studies. One of the models frequently used in experimental studies is the model of prenatal stress (Kuneninn et al., 2003) based on the probability of cognitive or neurodevelopmental disorders as well as abnormal biochemical parameters in animals. This model is based on the assumption that the exposure of pregnant females to intense stress during the third trimester leads to a series of changes in cerebral cytoarchitecture in the developing foetus, ultimately gen-
∗ Corresponding author. E-mail address: [email protected] (E. Nowakowska). http://dx.doi.org/10.1016/j.etap.2016.07.007 1382-6689/© 2016 Elsevier B.V. All rights reserved.
erating cognitive (e.g. spatial memory disorder, impaired object and social recognition, increase locomotor acitity) and biochemical disorders (e.g. GABAergic and presynaptic protein dysregulation) observed in their offspring (Kuneninn et al., 2003; Koenig et al., 2005). In this context animal model of schizophrenia applies only to offspring (not for pregnant mothers). Neurodevelopmental lesions are primarily related to the increasing level of the stress hormone (corticosterone) observed in the pregnant mother; corticosterone crosses the placental barrier and leads e.g. to neural atrophy, especially in the hippocampal region (Koenig et al., 2005). We have proven in our studies that animals stressed prenatally (during fetal development) present with cognitive function disorders (Ratajczak et al., 2012; Ratajczak et al., 2013b; Nowakowska et al., 2014) as well as increased basal level of corticosterone (Ratajczak et al., 2013b) and decreased level of brain derived neutorhropic factor (BDNF) (Nowakowska et al., 2014). The efficient treatment of schizophrenia includes the use of drugs acting on the Central Nervous System (CNS) neurotransmitters, dopamine (DA) D2 receptors in particular (Barnes, 2011). It is estimated that the level of dopamine in subcortical structures increases in schizophrenia, which is the cause of clinically assessed positive symptoms of schizophrenia. On the other hand, DA transmission in the cerebral cortex is weakened, which determines the
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presence of negative symptoms of schizophrenia (Winterer and Weinberger, 2004). With development of neuropsychiatry and introduction of new neuroleptics, the important role of noradrenaline (NA) and serotonine (5-HT), apart from DA, in the course of schizophrenia has been confirmed (Huang et al., 2008). Olanzapine (OLA) and aripiprazole (ARI) are atypical neuroleptics with a mechanism of action reducing positive and negative schizophrenia symptoms (Jafari et al., 2012; Hirose and Kikuchi, 2005). ARI is thought to be a partial agonist of D2 and D3 dopamine receptors and 5-HT1A serotonin receptors as well as an antagonist of 5-HT2A and 5-HT6 receptors (Hirose and Kikuchi, 2005; Burda et al., 2011). On the other hand, OLA is a typical antagonist of D2 , 5HT2A , 5-HT2B , 5-HT2C receptors as well as H1 histamine receptors and ␣1 adrenergic receptors. Due to the receptor profile of those drugs, their antagonistic effect on D2 receptors in the mesolimbic system causes a reduction of positive schizophrenia symptoms (Hirose and Kikuchi, 2005). Our previous studies indicate that OLA and ARI reduce memory disorders in prenatally stressed animals and modify the levels of corticosterone and BDNF (Ratajczak et al., 2013b; Nowakowska et al., 2014), therefore it was interesting to find out whether neuroleptics also affect the levels of neurotransmitters (DA, NA, 5-HT) and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homo-vanillic acid (HVA) and 5-Hydroxyindoleacetic acid (5-HIAA) tested in the frontal cortex of prenatally stressed animals. 2. Materials and methods
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Fig. 1. Experiment schedule. Table 1 Schedule of stress exposures applied in the prenatal stress paradigm. Gestation day
Morning
Midday
Evening
14 15 16 17 18 19 20 21
metal tube 60 min cold exposure swim 15 min open social stress 12 h metal tube 60 min
swim 15 min
metal tube 60 min fast overnight swim 15 min lights on overnight
swim 15 min
metal tube 60 min swim 15 min swim 15 min cold exposure 6 h metal tube 60 min
metal tube 60 min swim 15 min
2.1. Animals Timed pregnant Wistar female rats (15) were purchased from Poznan University of Medical Sciences, Poznan, Poland (licensed by the Ministry of Agriculture in Warsaw, Poland) and arrived at our animal facility on day 2 of gestation. The pregnant animals were housed individually in a light- (lights on 07.00–19.00 h), temperature- and humidity-controlled animal facility. The dams had free access to rat chow (Labofeed B) and water. The total number of animals in the study was 51 (15 females and 36 offspring males). The male rats (36 animals) were divided into three groups – non stressed control group (NSCG) receiving ARI or OLA – (12 non-prenatally stressed rats, offspring of the 5 non-stressed females), prenatally stressed group (PSG) receiving ARI or OLA (12 prenatally stressed rats, offspring of the 5 prenatally stressed females) and “saline” group receiving sodium chloride NaCl dissolved in 1% carboxymethylcellulose vehicle in the 1:1 ratio (not ARI or OLA) (12 non-prenatally stressed rats, offspring of the 5 non-stressed females). Fig. 1 contain schematic picture of conducted experiments. All procedures related to the use of rats in these experiments were conducted with due respect to ethical principles regarding experiments on animals. The study protocol was approved by the Local Ethics Committee for Research on Animals in Poznan. 2.2. Drugs Pure carboxymethylcellulose was obtained from Koch-Light Laboratories (London, England), ARI – Otsuka Pharmaceutical Europe, Bristol-Myers Squibb (Warsaw, Poland), OLA – Zyprexa Eli Lilly (Warsaw, Poland). The rats were administered ARI (1.5 mg/kg ip), OLA (0.5 mg/kg ip) or the vehicle intraperitoneally (ip). ARI and OLA were suspended in saline solution (0.5% carboxymethylcellulose) (2 ml, ip). The controls were given saline solution according to the same schedule. Separate groups of animals were used for different tests.
ARI and OLA dose which was used in the study were calculated based on the previous studies (Ratajczak et al., 2012; Ratajczak et al., 2013b; Nowakowska et al., 2014; Burda et al., 2011; Czubak et al., 2010). All the chemicals used for high performance liquid chromatography (HPLC) were from Merck (Warszawa, Poland). 2.3. Prenatal stress procedure (Kuneninn et al., 2003) Beginning on day 14 of gestation, the pregnant dams were exposed to a repeated variable stress paradigm until delivery of their pups on gestational day 22 or 23 as previously described. The stresses used in this paradigm consisted in: (1) restraint in metal tube for 1 h, (2) exposure to a cold environment (4 ◦ C) for 6 h, (3) overnight food deprivation, (4) 15 min of swim stress in water of room temperature, (5) lights on for 24 h, and (6) social stress induced by overcrowded housing conditions during the dark phase of the cycle. A typical schedule of the stress applications is presented in prenatal stress table (Table 1). Pregnant control dams remained in the animal room from gestational days 14–21 and were only exposed to normal animal room husbandry procedures. All dams delivered their pups vaginally. At weaning, male offspring from each litter were placed with same sex litter mates per cage with free access to rat chow and water. The animals were exposed to normal animal room procedures from that point onward until experimental use on post-natal day 90. 2.4. Microdialysis Rats were anesthetised with ketamine (75 mg/kg im) and xylazine (10 mg/kg im), placed in a stereotactic apparatus (David Kopf Instruments, Tujunga, CA, USA) and subsequently vertical microdialysis probes were implanted in the rat frontal cortex with coordinates (mm) A + 2.8, L + 0.8, V − 6.0 from the dura. Twenty four hours after implantation, probe inlets were connected to a syringe
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DA (% of basal level)
500
naive NSCG
*^
schi PSG
400
sal-na ive saline
300
*^
OLANZAPINE 0,5 mg/kg 200 100
*^
*^ *^
^
^
20
40
0 -40
-20
0
60
*^
^
^
^
80
100
120
min Fig. 2. Effect of olanzapine (0.5 mg/kg) on DA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
450
naive NSCG
NA (% of basal level)
400 350 300 250
^
schi PSG
sal-na ive saline OLANZAPINE 0,5 mg/kg
200
^
*^ ^
*^ ^
*^
150
^
*^
*^
100
^
*^
50 0 -40
-20
0
20
40
60
80
100
120
m in Fig. 3. Effect of olanzapine (0.5 mg/kg) on NA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
pump (CMA, Sweden) which delivered an artificial CSF (aCSF) composed of (mM): NaCl 147, KCl 4.0, CaCl2 1.2, MgCl2 1.0 at a flow rate of 2 l/min. Baseline samples were collected every 20 min after the washout period. Appropriate drugs were then administered and dialysate fractions were collected for 120 min. At the end of the experiment, the rats were sacrificed and their brains were histologically examined to validate probe placement. 2.5. Analytical procedure Dopamine (DA), serotonin (5-hydroxytryptamine, 5-HT), noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by HPLC with coulochemical detection. Chromatography was performed using the Ultimate 3000 System (Dionex, USA), coulochemical detector Coulochem III (model 5300, ESA, USA) with a 5020 guard cell, a 5014B microdialysis cell and a Hypersil Gold-C18 analytical column (3 × 100 mm). The mobile phase was composed of 0.05 M potassium phosphate buffer adjusted to pH = 3.6, 0.5 mM EDTA, 16 mg/L 1-octanesulfonic acid sodium salt, and a 2.1% methanol. The flow rate during analysis was 0.7 ml/min. The applied potential of a guard cell was +600 mV, while those of microdialysis cell was E1 = −50 mV, E2 = +300 mV
and a sensitivity was set at 50 nA/V. The chromatographic data were processed by Chromeleon v. 6.80 (Dionex, USA) software run on a PC computer. 2.6. Statistical analysis The statistical significance was calculated using repeatedmeasures ANOVA, followed by Tukey’s post-hoc test. The results were considered statistically significant when p < 0.05 or p < 0.01. Only time points after drug administration was calculated in the result section. 3. Results 3.1. Effect of olanzapine on DA, NA, 5-HT, DOPAC, HVA and 5-HIAA extracellular level in frontal cortex of NSCG and PAG rats Single treatment of olanzapine in dose of 0.5 mg/kg increased DA extracellular level in PSG rats from 20 to 120 min after administration compared to the saline treated rats (PSG vs. saline p < 0.01) (Fig. 2). Moreover DA increase was also observed in PSG rats compared to NSCG rats in 20–120 min of the observation (PSG vs. NSCG
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5-HT (% of basal level)
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OLANZAPINE 0,5 mg/kg
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m in Fig. 4. Effect of olanzapine (0.5 mg/kg) on 5-HT extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
DOPAC (% of basal level
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OLANZAPINE 0,5 mg/kg
0 -40
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20
min Fig. 5. Effect of olanzapine (0.5 mg/kg) on DOPAC extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
p < 0.01) (Fig. 2). The study also found significant decline of DA level observed from 80 to 120 min of fraction collection compared to the saline treated rats (NSCG vs. saline p < 0.01) (Fig. 2). Olanzapine in a dose of 0.5 mg/kg increased NA extracellular level in NSCG and PSG rats compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 3). After olanzapine administration (0.5 mg/kg) in the PSG rats it was also observed both decrease (20 min and 80–120 min after administration) and increase (40–60 min after administration) of NA level compared to the NSCG rats (PSG vs. NSCG p < 0.01) (Fig. 3). 5-HT extracellular level was slightly but significantly increased at 20, 40, 80 min and decreased at 100–120 min after olanzapine administration in NSCG rats compared to the saline treated rats (NSCG vs. saline p < 0.01) (Fig. 4). Olanzapine also potently increased extracellular 5-HT level in PSG rats from 20 to 120 min after administration compared to the NSCG rats (PSG vs. NSCG p < 0.01). There was also observed significant increase of 5-HT in PSG rats versus saline rats from 20–120 min after OLA administration (PSG vs. saline p < 0.01) (Fig. 4). Intraneuronal metabolite of DA, DOPAC was increased in both NSCG and PSG rats after olanzapine administration compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 4). There
was also significant increase of DOPAC in PSG rats versus NSCG rats after administration of OLA (PSG vs. NSCG p < 0.01) (Fig. 5). Extraneuronal metabolite of DA, HVA was increased in both, NSCG and PSG rats after administration of olanzapine compared to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 6). There was also significant increase of HVA in PSG rats versus NSCG after administration of OLA (PSG vs. NSCG p < 0.01) (Fig. 6). Serotonin metabolite, 5-HIAA was not affected by olanzapine administration in both, NSCG and PSG rats (Fig. 7). There was also no significant increase of 5-HIAA in PSG versus NSCG rats after administration of OLA (Fig. 7).
3.2. Effect of aripiprazole on DA, NA, 5-HT, DOPAC, HVA and 5-HIAA extracellular level in frontal cortex of NSCG and PSG rats Administration of aripiprazole (1.5 mg/kg) significantly increased DA extracellular level only in PSG rats from 20 to 120 min of the observation period in comparison to the saline treated rats (PSG vs. saline p < 0.01 or p < 0.05) (Fig. 8). There was also significant increase of DA level in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.01 p < 0.05) (Fig. 8).
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HVA (% of basal level)
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50 0 -40
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m in Fig. 6. Effect of olanzapine (0.5 mg/kg) on HVA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level. Pˆ < 0.01 versus saline treated naïve rats, * P < 0.01 versus naïve olanzapine treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HIAA (% of basal level
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OLANZAPINE 0,5 mg/kg
schi PSG
sal-na salineive 60 -40
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min Fig. 7. Effect of olanzapine (0.5 mg/kg) on 5-HIAA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 6 rats per group) and are expressed as percent of basal level.
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ARIPIPRAZOLE 1,5 mg/kg
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DA (% of basal level)
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min Fig. 8. Effect of aripiprazole (1.5 mg/kg) on DA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆ P < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
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m in Fig. 9. Effect of aripiprazole (1.5 mg/kg) on NA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HT (% of basal level)
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ARIPIPRAZOLE 1,5
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m in Fig. 10. Effect of aripiprazole (1.5 mg/kg) on 5-HT extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. ˆˆ P < 0.01 versus saline treated naïve rats, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
Aripiprazole (1.5 mg/kg) increased NA extracellular level in NSCG rats only at 40 min after administration compared to saline treated rats (NSCG vs. saline p < 0.01) (Fig. 9). There was also observed decrease in extracellular NA level in PSG rats in compared to the NSCG rats (PSG vs. NSCG p < 0.05) (Fig. 9). Administration of aripiprazole (1.5 mg/kg) increased 5-HT extracellular level in both NSCG and PSG rats from 20 to 120 min after administration in comparison to the saline treated rats (NSCG/PSG vs. saline p < 0.01) (Fig. 10). There was also significant increase of the 5-HT level in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.01) (Fig. 10). Aripiprazole administration (1.5 mg/kg) increased DOPAC extracellular level only in PSG rats from 20 to 120 min after administration in comparison to the saline treated rats (PSG vs. saline p < 0.05 or p < 0.01) (Fig. 11). There was also significant increase in DOPAC level in PSG rats versus NSCG rats (20–120 min) after administration of ARI (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 11). HVA extracellular level was significantly increased in both NSCG and PSG rats after treatment of aripiprazole in comparison to the saline treated rats (NSCG/PSG vs. saline p < 0.05 or p < 0.01) (Fig. 12). There was also significant decrease of HVA in PSG versus NSCG rats after administration of ARI from 60 to 120 min (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 12).
5-HIAA extracellular level was increased in PSG rats from 20 to 100 min after aripiprazole administration in comparison to the saline treated rats (PSG vs. saline p < 0.05 or p < 0.01) (Fig. 13). There was also significant increase (20–100 min) and decrease (120 min) of 5-HIAA in PSG rats versus NSCG rats after administration of ARI (PSG vs. NSCG p < 0.05 or p < 0.01) (Fig. 13). 4. Discussion Biochemical studies indicate that the functioning of neurotransmission systems is disturbed in schizophrenia, primarily the dopaminergic and serotoninergic systems, which was the basis of constructing theories on the genesis of the disease in the context of various neurotransmission systems. The dopamine theory of schizophrenia assumes that the clinical presentation of the disease includes increased DA activity in the CNS that can be reduced by antipsychotic drugs capable of reducing the level of dopaminergic activity in the limbic system’s structures (Podgrodzka and Jarema, 2010). According to this acknowledged idea developed for many years, the negative symptoms of schizophrenia are an effect of dopaminergic insufficiency of the mesocortical pathways and positive psychotic symptoms result from hyperactivity of the mesolimbic dopaminergic pathway (Siwek et al., 2010).
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schi PSG sal-na salineive
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m in Fig. 11. Effect of aripiprazole (1.5 mg/kg) on DOPAC extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
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sal-na salineive
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min Fig. 12. Effect of aripiprazole (1.5 mg/kg) on HVA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
5-HIAA (% of basal level
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naive NSCG
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min Fig. 13. Effect of aripiprazole (1.5 mg/kg) on 5-HIAA extracellular level in frontal cortex of naïve and schizophrenic rats. Values are mean ± SEM (n = 4 rats per group) and are expressed as percent of basal level. Pˆ < 0.05, ˆˆ P < 0.01 versus saline treated naïve rats, * P < 0.05, ** P < 0.01 versus naïve aripiprazole treated rats (repeated measures ANOVA and post hoc Tukey’s test).
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Diversely, the serotonin theory substantiated the efficiency of atypical antipsychotics in schizophrenia which affect dopamine receptors but also show strong antagonistic affinity with serotonin receptors (especially 5-HT2A ) (Krzystanek, 2007). Apart from the mechanism related to the antagonistic effect on D2 receptors as well as 5-HT2A and 5-HT2C serotonin receptors, found in all atypical antipsychotics, attention should be paid to a new neuroleptic, aripiprazole, which also is partially agonistic to D2 , D3 , and 5-HT1A receptors (Krzystanek, 2007). Mainly due to its mechanism of action, ARI has been called a stabiliser of the dopamine-serotonin system, acting antagonistically, weakening dopaminergic transmission, in case of dopamine receptor hyperactivity, and acting as an agonist, enhancing DA transmission, in conditions of insufficient dopaminergic activity (Burris et al., 2002). It should also be mentioned that ARI’s partial agonism of D2 receptors in combination with antagonism of 5-HT2A receptors minimises the risk of dopamine insufficiency in the nigrostriatal and tuberoinfundibular pathways, but also the risk of drug-induced parkinsonism or hyperprolactinaemia (Krzystanek, 2007). Obtained results indicate that the level of DA in non-stressed animals (NSCG) was regulated only by the administration of OLA. Conversely, in prenatally stressed rats (PSG), the administration OLA and ARI resulted in an increase of DA level which suggests an agonist effect on D2 receptors located in the frontal cortex (FC). Other studies indicate that ARI caused an increase of DA level or had no effect on the prefrontal cortex (PFC). In several studies, ARI did not cause a DA level increase (Newma-Tancredi et al., 2005), but Li et al. (2004) observed an increased DA level after administering ARI in the dose of 0.3 mg/kg. DA increase observed after OLA administration (OLA—typical antagonist of the D2 receptors) was observed by Bymaster et al. (1999) but other authors failed to obtain any DA level increase in the PFC (Rollema et al., 2000; Volonte et al., 1997). Literature indicate that OLA and ARI also induce the increase of DA levels in the occipital cortex and in the cerebellum (Devoto and Flore, 2006). Meltzer and McGurk (1999) also observed a DA increase in the medial prefrontal cortex (mPFC). Moreover changes in the level of DA in PFC could be related to the cytoarchitecture changes observed in the prenatally stressed rats. The level of DA directly affects the regulation of the level of its metabolites, DOPAC and HVA. The level of DOPAC is regulated by the level of DA throughout the brain (Devoto and Flore, 2006). Administration of antipsychotics can regulate the level of DA, DOPAC and HVA. Obtained results indicate that DOPAC level in NSCG was regulated only by OLA (increase). However, the administration of both OLA and ARI increased DOPAC level in prenatally stressed rats. Moreover, HVA level observed after the administration of OLA and ARI was higher in both NSCG and PSG. The results obtained by Semba et al. (1995) indicate that the administration of both OLA and ARI in doses of (2, 10, and 40 mg/kg) caused an increase of DOPAC concentration in mPFC and in the striatum. In the case of OLA administration, the DOPAC level increase may depend on the drug doses used (Jordan et al., 2004). Earlier studies indicate a strong antagonistic effect of OLA on presynaptic D2 receptors (Volonte et al., 1997; Li et al., 1998; Westerink et al., 1998). In the case of HVA, the studies by Jordan et al. (2004) indicate that both the administration of OLA and ARI can generate increased HVA level in the mPFC and in the striatum. Moreover, Jordan et al. (2004) demonstrated that HVA level observed after the use of OLA and ARI depended on the dose and the duration of drug administration. Single administration of both ARI and OLA in doses of 1 mg/kg, 10 mg/kg, and 20 mg/kg increased the level of this metabolite. Contrary, in groups of animals receiving drugs for 21 days, increased HVA level was observed only in rats receiving ARI (Jordan et al., 2004), which suggests the possibility of tolerance to OLA. Cognitive functions are regulated by the normal level of DA and NA in the CNS (Yamamoto and Horynkiewicz, 2004). Moreover, NA
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is considered to play a crucial role in defensive reactions related to strong stress which can generate disruptions in the level of NA (Yamamoto and Horynkiewicz, 2004). The level of NA is regulated mainly by DA synthesis, both in noradrenergic and dopaminergic neurons (Devoto and Flore, 2006). DA release increases the level of NA, which in turn can act back increasing DA activity (Pan et al., 1996). NA also has an indirect effect on DA reuptake in PFC (Moron et al., 2002). Obtained results indicate that NA level in NSCG was regulated by the administration of OLA and ARI (increased neurotransmitter level). In the case of PSG, the administration of OLA generated an NA level increase, while ARI decreased it. Comparing the efficiency of aripiprazole and clozapine Zocchi et al. (2005) indicated that increased NA level, coexisting with increased DA level, was observed after the use of clozapine in the dose of 10 mg/kg. No changes of NA level were observed after the use of ARI in doses (0.1 mg/kg, 0.3 mg/kg, 3.0 mg/kg, and 30 mg/kg). Blocking 5-HT2 receptors in combination with effect on other neurotransmission systems is believed to be responsible for the anti-deficit, anti-depressant, and pro-cognitive effect (Rybakowski, 2007). ARI is an antagonist of 5-HT2A and 5-HT2C receptors, and an agonist of 5-HT1A receptors (Lieberman, 2004), while OLA is a typical antagonist of 5-HT2A and 5-HT2C receptors. Patient’s mood, possibly regulated by the level of 5-HT and NA, is believed to ˛ 2012). be at the root of schizophrenia (Markowicz-Narekiewicz, Obtained results indicate that the level of 5-HT in the FC of both NSCG and PSG was regulated by the administration of OLA and ARI. According to Carli et al. (2011), the use of 3 mg/kg of ARI can cause an increase of 5-HT level in the mPFC. However, other investigators did not indicate changes of 5-HT level in the mPFC in mice following the administration of ARI (Volonte et al., 1997) and the hippocampus of rats (Assie et al., 2008). Bortolozzi et al. (2007) observed a decreased 5-HT level in the mPFC in mice due to reduced re-uptake of 5-HT. In turn, Knauer et al. (2008) believe the use of OLA can lead to rapid saturation of 5-HT2A receptors located in the FC, which can reduce 5-HT level. Saturation of 5-HT receptors was also observed following the administration of ARI in the studies of Natesan et al. (2006), but only when large doses of the drug were used (over 20 mg/kg). The level of serotonin directly affects the level of 5-HIAA, and obtained results indicate that ARI in PSG generated an increase in the level of 5-HIAA. The effect of changes in 5-HIAA level was not observed in NSCG. Literature reports state that the use of ARI can cause a decrease of 5-HIAA level in the mPFC and in the striatum. It was also found that 5-HIAA level in these structures was reduced after repeated administration, which can be due to sudden functional desynthetisation of 5-HT1A receptor somatodendrites (Kennett et al., 1987; Fuller and Perry, 1989). Through presynaptic activation of 5-HT1A receptors, ARI can reduce serotonin release and thereby reduce 5-HIAA level in the forebrain as was proposed by Jordan et al. (Jordan et al., 2004). However studies by Li et al. (1998) on the effect of OLA on the level of 5-HIAA indicate that regardless of the dose the drug affected 5-HIAA level in the PFC and the nucleus accumbens.
5. Conclusion Both OLA and ARI increase the level of DA and 5-HT as well as their metabolites (DOPAC, HVA) in the frontal cortex. Increased 5HIAA level was observed only following the administration of ARI, while OLA increased the level of NA. The differences in the observed levels of neurotransmitters and their metabolites were probably due to different mechanisms of action of the used neuroleptics. All the neuroleptics used act mainly on dopaminergic receptors. Differences in clinical profiles are the result of the degree to which they
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block particular subtypes of dopamine receptors. The dopaminergic system is a system of neuromodulation acting in midbrain cells on the mesolimbic region, striatum, and cortical areas (Rzewuska, 2009). The dopaminergic activity in the midbrain-cortex circuit allows regulation of many behaviours, that is cognitive functions, movement control, mood, or attention, so disorders of dopaminergic functions can lead to neurological and mental deficits observed in patients. Considering the complexity and episodic character of schizophrenia, aripiprazole seems to be efficient in reducing positive and negative disorders. Conflict of interest The authors declare that there are no conflicts of interest associated with this manuscript. Acknowledgment Project was funded by the National Science Centre—Cracow, Poland. References Assie, M.B., et al., 2008. The antipsychotics clozapine and olanzapine increase plasma glucose and corticosterone levels in rats; comparison with aripiprazole ziprasidone bifeprunox and F15063. Eur. J. Pharmacol. 592, 160–166. Barnes, T., 2011. Evidence-based guidelines for the pharmacological treatment of schizophrenia:recommendations from the British Association for Psychopharmacology. J. Psychopharmacol. 25 (5), 567–620. Bortolozzi, A., et al., 2007. Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT1A receptors but not 5-HT2A receptors. Int. J. Neuropsychopharmacol. 13, 1299–1314. Burda, K., et al., 2011. Influence of aripiprazole on the antidepressant anxiolytic and cognitive functions of rats. Pharmacol. Rep. 63, 898–907. Burris, K.D., et al., 2002. Aripiprazole a novel antipsychiotic is a high-affinity partial agonist at human dopamine D2 receptors. J. Pharmacol. Exp. Ther. 302 (1), 381–389. Bymaster, F., et al., 1999. Olanzapine: a basic science update. Br. J. Psychiatry Suppl. 37, 36–40. Carli, M., et al., 2011. Effects of aripiprazole olanzapine and haloperidol in a model of cognitive deficit of schizophrenia in rats:relationship with glutamate release in the medial prefrontal cortex. Psychopharmacology 214, 639–652. Czubak, A., et al., 2010. Effect of venlafaxine and nicotine on the level of neurotransmitters and their metabolites in rat brains. J. Physiol. Pharmacol. 61 (3), 339–346. Devoto, P., Flore, G., 2006. On the origin of cortical dopamine:is it a co-transmitter in noradrenergic neurons? Neuropharmacology 4, 115–125. Fuller, R.W., Perry, K.W., 1989. Effects of buspirone and its metabolite 1-(2-pyrimidinyl)piperazine on brain monoamines and their metabolities in rats. J. Pharmacol. Exp. Ther. 248, 50–56. Hirose, T., Kikuchi, T., 2005. Aripiprazole a novel antipsychotic agent:Dopamine D2 receptor partial agonist. J. Med. Invest. 52, 284–290. Huang, M., et al., 2008. Asenapine increases dopamine norepinephrine and acetylcholine efflux in the rat medial prefrontal cortex and hippocampus. Neuropsychopharmacol 33, 2934–2945. Jafari, S., et al., 2012. Novel olanzapine analogues presenting a reduced H1 receptor affinity and retained 5HT2A/D2 binding affinity ratio. BMC Pharmacol. 12, 8, http://dx.doi.org/10.1186/1471-2210-12-8. Jordan, S., et al., 2004. In vivo effects of aripiprazole on cortical and striatal dopaminergic and serotonergic function. Eur. J. Pharmacol. 483, 45–53. Kennett, G.A., et al., 1987. Single administration of 5-HT(1A) presynaptic but not postsynaptic receptor-mediated responses:relationship to antidepressant-like action. Eur. J. Pharmacol. 138, 53–60. Knauer, C.S., et al., 2008. Validation of a rat in vivo [(3)H]M100907 binding assay to determine a translatable measure of 5-HT(2A) receptor occupancy. Eur. J. Pharmacol. 591, 136–141. Koenig, J.I., et al., 2005. Prenatalexposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring:potential relevance to schizophrenia. Behav. Brain Res. 156 (2), 251–261.
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