| BackgroundOpioid dependence is a chronic and recrudescent encephalopathy, and is very harmful to organism. The problems of public health and society caused by opioid abuse are striking gradually. Relapse is the significant character of drug addiction and the key to treatment of drug addiction. The more and more proof indicates that main cause of relapse is permanent existence of addiction memory.Consolidation is the transition from short-term memory to long-term memory. Traditionally, consolidation has been viewed as an event that occurs once in the life of a memory, and consolidated memories are stable and highly resistant to disruption by amnestic influences such as protein synthesis inhibitor. But Recent research indicates that when consolidated memories are active they will become temporarily labile again and require a further consolidation process referred to as reconsolidation.ObjectiveAccording to knowledge about neuropsychology and psychotherapy, referring to the reconsolidation theories and methods about memory, through the model of conditional place preference(CPP), we used durg and shock to change the behavior of CPP model rats, then studied the glucose metabolism of the brain areas correlated with relapse, to search for the possibility of changing the addiction memory correlative with conditional cues(CS).Methods1. Drug treatment test: 24 adult female wistar rats were randomized into 3 equal groups: CPP model, drug treatment and normal control groups. The baseline preferences of rats were tested before training(d2), then rats accepted CPP training in CPP shuttle box for 6 consecutive days(d3-d8): rats were injected with morphine(rats of normal control group were injected with saline), then were placed into the white compartment for 1 hour in the morning, and in the afternoon were injected with saline then placed into the black compartment for 1 hour. In d9, the CPP value of every rat was tested. In the following 6 days(d10-d15), rats of drug treatment group were fed Qingfeng capsule solution in the morning. Finally, CPP value of every rat was tested again in d16, d21 and d26.2. Shock treatment test: 24 adult female wistar rats were randomized into 3 equal groups: CPP model, shock treatment and normal control groups. The baseline preferences of rats were tested before training(d2), then rats accepted CPP training in CPP shuttle box for 6 consecutive days(d3-d8):rats were injected with morphine(rats of normal control group were injected with saline), then placed into the white compartment for 1 hour in the morning, and in the afternoon were injected with saline then placed into the black compartment for 1 hour. In 69, the CPP value of every rat was tested. In d10, rats of shock treatment group accepted shock treatment(100v, 25mA, 1s) in the morning. Finally, CPP value of every rat was tested again in d16,d21 and d26.3. Glucose metabolism test: 18 adult female wistar rats were randomized into 6 equal groups: CPP model, shock control, shock treatment, blank control, drug treatment and normal control groups. The rats of CPP model, shock treatment, drug treatment and normal control groups accepted CPP training and were treated by the same methods used in drug and shock treatment test. At the same period, the other rats did not accepted any treatment except that shock control group's rats accepted shock treatment (100v, 25mA, 1s) in d10. Then in d26, after were fasted for 8 hours, all rats except blank control group were injected through vena caudalis with 0.5ml 2—NBDG (0.25mg/ml) per rat and placed into the white compartment for 0.5 hour. After every rat was anesthetized in chloral hydrate, rat's brain was harvested and freezed quickly with liquid nitrogen, then cut into sections of 10 um in thickness with a freezing microtome. The fluorescent pictures of sections were taken using Kodak 2000MM imaging system and analyzed to obtain mean gray scale of area of interest (MAOI) and of the picture(MTOTAL), in this experiment, areas of interest are caudate putamen(CPu), nucleus accumbens(NAc), septal nucleus(SPN), cornu ammonis (CA), cingulate gyrus (CG), amygdala (AMG) and ventral tegmental area (VTA).4. Statistical method: Statistical analysis was performed by using the program SPSS13.0. In all statistical tests, significance level was set to p<0.05. The CPP data of all groups and each group were analysed by repeated measures ANOVA, and the data at each time were analysed by one-way ANOVA respectively, all ANOVA followed by multiple post-hoc tests (Fisher's LSD). Imaging data, (MA0I-MT0TAL)/MT0TA L as the dependent variable, were analysed by two-way ANOVA, and then performed one-way ANOVA in each AOI followed by multiple post-hoc tests (Fisher's LSD).Results1. CPP Behavior of drug treatment: The data satisfied Mauchly's test ofsphericity (P=0.623). Repeated measures ANOVA revealed significant effect of time or grouping factor (F=122.101, P =0.000; F=193.093, P=0.000) and crossover effect of time and grouping factor (F=52.186, P=0.000). CPP of drug treatment group had significant decrease compaired with CPP model group.Time factors had significant effect in all groups(F=5.872, 238.689,58.301; P= 0.001, 0.000, 0.000). In normal contrl group, the CPP value presented increase tendency, the increase in d21 or d26 compaired with d2 were all statistically significant(P=0.022, 0.001). In CPP model group, CPP value in d9 was the peak, then presented decrease tendency, and the decrease in d21 or d26 compaired with d9 were all statistically significant(P=0.012, 0.001), but in d9, d16, d21and d26 still more than d2 significantly. In drug treatment group, CPP value in d9 was the peak, and d9 as the reference, the decrease in d16, d21 and d26 were all statistically significant, but when d2 as the reference, the CPP expression in d26 presented no significant difference(P=0.141).In d2, the difference among the groups was not significant(F=0.196, P=0.823), indicating that initial grouping was balanced. In the other testing days, there were all significant differences among the groups(F= 177.136, 173.014, 113.99, 64.848; P were all 0.000). Multiple post-hoc tests (Fisher's LSD) indicated that, in d9, the difference between CPP model and drug treatment group was not significant (F=0.124), the CPP walue of CPP model or drug treatment group was significantly more than normal control group(P were all 0.000); in d16, d21 or d26, drug treatment or normal control group was significantly less than CPP model group(P were all 0.000), and the P values between drug treatment and normal control group were 0.001, 0.007, 0.735 respectively.2. CPP Behavior of shock treatment: The data satisfied Mauchly's test of sphericity (P=0. 272). Repeated measures ANOVA revealed significant effect of time or grouping factor (F= 174.917, P = 0.000; F = 267.121, P = 0.000) and crossover effect of time and grouping factor(F= 120.470, P=0.000). CPP of drug treatment group had significant decrease compaired with CPP model group.Time factors had significant effect in all groups(F=5.872, 238.689, 58.301; P= 0.001, 0.000, 0.000). In normal contrl group, the CPP value presented increase tendency, the increase in d21 or d26 compaired with d2 were all statistically significant(P=0.022, 0.001). In CPP model group, CPP value in d9 was the peak, then presented decrease tendency, and the decrease in d21 or d26 compaired with d9 were all statistically significant(P=0.012, 0.001), but in d9, d16, d21and d26 still more than d2 significantly. In drug treatment group, CPP value in d9 was the peak, and d9 as the reference, the decrease in d16, d21 and d26 were all statistically significant, but when d2 as the reference, the CPP expression in d26 presented no significant difference(P=0.141).In d2, the difference among the groups was not significant (F=0.528, P= 0.597) , indicating that initial grouping was balanced. In the other testing days, there were all significant differences among the groups(F= 212.699, 465.762, 256.460, 108.043; P were all 0.000). Multiple post-hoc tests (Fisher's LSD) indicated that, in d9, the CPP value of CPP model or shock treatment group was significantly more than normal control group(P were all 0.000); in d16, d21 or d26, shock treatment or normal control group was significantly less than CPP model group(P were all 0.000).3. Imaging analytic result: The difference among groups or brain areas was all statistically significant(F=24.783, P=0.000; F= 186.352, P=0.000), and there was significant crossover effect between grouping and brain area factors(F= 16.017, P=0.000). The simple effect of grouping factor in each brain area was as follows:CPu: The relative fluorescence intensity of CPP model or shock control group was all higher than normal control group (P=0.000, 0.017), but for shock treatment, blank control, drug treatment groups, the differences were not significant compared with normal control group(P=0.471, 0.896, 0.384); compared with CPP model group, shock treatment and drug treatment groups were all lower(P were all 0.000).NAc: The relative fluorescence intensity of CPP model was higher than normal control group(P=0.000, 0.017), but for the other groups, the differences were not significant compared with normal control group(P=0.471, 0.896, 0.384); shock treatment and drug treatment groups were all lower than CPP model group (P=0.000, 0.037).SPN: The relative fluorescence intensity of CPP model was higher than normal control group(P=0.000, 0.017), but for the other groups, the differences were not significant compared with normal control group(P=0.471, 0.896, 0.384); compared with CPP model group,shock treatment and drug treatment groups were all lower(P = 0.000,0.001).CG: The relative fluorescence intensity of CPP model, shock control and drug treatment group was all higher than normal control group(P=0.018, 0.004, 0.001), but for shock treatment and blank control groups, the differences were not significant compared with normal control group(P=0734, 0.920); drug treatment group was lower than CPP model group (P= 0.040).CA: The relative fluorescence intensity of CPP model, shock control, shock treatment and blank control groups was all higher than normal control group (P=0.000,0.028,0.011,0.000), but for drug treatment group, the difference was not significant compared with normal control group (P=0.524); compared with CPP model group, shock treatment and drug treatment groups were all lower(P were all 0.000).AMG: The relative fluorescence intensity of CPP model or shock control group was all lower than normal control group (P=0.000, 0.047), but for shock treatment, blank control, drug treatment groups, the differences were not significant compared with normal control group(P=0.292, 0.206, 0.377); compared with CPP model group, shock treatment and drug treatment groups were all higher(P=0.000, 0.001).VTA: The relative fluorescence intensity of CPP model or blank control group was all different significantly compared with normal control group(P=0.012, 0.000), but for shock treatment, shock control, drug treatment groups, the differences were not significant (P=0.271, 0.702, 0.954); compared with CPP model group, shock treatment and drug treatment groups were all lower(P=0.004,0.010).Conclusions1. Normal rats' aversion to white box decreased statistically as time extension.2.After CPP training for 6 days, CPP model rats expressed significant preference for white box, indicating the model-making succeeded. The CPP model rats' preference for white box decreased in 18 days, but still was significant.3. The preference's expression of CPP model rats was suppressed after rats had accepted Qingfeng capsule or shock treatment, and the suppression lasted a long time after the treatment finished.4. In d26,when CS was presented, the glucose metaboilic levels of CPP model rats in CPu,NAc,SPN,CG,CA and VTA were higher, but in AMG were lower than normal rats, indicating that glucose metabolism of model rats in each brain area were abnormal. The glucose metaboilic levels of the CPP model rats that had accepted shock treatment increased in CPu,NAc,SPN,CG,CA,VTA and decreased in AMG, showed that the abnormity of glucose metabolism was corrected, and the glucose metabolism of AOI eccept CA got back to the levels of normal rats. The levels of rats that had accepted drug treatment increased in CPu,NAc,SPN,CA,VTA and decreased in AMG, showed that the abnormity of glucose metabolism of AOI except CG was corrected and got back to the levels of normal rats.5. Taken together, these results suggested that: After the CPP model rats accepted shock or drug treatment, the rats' behavior and glucose metabolism of AOI went back to levels of normal rats, and changes lasted a long time after the treatment finished. So we could think well-foundedly that the treatment effects were based on the changes of addiction memory. |
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