MK-801, a NMDA receptor antagonist, increases phosphorylation of histone H3 in the rat medial prefrontal cortex

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MK-801, a NMDA receptor antagonist, increases phosphorylation of histone H3 in the rat medial prefrontal cortex

Marzena Maækowiak, Rafa³ Guzik, Dorota Dudys, Ewelina Bator, Krzysztof Wêdzony

Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Correspondence: Krzysztof Wêdzony, e-mail: nfwedzon@cyf-kr.edu.pl

Abstract:

Background: The present study investigated whether MK-801, when given in doses that cause psychomimetic effects in rats, could alter the phosphorylation of histone 3 (H3) at serine 10 (H3S10p) and the acetylation of H3 at lysine 14 (H3K14ac) in the medial prefrontal cortex (mPFC). These posttranslational modifications of H3 promote chromatin relaxation and increase the probability of gene expression.

Methods: Stereological counting, immunoblot analysis and confocal laser scanning microscopy.

Results: Treatment with MK-801 (0.4 mg/kg) evoked a time-dependent increase in the number of H3S10p positive nuclei in both the II/III and V/VI layers of the mPFC, reaching the peak of activation 30 min after injection. MK-801 treatment (0.4 mg/kg) failed to alter H3K14ac. These effects were confirmed by immunoblot analysis on tissue samples from the mPFC. Analysis of cortical cells expressing H3S10p positive nuclei revealed that constitutive and MK-801-induced expression of H3S10p was observed only in neurons and not in glia cells (H3S10p colocalized with NeuN but not with S-100b). Moreover, it has been found that H3S10p is ex- clusively present in pyramidal (glutamate-positive) but not in cortical GABA-ergic interneurons (GABA-positive). The effects of MK-801 can be attenuated or blocked by the neuroleptic drug risperidone. In the cortical layer II/III, risperidone was effective at doses of 0.2 and 1 mg/kg, while it was only active at a dose of 1 mg/kg in the V/VI layer. Again, these stereological data were confirmed by immunoblot analysis.

Conclusions: Our results indicate that MK-801 may increase the transcriptional activity of mPFC via the activation of the epigenetic program associated with H3S10p phosphorylation during the course of experimental psychosis.

Key words:

epigenetics, histone 3, medial prefrontal cortex, MK-801, psychosis, phosphorylation, pyramidal neurons, schizophrenia

Introduction

In recent years, there has been a growing amount of evidence linking schizophrenia with copy number variations, microdeletions, and polymorphisms of candidate genes; however, the straightforward genetic causes of the illness are still unclear [22]. Non-Men-

delian inheritance patterns and concordance rates less than 70% among monozygotic twins have inclined re- searchers to look for alternative causes of schizophrenia [20]. These alternative mechanisms may be based on epigenetic factors, which are defined as heritable changes in gene expression and function without any alteration of the DNA sequence [30, 35].

Pharmacological Reports 2013, 65, 11121123 ISSN 1734-1140

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Two major mechanisms are engaged in the epigenetic control of gene expression: the methylation of DNA and the posttranslational modification of histone (H) proteins [30, 35].

Clinical data indicate that schizophrenic patients treated with L-methionine, which is converted to SAM – to attenuate dopaminergic neurotransmission – expe- rience a worsening of symptoms [10]. This effect has been linked with an increase in DNA methylation and the suppression of the expression of certain candidate genes, such as reelin and 67 KD glutamic acid decar- boxylase (GAD67) [15]. Deficits in GAD67 mRNA are considered to be among the most frequently repli- cated findings in schizophrenia postmortem brain [24].

Also mice treated with L-methionine may exhibit behavioral deficits that resemble schizophrenic endophe- notypes [46]. Data collected postmortem provide further evidence that the reelin [1, 11], sex-determining region Y-box containing gene 10 (SOX10) [17] promoters are hypermethylated while membrane-bound catechol-O-methyltransferase (MB-COMT) is hypo- methylated [38] during the course of schizophrenia.

Enhanced DNA methylation has also been correlated with the enhanced expression of DNA-methyltransferase 1 (DNMT1) in GABA-ergic interneurons in the mPFC of psychiatric subjects [42, 47].

Histone modifications have also been implicated in schizophrenia. The histone deacetylase complex (HDAC1) [30], which catalyzes the removal of acetyl groups and leads to the condensation of the chromatin and the repression of gene expression [30], has been shown, postmortem, to be elevated in the brains of schizophrenia patients [23, 30, 45]. Other changes have also been observed, such as the decreased methylation of histone 3 (H3) at lysine 4 (H3K4) at the GAD67 promoter (specifically a shift from the H3K4me3 to the H3K27me3 mark in chromatin sur- rounding the transcription start site of GAD1 gene, encoding GAD67) [16, 30]. Also high levels of H3- (methyl) arginine 17 are associated with down- regulated metabolic gene expression in the prefrontal cortex of a subset of subjects with schizophrenia [5, 30]. For example, high levels of methylated H3 at arginine 17 (H3R17), phosphorylation of H3 at serine 10, and acetylation of H3 at lysine 14 have been observed in postmortem schizophrenic brains relative to non-psychiatric control brain samples [5, 30].

So far it is not known whether above modifications of H3 are linked with NMDA receptor hypofunction in the mPFC and subsequent cognitive symptoms as-

sociated with schizophrenia. Above hypofunction has been modeled both in humans and experimental animals by administering NMDA receptor antagonists, such as phencyclidine and ketamine in humans [18, 34] and MK-801 in rodents [34, 52]. In humans, phencyclidine and ketamine produce transient schizophrenia-like symptoms in healthy individuals and re- instate the pre-existing symptoms of stabilized schizophrenia patients [18]. In rodents, NMDA receptor antagonist treatment results in mPFC-dependent deficits in working memory and set-shifting tasks [2, 37, 41, 52]. Thus high degree of translation of clinical data on experimental animals inclined us to investigate whether MK-801 treatment can result in altered phosphorylation and acetylation of H3 in the medial prefrontal cortex (mPFC) of rat brain. We used here MK-801 in a dose of 0.4 mg/kg, a dose which in the strain of Wistar rats induced strong cognitive deficits observed as deficits of sensorimotor gating and decreased efficacy of working memory [52, 53]. To an- swer the given experimental question, we measured the expression of H3S10p and H3K14ac in the mPFC after MK-801 treatment using immunohistochemistry, stereology, and immunoblot analysis. In consecutive sets of experiments, we investigate the phenotypes of cortical cells constitutively expressing H3S10p and H3K14ac before and after the administration of MK- 801. In the final sets of experiments, we investigate whether the effects of MK-801 on the expression of H3S10p can be rescued by treatment with risperidone, a clinically effective neuroleptic drug [33]. The above strategy is based on the assumption that the phosphorylation of the N-terminal tail of H3 at serine 10 (H3S10p) has been shown to be involved in transcriptional activation in post-mitotic cells, such as neurons [30].

Materials and Methods

Animals

All experiments were performed on male Wistar rats (200–300 g), obtained from Charles River Laborato- ries, Inc. (Germany). The rats were housed under controlled conditions with a 12 h light/dark cycle (lights on at 7:00 h), a set temperature of 22 ± 2°C, and free access to standard laboratory food and water. Experi- mental procedures were approved by the Animal Care and Use Committee at the Institute of Pharmacology,

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Polish Academy of Sciences, and were carried out in accordance with the European Council Guide for the Care and Use of Laboratory Animals (86/609/EEC) and with national law.

Drugs and treatment

Rats were injected intraperitoneally (ip) with MK-801 ((5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]

cyclohepten-5,10-imine maleate, Tocris) at a dose of 0.4 mg/kg or with the vehicle (0.9% NaCl). The rats were treated with the drug followed 0.5, 2, or 4 h by perfusion or 0.5 h before decapitation. Risperi- done (3-{2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-pipe- ridinyl]ethyl}-6,7,8,9-tetrahydro-2-methyl-4H-pyrido- [1,2- a]pyrimidin-4-one, RIS, Janssen, Beerse, Belgium) was suspended in 1% Tween 80; the vehicle control or the drug was injected ip at doses of 0.2 or 1.0 mg/kg 1 h before MK-801 administration. All drugs were administered in a volume of 2 ml/kg. Each animal was habitu- ated to the experimental conditions by handling, once a day, for three days prior to the experiment. All experiments were performed between 9:00 and 14:00 h.

Immunoperoxidase staining Tissue preparation

The rats were deeply anesthetized using sodium pento- barbital (100 mg/kg, ip) and transcardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). After a 24 h post-fixation period, 50 µm thick sections were cut through the entire mPFC using a VT-1000S vibra- tome (Leica Microsystems, Heidelberg, Germany).

Staining

Free floating sections were processed following the procedure described by Mackowiak et al. [28, 29]. All steps were carried out at room temperature (RT) unless otherwise indicated. The sections were rinsed in 0.01 M PBS and incubated for 1 h in a blocking buffer containing 5% normal goat serum (Vector Laborato- ries) and 0.3% Triton X-100 diluted in 0.01 M PBS.

Next, the sections were incubated for 48 h at 4°C with the primary rabbit polyclonal anti-phospho (Ser-10) or the anti-acetyl (Lys-14) histone H3 antibodies (anti-H3S10p, anti-H3K14ac, 1 : 1000, Millipore). Sub- sequently, the brain sections were rinsed in 0.01 M PBS and incubated for 1 h at RT with a biotinylated

secondary antibody (1 : 200, Vector Laboratories).

Both the primary and secondary antibodies were diluted in 0.01 M PBS containing 0.3% Triton X-100 and 3% goat serum. To exclude endogenous peroxidase activity, the sections were incubated for 20 min in 0.3% H₂O₂diluted in 0.01 M PBS containing 0.3%

Triton X-100. Then, the sections were rinsed in 0.01 M PBS and incubated for 1 h at RT with avidin–biotin horseradish peroxidase complex (1 : 170, Vectastain Elite ABC Kit, Vector Laboratories) diluted in 0.01 M PBS containing 2% NaCl. Finally, to trigger a color reaction, the sections were dipped in a 0.02% solution of 3,3’-diaminobenzidine tetrahydrochloride (DAB) with 0.03% NiCl₂diluted in 0.01 M PBS. After incu- bating for 5 min, H₂O₂was added to a final concentration of 0.07%, and the sections were incubated for another 3 min in the resultant DAB-Nickel-H₂O₂ solution. This resulted in a black color in the nuclei that were positive for H3S10p or H3K14ac. At the end of the protocol, the sections were mounted, air-dried, de- hydrated with ascending alcohols, cleared in xylene, and mounted using Permount Mounting Medium (EMS, Hatfield, PA, USA).

Quantitative evaluation of staining

The number of immunopositive cells in the mPFC was estimated using unbiased stereological methods [28, 29]. Every fifth section from systematic random sampling along the anteroposterior axis of the mPFC (beginning at 2.80 and finishing at 1.20 ± 0.10 mm from the bregma, according to the atlas of Paxinos and Watson [39]), was analyzed with a 63×/1.4–0.7 lens attached to a Leica DM6000 B microscope using the Stereo Investigator software (MBF Bioscience, Magdeburg, Germany); a total of 6 sections were ex- amined per animal. The optical dissector dimensions were 40 × 40 × 15 µm (X, Y, Z, respectively) [28, 29].

Immunofluorescence staining

Tissue preparation

Rats were deeply anesthetized with 100 mg/kg sodium pentobarbital and transcardially perfused with 0.9% NaCl, followed by perfusion with 0.2% glu- taraldehyde in a solution of 4% PFA in 0.1 M PBS.

After 3–4 h of post-fixation, the brains were sectioned as described above.

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Staining

The sections were rinsed in 0.01 M PBS and incubated for 1 h at RT in a blocking buffer containing 5%

normal donkey serum and 0.3% Triton X-100 in 0.01 M PBS. Next, the sections were incubated for 48 h at 4°C with the primary rabbit polyclonal anti- H3S10p antibody (1 : 500, Millipore) and with the one of the following primary antibodies: monoclonal mouse anti-S-100b (1 : 1000, Sigma), monoclonal mouse anti-neuronal nuclei (NeuN, 1 : 1000, Millipore), or monoclonal mouse anti-g-aminobutyric acid (GABA, 1 : 500, Sigma) diluted in the blocking buffer. In the case of the glutamate staining protocol, primary antibodies were applied sequentially: first, the sections were incubated in the blocking buffer without Triton X-100 for 1 h at RT and incubated for 48 h at 4°C with the primary monoclonal mouse anti-glutamate antibody (Glu, 1 : 5000, Swant, Bellinzona, Switzer- land) diluted in the blocking buffer without Triton X-100. The sections were then washed in 0.01 M PBS (3 × 10 min at RT) and incubated for 48 h at 4°C with the anti-H3S10p antibody (1 : 500) diluted in the blocking buffer. Finally, the sections from all of the immunostaining techniques were washed in 0.01 M PBS (3 × 10 min at RT) and incubated for 24 h at 4°C in a mixture of secondary antibodies (Cy3-conjugated anti-rabbit IgG, 1 : 300 and Alexa488-conjugated anti-mouse IgG, 1 : 200, Jackson ImmunoResearch, West Grove, PA, USA) diluted in the blocking buffer.

The sections were then washed in 0.01 M PBS, mounted, and coverslipped.

Quantitative evaluation of staining

To examine the phenotype of the cells expressing the active form of histone H3, double-labeled slices were analyzed using the DMRXA2 TCS SP2 confocal laser scanning microscope driven by the confocal software (Leica Microsystems, Wetzlar, Germany) using a 63×/

1.4–0.7 oil objective lens. Applying either argon or GreNe lasers, two laser lines emitting at 488 or 543 nm were used to excite the Alexa488- or Cy3- conjugated antibodies, respectively. The background noise for each confocal image was reduced by averaging 4 scans per line and 4 frames per image. The speed of scanning was 800 lines per second.

Two sections, with 150 µm distance between one another, were chosen from the mPFC of each animal (AP co-ordinates: between 2.80- and 2.20 mm from

the bregma according to the Paxinos and Watson sterotaxic atlas) [39]. In every section, 12 regions were selected and submitted for analysis; these sections included 3 regions from layers II/III and 3 regions from layers V/VI in each hemisphere. Each region was 140.03 µm long and 140.03 µm wide. The colocalization of H3S10p with different cell markers was confirmed or ruled out using 2D stacks con- structed with 10 to 15 images taken 1 µm apart. The number of H3S10p-positive nuclei and cells expressing particular phenotypic markers, as well as the number of colocalizations that occurred between them, were counted using the Leica Microsystems software.

Immunoblotting

Tissue preparation

The brains were removed after decapitation and cooled on ice, followed by mPFC dissection. The mPFCs from 3 animals for each experimental group were pooled to- gether. The isolated tissue was homogenized for 5 min in ice-cold buffer (100 µl buffer per 15 mg tissue) containing 250 mM sucrose, 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 0.9 mM sodium butyrate, 1.0 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 1% protease inhibitor mixture (Sigma) using a Tissuelyser kit (Qiagen).

Histone extraction

All steps were carried out on ice unless otherwise indicated. Tissue homogenates were centrifuged at 7,700 × g for 2 min. The supernatant (cytoplasmic fraction) was aspirated and stored at –30°C for other investigations. To perform the acid extraction of the histones, the remaining pellet (nuclear fraction) was resuspended and incubated for 1 h in 1 ml of 0.2 M H₂SO₄. Then, the samples were centrifuged at 16,000

× g for 10 min, and the supernatant was transferred to a fresh tube where the proteins were precipitated by the addition of 350 µl of 100% trichloroacetic acid with 4 mg/ml deoxycholic acid (Na⁺salt, Sigma). The precipitation lasted 45 min. Precipitated proteins were collected by centrifugation at 16,000 × g for 30 min.

The supernatant was discarded, and the pellet containing the histones was washed for 5 min with 1 ml of acidified acetone (0.1% HCl), then centrifuged at 16,000 × g for 5 min; the pellet was then washed with 1 ml of pure acetone for 5 min and centrifuged again

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at 16,000 × g for 5 min. Finally, the samples were left at RT for 20 min to evaporate the acetone. The resulting purified proteins were resuspended in 30 µl of 10 mM Tris (pH 8) and stored at –30°C.

Immunoblotting

Protein concentrations were determined using the BCA Protein Assay Kit (a modification of the Lowry assay, Sigma, St. Louis, MO, USA). Samples with equal protein content were adjusted to the final concentration with homogenization buffer containing 2%

SDS, 8% glycerol and 2% 2-mercaptoethanol with bromophenol blue as a pH marker and were then boiled for 8 min. Protein extracts (20 µg) were separated on 15% SDS-PAGE gels and transferred onto ni- trocellulose membranes using an electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were stained with Ponceau S to confirm equal loading and efficient transfer to the membranes. The blots were then incubated over- night at 4°C with the primary anti-H3S10p antibody (1 : 500). Immune complexes were detected using a peroxidase-conjugated secondary antibody (1 : 1000, anti-rabbit IgG, Roche Diagnostics, GmbH, Mann- heim, Germany). The reaction was visualized using ECL (Lumi-LightPlus Western Blotting Kit, Bio-Rad Laboratories). Chemiluminescence was recorded and evaluated with a luminescent image analyzer (Fuji- film LAS-4000 mini, Fujifilm Corporation, Tokyo, Japan). The relative levels of immunoreactivity were quantified using the Image-Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA). Using the

same software, the molecular weights of the immuno- reactive bands were calculated on the basis of the mi- gration of the molecular weight markers (Roche). All values are expressed as the percentage of the vehicle- treated control. To normalize for the small variations in loading and transfer, the ratio of the specific protein level to the total histone H3 (anti-H3 antibody, 1 : 10,000, Millipore) level was calculated for each sam- ple. Representative pictures illustrating separation of H3S10p or H3K14ac and H3 were prepeared in Adobe Photoshop program.

Statistics

The results are presented as the mean ± standard error of the mean (SEM). Statistical evaluation was performed using a two- or one-way analysis of variance (ANOVA) followed by the Duncan or Tukey post-hoc test, respectively, using the Statistica software pack- age (StatSoft, Inc., Tulsa, OK, USA). Time and treatment (MK-801) or pretreatment (RIS) and treatment (MK-801) were regarded as independent variables.

Results

The effect of MK-801 on the number of H3S10p positive nuclei in the mPFC

Using an antibody that specifically recognizes the N-terminal tail of H3 phosphorylated at Ser 10 (H3S10p), we noticed the constitutive presence of

Fig. 1. The effect of MK-801 on the number of nuclei positive for the presence of phosphorylated H3 (H3S10p) in the medial prefrontal cor- tex (mPFC) of rats. A and B: photomicrographs showing the expression of H3S10p in the mPFC analyzed 0.5 h after administration of the vehi- cle (VEH) or MK-801 (0.4 mg/kg ip), respec- tively. Insets in A and B are a high power visu- alization of the H3S10p positive nuclei in the VEH and MK-801 treated animals. Abbrevia- tions for A and B: CC – corpus callosum; II/III V/VI – layers of the mPFC. Scale bar = 50 µm. C and D: the number of H3S10p positive nuclei in cortical layers II/III and V/VI measured stereologically at different time points after admini- stration of MK-801 (0.4 mg/kg ip). Data are given as the mean ± SEM; n = 6 rats per treatment and time point. Asterisks indicate statistical significance (p < 0.05) compared with the appropriate control group and time point, as determined by a two-way ANOVA followed by the Duncan test

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H3S10p immunostaining in the nuclei of some cells in the whole mPFC. A substantial H3S10p immunoreactivity was observed in layers II/III, while layers V/VI had only moderate H3S10p immunoreactivity. Almost no staining appeared in layer I (Fig. 1).

MK-801 treatment (0.4 mg/kg) evoked a time- dependent increase in the number of H3S10p positive nuclei in both the II/III and V/VI layers of the mPFC [treatment × time = F (2, 30) = 3.833, p < 0.03; and F (2, 30) = 5.131, p < 0.01, respectively]. Post-hoc analysis revealed a significant increase in the number of H3S10p positive nuclei 0.5 h after MK-801 ad-

ministration (p < 0.0008 for layers II/III and p <

0.0002 for layers V/VI) (Fig. 1). The increased level of H3S10p immunostaining was also observed 2 h after MK-801 administration (Fig. 1) but was not statistically significant (p = 0.17 for layers II/III and p = 0.09 for layers V/VI).

The effect of MK-801 on the number of nuclei and expression of H3K14ac protein in the mPFC

Using an antibody that specifically recognizes the N- terminal tail of H3 acetylated on Lys 14 (H3K14ac), we noticed the constitutive presence of H3K14ac im-

Fig. 2. The effect of MK-801 on the number of nuclei positive for the presence of acetylated H3 (H3K14ac) in the medial prefrontal cortex (mPFC) of rats. A and B: photomicrographs showing the expression of H3K14ac in the mPFC analyzed 0.5 h after administration of the vehicle (VEH) or MK-801 (0.4 mg/kg ip), re- spectively. Insets in A and B are a high power visualization of the H3K14ac positive nuclei in the VEH and MK-801 treated animals. Abbre- viations for A and B: CC – corpus callosum; II/III and V/VI – layers of the mPFC. Scale bars = 50 µm. C: the number of H3K14ac positive nuclei in cortical layers II/III and V/VI measured stereologically. D: expression of H3K14ac in the mPFC as measured by immunoblot. All analyses occurred 0.5 h after administration of MK-801 (0.4 mg/kg ip). Data are given as the mean ± SEM; n = 6 rats per group. Statistical significance was determined using a two-way ANOVA

Fig. 3. Confocal microscope and two-di- mensional (2D) analysis of cells in the medial prefrontal cortex (mPFC) of rats (control, vehi- cle treated) expressing H3S10p. A: NeuN (blue) and H3S10p (red) staining. B: S-100b (blue) and H3S10p (red) staining. C: glutamate (green) and H3S10p (red) staining. D: GABA (green) and H3S10p (red) staining. A, B, C and D are single high-resolution scans (x, y axis), while the orthogonal images (A1, A2; B1, B2;

C1, C2 and D1, D2, respectively) were con- structed by averaging 10 to 20 optical sections taken 0.5 µm apart (y, z and x, z axis for A1, B1, C1, D1 and A2, B2, C2, D2, respectively).

Scale bars = 50 µm, 63´ objective

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munostaining in the nuclei of some but, presumably, not all cells in the whole mPFC. Substantial H3K14ac immunoreactivity was observed in all layers of the mPFC, including layer I (Fig. 2), as well as in the ad- jacent corpus callosum. Stereological analysis revealed that 0.4 mg/kg MK-801 given 0.5 h before perfusion did not significantly alter the number of H3K14ac positive nuclei in layers II/III and V/VI of the mPFC [F (1, 10) = 3.85, p = 0.078 and F (1, 10) = 3.8, p < 0.08, respectively]. Immunoblot analysis demonstrated that treatment with 0.4 mg/kg MK-801 for 0.5 h before decapitation failed to alter the expression of H3K14ac protein in the whole mPFC [F (1, 6)

= 0.25, p = 0.63); Fig. 2].

Phenotype of the H3S10p positive cells in the mPFC

The phenotype of cells demonstrating constitutive (vehicle treated) or MK-801 induced H3S10p immunopositive material was studied 0.5 h after injection. Double immunofluorescence labelling for H3S10p and NeuN, a protein expressed in mature neurons [36], showed that all of the observed H3S10p positive nuclei were present in NeuN positive cells (Fig. 3). This pattern of immunostaining was found in both the control group (vehicle-treated) and after MK-801 treatment. These results were further confirmed by double immunofluorescence labelling of

Tab. 1. Colocalization of phosphorylated histone 3 at serine 10 (H3S10p) with neuronal marker (NeuN), glutamatergic and GABA-ergic neu- ronal markers (glutamate and GABA, respectively), and glia cell marker (S-100b) in the medial prefrontal cortex of control (VEH) and MK-801 treated rats

NeuN + H3S10p

Treatment¯/marker®

Cell number % colocalization

NeuN H3S10p NeuN + H3S10p [(NeuN+ H3S10p)/

NeuN]´ 100 [(H3S10p+ NeuN)/

H3S10p]´ 100

Veh 668 ± 18 37 ± 7 37 ± 7 5.5 ± 1.1 100 ± 0

MK-801 752 ± 92 113 ± 9* 113 ± 9* 1.2 ± 1.0* 100 ± 0

S-100b + H3S10p

S-100b H3S10p S-100b + H3S10p [(S-100b + H3S10p)/

S-100b] ´ 100

[(H3S10p + S-100b)/

H3S10p]´ 100

Veh 180 ± 13 36 ± 2 0 ± 0 0 ± 0 0 ± 0

MK-801 210 ± 15 105 ± 6* 0 ± 0 0 ± 0 0 ± 0

Glu + H3S10p

GLU H3S10p GLU + H3S10p [(GLU + H3S10p)/

GLU]´ 100

[(H3S10p + GLU)/

H3S10p]´ 100

Veh 476 ± 42 43 ± 8 40 ± 7 8.4 ± 2 93.0 ± 2

MK-801 435 ± 35 113 ± 7^* 106 ± 8^* 24.4 ± 1^* 93.8 ± 2

GABA + H3S10p

GABA H3S10p GABA + H3S10p [(GABA + H3S10p)/

GABA]´ 100

[(H3S10p + GABA)/

H3S10p]´ 100

Veh 197 ± 26 29 ± 4 0 ± 0 0 ± 0 0 ± 0

MK-801 176 ± 31 101 ± 10* 0 ± 0 0 ± 0 0 ± 0

Colocalization of H3S10p with neuronal markers was analyzed 30 min after administration of the vehicle (Veh) or MK-801 (0.4 mg/kg ip). Data are given as the mean ± SEM. Each experimental group consisted of 4 rats and the resulting cell number is a sum of the cells from 2 sections of each animal. A one-way ANOVA followed by the Duncan test was used to determine statistical significance (p < 0.05). Asterisks indicate statistically significant differences compared to the control group

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H3S10p and S-100b, a marker of glial cells. We ana- lyzed 36 H3S10p positive nuclei and 180 S-100b positive cells in the vehicle-treated animals and 96 H3S10p positive nuclei and 192 S-100b positive cells in the MK-801 treated animals. Cells positive for the

presence of both H3S10p and S-100b proteins were not observed in either the vehicle-treated or in the MK-801 injected animals (Fig. 3). The above findings suggest that the expression of H3S10p protein is observed constitutively (vehicle) or after MK-801 only in the nuclei of neurons.

Subsequent immunohistochemical experiments showed that H3S10p positive nuclei are observed in glutamate-positive but not in GABA-positive cells in both the vehicle-treated and MK-801-treated animals (Tab. 1). As shown in Table 1, the percentage of glutamate cells positive for the presence of H3S10p proteins was statistically higher after MK-801 treatment com- pared to the respective controls [24% vs. 9%, respec- tively; F (1, 6) = 27.58, p < 0.002], which suggests that MK-801 increases the number of H3S10p positive nuclei only in some, but not all, glutamatergic neurons.

The impact of risperidone on MK-801 induced expression of H3S10p – immunohistochemistry and immunoblot studies

Using immunohistochemical and immunoblot analyses, the effect of pretreatment with either 0.2 mg/kg or 1.0 mg/kg risperidone (RIS) on the MK-801-induced H3S10p expression was analyzed 0.5 h after MK-801 injection; the time point of the highest increase in the

Fig. 4. The impact of risperidone (RIS) on the MK-801 induced in- crease in the number of H3S10p-positive nuclei in the medial prefron- tal cortex (mPFC) of rats. RIS was given ip at doses of 0.2 or 1.0 mg/kg 1 h before MK-801 treatment (0.4 mg/kg, ip), and the number of H3S10p-positive nuclei was analyzed stereologically 0.5 h after vehicle (VEH) or MK-801 administration. A: layers II/III of the mPFC. B: layers V/VI of the mPFC. Data are given as the mean

± SEM; n = 6 rats per group. A two-way ANOVA followed by the Dun- can test was used for post-hoc comparison; asterisks indicate statis- tical significance (p < 0.05) compared with the control group (VEH + VEH), while hashes indicate statistical significance compared with the MK-801 group (VEH + MK-801). C: representative photomicro- graphs showing the impact of the vehicle (C₁), MK-801 (C₂), RIS (C₃), and RIS + MK-801 (C₄) on the expression of H3S10p – positive nuclei in the mPFC; for the sake of clarity, only samples treated with 1.0 mg/kg RIS are shown. Abbreviations for C₁₄: CC – corpus callosum; II/III and V/VI – layers of the mPFC. Scale bars = 50 µm

Fig. 5. The impact of risperidone (RIS) on the MK-801 induced an in- crease in the expression of H3S10p in the medial prefrontal cortex of rats. RIS was administered at a dose of 1.0 mg/kg (ip) 1 h before MK-801 treatment (0.4 mg/kg, ip), and rats were decapitated 0.5 h after MK-801 administration. Data are expressed as the mean ± SEM;

n = 4 rats per group. A two-way ANOVA followed by the Duncan test was used for post-hoc comparison; asterisks indicate statistical sig- nificance (p < 0.05) compared with the control group (VEH + VEH), while hashes indicate statistical significance compared with the MK-801 group (VEH + MK-801). Representative blots are shown

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number of H3S10p positive nuclei observed in both the II/III and V/VI layers of the mPFC (Fig. 1).

As shown in Figure 4, immunohistochemical analysis showed that RIS attenuated the MK-801- induced increase in the number of H3S10p positive nuclei in layers II/III and V/VI of the mPFC in a dose-dependent manner ([RIS vs. MK-801; F (2, 30)

= 4.27, p < 0.02 and F (2, 30) = 3.10, p < 0.05, respec- tively]. The post-hoc analysis revealed that in layers II/III, 0.2 mg/kg and 1.0 mg/kg RIS inhibited the effects of MK-801 (p < 0.001 and p < 0.00008, respectively), while in layers V/VI, only 1.0 mg/kg RIS was effective (p = 0.25 for 0.2 mg/kg and p < 0.002 for 1.0 mg/kg). RIS alone, at both doses, did not have an effect on the number of H3S10p positive cells in the mPFC (Fig. 4).

The antibody used in the present study recognized a band on the immunoblots with a molecular weight of approximately 17 kDa (Fig. 5), which corre- sponded to the calculated molecular weight of H3S10p. In the immunoblot experiments, RIS was used only at a dose of 1 mg/kg, which was the most effective dose in the immunohistochemical studies (Fig. 4). Similar to the results shown above, RIS administration blocked the effects of MK-801 injection on the expression of H3S10p protein in the mPFC [F (1, 12) = 6.16, p < 0.03; Fig. 5].

Discussion

In the present study, we demonstrated that administration of MK-801 leads to an enhancement of H3 phosphorylation in the mPFC. Such an enhancement was observed predominantly, if not exclusively, in glutamatergic pyramidal neurons but not in GABA-ergic interneurons. It has also been observed that MK-801 (in analyzed time windows) does not operate via the acetylation of H3, which is another form of posttranslational modification that is also involved in the relaxation of chromatin and the acceleration of gene expression [30]. Interestingly, the effects of MK-801 were dose dependently attenuated by RIS, an atypical neuroleptic drug effective against both positive and negative symptoms of schizophrenia [32, 33]; these symptoms are dependent on the activation of subcortical and cortical brain regions, respectively [32, 33].

Noncompetitive antagonists of NMDA receptors are used as a pharmacological model of schizophrenia

[18, 34]. Administering ketamine or phencyclidine to healthy volunteers evokes transient symptoms and can exaggerate pre-existing symptoms in schizophrenic patients [18, 34]. In experimental animal models, MK-801 treatment can evoke the disruption of sensorimotor gating, efficacy of working memory, social withdrawal, and psychomotor agitation, among other symptoms [19, 34, 52]. The aforementioned effects are antagonized by clinically effective neuroleptic drugs [32, 33]. It is conceivable that psychotomimetic effects of MK-801 may result from the activa- tion of gene expression via the phosphorylation of H3 at Ser 10 [30, 31].

So far, the mechanism activated by MK-801 that leads to the enhancement of H3S10p is not clear. The first mechanism that has to be considered is an activity dependent increase in H3 phosphorylation to com- pensate for the increased activity of the mPFC. This increased activity of the mPFC is not limited to the effects of MK-801 [14] but has also been demonstrated for phencyclidine and ketamine [2, 21, 55]. Activa- tion of the mPFC after ketamine has been observed in human subjects in neuroimaging experiments [9, 48].

More detailed studies have revealed anatomically specific activation of the pyramidal glutamatergic output neurons and the inhibition of cortical GABA-ergic interneurons [14].

For explanation of anatomical selectivity i.e., expression of H3S10p in pyramidal but not GABA-ergic cells after MK-801, a direct link between neuronal activity and expression of H3S10p is required. For example, Wittmann et al. [54] on cultured hippocampal neurons demonstrated that an increase in the neuronal activity dramatically increases the number of infolds in the nucleus, predominately spherical at rest via a process that requires the ERK-MAP kinase pathway and new protein synthesis. An increase in phosphorylation of histone H3 on serine 10 (H3S10p) was predominately visible in neurons with infolded nuclei compared with neurons with spherical nuclei. Above data suggest that expression of H3S10p is neuronal activity dependent and that there is a potential func- tional link between H3S10p and enhanced neuronal activity. As it has been mentioned above, after MK- 801 there is an increase in the activity of the principal neurons and decreased activity in inhibitory interneurons [2, 14, 21, 55].

Alternatively, MK-801 treatment may activate a specific neurotransmitter pathway, leading to an increase in H3S10p. NMDA receptor antagonists,

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among others, enhance the release of glutamate [27], dopamine [50, 51], and serotonin [27]. Dopamine is of special interest because it has been recently demonstrated in mice that the activation of dopamine D1 re- ceptors via ERK and direct or indirect activation of MSK-1 can enhance the phosphorylation, but not the acetylation, of H3 [8]. Additionally, this led to the induction of early genes, such as c-Fos and Zif 268, in mice striatum [8]. It is not yet clear whether the above mechanism may be directly translated into the mPFC.

While some data show that MK-801 may enhance the phosphorylation of ERK at certain doses and time points and brain region [3, 13], other data clearly show that an MK-801 alone is ineffective and de- creases elevation in ERK phosphorylation induced by morphine, ethanol etc. [6, 25], suggesting that a different mechanism may be at play. It is worth noting that MK-801 and other NMDA receptor antagonists are capable of inducing c-Fos expression only in the mPFC and not the striatum [21, 49, 56]. On the other hand, a dopaminergic mechanism cannot be fully ruled out because we observed that RIS blocked the MK-801-enhanced expression of H3S10p. RIS oper- ates as an antagonist of the dopaminergic D2 and serotonin 5HT2A receptors [33]. Thus, the engagement of dopamine and serotonin in the remodelling of the chromatin structure cannot be ruled out just yet [12, 30]. Here, we deliberately used RIS because it is clinically effective and attenuates both the positive and negative symptoms of schizophrenia; as such, RIS en- gages both the subcortical and cortical regions of the brain [33]. Secondly, as an atypical neuroleptic is capable to attenuate NMDA antagonists-induced change the firing neurons of the rat mPFC, the expression of c-Fos [21, 27], and, as demonstrated here, can enhance H3 phosphorylation. Opening the chromatin structure enables transcriptional factors to access the DNA, allowing the transcription of early response genes, such as c-Fos [8, 30]. c-Fos is deliberately mentioned here because NMDA antagonists evoke ro- bust expression of this gene predominantly in the mPFC, and this effect is antagonized by neuroleptic drugs. Interestingly, in the mPFC, c-Fos is induced in glutamatergic pyramidal output neurons [21, 27].

A similar pattern of induction has been observed for phosphorylated H3 (the present study). Although it is not clear whether the induction of H3 and c-Fos are cause dependent in our study, the data available in the literature support such suggestion.

Interestingly, RIS failed to alter the constitutive expression of H3S10p and dose-dependently attenuated or abolished the induction of H3S10p evoked by MK-801. This ineffectiveness of RIS is in sharp contrast with data demonstrating that haloperidol induced H3S10p in mice striatum via a dopamine D2 receptor dependent mechanism [7]. There are a few possible explanations of the above phenomenon. First, both neuroleptic drugs have different pharmacological pro- files. Haloperidol’s main mechanism of action is to block the D2 receptors [33]. RIS, in addition to blocking the D2 receptors, is also an antagonist of the serotonin 5-HT2A receptors [33, 40]. Therefore, the an- tagonism of the 5-HT2A receptors may account for the observed differences. Second, we looked at the alteration of H3 phosphorylation in the mPFC but not in the striatum. Haloperidol increases the expression of H3S10p in the striatum by engaging the dopamine D2 receptors [7]. It is important to mention that the popu- lation of cells controlled by the D2 and D1 receptors in the striatum are separated and form distinct out- puts, striopallidal and strionigral, respectively – for recent review see [26]. In contrast to the striatum, in the mPFC, the D1 and D2 receptors are not separated and situated on both principal output neurons and interneurons [43, 44]. Thus, in contrast to the striatum, the final effect in the mPFC (e.g., H3 phosphorylation) is a result of activation and deactivation.

Clinical data supporting the potential involvement of histone modifications in schizophrenia are minimal.

It has been found in a subpopulation of schizophrenic patients that H3 methylation is significantly higher than in the respective controls [5]. Enhanced methylation at H3R17 promotes an open chromatin state [4, 30]. Subsequent studies have revealed that during the course of schizophrenia, there is altered level of H3K4me3 and H3K27me3 [15, 16]. Moreover, increased HDAC1 activity has been noticed [45]. Inter- estingly, alteration of trimethylated form of H3 has been linked with GAD 67 expression, which is an en- zyme responsible for the production of GABA; GAD 67 expression has been shown to be decreased in the prefrontal cortex of patients with schizophrenia [5].

Our present results indicate that epigenetic mechanisms may be associated with experimentally induced psychosis, at least acute phase, and that above effect is sensitive to neuroleptic drugs.

Acknowledgments:

Supported by the statutory activity of IP PAS and grant NN401 066938 to MM by the MS&HE.

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Received: July 24, 2013; in the revised form: September 18, 2013;

accepted: September 20, 2013.