Accepted Manuscript Title: Alterations in the hippocampal phosphorylated CREB expression in drug state-dependent learning Author: Sakineh Alijanpour Ameneh Rezayof Houri Sepehri Ladan Delphi PII: DOI: Reference: S0166-4328(15)30030-9 http://dx.doi.org/doi:10.1016/j.bbr.2015.06.003 BBR 9642 To appear in: Behavioural Brain Research Received date: Revised date: Accepted date: 12-2-2015 1-6-2015 3-6-2015 Please cite this article as: Alijanpour Sakineh, Rezayof Ameneh, Sepehri Houri, Delphi Ladan.Alterations in the hippocampal phosphorylated CREB expression in drug state-dependent learning.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2015.06.003 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. Alterations in the hippocampal phosphorylated CREB expression in drug state-dependent learning Sakineh Alijanpour, Ameneh Rezayof, Houri Sepehri, Ladan Delphi Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran Correspondence to: A. Rezayof, PhD. Professor, Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-61112483 e-mail: rezayof@khayam.ut.ac.ir Highlights ► Passive avoidance learning increased phosphorylation of CREB in the hippocampus. ► Ethanol or WIN-induced amnesia decreased hippocampal p-CREB levels. ► Hippocampal p-CREB levels were increased in ethanol state-dependent learning (STD). ► Cross STD between the drugs increased hippocampal p-CREB/CREB ratio. ► Hippocampal p-CREB is a marker associated with drug-induced STD. Abstract The present study investigated the possible alterations of hippocampal CREB phosphorylation in drug state-dependent memory retrieval. One-trial step-down passive avoidance task was used to assess memory retrieval in adult male NMRI mice. Pre-training administration of ethanol (1 g/kg, i.p.) induced amnesia. Pre-test administration of ethanol (1 g/kg, i.p) or nicotine (0.7 mg/kg, s.c.) reversed ethanol-induced amnesia, indicating ethanol- or ethanol-nicotine induced state-dependent learning (STD). Using Western blot analysis, it was found that the p-CREB/CREB ratio in the hippocampus increased in the mice that showed successful memory retrieval as compared with untrained mice. In contrast, pre-training administration of ethanol (1 g/kg, i.p.) decreased the hippocampal p-CREB/CREB ratio in comparison with the control group. The hippocampal p-CREB/CREB ratio enhanced in ethanoland ethanol-nicotine induced STD. Moreover, memory impairment induced by pre-training administration of WIN (1 mg/kg, i.p.) improved in the animals that received pre-test administration of WIN (1 mg/kg, i.p.), ethanol (0.5 g/kg, i.p.) or nicotine (0.7 mg/kg, s.c.), suggesting a cross STD between the drugs. The p-CREB/CREB ratio in the hippocampus decreased in the of WIN-induced amnesia and STD groups in comparison with the control group. In addition, cross state-dependent learning between WIN and ethanol or nicotine was associated with the increase of the hippocampal p-CREB/CREB ratio. It can be concluded that phosphorylation of CREB in the hippocampus is a critical event underlying the interaction of coadministration of drugs on memory retrieval in passive avoidance learning. Keywords: Drug state-dependent learning; Hippocampus; p-CREB/CREB ratio 1. Introduction Long-term potentiation (LTP) is long-lasting alterations in the synaptic strength which occurs during learning and memory processes [1]. The basic mechanisms underlying LTP consist of protein phosphorylation and de novo protein synthesis [2]. Neural pathways implicated in LTP induction modulate the changes in the functions of many transcription factors such as cAMPresponsive element binding protein [CREB; 3]. Multiple kinases including protein kinase A (PKA), calcium calmodulin-dependent protein kinase (CAMKII or CAMKIV) and mitogen activated protein kinases (MAPKs) can phosphorylate CREB as an active transcriptional factor [4, 5]. There is increasing evidence that phosphorylated CREB (p-CREB) elevation in the hippocampus may be associated with memory formation [6]. Hippocampal p-CREB function has been suggested to participate in fear memory [7], spatial memory [8], passive avoidance [9], and reward-related learning [10]. Exposure to drugs of abuse induces the enhancement of dopamine transmission in the mesocorticolimbic pathways which originate from the ventral tegmental area (VTA) and sends its projections to several brain areas including the nucleus accumbens (Nac), the prefrontal cortex (PFC) and the hippocampus [11]. Drug addiction and memory formation may share common molecular and cellular substrates to induce the alterations in synaptic plasticity [12]. Ethanol administration has been reported to impair cognitive functions [13, 14] through GABAA [15] and NMDA [16] receptors that exist in high proportions in the hippocampus. In addition, previous studies have highlighted the role of cAMP signaling pathway and the augmentation of CREB levels in ethanol tolerance and dependence [17, 18]. It should be noted that memory retrieval in ethanol-induced state dependent learning (STD) is possible unless the subject is situated in a state similar to the acquisition phase [19, 20]. Interestingly, nicotine can also reverse the amnesic effect of ethanol-induced memory impairment which may lead to the induction of cross STD between ethanol and nicotine [21]. Substantial evidence has shown that memory-facilitating effect of nicotine on learning and memory processes is mainly mediated via central α 7 and α 4ß 2 nicotinic acetylcholine receptors [nAChRs; 22] which trigger the activation of kinases such as PKA [23]. CREB phosphorylation was found to increase in the nicotine-related reward as measured in conditioned place preference (CPP) paradigm [24]. In another study, Kenney et al. reported that the effect of nicotine on hippocampal-based memory is assossiated with the increase of CREB phosphorylation [25]. On the other hand, cannabis, a derivative of cannabis sativa, is often co-administrated with ethanol [26] or nicotine [27]. Acute administration of cannabis can produce memory deficits in humans [28] and laboratory animals [29]. Cannabinoids interact with two members of the superfamily of Gi/Go-coupled receptors, cannabinoid receptors-1 (CB1) and CB2 receptors. Cannabinoid CB1 receptors (CB1Rs) which are widely distributed in the hippocampus play a critical role in mediating memory consolidation and retrieval [30]. The activation of CB1Rs impairs hippocampal synaptic plasticity by reduction of CREB phosphorylation [31]. In view of the fact that drug addiction affects the various stages of learning and memory processes and also the implication of CREB as a key protein in memory formation and drug reward, the aim of the present study was to examine the possible alterations of hippocampal CREB phosphorylation in drug state-dependent memory retrieval. 2. Materials and methods 2.1. Animals Male NMRI mice (160 animals, weighing 20-30 g) were obtained from Pasteur Institute, Iran and maintained in a room with 12 h alternating light/dark cycle, with light beginning at 7:00 a.m and in a controlled temperature (22 ± 2 °C). Food and water were available ad libitum. Ten mice were used in each experiment and housed as a group in a separate cage. Each animal was used once. The mice were acclimated to the testing room for at least 30 min before behavioral assay. All procedures for the treatment of animals were approved by the Research and Ethics Committee of the School of Biology, University of Tehran and were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Moreover, all efforts were made to minimize the number of animals used and limiting their suffering. 2.2. Passive avoidance apparatus Step-down type passive avoidance was used as a measure of memory retention. This apparatus was a black Plexiglas (30 × 30 × 40 cm high) with a grid floor consisting of parallel stainless steel rods (0.3 cm diameter spaced 1 cm apart). A wooden platform (4 × 4 × 4 cm) was fixed at the center of the grid floor. The test consisted of a training session and a retention session carried out 24 h after the training. During the training sessions, each animal was placed on the platform and its latency to step down on the grid with all four paws was measured. When it stepped down and placed its four paws on the grid floor, electric shocks (1 Hz, 0.5 s, 45 V DC) were delivered for 15 s using an isolated stimulator (Borj sanat, Tehran, Iran). If any animal stayed on the platform for more than 20 s or stepped up to the platform before the end of 15 s of electric shocks, it was omitted from the experiments. The retention test was carried out in a similar manner to that of training session except that no shock was applied. An upper cut-off time of 300 s was set. It is important to note that all animals were sacrificed immediately after the retention test and their hippocampi were extracted in minor time [32] and stored at –80˚ C until ready to homogenize. 2.3. Design of experiments In this study, the animals were divided into three main experimental groups. In each series of experiments, we used two types of control groups. One naïve control group was habituated and handled in the experimental room without being given any treatment (Intact group). The other control group received saline or vehicle (10 ml/kg) 30 min before training (pre-training) and testing (pre-test) sessions (Saline/saline or vehicle group). Since environmental conditions may affect the hippocampal plasticity [33], the same social and physical laboratory conditions were used for the intact, saline and drug-treated animals. The experimental schedule of experiment 1 consisted of three groups of animals which received pretraining ethanol (1 g/kg, i.p.), followed by different doses of pre-test ethanol (0, 0.25 and 1 g/kg, i.p.) in order to produce ethanol-induced state-dependent learning (STD; Fig. 1). The aim of experiment 2 was to evaluate the cross STD between ethanol and nicotine. So, three groups received ethanol (1 g/kg, i.p.) 30 min before training and different doses of nicotine (0, 0.3 and 0.7 mg/kg, s.c.) 30 min before testing (Fig. 2). In the third experiment, three groups of animals received pre-training injection of WIN (1 mg/kg, i.p.), followed by pre-test administration of different doses of WIN55, 212-2 mesylate (WIN; 0, 0.1, 1 mg/kg, i.p.) for producing WINinduced STD. Also, the other four groups which were used for the evaluation of cross STD between WIN and nicotine or ethanol received a dose of 1 mg/kg of WIN 30 min before training, followed by different doses of ethanol (0.25 and 1 g/kg, i.p.) or nicotine (0.3 and 0.7 mg/kg, s.c.) 30 min prior to testing (Fig. 3). Immediately after measuring the step-down latency, the animals were sacrificed for their hippocampi to be extracted and collected; the p-CREB/CREB ratio was evaluated via western blotting assay. 2.4. Western Blot Analysis Mouse hippocampal tissues were homogenized in lysis buffer containing complete protease and phosphatase inhibitor cocktail (Inhibitor complete mini; Roche Diagnostics, Mannheim, Germany) on the ice and centrifuged at 12000 rpm for 10 min at 4 °C. Supernatant were analyzed for protein concentration by Bradford’s assay [34]. Then, the total proteins were resolved by 12 % SDS-PAGE gels electrophoresis. A semi-dry electroblotting method was used to transfer the proteins onto PVDF membrane. The blots were blocked in 2% skim milk dissolved in Tris-buffered saline with tween 20 (TBST) and then incubated with mouse anti CREB, p-CREB or β–actin antibody (1/1000) overnight at 4 °C. The following day, after being washed completely with TBST, the blot was incubated in the secondary HRP-conjugated antirabbit antibody (1/3000) for 1 h at room temperature. The blots were washed again and wrapped in plastic foil and exposed to X-ray film. Immunoreactive polypeptides were detected by ECL reagents and subsequent autoradiography. Quantification of the results was performed by densitometric scan of the films. Data analysis was done by Image J, measuring integrated density of bands after background subtraction. It should be considered that CREB and p-CREB runs with a mass of 43 KDa when resolved with SDS-PAGE gel electrophoresis, while β–actin has a molecular weight of 45 KDa. 2.5. Drugs Ethanol (Merck, Germany) and nicotine hydrogen tartrate (Nicotine; Sigma, Poole, Dorset, UK) were dissolved in sterile 0.9% saline and then the pH of nicotine solution was adjusted to 7.2 with NaOH (0.1 normal solution). WIN55, 212-2 mesylate (WIN; Tocris, Bristol, UK), a mixed CB1/CB2 receptors agonist, was dissolved in dimethylsulfoxide (DMSO; up to 10% v/v) and sterile 0.9% saline and a drop of Tween 80, which also was used as vehicle. Control animals received either saline or vehicle. All injections were administrated intraperitoneally (i.p.) or subcutaneously (s.c.) at a volume of 10 ml/kg. Antibodies directed against cAMP-responsive element binding (CREB) protein, phosphorylated-CREB (p-CREB) and β–actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Electrochemiluminescence (ECL) kit was provided by Amersham Bioscience (Piscataway, NJ, USA). Poly vinylidene fluoride membrane (PVDF) was purchased from Millipore (Billerica, MA, USA). 2.6. Data analysis Data from step-down latencies are presented as the median and interquartile range. The obtained data were analyzed by using the Kruskal–Wallis non-parametric one-way analysis of variance (ANOVA) followed by a two-tailed Mann–Whitney U-test. Also, the obtained data from western blotting assay were analyzed using one-way analysis of variance (ANOVA) and are expressed as means ± SEM. Post-hoc analysis was performed for the paired comparisons. In all statistical evaluations p < 0.05 was used as the criterion for statistical significance. 3. Results 3.1. Changes of the hippocampal p-CREB/CREB ratios in ethanol-induced amnesia and statedependent learning Fig. 1A shows the effect of pre-training and pre-test administration of ethanol on memory retrieval in the passive avoidance learning test. Kruskal-Wallis non parametric ANOVA showed that pre-training administration of ethanol (1 g/kg, i.p.) impaired memory retrieval, while pre-test administration of the same dose of ethanol (1 g/kg, i.p) significantly reversed the memory impairment, indicating ethanol-induced state-dependent learning [H(3)= 28.12, P<0.001]. As shown in Fig. 1B, the hippocampal p-CREB/CREB ratios were measured in ethanol-induced amnesia and state-dependent learning via western blot analysis. The p-CREB/CREB ratio in the hippocampus increased by about 1.43 fold in the mice that showed successful memory retrieval (control group) as compared with untrained mice (intact group). In contrast, pre-training administration of ethanol (1 g/kg, i.p.) decreased the hippocampal p-CREB/CREB ratio (32.59%, P<0.001) in comparison with the control group. Moreover, the densitometric analysis revealed that p-CREB/CREB ratio in the hippocampus (1.72 fold, P<0.001) increased in ethanol-induced state-dependent learning (ethanol/ethanol group) as compared with ethanol-induced amnesia (ethanol/saline group). 3.2. Changes of the hippocampal p-CREB/CREB ratio in cross state-dependent learning between ethanol and nicotine Fig. 2A shows the effect of the co-administration of ethanol and nicotine on memory retrieval. Kruskal-Wallis non parametric ANOVA [H(3)= 26.35, P<0.001)] revealed that pre-test administration of nicotine (0.7 mg/kg, s.c.) reversed ethanol-induced amnesia which was produced by pre-training administration of ethanol, suggesting a cross state-dependent learning between ethanol and nicotine. Mice in these groups were used to evaluate the hippocampal p- CREB/CREB ratios by western blot analysis (Fig. 2B). The results indicated that p-CREB/CREB ratio increased in the animals that showed successful memory retrieval (control group) as compared to intact group (1.26 fold, P<0.001). In contrast, pre-training administration of ethanol (1 g/kg, i.p.) led to the decrease of the hippocampal p-CREB/CREB ratio in comparison to the control group (43.42%, P<0.001). In addition, the hippocampal p-CREB/CREB increased by about 2.79 fold in the animals which received pre-training injection of ethanol and pre-test injection of 0.7 mg/kg of nicotine as compared with the animals that received ethanol before training (P<0.001; Tukey’s test). 3.3. Changes of the hippocampal p-CREB/CREB ratio in WIN-induced amnesia and cross statedependent learning between WIN, ethanol or nicotine Fig. 3A shows the effects of WIN (1 mg/kg, i.p.) with or without ethanol or nicotine on memory retrieval in passive avoidance task. Kruskal–Wallis nonparametric ANOVA [H(7)= 46.9, P<0.001)] revealed that the memory impairment induced by pre-training administration of WIN (1 mg/kg, i.p.) improved in the animals that received pre-test administration of WIN (1 mg/kg, i.p.), ethanol (0.5 g/kg, i.p.) or nicotine (0.7 mg/kg, s.c.), suggesting cross statedependent learning between the drugs. The ratios of the hippocampal p-CREB/CREB in WINinduced amnesia and WIN-ethanol or -nicotine cross state-dependent learning are presented in figure 3B. The densitometric analysis showed that the p-CREB/CREB ratio in the hippocampus increased (1.25 fold, P<0.001; Fig 3B) in the animals that showed successful memory retrieval (control group). The p-CREB/CREB ratio in the hippocampus decreased in the animals that received pre-training administration of WIN (57.98%, P<0.001) as compared with the control group. The analysis revealed no significant changes (P>0.05) in hippocampal p-CREB/CREB ratio in the animals that received pre-training WIN (1 mg/kg, i.p.), followed by pre-test administration of the same dose of WIN (1 mg/kg, i.p.) in comparison with the animals that showed WIN-induced amnesia. In addition, the analysis revealed that cross state-dependent learning between WIN and ethanol (1 g/kg, i.p.; 1.78 fold, P<0.001) or nicotine (0.7 mg/kg, s.c.; 2.46 fold, P<0.001) was associated with the increase of the hippocampal p-CREB/CREB ratio. 4. Discussion The present study examined the possible alterations of the hippocampal p-CREB/CREB ratio in the effects of co-administration of ethanol, nicotine and/or cannabis on memory formation. The results showed that pre-training systemic administration of ethanol impairs memory retrieval in passive avoidance task and induced amnesia. Previously, the amnesic effect of pre-training acute ethanol administration had been observed in different memory assay models such as Morris water maze [MWM; 35], object recognition [36] and passive avoidance task [37]. Also, the results revealed that pre-test administration of ethanol improved memory impairment and produced drug state-dependent memory retrieval (STD). We found that hippocampal pCREB/CREB ratio decreased in ethanol-induced amnesia, while it significantly increased in ethanol-STD. Interestingly, the elevation of p-CREB/CREB ratio in ethanol-STD was even much more than control group. It is important to note that passive avoidance learning in control groups enhanced hippocampal p-CREB/CREB ratio in comparison with intact animals. Although pretraining and pre-test administration of saline, vehicle or DMSO had the same effect on the passive avoidance task and induced successful memory retrieval in saline/saline, saline/vehicle and vehicle/vehicle control groups in all experiments, the hippocampal levels of p-CREB and CREB in these groups were different in comparison with one another. This finding was unexpected and suggests that the use of different solvents may have different effects on hippocampal signaling pathways. Further studies, which take these variables into account, should be undertaken. In agreement with our results, Cammarota et al. reported that memory formation of one-trial avoidance learning activates hippocampal PKA/CREB signaling pathway [9]. A quantitative immunocytochemistry study in rats’ hippocampus showed that training in a social transmission of food preference task increased the number of p-CREB positive neurons [38]. Although treatment with acute ethanol increased p-CREB in the striatum, the total CREB didn’t change in this region [39]. On the other hand, ethanol consumption and withdrawal reduced CREB phosphorylation in the amygdala [40] and the cortex [41], Thus, one may suggest that pCREB levels may be changed in ethanol-related behaviors and it is likely that hippocampal CREB signaling cascades are involved in mediating the effects of ethanol on learning and memory processes. The present results also indicated that pre-test administration of nicotine improved ethanol-induced memory impairment, suggesting that there is a cross STD between ethanol and nicotine. Another important finding was that ethanol-nicotine cross STD significantly increases the p-CREB/CREB ratio in the hippocampus. Duka et al. [42] and Nakagawa and Iwasaki [43] reported that ethanol-induced amnesia can be reversed by the administration of the same dose of drug in human and laboratory animals. Previous findings in our laboratory have also indicated that different neurotransmitter systems such as dorsal hippocampal glutamatergic, nitric oxide and dopaminergic systems contribute to ethanol-induced STD [44-46]. Signaling pathways triggered with these neurotransmitter systems play an important role in mediating CREBdependent gene expression in neurons [47-49]. CREB acts as a critical transcription factor to induce long-term potentiation (LTP) related to acute nicotine administration [50]. Association between nAchRs activation and CREB phosphorylation has been proposed in various reward- related brain regions such as the ventral tegmental area (VTA) and the nucleus accumbens [Nac; 51, 24]. Several studies have revealed that CREB activity changes in the nicotine-CPP [52] and withdrawal [53]. Therefore, a possible explanation for the cross-STD between ethanol and nicotine can be found in enhanced p-CREB signaling response in the hippocampus. The present data also showed that pre-training exposure to WIN55, 212-2 (WIN), a CB1/CB2 receptor agonist, had an impairing effect on memory retrieval. Similar to ethanol results, we observed a decrease in hippocampal p-CREB/CREB ratio following pre-training administration of WIN. The disruptive effects of cannabis on learning and memory processes have previously been shown in human [54]. The neurocognitive deficits observed with cannabis may be due to alterations of hippocampal cholinergic [55] and glutamatergic [56] neurotransmissions. The current study also revealed that WIN-induced STD is related to the decrease of p-CREB/CREB ratio in the hippocampus. Fan et al. reported that repeated exposure to THC, a CB1 receptor agonist, decreased hippocampal CB1 receptor-dependent p-CREB and also inhibited LTP [31]. Spatial memory and object recognition-deficit induced by CB1Rs agonist diminished CREB phosphorylation in the hippocampus [57]. In contrast to the mentioned results, it has been shown that acute exposure to THC increased p-CREB in the rat hippocampus [58] and the cerebellum [59]. Further research regarding the role of hippocampal CB1 receptor mechanisms in WIN-induced amnesia would be interesting. In accordance with our previous study [60], the present results indicated that pre-test administration of ethanol or nicotine reversed WIN-induced amnesia and produced WIN-ethanol and WIN-nicotine cross STD respectively. The most interesting finding was that the cross STD between WIN and ethanol or nicotine increased the hippocampal p-CREB/CREB ratio. There is a large volume of published studies suggesting the interaction between ethanol or nicotine application and cannabinoid system in the synaptic plasticity and memory formation. For example, Basavarajappa et al. showed that ethanol inhibits glutamate neurotransmission in the CA1 region of dorsal hippocampus through the activation of CB1 receptors and enhancement of endocannabinoid production [61]. Also, Thanos et al. findings in CB1Rs knockout mice showed the reduction of ethanol reward [62]. Systemic administration of CB1Rs antagonist prevented nicotine-induced sensitization [63], self-administration [64] and conditioned place preference [CPP; 65]. In view of the fact that CB1 receptor signaling mediates ethanol or nicotine rewarding effects [66, 67], a functional interaction between the drugs in STD which is a reward-related learning [68] can be highly likely. It should be highlighted that the hippocampus is a key region in reward-related learning [69] and CREB as a critical transcriptional factor is involved in converting short-term (STM) to long-term memory (LTM). It has also been shown that exposure to abuse drugs overexpressed CREB levels in the Nac which leads to the reduction of rewarding effects [70]. Since STD seems to be related to the rewarding effects of abuse drugs, one may suggest that cross STD between the drugs may be due to the induction of the same rewarding physiological conditions during the acquisition and memory retrieval phases. 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[70] Carlezon WA Jr, Thome J, Olson VG, Lane-Ladd SB, Brodkin ES, Hiroi N, Duman RS, Neve RL, Nestler EJ. Regulation of cocaine reward by CREB. Science 1998;282:2272-5. Legends Fig. 1. Changes of the hippocampal p-CREB/CREB ratios in ethanol-induced amnesia and statedependent learning. Fig. 1A) five groups of animals were used. One group had no treatment and was used as an intact control group and the other control group received saline (10 ml/kg) 30 min before training (pre-training) and testing (pre-test) sessions. The other three groups received pre-training ethanol (1 g/kg, i.p.), and different doses of pre-test ethanol (0, 0.25 and 1 g/kg, i.p.). The step-down latency was measured 30 minutes after the last injection in all animals. Each value represents the median and interquartile ranges of 10 mice. ***P<0.001 compared with saline/saline and +++P<0.001 compared with ethanol/ethanol. Fig. 1B) Western blotting of CREB, p-CREB and β–actin proteins are represented in the above panels. The mean hippocampal p-CREB/CREB ratio calculated from densitometric quantification of the corresponding bands were shown in the bottom graph. Each bar shows the mean ± SEM for 3 mice. ***P<0.001 in comparison with intact group. +P<0.05 and +++P<0.001 compared with control group. ###P<0.01 compared with ethanol/saline group. Fig. 2. Changes of the hippocampal p-CREB/CREB ratio in cross state-dependent learning between ethanol and nicotine. A) Five groups of animals were used. One group was used as an intact control group. The control group received saline (10 ml/kg) 30 min before training and vehicle (10 ml/kg) before testing. Three groups received ethanol (1 g/kg) 30 min before training and different doses of nicotine (0, 0.3 and 0.7 mg/kg) 30 min before testing. ***P<0.001 compared with saline/vehicle group and +++ P<0.001 compared with ethanol/vehicle group. B) Western blotting of CREB, p-CREB and β–actin proteins are represented in the above panels. The bottom graph shows mean hippocampal p-CREB/CREB ratio calculated from densitometric quantification of the corresponding bands. Each bar shows the mean ± SEM for 3 mice. ***P<0.001 in comparison with intact group. +++ P<0.001 compared with saline/vehicle group. ###P<0.001 compared with ethanol/vehicle group. Fig. 3. Changes of the hippocampal p-CREB/CREB ratio in WIN-induced amnesia and cross state-dependent learning between WIN, ethanol or nicotine. A) One group was used as intact control group. The control group received saline (10 ml/kg) 30 min before training and vehicle (10 ml/kg) before testing. Three groups of animals received pre-training injection of WIN (1 mg/kg, i.p.), and pre-test administration of different doses of WIN (0, 0.1, 1 mg/kg). The other four groups received WIN (1 mg/kg) 30 min before training and different doses of ethanol (0.25 and 1 g/kg, i.p.) or nicotine (0.3 and 0.7 mg/kg), 30 min prior to testing. ***P<0.001 compared with vehicle/vehicle group. ++ P<0.01 and +++ P<0.001 compared with WIN/vehicle group. B) Western blotting of CREB, p-CREB and β–actin proteins are represented in the above panels. The bottom graph shows the mean hippocampal p-CREB/CREB ratio calculated from densitometric quantification of the corresponding bands. Each bar shows the mean ± SEM for 3 mice. ***P<0.001 in comparison with intact group. group. ### P<0.01 compared with WIN/vehicle group. +++ P<0.001 compared with vehicle/vehicle Pre-training treatment A Latency to step-down (s) 350 Saline (10 ml/kg) Ethanol (1 g/kg) +++ 300 Fig. 1 250 200 150 100 * 50 0 *** 0 0.25 Saline (10 ml/kg) 1 Ethanol (g/kg) Pre-test treatment p-CREB 43 KDa CREB 43 KDa ß-actin 45 KDa B + 1.8 *** 1.6 *** 1.4 + 1.2 +++ 1.0 0.8 0.6 0.4 0.2 e /S al in e Sa lin Et ha Sa nol lin (1 e g/ kg )/ Et Eth ha an no ol l ( (1 0. g 25 /k g/ g) / kg ) Et Et ha n ha o no l (1 l( g 1 /k g/ g) kg / ) 0.0 In ta ct p-CREB/CREB ratio ( Arbitrary unit) ### Fig.1 A Pre-training treatment Saline (10 ml/kg) Ethanol (1 g/kg) +++ 300 250 200 150 100 *** * 50 0 0 0.3 Vehicle (10 ml/kg) 0.7 Nicotine (mg/kg) Pre-test treatment p-CREB 43 KDa CREB 43 KDa ß-actin 45 KDa B ### 1.4 +++ *** 1.2 1.0 *** 0.8 +++ 0.6 +++ 0.4 0.2 g) / N Eth ic a ot no in l e (1 (0 g .3 /k m g) g/ / kg E ) N th ic a ot no in l e (1 (0 g .7 /k m g) g/ / kg ) ha n Ve ol hi (1 cl g/ e k Et Sa lin e /V ta c t eh ic le 0.0 In p-CREB/CREB ratio ( Arbitrary unit) Latency to step-down (s) 350 Fig. 2 Pre-training treatment A Latency to step-down (s) 350 Vehicle (10 ml/kg) WIN 55, 212-2 (1 mg/kg) ++ 300 +++ ++ 250 200 150 100 *** 50 0 0 Vehicle (10 ml/kg) 0.1 1 0.25 WIN 55, 212-2 (mg/kg) 0.3 0.5 Ethanol (g/kg) 0.7 Nicotine (mg/kg) Pre-test treatment p-CREB 43 KDa CREB 43 KDa ß-actin 45 KDa B Pre-training: WIN 55,212-2 (1 mg/kg) 1.4 1.2 ### *** *** ### 1.0 +++ 0.8 0.6 +++ +++ +++ +++ +++ 0.4 0.2 g) (1 Et m ha g/ kg no ) l( 0. 25 Et g ha /k g) no l( N 0 ic 5 ot g/ in kg e ) (0 . 3 N m ic g/ ot kg in e ) (0 .7 m g/ kg ) m g/ k IN .1 (0 W IN W Ve hi cl e 0.0 In Ve ta ct hi cl e/ Ve hi cl e pCREB/CREB ratio (an arbitrary unit) 1.6 Fig. 3