Involvement Of Phosphodiesterase-4Mediated Signaling Pathway In Alcohol Dependence

Objective:Alcohol abuse and addiction has become a worldwide problem which compromises both individual and society development. It not only causes harm far beyond the physical and psychological health of the drinker, but also can put their family and the whole society at risk. Alcohol dependence is a complicated and chronic disease, starting from priming drinking to maintaining drinking, finally leading to relapse, which is accompanied by the neuroadaptive changes of neurotransmitters, ion channels and receptors mediated signal pathways. As known, cyclic adenosine monophosphate (cAMP) is one of the most important second messengers, cAMP-mediating signal cascade plays a critical role in the biological and pharmacological effects of alcohol. As shown in a number of studies, acute alcohol exposure elevates adenylyl cyclase activity and increases cAMP level and cAMP dependent signal. Following chronic and repeated drinking, neuroadaptive changes happen to cAMP signal pathway, chronic alcohol exposure desensitize cAMP dependent pathway to alcohol challenge, which might be relative to acute alcohol withdrawal symptoms. Moreover, it has shown that the basal levels of phosphorylation of cAMP-response element binding protein (pCREB) and brain derived neurotrophic factor (BDNF) lower in amygdala trigger the animals to drink more alcohol, which might be due to the anxiety behaviors induced by decreased pCREB and/or BDNF, while reversal the decreased pCREB and BDNF by pharmacological methods or lentivirus can significantly reduce alcohol intake and anxiety. Intracellular cAMP level is regulated by AC and phosphodiesterases (PDEs). So far, AC has been shown to be involved in the adaptive changes of cAMP during acute alcohol exposure, chronic consumption, alcohol withdrawal and relapse, but little is known about the role of PDEs in these processes. Phosphodiesterase-4(PDE4), one of the most important PDEs which regulate cAMP in the brain, is concerned with Alzheimer’s disease, stroke and depression, esc. Rolipram, a selective PDE4inhibitor can ameliorate the cognitive impairments, and reverse anxiety and depressive symptoms, which are the main factors leading to relapse after long-term abstention in alcoholics. Moreover, rolipram has been shown to reduce alcohol intake and preference in mice. Therefore, based upon the theories that cAMP dependent signal pathway plays a critical role in alcohol dependence and PDE4regulates intracellular cAMP level, this project focuses on investigating whether PDE4is involved in the development of alcohol dependence. We used rolipram as an instrumental drug and adopted several animal behavioral tests and biological methods to determine:1. whether PDE4regulates voluntary alcohol drinking and acute alcohol withdrawal sysptoms;2. the role of PDE4in reward effects and positive reinforcement effects of alcohol;3. the role of PDE4in stress induced alcohol relapse.Methods:1. Role of PDE4in alcohol reward and reinforment effects.①Alcohol induced conditioned place preference (CPP) contains three phases:pre-test (1d), conditioning (2-9d) and test (10d). Based upon the results of pre-test, male C57BL/6J mice (8w year-old) were assigned randomly and equally into vehicle+saline (Veh+Sal), vehicle+EtOH (Veh+EtOH), rolipram0.5mg/kg+saline (Rol+Sal), rolipram0.5mg/kg+EtOH (Rol+EtOH), vehicle+saline+rolipram0.5mg/kg (Veh+Sal+Rol) and vehicle+EtOH+rolipram0.5mg/kg (Veh+EtOH+Rol). Veh+Sal, Veh+EtOH, Rol+Sal and Rol+EtOH were used to analyze the effects of rolipram on acquisition of CPP, for which rolipram was given daily during conditioning; while Veh+Sal, Veh+EtOH, Veh+Sal+Rol and Veh+EtOH+Rol to analyze the effects of rolipram on expression of CPP, for which rolipram was only given30min prior to test. The data was shown as duration in the drug paired box/duration of both in drug paired box and in saline paired box.②Alcohol induced locomotor sensitivity includes four phases:baseline (1-2d), acute stimuli of2.0g/kg EtOH (3d), reinforcement with2.5g/kg EtOH (4-13d) and test with2.0g/kg EtOH (14d). According to the baseline results, C57BL/6J mice were randomly and equally assigned into vehicle+saline (Veh+Sal), vehicle+EtOH (Veh+EtOH), rolipram0.5mg/kg+saline (Rol+Sal), rolipram0.5mg/kg+EtOH (Rol+EtOH), vehicle+saline+rolipram0.5mg/kg (Veh+Sal+Rol) and vehicle+EtOH+rolipram0.5mg/kg (Veh+EtOH+Rol). Veh+Sal, Veh+EtOH, Rol+Sal and Rol+EtOH were used to analyze the effects of rolipram on acquisition of sensitivity, for which rolipram was given daily during reinforcement; while Veh+Sal, Veh+EtOH, Veh+Sal+Rol and Veh+EtOH+Rol to analyze the effects of rolipram on expression of sensitivity, for which rolipram was only given30min prior to test. To test whether rolipram affected the absorbrance and metabolism of alcohol, blood was collected to assay the blood alcohol concentration, which was carried out with headspace gas chromatography.2. Role of PDE4in voluntary alcohol drinking and acute alcohol withdrawal and the effects of rolipram. After habituation to the new environment for1w, male C57BL/6J mice (8week-old) were housed individually, and allowed to free access to10%alcohol (v/v) for3w. According to alcohol intake and alcohol preference, mice were randomly and equally assigned to three groups:Vehicle group (1%DMSO) and rolipram treated group with the dose of0.25and0.5mg/kg respectively. Two bottle choice test and drink in dark test were used to determine the effects of rolipram on voluntary alcohol drinking. Open field test and light/dark box test were adopted to investigate if rolipram is effective to anxiety induced by12-h alcohol withdrawal. All the mice were allowed to re-access to10%alcohol for3additional days after the behavioral tests, then were decapitated and the prefrontal cortex, striatum and amygdala were dissected for further molecular analysis. Variants of PDE4mRNA were detected by RT-PCR, while levels of pCREB, pERKl/2and pGSK-3β were analyzed by western blotting. PDE4activity was assayed by HPLC.3. Role of PDE4in stress induced alcohol relapse. After habituation to the new environment for1w, male C57BL/6J mice (8week-old) were housed individually, and allowed to free access to10%alcohol (v/v) for3w. According to alcohol intake and alcohol preference, mice were randomly and equally assigned to different groups: Non-EtOH+Non-stress, Non-EtOH+Stress, EtOH+Non-stress, EtOH+Stress and Rolipram0.5mg/kg+EtOH+Stress (Rol+EtOH+Stress). After alcohol withdrawing for1w, foot shock was performed for15times in10min, two bottle choice was used to assess the effects of acute stress on alcohol drinking behavior. To estimate the effects of rolipram on acute stress induced alcohol drinking, mice were given vehicle or rolipram respectively30min prior to foot shock according to the assignment. Open field test and light/dark box test were preformed to determine whether foot shock affected the locomotor activity and anxious behavior. After all the behavioral tests were finished, mice were decapitated, and NAc, striatum and prefrontal cortex were dissected. PDE4variants mRNA in different brain regions was detected by RT-PCR.Results:1.①Based upon the results we got during the pre-conditioning, the non-preference white box was assigned as the drug-paired box, while the black box as the saline-paired box. Following4cycles of training in8days, mice spent more time in the drug-paired box, which implies mice preferred the drug-paired box. Rolipram with the dose of0.5mg/kg prevented the acquisition of CPP, but did not affect the expression of CPP.②In the alcohol induced locomotor sensitivity, acute alcohol (2.0g/kg) stimuli increased locomotor activities. Mice were reinforced with high concentration alcohol (2.5g/kg) for10days; later low concentration alcohol (2.0g/kg) increased more significantly locomotor activities than the acute stimuli. Treatment of rolipram during the reinforcement phase significantly prevented the acquisition of locomotor sensitivity, but did not show any effects on the expression of locomotor sensitivity. However, rolipram did not affect the blood alcohol concentration. No matter when the rolipram was given. Therefore, rolipram did not affect the absorbance and metabolism of alcohol, but interfered with the rewarding and reinforcement effects of alcohol.2. Training for drinking alcohol for3w significantly elevated alcohol intake and preference to a high and steady level. In two bottle choice, rolipram dose dependently decreased alcohol intake and alcohol preference, but not affect the total fluid intake, implying the decreased-alcohol intake was not due to inhibitive locomotor activity induced by rolipram. Drink in dark imitated the binge drinking in human beings to provide the mice20%alcohol for4h after the lights off. Mice consumed alcohol4.31±0.56g/kg per4h, which were significantly reversed by rolipram at dose of both0.25and0.5mg/kg. In the open field test,12-h alcohol withdrawal induced lesser crossings in the central zone, but had no effects on the total horizontal crossings and rears, comparing with alcohol naive mice. Moreover, both of duration of the light box and transitions between the light and the dark boxes reduced without affecting the latency escaped from light chamber into dark chamber after alcohol withdrawal for12h. Taken together, these results suggest that alcohol withdrawal induced anxiety-like behavior. Only rolipram at dose of0.5mg/kg ameliorated the anxiety-like symptoms. Chronic alcohol exposure significantly elevated PDE4A mRNA in striatum, but had no effects on the others. Chronic alcohol exposure increased PDE4activity in striatum accompanied by decreased phosphorylation of CREB, ERK1/2and GSK-3β, while chronic alcohol drinking had little effects on phosphorylation of CREB and ERK1/2levels in prefrontal cortex. Rolipram at dose of0.5mg/kg significantly reversed the increased PDE4activity in alcohol-drinking mice, also increased the levels of pGSK-3β in striatum without affecting pCREB and pERKl/2.3. Training for drinking alcohol for3w significantly elevated alcohol intake and preference to a high and steady level. The acute stress (foot shock) significantly increased alcohol intake and preference in chronic drinking mice compared with non-stressed mice. Rolipram given30min prior to foot shock significantly decreased the increased alcohol intake and preference induced by stress, but not the total fluid intake. Moreover, stress significantly shortened the duration in the light box and decreased the transitions between the light and dark box without affecting the latency escaped into the dark box, which were much more significant in chronic alcohol drinking mice. While chronic alcohol drinking had no effect on anxiety-like behavior. Neither stress nor chronic alcohol drinking altered the lomocotor activity in mice. Rolipram significantly reversed the anxiety induced by acute stress in chronic alcohol drinking mice without altering the locomotor activity. Foot shock significantly increased PDE4A,4B,4D mRNA in NAc and PDE4A,4D mRNA in striatum, and also increased PDE4A mRNA in prefrontal cortex in mice with alcohol history, which were significantly reversed by rolipram. Either chronic alcohol drinking or stress only elevated PDE4D mRNA in striatum.Conclusion:. cAMP is one of the most important second messengers; cAMP-mediating signal pathway has been involved in alcohol dependence. Our results suggested that PDE4, the most important phosphodiesterase in regulating stabilization of cAMP level in brain, plays the critical role in alcohol dependence. Our results have shown that chronic alcohol drinking significantly changed PDE4mRNA and elevated PDE4activity in striatum, and also decreased the phosphorylation of CREB, ERK1/2and GSK-3β,which might be the underlying mechanism which promotes and maintains alcohol drinking behavior. Levels of PDE4mRNA in brain were altered inconsistently by acute stress after long-term abstention, which was much more potential in NAc and striatum. This might be relative to the increased alcohol intake and preference and anxiety-like behavior induced by acute stress. Rolipram, the selective PDE4inhibitor significantly interrupted the rewarding and reinforcement effects of alcohol in alcohol induced conditioned place preference and locomotor sensitivity, implying enhanced cAMP dependent signal would reverse the rewarding and reinforcement effects of alcohol. Rolipram also decreaed alcohol intake and preference and reversed the anxious state triggered by alcohol withdrawal or acute stress. Therefore, PDE4-cAMP signal pathway plays a great role in alcohol dependence. However, PDE4contains four isoforms, most of which were rich in brain except that PDE4C expresses highly in peripheral tissue. There is little known about which of PDE4variants contribute to the development of alcohol dependence, thus more researches should be done to determine the role of each PDE4isoforms in alcohol dependence. Taken tegather, this study suggests that PDE4-cAMP dependent signal pathway is critical in the development of alcohol dependence. Thus, this study not only exaggerates the role of PDE4-cAMP-PKA signal pathway in alcohol dependence, but also supports PDE4inhibitor as a novel pharmacotherapy for alcohol abuse and dependence.