| Objective: Prenatal morphine exposure will not only seriously affect physical and mental health,but also leads to lasting effects,and even affect the brain structure and behavior in the next generation.Relevant literatures have showed that embryonic passive exposure to morphine in newborn rats there will be changes in behavior and brain neurotransmitters,including morphine psychological dependence;and we have found that exposure to morphine during the third trimester of pregnancy increase the opioid receptors in the accumbens nucleus and amygdala,Both the nucleus accumbens and central amygdala play important roles in brain reward pathways closely related to drug addiction.Therefore,we hypothesized that prenatal morphine exposure could increase the susceptibility to addiction in offspring rats.In this study,conditioned place preference model was used to detect the susceptibility to addiction in offsprings,and further explore the mechanism through detecting NR2 A and NR2 B expression in the nucleus accumbens.The purpose mainly include:(1)To establish morphine exposure model during the third trimester of pregnancy;(2)To explore weight and mortality in offsprings during the third trimester of pregnancy;(3)To explore the effects of addiction susceptibility in offsprings during the third trimester of pregnancy exposure to morphine;(4)To detect the level of morphine exposure in the late pregnancy on the expression of 2A and 2B subunit-containing N-methyl-D-aspartate receptor(NR2A and NR2B)in the nucleus accumbens of offspring rats,and further to study the neurobiological mechanism of increased addiction susceptibility in offspring rats in the third trimester of pregnancy morphine exposure.Methods: The pregnant 14 th Spraguee-Dawley rats were randomly divided into two groups,control group(group C)and morphine group(group M).The offspings of M group were injected with morphine between neck and back subcutaneous during pregnant days 14-20 and every day 2 times(9:00 am and5:00 PM),the first dose of morphine for 2 mg / kg,daily increasing 1 mg/kg,maintain to 6 mg / kg;group C used in much the same way as with physiologicalsaline instead of morphine.(1)the weight of the two groups of offspring at birth,4 weeks and 8 weeks of weight were weighed and the mortality rate was 3 days,4 weeks and 8 weeks after birth;(2)Offsprings were randomly selected from the two groups and normally fed to 8 weeks : 1)The pre-experimental stage(1-3d)Offsprings in each group were in box on both sides to free movement for 15 min,once daily,consecutive for 3 days to adapt the environment of experiment box;the mean value of the residence time in the 2th and 3th days to record as CPP baseline value(in seconds).Whichstay long time for preference box,stay short time for the medicine box with(after the rats injected with morphine into this side)to train rats.2)Training stage(4-10d)Each offspring rats was established by subcutaneous injection of morphine into medicine side for 45 min and in 8h intervals with the same method of injection with the same volume of saline into the preference side for 45 min.The dose of morphine was constant 3mg/kg,continuous training for 7 days.3)Testing stage(11d)Each group offsprings were freely active on both sides of the box for 15 min,the retention time in the medicine side was recorded as CPP testing value(in seconds).The susceptibility of the offspring rats can be evaluated by CPP score(CPPtesting value minus CPP baseline).According to the size of the CPP score and theproportion in 20%,60% and 20%,group M was divided into the high preferencegroup(H),the middle preference group and the low preference group(L).In order to compare the differences in susceptibility to morphine addiction,we compared the high preference group(H)and the control group(L)with the control group(C).(3)Ten offspring rats were selected from H,L,C group and sacrificed after the test,then we removed the brain and the nucleus accumbens was isolated.To detect the expression of NR2 A and NR2 B in the nucleus accumbens by immunohistochemistry.To explore the expression of NR2 A and NR2 B in the nucleus accumbens by westernblotting.Results:(1)The weight of offsprings in each group was found: the birth weight of M group was less than N group(P<0.01),and the weight of M group was less than N group(P<0.01)at 4 weeks and 8 weeks after birth.The mortality rate of M group was significantly higher than that of group N(P<0.01)which born for 3 days,4 weeks and 8 weeks.(2)After continuous morphine training for 7 days,the residence time of medicine side in offsprings of group M significantly prolonged compared with group C(P < 0.01);CPP score of group H was significantly higher than that of group L and group C(P < 0.01,P < 0.01),and the difference was statistically significant.(3)Immunohistochemistry showed that the expression of NR2 A and NR2 B in group H was significantly higher than that of group L and group C(P<0.01).(4)Westernblotting suggested that the expression of NR2 A and NR2 B in group H was significantly higher than that of group L and group C(P<0.01).Conclusions:(1)Late gestational morphine exposure can lead to the decrease of body weight and increase the mortality in different stages of the growth and development process in offsprings.(2)Late gestational morphine exposure has a significant enhancement effect on the susceptibility to addiction in offsprings.(3)Increased susceptibility of morphine-induced addiction was found in offspring rats undergone late gestational morphine exposure may be associated with up-regulated NR2 A and NR2B level. |
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