Repeated co-treatment with antidepressants and risperidone increases BDNF mRNA and protein levels in rats

Accepted Manuscript Title: Repeated co-treatment with antidepressants and risperidone increases BDNF mRNA and protein levels in rats Authors: Zofia Rog´oz˙ , Katarzyna Kami´nska, Patrycja Pa´nczyszyn-Trzewik, Magdalena Sowa-Ku´cma PII: DOI: Reference:

S1734-1140(16)30398-X http://dx.doi.org/doi:10.1016/j.pharep.2017.02.022 PHAREP 670

To appear in: Received date: Accepted date:

22-11-2016 24-2-2017

Please cite this article as: Zofia Rog´oz˙ , Katarzyna Kami´nska, Patrycja Pa´nczyszyn-Trzewik, Magdalena Sowa-Ku´cma, Repeated co-treatment with antidepressants and risperidone increases BDNF mRNA and protein levels in rats, http://dx.doi.org/10.1016/j.pharep.2017.02.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Article

Title: Repeated co-treatment with antidepressants and risperidone increases BDNF mRNA and protein levels in rats Authors: Zofia Rogóż1, 3, Katarzyna Kamińska1, Patrycja Pańczyszyn-Trzewik2, Magdalena Sowa-Kućma2 Institute of Pharmacology, Polish Academy of Sciences, 1Department of Pharmacology, 2Department of Neurobiology, 31-343 Kraków, Smętna street 12, Poland, 3The Podhale State Higher Vocational School, 34-400 Nowy Targ, Kokoszków street 71, Poland

Running title: Co-treatment with antidepressants and risperidone and BDNF mRNA and protein levels

Correspondence: Zofia Rogóż, e-mail: [email protected]

1

ABSTRACT Background: Recently, several clinical studies have suggested a beneficial effect of a combination of antidepressants (ADs) with antipsychotic drugs in drug-resistant depression. Moreover, preclinical and clinical studies indicated a role of brainderived neurotrophic factor (BDNF) in the pathology of depression, as well as in the mechanism of action of ADs. Methods: In the present study, we investigated the effect of repeated administration of ADs, escitalopram, fluoxetine or mirtazapine and a low dose of risperidone (an atypical antipsychotic drug) given separately or in combination, on the mRNA and protein levels of BDNF or cAMP response element binding (p-CREB) in the hippocampus and frontal cortex of male Wistar rats. ADs were given repeatedly (once daily for 14 days), separately or in combination with a low dose of risperidone. The tissue for biochemical assays was dissected 24 h after the last dose of ADs. Results: The obtained results showed that repeated co-treatment with an inactive dose of risperidone and escitalopram or mirtazapine but not fluoxetine increased the BDNF mRNA expression in the hippocampus and frontal cortex. Moreover, combined treatment with an inactive dose risperidone and escitalopram elevated the protein levels of p-CREB in the frontal cortex. While, co-treatment with risperidone and fluoxetine or mirtazapine increased the protein levels of BDNF and p-CREB in both examined regions of the brain. Conclusions: Our present findings suggest that enhancement levels of BDNF may be essential for the therapeutic effect of co-treatment with ADs and a low dose risperidone in patients with drug-resistant depression.

Keywords: Escitalopram; Fluoxetine; Mirtazapine; Risperidone; BDNF

2

Introduction Recent preclinical and clinical studies indicated an important role of brainderived neurotrophic factor (BDNF) in the pathology of depression as well as in the mechanism of action of antidepressant drugs (ADs) [e.g. 1, 2, 3]. It was shown that stress decreased the expression of BDNF in the hippocampus [4], which is the region of the brain important in emotional cognition and memory processing [5], and this effect might contribute to the observed atrophy of stress-vulnerable neurons in this region of the brain [1, 6, 7]. The prefrontal cortex is also essential to emotional processing, and in patients with major depressive disorder (MDD) this region was also shown to decrease in volume, what correlated with the reduction BDNF levels [2, 3]. The above data suggested that both stress and MDD affected BDNF expression in limbic brain regions. Furthermore, studies using a postmortem human brain tissue demonstrated an increase in the hippocampal BDNF immunoreactivity in patients treated with ADs compared to untreated subjects [8]. In addition, some earlier clinical studies have shown that serum BDNF levels are reduced in depressed patients and it can be normalized by successful treatment with ADs [2, 9]. Moreover, behavioral studies showed that local infusion of BDNF into the midbrain and hippocampus evoked antidepressant-like activity in animal tests of depression [10, 11], what suggests that the increased expression of endogenous BDNF may have an antidepressant-like activity. In contrast, in BDNF knockout mice, the loss of forebrain BDNF attenuated the antidepressant-like effect of desipramine in the forced swim test (FST) [12]. In addition, recent studies demonstrated that repeated (but not acute) administration of different ADs increased BDNF and trkB (a BDNF receptor) mRNA in the hippocampus, whereas electroconvulsive shock (ECS) and tranylcypromine (a monoamine oxidaze inhibitor (MAOI)) induced a similar effect in the hippocampus and cerebral cortex [13, 14].

3

Several clinical reports suggested a beneficial effect of the addition of a low dose of an atypical antipsychotic drug (e.g., risperidone) to the ongoing treatment with ADs, especially SSRI on the therapy of drug-resistant depression [15 -19]. It is known that risperidone, like other atypical antipsychotic drugs, evokes minimal extrapyramidal side-effects compared to classic antipsychotics (e.g. haloperidol) [20]. This drug is more potent in the binding to serotonin 5-HT2A than dopamine D2 receptors. Affinities of risperidone for serotonergic 5-HT2A, dopaminergic D2, adrenergic α1, α2A, and histaminergic H1 receptors (Ki value) are 0.81, 6.4, 2.5, 10, and 33 nM, respectively [21]. Our earlier observations showed that risperidone at a low dose enhanced the antidepressant-like effect of ADs in the FST in rats [22, 23]. Thus, to understand the mechanism of clinical efficacy of a combination of an AD and risperidone in the therapy of drug-resistant depression, and considering that the most potent effect of ADs on BDNF expression was found after repeated treatment, in the present study we investigated the effect of repeated administration of ADs: SSRI escitalopram (ESC) and fluoxetine (FLU) or mirtazapine (MIR, a noradrenergic and specific serotonergic antidepressant with an agonist action at 5HT1A receptor and an antagonist at the central α2-auto- and hetero-adrenoreceptors) [ 24] and a low dose of risperidone, given separately or in combination, on the mRNA and protein levels of BDNF or cAMP response element binding (p-CREB) in the rat hippocampus and frontal cortex. The effect of repeated co-treatment with ESC, FLU or MIR and risperidone on the mRNA and protein levels of BDNF or pCREB in the hippocampus and frontal cortex has not been studied, yet.

Materials and methods

Animals

4

The experiments were carried out on male Wistar rats (250- 270 g) (Charles River Laboratories, Sulzfeld, Germany). The animals were housed 4 per cage (57 cm × 35 cm × 20 cm) in a colony room kept at 22 ± 1oC with a 40-50% humidity, on a 12-h light-dark cycle (the light on at 7 a.m.). The rats had free access to food and water before the experiments. All the experiments were conducted during the light phase and were carried out according to the procedures approved by the Animal Care and Use Committee at the Institute of Pharmacology, Polish Academy of Sciences, Cracow.

Drug administration

Escitalopram oxalate (ESC), fluoxetine hydrochloridr (FLU) (Tocris Bioscience, Bristol, UK) were dissolved in a 0.9% NaCl, while risperidone (RIS) and mirtazapine (MIR) ( Tocris Bioscience, Bristol, UK) was dissolved in 0.1 M tartaric acid and the solution were adjusted to pH 6-7 with 0.1 N NaOH. ESC (2.5, 5 or 10 mg/kg, ip),

FLU or MIR (5 mg/kg, ip) in a volume of 2 ml/kg were given

repeatedly (once daily for 14 days), separately or jointly with risperidone (0.05 or 0.1 mg/kg ip). Risperidone was administered 30 min after AD injection. The tissue (hippocampus and frontal cortex) for biochemical assays was dissected 24 h after the last dose of ADs.

BDNF expression analysis (Real-Time PCR)

The procedure for determining BDNF mRNA levels was carried out according to Pochwat et al. [25]. Briefly, total RNA was extracted using the TRIzol reagent (Invitrogen, UK) and precipitated with ethanol. After dissolving in water, RNA (1 μg) was reverse-transcribed to cDNA using High Capacity cDNA Reverse Transcrition kit with Rnase inhibitor and random hexamers (Life Technologies, CA). The BDNF mRNA level was determined by RealTime PCR using pre5

designed TaqMan Gene Expression Assays (Life Technologies, CA) on a CFX96 Real-Time System (BioRad) using 96-well optical plates (Bio-Rad). The used gene expression assays were: BDNF [Rn02531967 s1, RefSeq: NM 012513.3] and Gapdh (glyceraldehyde-3-phosphate dehydrogenase) [Rn99999916 s1, RefSeq: NM 017008.3] as endogenous control. Each cDNA sample was run in triplicate and no template control wells were included on each plate to check for contamination. The PCR reaction mix consisted of sterile water, TaqMan Gene Expression Master Mix (20 μmol/l) (2 × concentration, contains AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, ROX passive reference and optimized buffer components) (Life Technologies, CA) and TaqMan Gene Expression Assay (20 × mix). The Real-time PCR program included a holding stage at 50°C for 2 min and 9° C for 10 min, and cycling 40 times at 95°C for 15 s, and 60°C for 1min. The Delta–Delta Comparative Threshold method was used to quantify the fold change between the samples.

BDNF and p-CREB protein analysis (Western blot)

The tissue samples were prepared as published previously [25]. The samples containing 50 μg of protein were fractioned by 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. After blocking of the non-specific binding, the membranes were incubated overnight at 4oC with polyclonal rabbit anti-BDNF or anti-p-CREB antibodies (1:200 dilution, Santa Cruz Biotech., USA). Then the membranes were washed in TBST and incubated for 60 min at RT with a goat HRPconjugated anti-rabbit IgG (1:7.000dilution, ROCHE). After that, blots were washed in TBST and developed using enhanced chemiluminescence reaction (BM Chemiluminescence Western Blotting Kit, Roche). The BDNF and p-CREB signals were visualized and quantified with the FUJI-LAS 4000 System and FUJI Image Gauge software. To confirm equal loading of the samples on the gel, the membranes were incubated for 30 min with mouse anti-β-actin antibody (1:1.000 dilution, Santa 6

Cruz Biotechnology, USA) and then processed as described above. The density of each BDNF and p-CREB protein band was normalized to the density of β-actin band.

Statistical analysis All results are shown as the percent of control (mean ± SEM). Each group consisted of 7-8 rats. The statistically significant differences between the studied groups were calculated using a two-way ANOVA followed by the NewmanKeuls test. p < 0.05 was considered statistically significant.

Results The effects of repeated treatment with ADs (ESC, FLU or MIR) and risperidone, administered separately or in combination, on the BDNF mRNA expression in the hippocampus and frontal cortex In the hippocampus (Fig. 1A - left panel), repeated administration of ESC only at the highest dose (10 mg/kg, but not at doses of 2.5 and 5 mg/kg) significantly elevated BDNF mRNA level (by 360% vs. vehicle-treated group, p = 0.00017). Co-administration of ESC (5 mg/kg) and risperidone at a lower dose (0.05 mg/kg) or ESC at both doses (2.5 or 5 mg/kg) and risperidone at a higher dose (0.1 mg/kg) evoked a statistically significant increase in the level of BDNF mRNA vs. treatment with vehicle (ESC 5 + RIS 0.05 by 263%, p = 0.007160; ESC 2.5 + RIS 0.1 by 303%, p = 0.000544; ESC 5 + RIS 0.1 by 338%, p = 0.000164 In the hippocampus, a two-way ANOVA demonstrated a significant effect of ESC treatment [F(3,63) = 12.554, p = 0.0001], a significant effect of risperidone [F(2,63) = 9.38, p = 0.00027] and a significant interaction [F(4,63) = 2.67, p = 0.040]. In the frontal cortex (Fig.1A - right panel), repeated administration of ESC (2.5, 5 and 10 mg/kg) or risperidone (0.05 and 0.1mg/kg) did not change the level 7

of BDNF mRNA in a statistically significant manner compared to the vehicletreated group. Co-treatment with ESC (5 mg/kg) and risperidone (0.05 mg/kg) or ESC at the lower dose (2.5 mg/kg) with risperidone at the higher dose (0.1 mg/kg) induced a more potent increase in the level of BDNF mRNA compared to the control group (ESC 5 + RIS 0.05 by 188%, p = 0.007421; ESC 2.5 + RIS 0.1 by 175%, p = 0.033283. In the frontal cortex, two-way ANOVA indicated a significant effect of ESC [F(3,62) = 5.752, p = 0.002) or risperidone [F(2,62) = 6.236, p = 0.003] and a significant interaction [F(4,62) = 2.987, p = 0.025]. Repeated administration of FLU (5 mg/kg, but not risperidone, 0.05 or 0.1 mg/kg) induced a significant increase in the BDNF mRNA level in the frontal cortex compared to the vehicle-treated group (by 189% of control, p = 0.016, Fig. 1B - right panel). On the other hand there were no changes in the hippocampus (Fig 1B - left panel). In the hippocampus, two-way ANOVA indicated no significant effect of FLU [F(1,40) = 0.688, p = 0.412) or risperidone [F(2,40) = 0.486, p = 0.619] and no significant interaction [F(2,40 ) = 1.066, p = 0.354]. In the frontal cortex, two-way ANOVA indicated a significant effect of FLU [F(1,40) = 4.391, p = 0.043), no significant effect of risperidone [F(2,40) = 0.232, p = 0.794] and a significant interaction [F(2,40) = 3.804, p = 0.031]. MIR (5 mg/kg) did not change the level of BDNF mRNA in the hippocampus (Fig. 1C - left panel) and frontal cortex (Fig. 1C - right panel). Co-treatment with MIR (5 mg/kg) and risperidone only at the higher dose (0.1 mg/kg) induced a statistically significant increase in the level of BDNF mRNA in both examined regions of the brain compared to control group. (Fig. 1C).

8

In the hippocampus, two-way ANOVA indicated no effect of MIR [F(1,40) = 3.357, p = 0.074], a significant effect of risperidone [F(2,40) = 3.683, p = 0.034] and an interaction of borderline significance [F(2,40) = 3.083, p = 0.059]. In the frontal cortex, two-way ANOVA indicated a significant effect of MIR [F(1,40) = 26.2, p = 0.00001], a significant effect of risperidone [F(2,40) = 4.08, p = 0.024] and no significant interaction [F(2,40) = 1.12, p = 0.336].

The effects of repeated treatment with ADs (ESC, FLU or MIR) and risperidone, administered separately or in combination, on the protein level of BDNF in the hippocampus and frontal cortex

Repeated administration of ESC only at the highest dose (10 mg/kg, but not at doses of 2.5 and 5 mg/kg) induced a significant increase of the BDNF protein level in the hippocampus (by 155% of control, p = 0.018451, Fig. 2A - left panel), but not in the frontal cortex (Fig. 2A - right panel). In the hippocampus, two-way ANOVA indicated significant effect of ESC [F(3,69) = 3.764, p = 0.015)]or risperidone [F(2,69) = 4.386, p = 0.016] and an interaction of borderline significance [F(4,69) = 2.425, p = 0.056]. In the frontal cortex, two-way ANOVA indicated a significant effect of ESC [F(3,69) = 3.449, p = 0.021], but no effect of risperidone [F(2,69) = 0.342, p = 0.72] and no significant interaction [F(4,69) = 0.64, p = 0.433]. As shown in Fig. 2B, FLU (5 mg/kg) did not change the BDNF protein level in the hippocampus and frontal cortex. Repeated co-treatment with FLU (5 mg/kg) and risperidone at the both doses (0.05 and 0.1 mg/kg) increased the BDNF protein levels in the hippocampus (FLU 5 + RIS 0.05 by 219 % of control, p = 0.000567 or FLU 5 + RIS 0.1 by 242 % of control, p = 0.000177.

9

Moreover, co-administration of FLU (5 mg/kg) and risperidone at the higher dose (0.1 mg/kg) induced also an increase in the BDNF protein levels in the frontal cortex (by 169% of control, p = 0.008521. In the hippocampus, two-way ANOVA indicated a significant effect of FLU [F(1,40) = 28.2, p = 0.000001], a significant effect of risperidone [F(2,40) = 7.02, p = 0.002] and no interaction [F(2,40) = 2.6, p = 0.087]. In the frontal cortex, two-way ANOVA indicated a significant effect of FLU [F(1,40) = 9.34, p = 0.004], no effect of risperidone [F(2,40) = 2.473, p = 0.097] and no significant interaction [F(2,40) = 0.889 p = 0.419]. MIR (5 mg/kg) administered repeatedly did not change the BDNF protein levels in both investigated regions of the brain. Co-treatment with MIR (5 mg/kg) and risperidone at the higher dose (0.1 mg/kg) increased the BDNF protein levels in the hippocampus (by 170% of control, p = 0.010735, Fig. 2C - left panel) and in the frontal cortex (by 182% of control, p = 0.002382, Fig. 2C - right panel). In the hippocampus, two-way ANOVA indicated that there was a significant effect of MIR [F(1,40) = 9.108, p = 0 .004], no effect of risperidone [F(2,40) = 2.474, p = 0.096] and no significant interaction [F(2,40) = 1.379, p = 0.263]. In the frontal cortex, two-way ANOVA indicated a significant effect of MIR [F(1,40) = 4.426, p = 0.00046], no effect of risperidone [F(2,40) = 2.28, p = 0.1115] and no significant interaction [F(2,40) = 1.37, p = 0.266].

The effects of repeated treatment with ADs (ESC, FLU or MIR) and risperidone, administered separately or in combination, on the protein level of p-CREB in the hippocampus and frontal cortex

As shown in Fig. 3A, repeated treatment with ESC (2.5, 5 and 10 mg/kg) did not change the p-CREB protein levels both in the hippocampus and frontal cortex, while repeated co-treatment with ESC (5 mg/kg) and risperidone (0.1 mg/kg) 10

increased the p-CREB protein level only in the frontal cortex, by 151% of control, p = 0.041094. In the hippocampus, two-way ANOVA indicated a significant effect of ESC [F(3,69) = 3.352, p = 0.024], no effect of risperidone [F(2,69) = 2.705, p = 0.074] and no significant interaction [F(4,69) = 1.604, p = 0.183]. In the frontal cortex, two-way ANOVA indicated a significant effect of ESC [F(3,70) = 4.0667, p = 0.01], no effect of risperidone [F(2,70) = 2.032, p = 0.139] and an interaction of borderline significance [F(4,70) = 2.427, p = 0.056]. Repeated treatment with FLU (5 mg/kg) and risperidone (0.05 and 0.1 mg/kg) did not change the p-CREB protein levels in the hippocampus and frontal cortex. In the hippocampus (Fig. 3A - left panel) co-treatment with FLU (5 mg/kg) and risperidone (0.05 or 0.1 mg/kg) increased p-CREB protein levels (FLU 5 + RIS 0.05, by 177 % of control, p = 0.000291 or FLU 5 + RIS 0.1, by 186 % of control, p = 0.000175). Co-administration of FLU (5 mg/kg) and risperidone (0.1 mg/kg) also increased the p-CREB protein levels in the frontal cortex (by 171% of control, p = 0.046198) (Fig. 3A - right panel). In the hippocampus, two-way ANOVA indicated a significant effect of FLU [F(1,40) = 22.58, p = 0.00002] or risperidone [F(2,40) = 8.93, p = 0.001] and no significant interaction [F(2,40) = 0.54, p = 0.587]. In the frontal cortex, two-way ANOVA indicated a significant effect of FLU [F(1,40) = 8.749, p = 0.005], no effect of risperidone [F(2,40) = 2.166, p = 0.127] and no significant interaction [F(2,40) = 0.074, p = 0.929]. Repeated treatment with MIR (5 mg/kg) or risperidone (0.05 and 0.1 mg/kg) did not change the p-CREB protein levels in both investigated brain regions. In the hippocampus co-treatment with MIR (5 mg/kg) and risperidone (0.05 and 0.1mg/kg) increased the p-CREB protein levels (MIR 5 + RIS 0.05 by 155% of

11

control, p = 0.048896 or MIR 5 + RIS 0.1 by 164% of control, p = 0.03141), Fig. 3C - left panel). Moreover, repeated co-administration with MIR (5 mg/kg) and risperidone at the higher dose only (0.1 mg/kg) increased the p-CREB protein levels in the frontal cortex (MIR 5 + RIS 0.1 by 158% of control, p = 0.0011148), Fig. 3C right panel). In the hippocampus, two-way ANOVA indicated a significant effect of MIR [F(1,40) = 7.835, p = 0.08] or risperidone [F(2,40) = 3.857, p = 0.029] and no significant interaction [F(2,40) =1.874, p = 0.166]. In the frontal cortex, two-way ANOVA indicated a significant effect of MIR [F(1,40) = 5.66, p = 0.022] or risperidone [F(2,40) = 5.593, p = 0.007] and no significant interaction [F(2,40) = 4.326, p = 0.020]. In Fig. 4 showed the representative immunoblots of BDNF, p-CREB and βactin in the hippocampus and frontal cortex of rats treated with saline (CON), risperidone (RIS, 0.05 or 0.1 mg/kg) and/or fluoxetine (FLU, 5 mg/kg). A total of 50 µg of protein was loaded to each well.

Discussion Clinical studies suggested a beneficial effect of the addition of a low dose of an atypical antipsychotic drug (e.g. risperidone) to the ongoing treatment with ADs, especially SSRI, on the therapy of drug-resistant depression [15 -19]. In those studies, patients with major depression who did not respond to AD monotherapy [citalopram, FLU, paroxetine (20 mg/day), ESC (10 mg/day), MIR (30 mg/day) or sertraline (100 mg/day) and fluvoxamine (150 mg/day)] were randomized to risperidone (0.5-3 mg/day) in a four-week, double-blind, placebo controlled treatment trial. These data indicated that augmentation of an AD therapy with risperidone in patients with difficult-to-trial depression leads to more rapid response, a higher remission rate and better quality-of-life. 12

Moreover, since preclinical and clinical studies indicated an important role of BDNF in the pathology of depression as well as in the mechanism of action of ADs [e.g. 1, 2, 3], in the present study, we investigated the effect of repeated administration of ADs (ESC, FLU or MIR) and a low dose of risperidone, administered separately or in combination, on the BDNF mRNA and BDNF or pCREB protein levels in the rat hippocampus and frontal cortex. The obtained results indicate that only ESC (but not FLU or MIR) given repeatedly increased BDNF gene expression in the hippocampus, while FLU increased this effect only in the frontal cortex. Similarly, our earlier experiment also showed that FLU at a dose of 5 mg/kg increased the BDNA mRNA only in the cerebral cortex (but not in the hippocampus), while its higher dose (10 mg/kg) it increased BDNF gene expression in both examined brain regions [26]. MIR also at the lower dose (5 mg/kg) in the present and earlier study did not change the BDNF mRNA in the studied brain regions, while its higher dose (10 mg/kg) increased the BDNF gene expression in both examined brain regions [27]. Combined treatment with an inactive dose of ESC or MIR (but not FLU) and risperidone induced a more potent increase in the BDNF gene expression in both examined brain regions, compared to the treatment with either drug alone. Furthermore, ESC (but not FLU or MIR) administered repeatedly increased the BDNF protein level only in the hippocampus. Co-administration with an inactive dose of FLU or MIR (but not ESC) and risperidone induced an increase in the BDNF protein level in both brain regions studied, compared to the treatment with either drug alone. In addition, neither repeated treatment with ADs (ESC, FLU, MIR) nor risperidone changed in a statistically significant manner the levels of p-CREB protein in the hippocampus and frontal cortex. Moreover, combined treated with an inactive dose of the ADs studied (ESC, FLU, MIR) and risperidone induced a more potent increase in the p-CREB protein levels in both examined brain regions, compared to the treatment with either drug alone. 13

The increase in the BDNF mRNA expression in the rat hippocampus and cortex has been demonstrated after repeated treatment with ADs by other authors, but those studies indicated that the changes were not common to all ADs. For example, tranylcypramine and electroconvulsive shock (ECS) increased the level of BDNF, while desipramine, FLU and ESC had diverse effects [3, 14].The changes in the BDNF protein level have also been described, e.g. tranylcypromine and ECS increased it, but FLU and desipramine were without effects [14, 28]. Moreover, Nibuya et al [13] have shown that repeated administration of ADs from different pharmacological groups or ECS application increased the expression of CREB mRNA in the hippocampus, and suggested that transcription factor was a common intracellural target of ADs. It is known that BDNF binds to trkB receptors in the brain, and after activation by ligand-dependent autophosphorylation, this tyrosine kinase initiates a variety of intracellural signaling cascades, thereby inducing the MEK-ERK pathway and the downstream activation of RSK2, which can phosphorylate CREB. A connection between CREB and BDNF was suggested by the findings in which AD-mediated up-regulation of BDNF was blockaded in CREB-deficient mice [29]. It was shown earlier that repeated administered of FLU to rats increased the phosphorylated CREB (p-CREB) levels in the amygdala, cortex, dentate gyrus and hypothalamus, but desipramine increased the level of pCREB only in the dentate gyrus [30]. The following study showed that repeated treatment with FLU elevated p-CREB levels in the prefrontal and frontal cortex to a greater extent than did the selective noradrenaline reuptake inhibitors desipramine or reboxetine, and that activation of the Ca2+ - CaM kinase IV and MAP kinase cascades contributed more to that increase in p-CREB levels than did activation of the cAMP-PKA pathway [31]. Thus, the data suggest that the activity of ADs from different pharmacological groups in the brain depends on which postsynaptic receptors and associated signal transduction pathways are activated in various brain regions, thus, the effect on CREB activity may differ [31, 32]. Our present data indicated that combined treatment with ESC and risperidone increased the levels of 14

p-CREB protein to a greater extent in the cerebral cortex than in the hippocampus. On the other hand, co-administration with FLU or MIR and risperidone evoked a more potent increase in the p-CREB levels in the hippocampus than in the cerebral cortex. Moreover, it was demonstrated that repeated treatment with ESC (10 mg/kg) regulated intracellular pathway linked to neuroplasticity at both evaluated time points in an area-specific manner. For example, 7 days of treatment with ESC activated intracellural pathways linked to BDNF and increased the levels of proBDNF in the rat prefrontal cortex. On the other hand, 21 days of treatment with ESC decreased CREB/BDNF signaling, but increased p38 levels in the rat hippocampus. The data suggest that efficacy of ESC may be mediated by early and late effects on synaptic plasticity in selective brain areas [33]. In addition, it was shown that BDNF affected the function of serotonergic neurons. BDNF and TrkB are expressed in the raphe nucleus, and they may mediate the effects on these neurons [34]. It was also shown that BDNF from the hippocampus could be retrogradely transported to the raphe nucleus neurons, what suggests that a complex interaction determines how this neurotrophin can affect serotonergic function. For example, mice heterozygous for BDNF show changes in later life in serotonergic innervations in the cortex, hypothalamus, and hippocampus that could be linked to depression. These changes in serotonergic innervations involve its depletion in the cortex and the hypothalamus, and enhancement in the hippocampus. The following study showed that this effect of BDNF on serotonin function was region-specific, since BDNF heterozygous mice displayed blunted serotonin outflow in the ventral hippocampus after treatment with SSRIs. These data suggested that BDNF might serve to provide functional modulation of the serotonergic system at the level of both the receptor and the transporter [see Review Autry and Monteggia [3]. Our previous data also showed that co-treatment with ADs which enhanced serotonergic

transmission

(ESC,

FLU,

MIR)

and

risperidone

enhanced 15

antidepressant-like effect in the FST, and among other mechanisms, serotonin 5HT1A receptor may be involved in this effect [26, 27]. In summary, our present findings suggest that enhancement of the BDNF levels may be essential for the therapeutic effect of co-treatment with ADs and a low dose risperidone in patients with drug-resistant depression.

Conflict of interest: None declared.

Funding This study was financially supported by grant POIG 01.01.02-12-004/09-00 and partly by statutory funds of the Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland.

16

References 1. Castrén E, Rantamaki T. The role of BDNF and its receptors in depression and antidepressant drug action: reactivation of developmental plasticity. Dev Neurobiol 2010;70(5):289-97. 2. Satomura E, Baba H, Nakano Y, Maeshima H, Suzuki T, Arai H. Correlations between brain-derived neurotrophic factor and clinical symptoms in medicated patients with major depression. J Affect Disord, 2011;135(1-3):332-5. 3. Autry AE, Monteggia LM. Brain-derived neurotrophic factor and neuropsychiatric disorders. Pharmacol Rev 2012;64(2):238-58. 4. Smith MA, Makino S, Kvetnansky R, Post MN. Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J Neurosci 1995;15(3):1768-77. 5. McEven BS. Glucocorticoids, depression, and mood disorders: structural remodeling in the brain. Metabolism 2005; 54(5 Suppl 1):20-3. 6. Bremner JD, Narayan M, Anderson ER, Staib LH, Miller HL, Charney DS. Hippocampal volume reduction in major depression. Am J Psychiatry 2000;157(1):115-8. 7. Duman RS. Depression: a case of neuronal life and death. Biol Psychiatry 2004;56 (3):140-5. 8. Chen B, Dowlatshahi D, MacQueen GM, Wong J-F, Young LT. Increased hippocampal BDNF immunoreactivity in subject treated with antidepressant medication. Biol Psychiatry 2001;50(4):260-5. 9. Lee BH, Park YM, Um TH, Kim S. Lower serum brain-derived neurotrophic factor levels are associated with failure to achieve remission in patients with major depression after escitalopram, treatment. Neuropsychiatr Dis Treat 2014;10(1):1393-98. 10. Siuciak JA, Lewis DR, Wiegand SJ, Lindsay RM. Antidepressant-like effect of brain–derived neurotrophic factor (BDNF). Pharmacol Biochem Behav 1997;56(1):131-7.

17

11. Shirayama Y, Chen AC, Nakagawa S, Russell DS, Duman RS. Brainderived neurotrophic factor produced antidepressant effects in behavioural models of depression. J Neurosci 2002; 22(8):3251-61. 12. Monteggia LM, Barrot M, Powell CM, Berton O, Galanis V, Gemelli T et al. Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc Natl Acad Sci USA 2004; 101(29):10827-32. 13. Nibuya M, Morinobu S, Duman RS. Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J Neurosci 1995; 15(11):7539-47. 14. Jacobsen JPR, Mørk A. The effect of escitalopram, desipramine, electroconvulsive seizures and lithium on brain-derived neurotrophic factor mRNA and protein expression in the rat brain and the correlation to 5-HT and 5HIAA levels. Brain Res 2004; 1024(1-2):183-92. 15. O’Connor M, Silver H. Additing risperidone to selective serotonin reuptake inhibitor improves chronic depression. J Clin Psychopharmacol, 1998;18(1):89-91. 16. Ostroff RB, Nelson J. Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J Clin Psychiatry, 1999;60(4):256-9. 17. Hirose S, Ashby CR. An open pilot study combining risperidone and selective serotonin reuptake inhibitor as initial antidepressant therapy. J Clin Psychiatry, 2002;63(8):733-6. 18. Rapaport MH, Gharabawi GM, Canuso CM, Mahmoud RA, Keller MB, Bossie CA et al. Effects of risperidone augmentation in patients with treatment-resistant depression: results of open-label treatment followed by double-blind continuation. Neuropsychopharmacology, 2006;31(11):2505-13. 19. Keitner GI, Garlow SJ, Ryan CE, Ninan PT, Solomon DA, Nemeroff CB et al. A randomized, placebo-controlled trial of risperidone augmentation for patients with difficult-to-treat unipolar, non-psychotic major depression. J Psychiatry Res, 2009:43(3):205-14. 20. Kumar R, Palit G, Dhawan BN. Comparative behavioral effects of typical and atypical antipsychotic drugs in rhesus monkey. Eur J Pharmacol 2003;462:133-8. 18

21. Fernández J, Alonso JM, Andrés JI, Cid JM, Diaz A, Iturino L et al. Discovery of new tetracycline tetrahydrofuran derivatives as potential broadspectrum psychotropic agents. J Med Chem 2005;48(6):1709-12. 22. Rogóż Z, Kabziński M, Sadaj W, Rachwalska P, Gądek-Michalska A. Effect of co-treatment with fluoxetine or mirtazapine and risperidone on the active behaviors and plasma corticosterone concentration in rats subjected to the forced swim test. Pharmacol Rep, 2012;64(6):1391-9. 23. Kamińska K, Rogóż Z. The antidepressant- and anxiolytic-like effects following co-treatment with escitalopram and risperidone in rats. J Physiol Pharmacol 2016;67(3):471-80. 24. Croom KF, Perry CM, Plosker GL. Mirtazapine: a review of its use in major depression and other psychiatric disorders. CNS Drugs, 2009;23(5):427-52. 25. Pochwat B, Sowa-Kucma M, Kotarska K, Misztak P, Nowak G, Szewczyk B. Antidepressant-like activity of magnesium in the olfactory bulbectomy model is associated with the AMPA/BDNF pathway. Psychopharmacology (Berl) 2015; 232(2):355-67. 26. Rogóż Z, Skuza G, Legutko B. Repeated co-treatment with fluoxetine and amantadine induces brain-derived neurotrophic factor gene expression in rats. Pharmacol Rep 2008;60(6):817-26. 27. Rogóż Z, Skuza G, Legutko B. Repeated treatment with mirtazapine induces brain-derived neurotrophic factor gene expression in rats. J Physiol Pharmacol 2005;56(4):661-71. 28. Altar CA, Whitehead RE, Chen R, Wortwein G, Madsen TM. Effect of electroconvulsive seizures and antidepressant drugs on the brain-derived neurotroppic factor protein in rat brain. Biol Psychiatry 2003;54(7):703-9. 29. Conti AC, Cryan JF, Dalvi A, Lucki I, Blendy JA. cAMP response elementbinding protein is essentials for the up-regulation of brain-derived neurotrophic factor transcription, but not the behavioral or endocrine responses to antidepressant drugs. J Neurosci 2002;22(8):3262-8. 30. Thome J, Sakai N, Shin K, Steffen C, Zhong YJ, Impey S et al. cAMP response element-mediated gene transcriptionis upregulated by chronic antidepressant treatment. J Neurosci 2000;20(11):4030-6. 19

31. Tiraboschi E, Tardito D, Kasahara J, Moraschi J, Pruneri P, Gennarelli M et al. Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascade. Neuropharmacology 2004;29(10):1831-40. 32. Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME. Ca2+ influx regulates BDNF transcription by a CREB family transcription factordependentmechanism. Neuron 1998;20(4):709-26. 33. Alboni S, Benatti C, Capone G, Corsini D, Caggia F, Tascedda F et al. Timedependent effects of escitalopram on brain derived neurotrophic factor (BDNF) and neuroplasticity related targets in the central nervous system of rats. Eur J Pharmacol 2010;643(2-3):180-7. 34. Merlio JP, Ernfors P, Jaber M, Persson H. Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system. Neuroscience 1992;51(3):513-32.

List of Figures Fig. 1. The effect of repeated treatment with escitalopram (ESC; 2.5, 5 or 10 mg/kg) (A), fluoxetine (FLU; 5 mg/kg) (B), mirtazapine (MIR; 5 mg/kg) (C) and risperidone (RIS; 0.05 or 0.1 mg/kg) administered separately or in combination, on the BDNF mRNA levels in the hippocampus and frontal cortex. The results are presented as the mean ± SEM of 7-8 animals/group. Statistical significance was evaluated using a two-way ANOVA followed by the Newman Keuls test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CON-treated group; ^p < 0.05, ^^^p < 0.001 vs. RIS 0.1; ap < 0.05 vs. ESC 5; cp < 0.05, ccp < 0.01 vs. MIR 5 20

Fig. 2. The effect of repeated treatment with escitalopram (ESC; 2.5, 5 or 10 mg/kg) (A), fluoxetine (FLU; 5 mg/kg) (B), mirtazapine (MIR; 5 mg/kg) (C) and risperidone (RIS; 0.05 or 0.1 mg/kg) administered separately or in combination, on the BDNF protein levels in the hippocampus and frontal cortex. The results are presented as the mean ± SEM of 7-8 animals/group. Statistical significance was evaluated using a two-way ANOVA followed by the Newman Keuls test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CON-treated group; ##p < 0.01 vs. RIS 0.05; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 vs. RIS 0.1; bbp < 0.01, bbb p < 0.001 vs. FLU 5; c p < 0.05 vs. MIR 5 Fig. 3. The effect of repeated treatment with escitalopram (ESC; 2.5, 5 or 10 mg/kg) (A), fluoxetine (FLU; 5 mg/kg) (B), mirtazapine (MIR; 5 mg/kg) (C) and risperidone (RIS; 0.05 or 0.1 mg/kg) administered separately or in combination, on the p-CREB protein levels in the hippocampus and frontal cortex. The results are presented as the mean ± SEM of 7-8 animals/group. Statistical significance was evaluated using a two-way ANOVA followed by the Newman Keuls test. *p <0.05, **p < 0.01, ***p < 0.001 vs. CON-treated group; #

p < 0.05, ###p < 0.01 vs. RIS 0.05; ^p < 0.05, ^^p < 0.01 vs. RIS 0.1; b p < 0.05

vs. FLU 5, cc p < 0.01 vs. MIR 5 Fig. 4. Representative immunoblots of BDNF, p-CREB and β-actin in the hippocampus and frontal cortex of rats treated with saline (CON), risperidone (RIS, 0.05 or 0.1 mg/kg) and/or fluoxetine (FLU, 5 mg/kg). A total of 50 µg of protein was loaded to each well

21

5

M IR

5

5

IS

5

1

0.

0. 0

+

750

0. 1

05

0. 1

0.

IS

R

IS

R

5

N

0. 05

IS

R

IS

FL U

O

C

0. 1

05

0.

IS

R

IS

R

+

+

5

5

FL U

FL U

5

R

0. 1

C O N

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 IS 2 ES .5 + 0. 1 C R 2. IS 5 0 ES .0 + R 5 C IS 5 ES + R 0.1 IS C 5 + 0.0 R 5 IS 0. 1

ES C

200

IS

R

R

+

+

1

05

5

0.

0.

IS

R

IS

IR

M

N

250

O

FL U

+

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 2 IS ES .5 + 0. 1 C R 2. IS 5 0 ES . + R 05 C IS 5 + 0 ES .1 R C IS 5 0 . + R 05 IS 0. 1

O N

300

C

1

5

C

0.

0. 0

5

IS

0. 05

B

IS

R

IS

FL U

R

IS

C ES C

***

R

+

R

1

0.

05

5

N

***

IR

5

+

IS

0.

R

FL U

O

C

BDNF mRNA level (% of control)

A

M

M IR

IR

M

R

IS

5

N

BDNF mRNA level (% of control) 400

R

IR

M

O

C

BDNF mRNA level (% of control)

Hippocampus Frontal Cortex

ESCITALOPRAM

500

*** ^ a

** * ** ^ a

100

0

FLUOXETINE

250

*

200

150

100

50

0

MIRTAZAPINE

*** ^^^

c

500

*^

cc

0

IR

M

5

5

5

0. 1

5

0. 0

IS

R

IS

R

+

+

IR

M

0. 1

0. 05

IS

R

IS

O N

200

C

0. 1

C

IS

R

5

0. 0

5

0. 1

05

0. 1

0.

IS

R

IS

R

+

+

5

0. 05

IS

R

IS

R

FL U

N

0. 1

05

O

C

IS

R

0. 1

0.

IS

IS

R

+

+

5

5

FL U

FL U

FL U

5

R

*** ##

R

+

IS

R

5

FL U

0. 05

5

N

B

IR

5

+

IR

M

IS

R

FL U

O

C

BDNF/b -actin (% of control) C N

O

N

O

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 IS 2. 5 ES + 0. 1 C R 2. IS ES 5 + 0.0 R 5 C IS 5 ES + R 0.1 IS C 5 + 0.0 R 5 IS 0. 1

ES C

C

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 IS 2 ES .5 + 0. 1 C R 2. IS 5 0 ES . + R 05 C IS 5 ES + R 0.1 IS C 5 + 0.0 R 5 IS 0. 1

ES C

BDNF/b -actin (% of control)

A

M

5

IR

M

IR

M

0. 1

0. 05

IS

R

IS

R

O N

C

BDNF/b -actin (% of control)

Hippocampus Frontal Cortex

200

ESCITALOPRAM

*

150

100

50

0

FLUOXETINE

300

*** ^^^

bb bbb

200

** ^

100

0

MIRTAZAPINE

250

*^c

** ^^

150

100

50

0

0.

1

05

5

0. 1

0. 05

IS

R

IS

R

+

+

5

5

IR

M

IR

M

0.

IS

R

IS

M IR

N

#

0.

1

0. 1

5

0. 0

IS

R

IS

R

+

+

5

5

U

FL

FL U

5

5

0. 0

IS

R

IS

R

U

FL

N

b

O

###

O

0. 1

5

1

***

C

IS

R

0. 0

0.

B

C

0. 1

C

IS

IS

R

+

+

5

5

U

FL

FL U

5

5

0. 0

IS

R

IS

R

U

N

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 IS 2 ES .5 + 0. 1 C R 2. IS 5 0 ES . + R 05 C IS 5 + 0. ES 1 R IS C 5 + 0.0 R 5 IS 0. 1

N

O

C C

ES

2. ES 5 C ES 5 C 10 R IS ES 0. C R 05 IS 2. 5 ES 0. + 1 C R 2. IS 5 ES + 0.0 R 5 C IS 5 ES + R 0.1 IS C 5 + 0.0 R 5 IS 0. 1

N

O

C C

ES

p-CREB/b -actin (% of control)

A

R

1

0. 05

0.

05

5

FL

O

C

p-CREB/b -actin (% of control) 200

R

IS

R

+

+

5

5

IR

M

IR

M

0.

IS

R

IS

R

M IR

N

O

C

p-CREB/b -actin (% of control)

Hippocampus Frontal Cortex

ESCITALOPRAM

200

*^

150

100

50

0

FLUOXETINE

250

*** ^^ b *

150

100

50

0

200

MIRTAZAPINE

*^

cc

** ^^

150

cc

100

50

0

Hippocampus

CON

FLU5

RIS0.05 RIS0.1

FLU5 + RIS0.05

Frontal Cortex FLU5+ RIS0.01

CON

FLU5

RIS0.05 RIS0.1

FLU5 + RIS0.05

FLU5+ RIS0.01 BDNF (14 kDa)

p-CREB (43 kDa) β-actin (42 kDa)