The Effects Of Maternal Rats Exposed To Chronic Unpredictable Stress And Fluoxetine Treatment Before Pregnancy On The Behavior And Neurobiology Of Themselves And Their Progeny

Backgrounds and objective: Maternal stress or depression has a negative impact on the behavior and is linked to an increased risk for affective disorders in their progeny. Follow-up on the progeny of depressed and non-depressed parents shows that parental depression is a strong and consistent risk factor for progeny's major depression disorder and anxiety disorders. And substance dependence in the progeny of depressed parents are about three times as high as that in the progeny of non-depressed parents. Multiple animal experiments have demonstrated that prenatal stess is related to the increased depression-like and anxiety-like behaviors in progeny. At present, most studies consist in pregnant CUS in internal and abroad. Psychical and social pregnant stress significantly heighten the CRH and corticosteroid of patients, to result in the unhealthy pregnancy including premature birth, low birth weight infant and hypertensive disorder complicating pregnancy. Moreover, CUS during pregnancy is close to the postpartum depression, and disbennifit for psychological, behavior and central system development in their progeny. However, only a few studies have investigated the impact of maternal depression before pregnancy on their offspring and the related neurobiological mechanisms in animal models.Fluoxetine, a kind of selective serotonin reuptake inhibitors (SSRIs), was widely used in the treatment of depression or anxiety, which increases the extracellular expression of serotonin by inhibiting its reuptake into the presynaptic cells.The present study was designed to determine the effects of maternal rats exposed to chronic unpredictable stress and fluoxetine treatment before pregnancy on the behavior and neurobiology of themselves and their progeny.Materials and methods: Virgin female Sprague–Dawley rats (SD, 180～220g), were brought into the laboratory from the Lab Animal Center of Shantou University Medical College (Shantou, China) one week before the experiment and housed individually in polycarbonate cages. The room temperature was maintained at 22±1℃with low humidity. Rats were maintained on a 12-h light/dark cycle with food and tap water ad libitum. Twenty-four rats were evenly assigned to one of the four groups, with six in each group: control plus normal sodium intraperitoneal injection group (CON+NS), control plus fluoxetine intraperitoneal injection group (CON+Fluoxetine), chronic unpredictable stress (CUS) exposed plus normal sodium intraperitoneal injection group (CUS+NS), and CUS exposed plus fluoxetine treatement group (CUS+Fluoxetine). NS group and Fluoxetine group received a daily intraperitoneal injection of 2.5 ml/kg normal sodium or 10 mg/kg fluoxetine (4mg/ml) for 21 days. CUS-NS group and CUS-Fluoxetine group received a daily intraperitoneal injection of 2.5 ml/kg normal sodium or 10 mg/kg fluoxetine half an hour before undergoing one of the stressors for 21 days. Rats assigned to the CUS-NS group and CUS-Fluoxetine group were subjected to 21-day CUS according to our previously reported protocol. To be completely unpredictable to the rats, the stressors were applied randomly with the last stressor being food and water deprivation. Sixteen days after the 21-day CUS, females were caged with sexually experienced males of the same strain (ratio 4:1) in the night. Pregnancy was confirmed by sperm-positive vaginal smear (Day 0). All female rats got pregnancy except one in CUS group. The pregnant female rats were housed individually. The pups were weaned at the age of 22±3 days.The second day after the last stressor and four weeks after parturition, the behaviors were test and were recorded with DigBehav Video Analysis System. After the last behavioral test, the rats were anesthetized with ether and were decapitated. The brains were removed from the skull and placed on ice, and the hippocampus was dissected to examine the expression of DCX by Western blot analysis.All the experimental protocols and procedures were approved by the Laboratory Animals Care and Use committee of Shantou University Medical College (Shantou, China).The statistical analysis was performed using Excel 2003 and the"Statistical Package for Social Sciences"(SPSS, Version 13.0). All data were reported as mean±SD. The methods of ANOVA, T-test, nonparametric tests and repeated measures analysis of variance were used for comparing. P<0.05 was considered to be statistical significance, and P<0.01 was deemed to be highly statistical significance.Results:1. Open field test1) Maternal rats:Among groups:Before the CUS, Con+NS group, Con+Flu group, Cus+NS group, and Cus+Flu group had no statistic difference in total distance, central distance and border distance(P>0.05). After the CUS, the total distance in Con+Flu group was higher than that in Con+NS group(P<0.05), and the total distance and border distance in Con+Flu group increased significantly compared with Cus+Flu group(P<0.01).Within groups:In Cus+Flu group, the total distance, central distance and border distance after the CUS decreased significantly when compared to those before the CUS(P<0.01). 2)Progeny: The total distance, central distance and border distance between female and male in one-month-progeny had no statistic difference (P>0.05). The total distance in the Cus+NS progeny were longer than that in the Con+NS progeny (P<0.05); the total distance in the Cus+Flu progeny was significantly longer than that in the Con+Flu progeny (P<0.01); there was no significant difference in the central distance in the Con+NS , Con+Flu, Cus+NS and Cus+Flu progeny (P>0.05);the border distance in the Cus+NS progeny increased significantly compared with the Con+NS progeny (P<0.05); the border distance in the Cus+Flu progeny was significantly longer than that in the Con+Flu progeny (P<0.01); the border distance in the Cus+NS progeny was shorter than that in the Cus+Flu progeny (P<0.05). The gender had effects on the total and border distance in two-month-progeny, with female longer than male (P<0.01); the gender also had a effect on the central distance, with female shorter than male (P<0.05); Maternal treatment had no effects on the total, central and border distance in two-month-progeny in the both genders(P>0.05).2. sucrose consumption test1) maternal rats:Among groups:Before the CUS, there were no significant difference in sucrose intake and sucrose consumption percentage among the four groups(P>0.05,ANOVA). After the CUS, the sucrose intake in the Cus+NS group was significantly lower than that in other three group(sP<0.05,ANOVA);there was no significant difference in the the sucrose consumption percentage in the four groups(P>0.05,nonparametric test).Within groups:In the Cus+Flu group, the sucrose intake after the CUS increased significantly when compared to that before the CUS. 2) Progeny: Gender had effects on the sucrose intake and the sucrose intake in one-month progeny, with female lower than male (P<0.05,independent sample t test); there was no difference in the sucrose consumption percentage between the male and female progeny(P>0.05,independent sample t test). Maternal treatments had no effects on the sucrose intake (P>0.05 , ANOVA) and the sucrose consumption percentage for one-month progeny(P>0.05,nonparametric test).There was no significant difference in the sucrose intake(P>0.05,independent sample t test) and the sucrose consumption percentage(P>0.05,nonparametric test) between the male progeny and the female progeny.The gender and four treatments with maternal rats had no effect on the the sucrose intake(P>0.05 , ANOVA) and the sucrose consumption percentage(P>0.05,nonparametric test).3. Forced swim test1) Maternal rats: There was no difference in rat immobility time in 15-min pretest swim (F(3,20)=0.452, P>0.05), but the immobility time had significant difference in 5-min test swim among the four groups after the 21-day stress (F(3,20)=4.755, P<0.05). When compared by Fisher LSD test for post-hoc, the immobility time in CUS+NS group was longer than that in the other three groups, and there was no difference among CON+NS group, CON+Fluoxetine group and CUS+fluoxetine group.2) Progeny: Gender had no effects on one-month and two-month old progeny in 5-min test swim(P>0.05); Maternal treatments had no effects on one-month old progeny in 5-min test swim(P>0.05); the immobility time in the Cus+NS two-month old progeny was longer than that in the Cus+Flu(P<0.05).4. Morris water maze1) Maternal rats:Among groups:During the place navigation for maternal rats, the escape latency for the Con+NS group, Con+Flu group, Cus+NS group and Cus+Flu group decreased with the training days increasing( P<0.05 ) , but the interaction between the time and group had no difference(P>0.05),showing that the effect of the time didn't change with the different groups; the result of the individual variation , the escape latency for the Cus+NS group was longer than that in the Con+NS group(P<0.05).In the spatial probe for maternal rats, the number of times that the Cus+NS group crossed was less than that in the Con+NS group(P<0.05).2)Progeny: The escape latency of searching hidden platform for one-month and two-month progeny in the place navigation decreased with the training days increasing (P<0.05),but the interaction between the time and gender had no statistic difference(P>0.05),illuminating that the effect of the time didn't change with gender; the result of the individual variation showed that the gender had no effect on the escape latency of searching the hidden platform for one- month and two-month progeny(P>0.05).The escape latency of searching the hidden platform for one-month and two-month progeny in the place navigation decreased with the training days increasing(P<0.05), but there was no statistic difference in the interaction between the time and group(P>0.05), illuminating that the effect of time didn't change with the group; the result of the individual variance displayed that the group had no effec(tP>0.05). In the spatial probe, the gender had no effect on the number of times that one-month and two-month progeny crossing the hidden platform(P>0.05,nonparametric test). There was no statistic difference in the number of times that one-month and two-month progeny in the four groups crossing the hidden platform(P>0.05).5. Western blot1) Maternal rats: After the CUS, there was no statistic difference in the expression of DCX and DCX/actin in the hippocampus among the four groups .2) Progeny:There was no statistic difference in the expression of DCX and DCX/actin between male and female one-month progeny in the hippocampus(P>0.05). The expression of DCX in the hippocampus for one-month progeny in the Cus+NS was lower than that in the Con+NS(P<0.05). Maternal treatments had no effects on one-month old progeny on the expression of DCX/actin in the hippocampus(P>0.05). There was no statistic difference in the expression of DCX and DCX/actin between male and female two-month old progeny in the hippocampus(P>0.05). Maternal treatments had no effects on the expression of DCX and DCX/actin in the hippocampus in two-month old progeny(P>0.05).Conclusions:1. Open field test: 21-day CUS results in depressive-like behavior of maternal rats:the total distance and border distance in the open field increased, fluoxetine of the preventive application reversed anxiety-like behavior resulted from the CUS, resulting in the spontaneous activity decreasing. The gender had no effect on the spontaneous activity of the one-month old progeny. After 21-day CUS, the anxiety-like behavior increased, explaining that the CUS exposing to maternal rats before pregnancy could cause anxiety of one-month old progeny, and the intraperitoneal injection of fluoxetine before pregnancy didn't completely reverse the anxiety-like behavior; however, the spontaneous activity of two-month old female progeny was easier to be anxiety than that of the male progeny,the central distance of the male progeny was longer than that in the female progeny ,we use the central distance as the probe behavior of rat,so we can draw the conclusion that the male progeny was easier to probe than the female progeny. The result of spontaneous activity in one-month and two-month progeny showed that the anxiety-like behavior might be alleviated with the age of progeny increasing.2. Sucrose consumption test: After 21-day CUS the sucrose intake for maternal rats decreasing displayed that maternal rats gave birth to depressive-like behavior.We use sucrose consumption test as anhedonia which is the depressive-like behavior. One-month old female progeny was easier to form the depressive-like behavior such as anhedonia than male progeny. The CUS exposing to maternal rats before pregnancy didn't result in anhedonia of one-month old progeny, and the preventive application of fluoxetine for maternal rats couldn't heighten the sucrose intake in one-month old progeny. There was no significant difference in sucrose intake and sucrose consumption percentage between two-month old male and female progeny, illuminating that the depressive-like behavior wasn't significant difference with the age increasing.3.Forced swim test: After 21-day CUS , the immobility time of maternal rats was significantly longer than that before CUS .There was no significant difference in the immobility time between gender and group in one-month old progeny, but the effect of the group on the immobility time in two-month existed that the action of the CUS exposing to maternal rats before pregnancy made the immobility time prolonging, displaying that the CUS exposing to maternal rats before pregnancy brought depressive-like behavior to progeny. The treatment of fluoxetine couldn't reverse the immobility time prolonging.4. Morris water maze: The CUS exposing to maternal rats before pregnancy induced the spatial memory acquired losing, and at the same time the number of times that rats crossed the platform significantly decreased in the spatial probe, explaining that the distill of memory for maternal rats in the CUS group was injured. The preventive application of fluoxetine might not improve the damaged spatial memory acquired and kept. The effect of the gender on the morris water maze wasn't great. The CUS exposing to maternal rats before pregnancy might make no difference in spatial memory acquired and distilled for progeny, the preventive application of fluoxetine to progeny might not boost the grade in morris water maze.5. Western blot: The regeneration of neuron in hippocampus of maternal rats before pregnancy exposed to CUS was not reduced,maybe it was related with that the give birth to a child and lactation could heighten the emotion of the maternal rat,this effect counteract the regeneration of the neuron in hippocampus decreasing which was induced by CUS. The CUS exposing to maternal rats before pregnancy brought on the regeneration of neuron in hippocampus decreasing for one-month old progeny, and the preventive application of fluoxetine didn't reverse it. The CUS exposing to maternal rats before pregnancy didn't result in the regeneration of neuron in hippocampus decreasing for two-month old progeny, and the ability of regeneration of neuron in hippocampus desreasing led to the difference not significantly along with the age increasing.