Behavior and Hippocampal Epac Signaling to Nicotine CPP in Mice

Abstract Tobacco use is a major challenge to public health in the United States and across the world. Many studies have demonstrated that adult men and women differ in their responses to tobacco smoking, however neurobiological studies about the effect of smoking on males and females were limited. Exchange protein directly activated by cAMP (Epac) signaling participates in drug addictive behaviors. In this study, we examined the hippocampal Epac signaling in nicotine-induced place conditioning mice. Nicotine at 0.2 mg/kg and 0.4 mg/kg induced a conditioned place preference (CPP) in male and female mice, respectively. After CPP, male mice presented less anxiety-like behavior as demonstrated by an open-field test. The hippocampal Epac2 protein was elevated in both male and female nicotine place conditioning mice. However, Rap1 protein was elevated and CREB phosphorylation was reduced in female nicotine place conditioning mice. Our data provide direct evidence that hippocampal Epac signaling is altered in nicotine-induced CPP mice. Pharmacology manipulation Epac signaling may open a new avenue for the treatment of nicotine abuse and dependence.


INTRODUCTION
Cigarette smoking is a leading cause of preventable death in the United States and worldwide. Approximately 15.5% of American adults are current cigarette smokers, with males (17.5%) having a slightly higher rate of use than females (13.5%) [1]. Although more men than women smoke cigarettes, some studies suggest that female cigarette smokers may be more susceptible to the negative health consequences of tobacco use. For example, females metabolize nicotine faster, more likely develop respiratory disorders, and have more difficulty with tobacco cessation than male cigarette smokers [2]. In addition, nicotine craving may be more severe in adolescent females than in adolescent males [3]. Therefore, it is important to identify the differences in behavior and biology related to nicotine exposure between males and females.
Nicotine acts on nicotinic acetylcholine receptor (nAChR) and affects multiple intracellular signaling molecules. For example, activation of α7 nAChRs increases intracellular cAMP levels via adenosine cyclase activation in hippocampal neurons [4]. Exchange protein directly activated by cAMP (Epac) is a rap1 guanine-nucleotide exchange factor activated by cAMP. Epac modulates GTPase and alters the activities of extracellular regulated kinase (ERK), cAMP response element binding protein (CREB) and gene expression [5]. Besides modulating cardiac and smooth muscle contraction, learning and memory, cell proliferation and differentiation, apoptosis, and inflammation [6], some evidence shows that Epac signaling is altered in response to nicotine exposure. A microarray study showed that nicotine selfadministration increases the medial prefrontal cortex (mPFC) Epac mRNA expression in rats [7].
Another study in humans revealed that Epac gene SNPs (rs2072115 and rs2074533) present modest association with smoking progression to nicotine dependence [8]. To date, the adaptation of Epac in response to nicotine dependence remains elusive.
The hippocampus receives extensive cholinergic innervation and expresses nAChRs at both pre-and postsynaptic compartments [9]. ACh neurotransmission modulates hippocampal-dependent function (e.g. learning and memory). Moreover, nAChR-mediated signaling is involved in the nicotine addictive behaviors [10]. Hippocampal Epac signaling regulates learning and memory [11]. Epac null mutation impairs long-term potentiation (LTP) and this impairment is correlated with a severe deficit in spatial learning and social interaction [12]. Therefore, we explored the interaction between nicotine use and hippocampal Epac signaling in this study in male and female mice.
Preclinical animal models are useful in examining the effect of biological responses on behaviors related to nicotine use and dependence. The conditioned place preference (CPP) task is a classical and widely used procedure to study the conditioned reward effects of addictive drugs including cocaine, morphine, ethanol and nicotine. Nicotineinduced CPP has been reported in rats [13] and mice [14]. In this study, we developed nicotineinduced CPP assay in male and female mice and

Chemicals and reagents
Nicotine was purchased from Sigma-Aldrich

Conditioned place preference test
The construction of CPP box [15] and training [16] have been described previously. In brief,

Open field test
One hour after CPP testing, the mouse was

Western blotting
Right the open field test, randomly assigned mice (n=4) were deeply anesthetized by isoflurane before decapitation. The hippocampus was quickly harvested and homogenized with RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing PMSF and phosphatase inhibitor on ice for 15 min.
Hippocampal lysates were centrifuged at 10,000 ×g for 10 min at 4 ℃ to collect supernatant.

Statistical analysis
Data were expressed as mean ± SEM. All data were normal distribution. CPP data were analyzed by a paired sample Student's t-test.
Locomotor data and WB data were analyzed with an independent sample Student's t-test (SPSS 16.0, IBM, New York). P < 0.05 was considered statistically significant.

Locomotor activity in mice after nicotine place conditioning
Locomotor activity was recorded in an open field arena for 1 hour after the CPP test. In male mice, there was no difference in total distance traveled (t(14)= -1.56, P = 0.14; Fig. 2A Fig. 2A) as compared to vehicle-treated mice (n = 12), but there were no differences in immobility (Fig. 2B), percentage of time in the central zone (Fig. 2C)

Discussion
The present study demonstrates that male and    sexually dimorphic response in rats [17] and mice [18]. Male rats developed a significant CPP to lower doses of nicotine than females, regardless of age [19]. In line with this, we found that female mice need a higher dose of nicotine to induce a CPP. Nicotine also provokes sex-specific behavioral and biological changes [20]. indicate that ovarian hormones, particularly estrogen, play a role in producing sex differences in drug abuse [22]. Future research is necessary to design more effective drug abuse treatment programs and resources that are sex-specific.
Epac2 is expressed predominately in the brain, whereas Epac1 is expressed in many peripheral tissues but its expression in the brain is very low. Our data reveal that the protein level of Epac2, but not Epac1, was increased in both male and female mice after nicotine place conditioning. The role of Epac in nicotine exposure has been previously implicated.
Nicotine self-administration increase the mPFC Epac mRNA expression in rats [7].

Disclosure
The authors have no actual or potential conflicts of interest.