CDNA Cloning And Identification Of Hippocampal Differentially Expressed Genes Correlated To Morphine Dependent Memory And Application Study In Rats

Drug addiction has two aspects: physical dependence and psychological dependence. The development of psychological dependence is a special aberrant process of learning and memory, learning and memory play a key role in drug addiction, which is the totally new consensus of current international drug addiction research. whether psychological dependence of drug abuser could be reduced or eliminated by blocking the process of long-term memory induced by repeatedly administered drugs to prevent relapse is a new perspective study field in drug addiction research.Exploring how to Block the process of learning and memory associated with drug addiction at genetic level and providing experimental data to prevent relapse is the direction of my doctoral research. The first step is to screen and identify genes correlated closely to the memory of drug addiction, which is also the main content of my study. Altered expression of genes is one of molecular mechanisms that should lead to relatively stable functional changes within CNS neurons, which would eventually lead to long-term behavioral plastic of drug addiction. This is also the molecular basis of memory of drug addiction. So using differentially expressed genes analysis technique to compare the difference of genes' expression in brain tissues associated closely with memory of drug addiction between normal and morphine dependent rats is an effective way to find out objective genes.Relapse can be triggered by three main events:(1) enviornmental stimuli(cues) related to previously drug taking.(2) in sight of or taking drug itself.(3) stress event. At the top of these three factors that can availably elicit relapse is the enviornmental cues related to previously drug taking. In animal experiment for relapse research, environmental stimuli can establish relation with drug injection through conditioned reflection and gain the ability for triggering relapse behaviour, which can induce conditioned place preference(CPP). CPP in nature is a kind of behavioral learning process of central nervous system(CNS),CPP emerging indicates that the memory associated with environment of drug taking has come into being.Firstly we established animal model of morphine dependent rats with CPP. Secondly differentially expressed genes cDNA library of hippocampus between normal and morphinedependent rats with CPP was constructed using suppression subtractive hybridization (SHH) technique. Primary screening of differentially expressed genes was done by reverse Northern hybridization. After observing cell location and expression of related genes by in situ hybridization , we blocked out expression of objective genes by hippocampal injection of antisense -oligoeoxynucleotide(AS-ODN).Then we recorded the changes of CPP behaviour in morphine dependent rats to identify the function of differentially expressed genes and bring experimental data for drug addiction therapy through genetic approach.1. Establishment of animal model of morphine dependent rats with CPPAt this part we combined manufacture of morphine physical dependence and training of CPP tightly, successfully established the animal model of morphine dependent rats with CPP. Furthermore, we have observed the full process of formation,maintenance and disappearance of CPP behaviour in morphine dependent rats. The results are as following:(1) 24h and 48h after morphine injection in rats with increasing dose from 10mg/Kg to 50mg/kg every time, two times a day for five days,the body weight decreased obviously compared with the control, at the same time, withdrawal symptoms such as diarrhea and irritation were emerged,which indicated that physical dependence had been produced successfully.(2) 48h after training,the average dwell time at white box in rats with morphine injection is increased significantly compared with the control, which indicated that the stable CPP behaviour can be induced by five days' training in rats.(3)CPP appeared at 48h after training ,reached the peak at 12d after training, decreased at 24d, complet...