2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Neurochemistry International 60 (2012) 55–67

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Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus Konstantinos Sarantis a, Katerina Antoniou b, Nikolaos Matsokis c, Fevronia Angelatou a,⇑ a

Department of Physiology, Medical School, University of Patras, 26500 Patras, Greece Department of Pharmacology, Medical School, University of Ioannina, 45110 Ioannina, Greece c Laboratory of Animal and Human Physiology, Department of Biology, University of Patras, 26500 Patras, Greece b

a r t i c l e

i n f o

Article history: Received 8 July 2011 Received in revised form 14 October 2011 Accepted 29 October 2011 Available online 7 November 2011 Keywords: Dopamine NMDA Phosphorylation ERK1/2 Histone modification Immediate early genes

a b s t r a c t Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the ‘‘spatial’’ novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation–acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The hippocampus is involved in memory formation and plays a significant role in associative memory networks and spatial memory (Moser and Paulsen, 2001; Lee and Kesner, 2002). Besides these roles, hippocampus has been identified as a detector of novel stimuli, where a comparison of incoming information with stored memories occurs (Lisman and Grace, 2005). These novel stimuli

Abbreviations: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxy-5methyl-4-isoxazole propionate; BSA, bovine serum albumin; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetate; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of Mr 32,000; ERK1/2, extracellular signal-regulated kinase 1/2; HPLC, High-Performance Liquid Chromatography; HVA, homovanillic acid; NMDA, N-methyl-D-aspartate; IEGs, immediate-early genes; PBS, phosphate-buffered saline; PFC, prefrontal cortex; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline. ⇑ Corresponding author. Tel.: +30 2610 969159; fax: +30 2610 997215. E-mail address: [email protected] (F. Angelatou). 0197-0186/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.10.018

induce the natural exploration behavior, where it has been demonstrated that this stimulus increases the release of dopamine in hippocampus and prefrontal cortex (PFC) (Ljungberg et al., 1992; Ihalainen et al., 1999; Li et al., 2003). Furthermore, PFC is a cortical area involved in working memory, in selecting and retaining information to produce executive control (Huang et al., 2004; Rinaldi et al., 2007). Dopaminergic innervation is critical for long term changes in synaptic efficacy in hippocampus and PFC (Gurden et al., 2000; Li et al., 2003; Huang et al., 2004; Granado et al., 2008), as well as for learning-associated immediate-early gene expression (Lisman and Grace, 2005; Granado et al., 2008). Many studies have highlighted the dopaminergic modulation on glutamatergic synaptic transmission, namely dopamine D1 receptor interaction with glutamate NMDA receptor in modulating NMDA receptor-mediated responses for long term plasticity in PFC and hippocampus (Gurden et al., 2000; Yang, 2000; Chen et al., 2004; Tseng and O’Donnell, 2004; Navakkode et al., 2007).

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K. Sarantis et al. / Neurochemistry International 60 (2012) 55–67

Epigenetic changes, which include posttranslational modifications of histones and DNA, are involved in transcriptional events in neurons responding to changes of the environment (Colvis et al., 2005; Levenson and Sweatt, 2005). Moreover, several studies have shown that changes in the expression of the immediate-early genes (i.e. c-Fos, zif-268) play a critical role in neuronal plasticity and memory consolidation processes (Guzowski et al., 2001; Crosio et al., 2003; Kubik et al., 2007). In addition, it has been demonstrated that c-Fos and zif-268 are implicated in glutamate- dopamine-mediated synaptic plasticity (Konradi et al., 1996; Moratalla et al., 1996; Pavón et al., 2006; Granado et al., 2008). Our previous findings have shown that a synergistic interaction between dopamine D1 and NMDA receptors occurs at the phosphorylation level of NMDA and AMPA receptor subunits in the PFC and hippocampus, which leads to a robust phosphorylation/ activation of ERK1/2 signaling (Sarantis et al., 2009). Therefore, our present study is going to further investigate this functional interaction in rats, following their exposure to a novel open field environment, which is known to evoke dopamine release in the hippocampus and PFC (Lisman and Grace, 2005). In particular, the NMDA and AMPA receptor subunits’ phosphorylation, ERK1/2 activation, immediate early genes c-Fos and zif-268 expression, as well as chromatin remodeling were examined following reaction to novelty. The aforementioned biochemical alterations were also estimated following habituation procedure of rats in the open field apparatus. Interestingly, the specific role of D1 and NMDA receptors on this cascade was examined for discriminating the role of D1/NMDA interaction on the novelty-induced ERK1/2 signaling activation and its possible involvement in IEGs expression and chromatin remodeling events required for the regulation of synaptic plasticity and memory consolidation. 2. Materials and methods 2.1. Animals Male Wistar rats 45–60 days old were housed in a room with a controlled light–dark cycle (12 h light–12 h dark) and free access to food and water. In the present work, all efforts were made to minimize animal suffering and to reduce the number of animals used, according to the European Communities Council Directive (86/609/EEC) guidelines and to the Greek National Laws (Animal Acts, PD 160/91) guidelines for the care and use of laboratory animals. 2.2. Behavioral analysis 2.2.1. Open field behavior-exposure to novelty All rats were handled for three consecutive days, in order to be familiarized to the operator. The rats were initially habituated to the experimental room for 1 h prior to the experiment. The rats were then introduced into the testing box, an open field box (40 cm  40 cm  40 cm) and their behavior was observed for a 15-min period. A number of variables such as vertical counts, frequency and duration of moving and resting time were registered. These variables reflect the spontaneous motor activity seen following the exposure to the novel environment (Thiel et al., 1999; Antoniou et al., 2008; Polissidis et al., 2010). The duration of this session was chosen because a short exposure to a novel environment recapitulates the reactivity of the rats to novelty (Antoniou et al., 2008; White et al., 2007). After the 15-min period, the rats become habituated and moving and rearing behavior were declined. Drug administration was performed 30 min prior to the introduction of the rats to the novel environment. The following drugs’ combinations were used: (a) saline, as a control, (b) selective

antagonist of dopamine D1 receptors SCH23390 (0.03 mg/kg body weight) (Tocris), (c) selective antagonist of NMDA receptors MK801 (0.01 mg/kg body weight) (Sigma), (d) combination of SCH23390 and MK801 (drug concentrations same as in b and c, e) the selective inhibitor of ERK1/2 signaling, SL327 (50 mg/kg) (Tocris), (f) the selective antagonist of acetylcholine muscarinic receptors, scopolamine (3 mg/kg body weight) (Sigma) and (g) combination of SCH23390, MK801 and scopolamine (drug concentrations as used in b, c and f). The dose of MK-801 (0.01 mg/kg) was carefully selected, in order not to induce behavioral hyperactivity, as it is well established by behavioral studies (Ouagazzal et al., 1993; Rung et al., 2005; Gururajan et al., 2010, 2011). 2.2.2. Habituation procedure Another set of rats was used for experiments concerning the habituation procedure. All animals were placed daily in the testing box for 15 min for three consecutive days. The fourth day, each rat was again placed in the testing box and was then removed following a 15 min habituation period, anaesthetized with ether and decapitated, and proceed as described below. 2.3. Neurobiological analysis 2.3.1. Neurochemical assay Two groups of rats (n = 12, exposed to novelty; n = 12, controls) were used for dopaminergic system component estimation in specific rat brain regions. Following decapitation, the brains were rapidly removed and specific brain regions, specifically the hippocampus, the prefrontal cortex and the striatum were dissected. The dissected tissues were weighted, homogenized and de-proteinized in 500 ll of 0.2 N perchloric acid solution (Merck KgaA, Darmstadt, Germany) containing 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA (both by Riedel-de Haën AG, Seelze, Germany). The homogenate was centrifuged at 14,000 rpm for 30 min at 4 °C and the supernatant was stored at 80 °C, until analysis. The analytical measurements were performed using a Schimanzu HighPerformance Liquid Chromatography (HPLC) system, as previously described (Dalla et al., 2004; Antoniou et al., 2008) with some minor modifications (Rentesi et al., 2010). All samples were analyzed within 1 month after homogenisation. The sensitivity of the assay was tested for each series of samples using external standards. In all samples, reverse phase ion pair chromatography was used to assay dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetate (DOPAC) and homovanillic acid (HVA). Additionally, the ratios of DOPAC/DA and HVA + DOPAC/DA were calculated as an index of DA turnover rate (Antoniou et al., 2008; Dalla et al., 2008), in order to have a better evaluation of the dopaminergic activity. 2.4. Tissue preparation The respective subsets of rats either following exposure to novelty, or after the habituation procedure, were anaesthetized with ether and decapitated. The brain was rapidly removed and submerged in cold (4 °C) ACSF solution, which contained: 124 mM NaCl, 4 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose; at pH 7.4, and using a McIlwain tissue chopper, three kinds of tissue preparations were prepared: (a) coronal slices (300–350 lm) containing all the regions of the prefrontal cortex (PFC) (dorsal, medial, lateral) (1.5–2.5 mm anterior to bregma), (b) coronal slices (300–350 lm) containing only the striatum and (c) after the two hippocampi were excised free, we prepared transverse 300–350 lm thick slices from the dorsal and the ventral third parts of the hippocampus extending more than 1 and less than 3.5 mm from each end of the structure. Then, the slices were instantly frozen in isopentane (Sigma), and stored in 80 °C for further processing.

K. Sarantis et al. / Neurochemistry International 60 (2012) 55–67

2.5. Western blotting The experiments were performed as previously described (Brooks-Kayal et al., 2001). Briefly, the slices were rapidly microdissected and then solubilized in 100 ll 1% SDS v/w with 4 ll Sigma Phosphatase Inhibitor Cocktail I, 4 ll Sigma Phosphatase Inhibitor Cocktail II, 4 ll Sigma Protease Inhibitor Cocktail, sonicated, and boiled for 10 min. Protein concentration was determined for each sample by using the BSA protein kit (Pierce, Rockford, IL) and spectrophotometry. Duplicated samples (50 lg of total protein) were separated on 7% polyacrylamide gel and then transferred to nitrocellulose. After 1 h of blocking in 10% nonfat dried milk at RT, the nitrocellulose was incubated overnight at 4 °C with the following antibodies: rabbit anti-PhosphoNR1(ser897) polyclonal antibody (1:200, Chemicon International, Temecula, CA), rabbit antiPhosphoNR2B(ser1303) polyclonal antibody (1:750, Chemicon International), rabbit anti-PhosphoGLUR1(ser831) polyclonal antibody (1:200, Chemicon International), goat anti-PhosphoGLUR1(ser845) polyclonal antibody (1:200, Santa Cruz), rabbit anti-PhosphoERK1/2 (Thr202/Tyr204) polyclonal antibody (1:750, Cell Signaling Technology), rabbit anti-Phospho-Thr34–DARPP-32 polyclonal antibody (1:500, Novus Biologicals). Total amounts of proteins were detected using mouse anti-NR1 polyclonal antibody (1:500, Chemicon International), rabbit anti-NR2B polyclonal antibody (1:1000, Chemicon International), mouse anti-GLUR1 polyclonal antibody (1:200, Santa Cruz), mouse anti-ERK1/2 monoclonal antibody (1:750, Cell Signaling Technology), and mouse anti-DARPP-32 (1:1000, BD Transduction Laboratories) monoclonal antibody diluted in 10% nonfat dried milk. The blot was rinsed with TBS–Tween and then incubated with goat anti-rabbit horseradish peroxidase-linked IgG or goat anti-mouse horseradish peroxidaselinked IgG for 1 h at RT, followed by the Enhanced Chemiluminescence detection system (Amersham). The analysis of total protein levels was performed on the same blots, without stripping of the nitrocellulose, since different secondary antibodies were used for the phosphorylated and the total levels of the proteins. The only exception was for the NR2B subunit of NMDA receptor, where the nitrocellulose was stripped using the Re-Blot Plus Mild Solution (Chemicon International) for approximately 15 min at room temperature (RT). Molecular masses were determined by comparison with prestained protein molecular weight marker standards from Biomol. Blots were reprobed with anti-tubulin mouse monoclonal antibody (1:20,000; T5168; Sigma) rinsed with TBS–Tween, and then incubated with anti-mouse antibody and normalized to verify equivalent protein loading. Luminescence from the blots was detected by exposing the membranes to Fuji-Hyperfilm for 30 s to 7 min, to ensure that we were operating within the linear range of the film, followed by digital scanning of the developed film in transparency mode. The scanned image of the membranes and band intensities were calibrated and quantified using NIH ImageJ software (version 1.34). For each animal, the values obtained from the experiments with phosphorylated and total ERK1/2 corresponded to the total of the bands intensities for both ERK1 and ERK2 (also called p44 and p42 MAP kinases, in relation to their molecular weights, 44 and 42 kDa, respectively). For each animal, the values of the phosphorylated levels of each protein examined were normalized with the respective total levels. Each experiment was performed two to four times. Statistical analysis used ANOVA followed by Tukey post hoc test. 2.6. Immunofluorescence After the 15-min novelty exposure, the animals were removed and replaced in their original cage, where they remained for 1 h. After that, the rats were removed, anaesthetized with ether, decapitated and proceed as described above in the preparation of the

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hippocampal slices. The frozen tissue slices were mounted in the chucks of a Bright microtome (Leica, CM1500) (at 18 °C) and sections of 10 lm from the transverse slices were thaw-mounted onto poly-L-lysine-coated microscope slides. The sections were fixed with 4% PFA for 10 min at room temperature, washed with PBS and permeabilized with 0.3% Triton-X 100 in PBS. After incubation for at least 3 h with blocking solution [0.1% Tween 20 in PBS, 10% fetal bovine serum and 3% bovine serum albumin (BSA, Sigma)], sections were labeled overnight at 4 °C with the following primary antibodies diluted in blocking solution: polyclonal rabbit anti-zif-268 (1:400) (Santa Cruz), polyclonal mouse anti-c-Fos (1:400) (Santa Cruz), rabbit anti-ser10-Lys-14 histone H3 (1:500) (Chemicon International). After washing, sections were incubated at room temperature for 1 h with secondary antibodies Alexa Fluor 488 and 568 goat anti-rabbit and -mouse (1:1000 and 1:2000, respectively). Nuclei were stained with Hoechst (1:1500) (Sigma). The specificity of the staining was determined by omitting the primary antibodies. Samples were mounted with MOWIOL (Calbiochem). Photos were taken with a Nikon Eclipse TE2000-U microscope and collected with the Nikon camera Digital Sight DS-L1. All images were processed with Adobe Photoshop software. 3. Results 3.1. Exposure to novelty induced an increase in the dopaminergic activity as detected by an increase in the turnover rate of dopamine DOPAC/DA in the PFC and hippocampus Our neurochemical analysis showed that the reaction to novelty induced a robust increase in the dopamine levels (39%) and in the DOPAC levels (61%), and a slight elevation in the HVA levels (16%), compared to the control levels. Moreover, novelty behavior induced a significant elevation in the dopamine turnover ratios (DOPAC/DA, 43% and HVA + DOPAC/DA, 31%) in the hippocampus compared to the control levels (Fig. 1). Similar results were also obtained from the prefrontal cortex, where novelty behavior evoked a robust increase in the dopamine levels (58%) and in the DOPAC levels (63%), and a slight elevation in the HVA levels (13%), compared to the control levels. Furthermore, novelty induced a significant elevation in the dopamine turnover ratios (DOPAC/DA, 42% and HVA + DOPAC/DA, 41%) compared to the control levels (Fig. 1). Conversely, there was no effect on the dopamine, the DOPAC and the HVA levels, as well as in the dopamine turnover ratio in the striatum of the rats exposed to novelty compared to the nonexposed ones (Fig. 1). 3.2. Novelty induced an increase in the phosphorylation level of NMDA and AMPA receptor subunits in hippocampus and PFC, which is dependent on both dopamine D1 and glutamate NMDA receptors The introduction of rats into a novel environment resulted in a significant increase in the phosphorylation level of NR1ser897 and NR2Bser1303 subunits of NMDA receptor, which was higher in the hippocampus (40% and 37%, respectively compared to control levels) than in the PFC (20% and 26%, respectively compared to control levels) (Figs. 2 and 3). Similarly, novelty stimulus caused significant elevations of the phosphorylation levels of GLUR1ser831 and ser845 subunit of AMPA receptor (32% and 39% in the hippocampus and 26% and 34% in the PFC, respectively compared to the control levels) (Figs. 2 and 3). The administration of either the specific antagonist of D1 receptor, SCH23390 or the specific antagonist of NMDA receptor, MK801 prior to the introduction of rats to the novel environment partly

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Fig. 1. Levels of DA, DOPAC and HVA (lg/g of tissue) were determined in the hippocampus, prefrontal cortex and striatum of the [control] and [novelty] groups. DOPAC/DA and HVA + DOPAC/DA turnover ratios were calculated as an index of dopaminergic activity. Results are shown in means ± SEM values (number of animals, N = 12) per group. ⁄ p < 0.05 significantly different compared with the [control] group. Statistical analysis used Student’s t-test (level of significance: ⁄p < 0.05).

impaired the novelty-induced increase in the phosphorylation level of NR1ser897 and NR2Bser1303 subunits of NMDA receptors in the hippocampus and PFC (Figs. 2 and 3). A similar effect was observed for the GLUR1 subunit of AMPA receptor too, with the SCH23390 or MK801 partially attenuating the novelty-induced increase in the phosphorylation levels of GLUR1ser831 and ser845 subunit of AMPA receptor in the same areas (Figs. 4 and 5). Interestingly, co-administration of the specific antagonists SCH23390 and MK801 prior to the introduction of rats to the novel environment resulted in the complete abolishment of the noveltyinduced increase in the phosphorylation level of the NR1ser897 and NR2Bser1303 subunits of NMDA receptors (Figs. 2 and 3), and of the GLUR1ser831 and ser845 subunit of AMPA receptor in the hippocampus and PFC (Figs. 4 and 5). This indicates that the full phosphorylation levels of NMDA and AMPA receptor subunits by the novelty stimulus in the hippocampus and PFC requires the simultaneous activation of both dopamine D1 and NMDA receptors. Interestingly, administration of the selective inhibitor of ERK1/2 signaling SL327 prior to the exposure of the rats to the novel environment resulted in the complete abolishment of the

novelty-induced elevation of the phosphorylation level of NR1ser897 and NR2Bser1303 subunits of NMDA receptors (Figs. 2 and 3), as well as that of GLUR1ser831 and ser845 subunit of AMPA receptor observed in the hippocampus and PFC (Figs. 4 and 5). This fact indicates that the novelty-induced increase of the phosphorylation level of NMDA and AMPA receptor subunits, which is mediated by dopamine D1/NMDA receptor interaction, involves the ERK1/2 signal transduction pathway.

3.3. Novelty stimulus resulted in the activation of ERK1/2, but not of DARPP-32 signaling, depending on both D1 and NMDA receptors in hippocampus and PFC The exposure of rats to the novel environment caused a robust phosphorylation/activation of ERK1/2 kinases in the hippocampus and PFC compared to the non-exposed animals (Figs. 6 and 7). Conversely, there was no effect on the phosphorylation levels of DARPP-32 in either the hippocampus or PFC (Figs. 6 and 7), indicating that the novelty stimulus activated the ERK1/2 signal transduction pathway, but not of the DARPP-32 one.

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Fig. 2. Western blot analysis of the phosphorylation level of (A) NR1(ser897) subunit and (B) NR2B(ser1303) subunit of NMDA receptor, normalized with the respective total protein levels in dorsal hippocampus in different animal groups, according to the drug treatment: [control], [novelty], [SCH23390], [MK801], [SCH23390 and MK801], [SL327] and [habituation]. Results are shown in means ± SEM values (number of animals, N = 8) per group of representative Western blots. ⁄p < 0.05 significantly different compared with the [control] group. #p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test.

The administration of either the selective antagonist of dopamine D1 receptor SCH23390, or the selective antagonist of NMDA receptor MK801 prior to the introduction of the rats to the novel environment, partially impaired the novelty-induced increase of the phosphorylation level of ERK1/2 kinase in both the hippocampus and PFC (Figs. 6 and 7). On the other hand, co-administration of the two specific antagonists SCH23390 and MK801 resulted in the complete abolishment of the novelty-induced elevation of the phosphorylation level of ERK1/2 kinase in both the hippocampus and PFC (Figs. 6 and 7), indicating that the novelty-induced full phosphorylation/activation of ERK1/2 signaling requires the activation of both dopamine D1 and glutamate NMDA receptors. As expected, the administration of SL327 prior to the introduction of rats

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to the novel environment inhibited the phosphorylation/activation of ERK1/2 in the hippocampus and PFC (Figs. 6 and 7). 3.4. Novelty stimulus had no effect on the phosphorylation level of NMDA and AMPA receptor subunits, nor on ERK1/2 and DARPP-32 signaling in striatum In contrast to the effects in hippocampus and PFC, the introduction of rats to a novel environment did not affect the phosphorylation level either of NR1ser897 and NR2Bser1303 subunits of NMDA receptor or of GLUR1ser831 and ser845 subunit of AMPA receptor in the striatum (Supplementary Fig. 1). Moreover, no phosphorylation/activation of either ERK1/2 kinase or DARPP-32 could be detected in the striatum (Supplementary Fig. 1). This fact indicates

K. Sarantis et al. / Neurochemistry International 60 (2012) 55–67

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that the novelty stimulus affects specifically the hippocampal and PFC brain areas by increasing the phosphorylation of the NMDA and AMPA receptor subunits and activating the ERK1/2 signaling pathway, but it had no effect in the striatum. 3.5. Novelty induced an increase in the protein expression levels of the immediate early-genes c-Fos and zif-268 which depends on D1/NMDA, muscarinic acetylcholine receptors and ERK1/2 signaling The exposure of the rats to the novel environment resulted in a significant elevation of the protein expression levels of the immediate early-genes c-Fos (36%) and of zif/268 (12%) compared to the nonexposed ones, which was restricted in the CA1 region of the hippocampus (Figs. 8 and 9). A strong c-Fos protein staining has been also observed in the CA3 region and the dentate gyrus of the hippocampus, but no significant differences between control

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Fig. 4. Western blot analysis of the phosphorylation level of (A) GLUR1(ser831) subunit and (B) GLUR1(ser845) subunit of AMPA receptors, normalized with the respective total protein levels in dorsal hippocampus in different animal groups, according to the drug treatment (see Fig. 2). Results are shown in means ± SEM values (number of animals, N = 8) per group of representative Western blots. ⁄ p < 0.05 significantly different compared with the [control] group. #p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test.

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Fig. 5. Western blot analysis of the phosphorylation level of (A) GLUR1(ser831) subunit and (B) GLUR1(ser845) subunit of AMPA receptors, normalized with the respective total protein levels in prefrontal cortex in different animal groups, according to the drug treatment (see Fig. 2). Results are shown in means ± SEM values (number of animals, N = 8) per group of representative Western blots. ⁄ p < 0.05 significantly different compared with the [control] group. #p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test.

and novelty conditions could be detected (data not shown). Moreover, strong zif/268 protein staining was also observed in the CA3 region, but not in the dentate gyrus of hippocampus. However, no significant differences between control and novelty conditions could be measured (data not shown). The administration of either the dopamine D1 receptor antagonist SCH23390 or the NMDA receptor antagonist MK801 down regulated the novelty-induced increase in the c-Fos protein, albeit not significantly, in the CA1 region of the hippocampus (Fig. 8). Coadministration of SCH23390 and MK801 significantly reduced the novelty-elicited elevation of the c-Fos protein, albeit not to the control levels (Fig. 8). This fact indicates that the full induction of the protein expression of c-Fos as elicited by the novelty stimulus requires the concomitant activation of D1 and NMDA receptors in rat hippocampus.

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Fig. 6. Western blot analysis of the phosphorylation level of (A) ERK1/2 and (B) DARPP-32, normalized with the respective total protein levels in dorsal hippocampus in different animal groups, according to the drug treatment (see Fig. 2). Results are shown in means ± SEM values (number of animals, N = 8) per group of representative Western blots. ⁄p < 0.05 significantly different compared with the [control] group. #p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test.

Furthermore, administration of the selective inhibitor of ERK1/2 signaling pathway SL327 caused also a partial, but significant, down regulation of the novelty-induced elevation of the c-Fos protein (Fig. 8). This fact indicates the involvement of ERK1/2 signal transduction pathway in the induction of the c-Fos protein observed after the novelty stimulus in hippocampus. Moreover, the administration of the selective antagonist of muscarinic acetylcholine receptors scopolamine also partially down regulated the increase of the c-Fos protein induced by novelty stimulus, compared to the control levels (Fig. 8). This fact indicates that besides the concomitant stimulation of D1/NMDA receptors, activation of the muscarinic acetylcholine receptors is also required for the full induction of the novelty-induced increase of the c-Fos protein levels in the CA1 region of the hippocampus. In line with the above, co-administration of the three specific antagonists SCH23390, MK801 and scopolamine resulted in the

Fig. 7. Western blot analysis of the phosphorylation level of (A) ERK1/2 and (B) DARPP-32, normalized with the respective total protein levels in prefrontal cortex in different animal groups, according to the drug treatment (see Fig. 2). Results are shown in means ± SEM values (number of animals, N = 8) per group of representative Western blots. ⁄p < 0.05 significantly different compared with the [control] group. #p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test.

complete abolishment of the novelty-induced increase of the c-Fos protein (Fig. 8), indicating that the full induction of c-Fos protein by the novelty stimulus depends on the concomitant activation of D1/ NMDA receptors, as well as on the muscarinic acetylcholine receptors. In addition, co-administration of SCH23390 and MK801 or administration of the selective inhibitor of ERK1/2 signaling SL327 induced a slight down regulation of the novelty-induced increase of the protein expression of zif/268 in the CA1 region of the hippocampus, albeit not significant (Fig. 9). Similarly, the administration of the selective antagonist of muscarinic acetylcholine receptors scopolamine failed to down regulate the elevation of zif/268 protein (Fig. 9). However, co-administration of the three specific antagonists SCH23390, MK801 and scopolamine caused the complete abolishment of the novelty-induced increase of zif/268 protein (Fig. 9). This fact indicates that the novelty-induced elevation of the protein expression of the zif/268 in hippocampus depends on the simultaneous activity of D1, NMDA and muscarinic receptors.

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Fig. 8. Representative images of anti-c-Fos (red) immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control] (A–C), [novelty] (D–F), [SCH23390 + MK801] (G–I), [SL327] (J–L), [scopolamine] (M–O) and [SCH23390 + MK801 + scopolamine] (P–R). A portion of the CA1 region of hippocampus is shown. Nuclei were stained with Hoechst (blue). Immunofluorescence, novelty test and drug treatment were performed as described in Section 2. (S) Quantification of the data obtained by the anti-c-Fos immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control], [novelty], [SCH23390], [MK801], [SCH23390 + MK801], [SL327], [scopolamine], and [SCH23390 + MK801 + scopolamine]. Results are shown as a fraction of c-Fos positive cells/Hoechst positive nuclei in means ± SEM values (number of animals, N = 6) per group. ⁄p < 0.05 significantly different compared with the [control] group. ^p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

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Fig. 9. Representative images of anti-zif-268 (green) immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control] (A–C), [novelty] (D–F) and [SCH23390 + MK801 + scopolamine] (G–I). A portion of the CA1 region of hippocampus is shown. Nuclei were stained with Hoechst (blue). Immunofluorescence, novelty test and drug treatment were performed as described in Section 2. (J) Quantification of the data obtained by the anti-zif268 immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control], [novelty], [SCH23390], [MK801], [SCH23390 + MK801], [SL327], [scopolamine], and [SCH23390 + MK801 + scopolamine]. Results are shown as a fraction of zif-268 positive cells/Hoechst positive nuclei in means ± SEM values (number of animals, N = 6) per group. ⁄p < 0.05 significantly different compared with the [control] group. ^p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

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Fig. 10. Representative images of anti-[P(ser10)-Ac(Lys14)H3 histone] (green) immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control] (A–C), [novelty] (D–F) and [SCH23390 + MK801 + scopolamine] (G–I). A portion of the CA1 region of hippocampus is shown. Nuclei were stained with Hoechst (blue). Immunofluorescence, novelty test and drug treatment were performed as described in Section 2. (J) Quantification of the data obtained by the anti-[P(ser10)-Ac(Lys14)H3 histone] immunofluorescence-staining in the CA1 region of the dorsal hippocampus in different animal groups, according to the drug treatment: [control], [novelty], [SCH23390], [MK801], [SCH23390 + MK801], [SL327], [scopolamine], and [SCH23390 + MK801 + scopolamine]. Results are shown as a fraction of [P(ser10)-Ac(Lys14)H3 histone] positive cells/Hoechst positive nuclei in means ± SEM values (number of animals, N = 6) per group. ⁄p < 0.05 significantly different compared with the [control] group. ^p < 0.05 significantly different compared with the [novelty] group. Statistical analysis used ANOVA followed by Tukey post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

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3.6. Novelty induced epigenetic changes in histone H3 restricted in the CA1 region of the hippocampus, depending on D1/NMDA and muscarinic acetylcholine receptors The introduction of rats into the novel environment induced epigenetic changes, by elevating the level of phosphorylation/acetylation of histone H3 which were restricted in the CA1 region of the hippocampus, (Fig. 10). In particular, novelty stimulus induced an increase (12%, compared to control levels) of the phosphorylation at serine 10/acetylation at lysine 14 of histone H3 [P(Ser10Ac(Lys14)-H3 histone], (Fig. 10). A strong [P(Ser10)-Ac(Lys14)-H3 histone] staining was also observed in the CA3 region and the dentate gyrus of hippocampus, but no significant differences between control and novelty conditions could be detected (data not shown). However, the administration of the dopamine D1 receptor antagonist SCH23390 or of the NMDA receptor antagonist MK801 failed to down regulate the novelty-induced increase of the ser10-Lys14-histone H3 levels (Fig. 10). Moreover, co-administration of SCH23390 and MK801 or of the selective inhibitor of ERK1/2 signaling, SL327 induced a slight down regulation of the novelty-induced phosphorylation/acetylation of histone H3 (Fig. 10). Similarly, the administration of scopolamine failed to decrease the elevation of the ser10-Lys-14-histone H3 levels induced by the novelty stimulus (Fig. 10). However, co-administration of the three specific antagonists SCH23390, MK801 and scopolamine caused the complete abolishment of the novelty-induced increase of the ser10-Lys-14-histone H3 levels (Fig. 10). This fact indicates that the novelty-induced elevation of the ser10-Lys-14-histone H3 levels in the hippocampus depends on all three receptor activity.

3.7. Habituation procedure abolished the novelty-induced elevation in the phosphorylation level of NMDA and AMPA receptor subunits in hippocampus and PFC Interestingly, habituation of rats into the testing box resulted in the complete suppression of novelty-induced increase in the phosphorylation level of NR1ser897 and NR2Bser1303 subunits of NMDA receptor, as well as of the GLUR1ser831 and ser845 subunit of AMPA receptor in both hippocampus and PFC (Figs. 1–4). This fact indicates that the increase of the phosphorylation level of NMDA and AMPA receptor subunits after the novelty stimulus could represent a ‘‘novelty index’’ or a ‘‘novelty indicator’’, which is abolished after habituation. In line with the above, habituation of the rats into the testing box caused the complete abolishment of novelty-induced elevation of the phosphorylation level of ERK1/2 kinases in hippocampus, as well as in the PFC (Figs. 5 and 6).

4. Discussion In this study, we examined for the first time the effects of exposure to novelty on the phosphorylation level of NMDA and AMPA receptor subunits, as well as on the activation of ERK1/2 signaling and the induction of epigenetic changes and immediate earlygenes’ (IEGs) protein expression in the rat hippocampus and PFC. We showed that the ‘‘spatial’’ novelty induced a significant increase in the phosphorylation level of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, especially in the PFC and hippocampus. These phenomena are both dependent on the concomitant activation of dopamine D1 and NMDA receptors. Additionally, novelty behavior was characterized by chromatin remodeling events (phosphorylation/acetylation of histone H3) and significant increases of the IEGs c-Fos and zif/268 protein expression, which both depended on the

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co-activation of D1, NMDA and the acetylcholine muscarinic receptors. Our procedure involved the introduction of the rats to a novel open-field environment, which reflects a context of spontaneous exploratory behavior. Rats tend to respond specifically to novel stimuli by increasing their exploratory behavior (Vago and Kesner, 2008) and this procedure has been shown to evoke dopamine release in the PFC and hippocampus (Ljungberg et al., 1992; Ihalainen et al., 1999; Li et al., 2003; Lisman and Grace, 2005). This is in line with our results showing elevated levels of the metabolite of DA (DOPAC), as well as increased dopamine turnover ratio in the PFC and hippocampus, but not in the striatum. This fact confirms that novelty status induces elevated dopaminergic activity in areas like PFC and hippocampus, known to be involved in cognitive process, such as novelty detection, as well as learning and memory. It is possible that this effect is mediated by a functional loop between the hippocampus and the ventral tegmental area (VTA), regulating the entry of information into long term memory (Lisman and Grace, 2005). According to our results, the robust increase in the phosphorylation level of the NMDA (NR1ser897 and NR2Bser1303) and AMPA (GLUR1ser831 and ser845) receptor subunits in the PFC and hippocampus, induced by exposure to novelty demands the co-activation of dopamine D1/NMDA receptors. This is in line with our previous study, showing that a strong synergistic interaction between dopamine D1 and NMDA receptors exists at the phosphorylation level of NMDA and AMPA receptor subunits in the PFC and hippocampus (Sarantis et al., 2009). Several studies have demonstrated that the phosphorylation of the serine residue ser-897 of NR1 subunit by PKA is essential for the exit of NR1 subunit from the endoplasmatic reticulum (Tingley et al., 1997; Scott et al., 2003), whereas phosphorylation of the serine residue ser-1303 of NR2B subunit by PKC and CaMKII potentiates NMDA receptor currents (Liao et al., 2001). Moreover, the phosphorylation of the serine residues ser-831 by PKC and ser845 by PKA has been implicated to enhanced AMPA receptor currents by increasing the channel conductance and open probability, respectively (Derkach et al., 1999; Banke et al., 2000). Phosphorylation of both of these serine residues has been suggested to mediate LTP expression (Lee et al., 2000; Lee, 2006). Thus, the noveltyinduced increase in the phosphorylation level of NMDA and AMPA receptor subunits, which requires D1 and NMDA receptor interaction seen here, may be the molecular mechanism by which dopamine D1 receptor enhances the NMDA- and AMPA receptormediated excitability of the PFC and hippocampal pyramidal neurons (Yang, 2000; Gonzalez-Islas and Hablitz, 2003; Tseng and O’Donnell, 2004). Furthermore, this molecular mechanism may underlie the dopamine facilitation of long lasting plasticity, such as LTP, in the hippocampus and PFC (Gurden et al., 2000; Huang et al., 2004; Lisman and Grace, 2005; Granado et al., 2008). In support of this, it has been shown that the natural ‘‘spatial’’ novelty has a facilitatory effect on the induction of hippocampal LTP ‘‘in vivo’’, mediated by the dopamine D1 receptor activation (Li et al., 2003). In total, we can suggest that the dopaminergic facilitatory effect of LTP induction in hippocampus, which can be stimulated by a natural novel stimuli (Li et al., 2003), may be due to the noveltyinduced elevation of the phosphorylation levels of the NMDA and AMPA receptor subunits seen in our study, mediated by the dopamine D1/NMDA receptor interaction. In support of this suggestion, our results further showed that habituation resulted in the complete abolishment of novelty-induced elevation of the phosphorylation levels of the NMDA and AMPA receptor subunits. This fact could reflect a regulatory role of the phosphorylation of the NMDA and AMPA receptor subunits as ‘‘novelty indicator’’, underlying the molecular mechanism by which discrimination between novel and familiar stimuli is mediated.

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According to our results, the exposure of rats to a novel environment causes a robust increase of the phosphorylation level of ERK1/2 kinases in the PFC and hippocampus, the full expression of which depends on the concomitant activation of dopamine D1 and NMDA receptors. However, the novelty exposure had no effect on the DARPP-32 signaling. This is in line with our previous findings, showing that the molecular mechanism underlying D1/NMDA receptor synergistic interaction involves ERK1/2, but not DARPP-32 signaling in the PFC and hippocampus (Sarantis et al., 2009). In accordance with the above, previous studies have also demonstrated that D1 receptor stimulation elevated the phosphorylation level of ERK1/2 in the rat PFC and hippocampus (Roberson et al., 1999; Nagai et al., 2007). The phosphorylation/activation of ERK1/2 signaling has been indicated to be essential for the induction of epigenetic changes (chromatin remodeling) and the regulation of neuronal gene expression, which are necessary for the regulation of synaptic plasticity (Thomas and Huganir, 2004; Levenson and Sweatt, 2005). Thus, the novelty-induced phosphorylation/activation of ERK1/2 signaling mediated by the D1/NMDA receptor interaction observed, may be essential for LTP, as well as for learning and memory processes. In accordance with the above suggestion, a brief introduction of rats into a novel environment, induced histone H3 modifications (i.e. phosphorylation at Ser10/acetylation at Lys14), as well as elevation of the IEGs cFos and zif/268 protein levels, restricted in the CA1 region of hippocampus. This phenomenon has been also observed in other behavioral processes, which include spatial watertask training (Morris water maze) (Guzowski et al., 2001; Chwang et al., 2007), and also stress (Chandramohan et al., 2007, 2008) and fear conditions (Chwang et al., 2006, 2007). Interestingly, according to our results, novelty-induced histone H3 modifications and c-Fos and zif/268 protein expression are dependent on D1/NMDA interaction, as well as on muscarinic acetylcholine receptors’ function. This is in line with a previous study showing that activation of dopamine D1 or muscarinic acetylcholine receptors by exogenous administration of the respective agonists induced chromatin remodeling and c-Fos transcription in hippocampal neurons (Crosio et al., 2003). Several studies indicate that cholinergic mechanisms play an important role in the ability of novelty-exposure to influence cortical and hippocampal immediate-early gene expression (Wirtshafter, 2005; Miyashita et al., 2009). More solid, scopolamine administration prior to the exposure greatly suppressed the novelty-induced c-Fos expression in the hippocampus and cortex (Wirtshafter, 2005). Several studies have demonstrated that the phosphorylation of serine-10 (Ser10) and acetylation of lysine-14 (Lys14) in the Nterminal of histone H3 are associated with the local opening of condensed chromatin, thereby permitting transcriptional induction of specific, formerly silent genes (Cheung et al., 2000a; Clayton et al., 2000; Nowak and Corces, 2000; Strahl and Allis, 2000; Jenuwein and Allis, 2001; Li et al., 2001). Ser10 phosphorylation has been shown to facilitate acetylation at Lys14 (Cheung et al., 2000b), and these modifications are associated with transcriptionally active chromatin (Cheung et al., 2000a; Strahl and Allis, 2000). Furthermore, it has been reported that one of the critical effector kinases downstream of the ERK1/2 are the MSKs (Mitogen and Stress-induced Kinases) and the ser10 residue of the N-terminal of H3 histone can be directly phosphorylated by them (Thomson et al., 1999; Chwang et al., 2007). Thus, as it is pointed out here, the novelty-induced phosphorylation/activation of histone H3 at the ser10 residue – the full expression of which is dependent on D1/NMDA receptor co-activation – could be mediated, at least in part, by the ERK1/2 signal transduction pathway. Subsequently, the robust activation of ERK1/2 signaling induced by the novelty exposure (mediated by D1/NMDA receptor interaction) seen in our study, could regulate a diverse set of targets in the nucleus, leading to up regulation of gene transcription, such as the c-Fos.

It is well known that these genes are required for synaptic plasticity and several forms of learning and memory consolidation (Crosio et al., 2003; Thomas and Huganir, 2004; Levenson et al., 2004; Levenson and Sweatt, 2005; Chwang et al., 2007). In accordance with the above, suppression of the IEGs c-Fos (Lamprecht and Dudai, 1996; Grimm et al., 1997; Morrow et al., 1999) and zif/ 268 (Jones et al., 2001) impairs long-term memory consolidation. Thus, novelty exposure through the activation of D1/NMDA and muscarinic acetylcholine receptors can induce the elevation of c-Fos and zif/268 expression, leading to events that are required for long term plasticity and memory. According to our results, the aforementioned novelty-induced alterations in chromatin remodeling and c-Fos and zif/268 proteins were restricted in the CA1 region of hippocampus. This could be explained by the role of the CA1 region of hippocampus as a ‘‘comparator’’, that computes novelty by comparing the incoming information (novelty) with stored memories (Lisman and Grace, 2005). In conclusion, our data provide clear evidence for ‘‘spatial’’ novelty-induced increases in the phosphorylation levels of the NMDA and AMPA receptor subunits and the ERK1/2 signal transduction pathway activation in the hippocampus and PFC, which are both dependent on the concomitant activation of the dopamine D1 and glutamate NMDA receptors. This D1/NMDA receptor synergistic interaction is required for the full expression of spatial novelty-induced epigenetic changes and gene expression, known to be involved in long term plasticity and learning and memory. Acknowledgments We would like to thank the Advanced Light Microscopy facility of the Medical School of University of Patras for obtaining the photos derived by the immunofluorescence experiments. We would also like to thank Dr. M. Spella for her contribution in the immunofluorescence experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuint.2011.10.018. References Antoniou, K., Papathanasiou, G., Papalexi, E., Hyphantis, T., Nomikos, G.G., Spyraki, C., Papadopoulou-Daifoti, Z., 2008. Individual responses to novelty are associated with differences in behavioral and neurochemical profiles. Behav. Brain Res. 187, 462–472. Banke, T.G., Bowie, D., Lee, H., Huganir, R.L., Schousboe, A., Traynelis, S.F., 2000. Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J. Neurosci. 20, 89–102. Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Rikhter, T.Y., Kelly, M.E., Coulter, D.A., 2001. Gamma-aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development. J. Neurochem. 77, 1266–1278. Chandramohan, Y., Droste, S.K., Reul, J.M., 2007. Novelty stress induces phosphoacetylation of histone H3 in rat dentate gyrus granule neurons through coincident signalling via the N-methyl-D-aspartate receptor and the glucocorticoid receptor: relevance for c-Fos induction. J. Neurochem. 101, 815–828. Chandramohan, Y., Droste, S.K., Arthur, J.S., Reul, J.M., 2008. The forced swimminginduced behavioural immobility response involves histone H3 phosphoacetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/ mitogen- and stress-activated kinase signalling pathway. Eur. J. Neurosci. 27, 2701–2713. Chen, G., Greengard, P., Yan, Z., 2004. Potentiation of NMDA receptor currents by dopamine D1 receptors in prefrontal cortex. Proc. Natl. Acad. Sci. USA 101, 2596–2600. Cheung, P., Allis, C.D., Sassone-Corsi, P., 2000a. Signaling to chromatin through histone modifications. Cell 103, 263–271. Cheung, P., Tanner, K.G., Cheung, W.L., Sassone-Corsi, P., Denu, J.M., Allis, C.D., 2000b. Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol. Cell 5, 905–915.

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