Effects Of Postpartum Estrogen Withdrawal On Depression-and Anxiety-like Behaviors And The Relevant Hippocampal Neurogenesis Mechanisms

Women tend to exhibit obvious symptoms of depression during times of acute hormonal changes.Postpartum depression(PPD)is a serious medical condition that affects approximately 10%-20%of mothers during the first 4 weeks after delivery.The levels of estrogen(E2)and progesterone(P4)during pregnancy increase 100-fold and 10-fold,respectively,compared with the menstrual cycle levels.When the placenta is expelled during delivery,the levels of E2 and P4 drop off rapidly,and remain low for a prolonged postpartum period.Thus,the onset of PPD is thought to arise,at least in part,from the dramatic fluctuations in the levels of the gonadal hormones during the postpartum period.Studies indicate that postpartum E2 treatments can reduce the depressive symptoms in women with PPD.In addition,the depression-like behaviors in rodents can be prevented by prolonging exposure to high levels of E2.All the results indicate that the postpartum decrease of E2 may play a role in the occurrence of PPD.However,the neurobiological mechanisms underlying E2 withdrawal-induced PPD still remain unclear.The hippocampus plays a role in the etiology of depressive disorders.The subventricular zone(SVZ)in hippocampal dentate gyrus(DG)in the adult mammalian retains the ability to produce and incorporate new neurons,which is refered to adult neurogenesis.A large body of evidence has established that the mammalian brain continuously produces newborn neurons in the hippocampal DG throughout adulthood,with a comparative rate as that of mature granular cells death.These newly generated neurons have similar morphological and functional characteristics as the established ones.They can connect with the hippocampal CA3 zone and the entorhinal cortex,and integrate into the hippocampal synaptic circuits and the neural network.Several lines of evidence suggest that the number of newborn neurons in the hippocampal DG significantly increase during pregnancy and then decline immediately after parturition.In the report of hormone-simulated pregnancy(HSP),the hippocampal cell proliferation was reduced on day 4 after estradiol benzoate(EB)withdrawal.Little studies have reported the relevance of hippocampal neurogenesis and PPD to date.Especially there is no clear report about the involvement of E2 in the regulation of hippocampal neurogenesis in PPD.E2 has been reported to enhance the phosphorylation of the NR2B subunit of the N-methyl-d-aspartate receptor(NMDAr)via Src-mediated signal transduction.The activation of NMDAr is important for the survival and circuit integration of the newborn neurons in the hippocampal DG.However,whether the E2 withdrawal after HSP can decrease the survival of newborn neurons of hippocampus through down-regulation of NMDAr,and whether the NR2B and Src signal transduction paly a role during the occurrence of PPD,both are not clear.The brain-derived neurotrophic factor(BDNF)also plays an important role during the course of adult neurogenesis.Whether the EB withdrawal after HSP can influence the BDNF level in brain needs further study.The present study employed HSP ICR mice as tools and focused on exploring the effects of EB on affective disorders and the associated regulating mechanisms of hippocampal neurogenesis,and also the relevant molecular mechanisms.This study can provide new theoretical basis for the treatment of postpartum women with PPD.Objective1.To explore the effects of postpartum EB withdrawal on affective behavious and hippocampal neurogenesis.2.To study the relevant molecular mechanisms and signal pathways affecting hippocampal neurogenesis after postpartum EB withdrawal.The First PartEffects of postpartum estrogen withdrawal on affective behaviors and hippocampal neurogenesis in mice Materials and Methods1.Animals:Female mice(ICR,Oriental Bio Service Inc.,Nanjing),weighing 32-35 g(15-16 weeks)at beginning of experiment,were used.The animals were maintained in a constant environmental condition(temperature 23±2?,humidity 55 ± 5%,12:12 h light/dark cycle)in the Animal Research Center of Nanjing Medical University.They had free access to food and water before and after all procedures.2.Establishment of the animal models:The mice were bilaterally ovariectomized(OVX mice)under isoflurane anesthesia.The sham operation(control mice)groups underwent the same procedure as the OVX mice,except that the bilateral ovaries were not excised.After 7 days of recovery,the OVX mice were subcutaneously(SC)injected with EB(0.5?g/day)and P4(0.8 mg/day)dissolved in 0.1 ml sesame oil at 0830 h daily for 16 days,and then treated with EB(10?g/day)alone for 7-11 consecutive days3.Experimental groups:(1)Control mice:the sham OVX mice were treated with vehicle(sesame oil).(2)HSP mice:the OVX mice were treated with EB and P4 for 16 days,and then with a high dose of EB alone for 12 days.(3)EW mice:the OVX mice were administered EB/P4 for 16 days,and then a high dose of EB alone for 7 days.(4)OVX mice:the mice were ovariectomized on the same day as the EB withdrawal after HSP in EW mice.(5)OVX/EB mice:the OVX mice were treated with a high dose of EB for 5 days.4.Behavioral examinations(performed on days 25-28 of the experimental procedure):(1)Open field test(OFT):to evaluate spontaneous locomotor activity;(2)Elevated plus maze test(EPM):to evaluate the anxiety-like behavious;(3)Forced swim test(FST):to evaluate the depressive-like behavious;(4)Tail suspension test(TST):to evaluate the depressive-like behavious;5.BrdU immunostaining was used to evaluate the proliferation of hippocampal neural stem cells.The mice were given three intraperitoneally(IP)injections of BrdU(50 mg/kg)with an interval of 8 h on day 1 or 17 of the experimental procesure.The hetereogeneous BrdU immunopositive(BrdU+)cells were examined on day 5 after EB withdrawal to determine the numbers of 28-day-old and 10-day-old BrdU+ cells,respectively.6.The examination of BrdU and NeuN double immune-fluorescence staining were used to evaluate the maturation of newborn neurons..7.The examination of the Doublecortin-positive(DCX+)cells and the density of DCX+ fibers per DCX+ cell were immunostained to quantify the dentritic growth of newborn neurons.Results and Summary1.Affective behaviors:(1)In OFT,the distances traveled by the EW mice,OVX mice,HSP mice or OVX/EB mice were not different from the control mice.(2)In EPM test,the EW mice spent less time in open arms than the control mice.By contrast,the HSP mice,OVX mice,and OVX/EB mice did not show any differences in the time they spent in the open arms compared with the control mice.(3)Compared with the control mice,the EW mice exhibited a significant increase in the immobility in the FST and TST,whereas the HSP mice,OVX mice and OVX/EB mice did not exhibit differences in the immobility time in the FST or TST.The above results indicated that EB withdrawal after HSP induces depression-and anxiety-like behaviors in mice.2.The survival and neurite growth of newborn neurons in hippocampal DG after the EB withdrawal:(1)Compared with the control mice,the numbers of 28-day-old BrdU+ cells and BrdU+/NeuN+ cells were decreased significantly in the EW mice,whereas they were not altered in the OVX mice.By contrast,the numbers of 28-day-old BrdU+ and BrdU+/NeuN+ cells in the HSP mice were increased significantly compared with the control mice,but not the OVX/EB-mice.(2)The number of 10-day-old BrdU+ cells was not altered in the EW mice or OVX mice,whereas it was increased in the HSP mice and OVX/EB mice.(3)The number of DCX+ cells in the EW mice and OVX mice was not different from the control mice,but was increased in the HSP mice or OVX/EB mice.(4)Compared with the controls,the density of DCX+ fibers per DCX+ cell was decreased in the EW mice and OVX mice,but was not altered in the HSP mice or OVX/EB mice.The above results indicated that EB withdrawal after HSP reduces the survival and neurite growth of newborn neurons.ConclusionEB withdrawal after HSP induces the impairment of hippocampal neurogenesis and the occurrence of depression-and anxiety-like behavious in mice.The Second PartThe molecular mechanisms of postpartum EB withdrawal affecting hippocampal neurogenesis in miceMaterials and Methods1.Animals:Female mice(ICR,Oriental Bio Service Inc.,Nanjing),weighing 32-35 g(15-16 weeks)at beginning of experiment,were used.The animals were maintained in a constant environmental condition(temperature 23 ± 2?,humidity 55±5%,12:12 h light/dark cycle)in the Animal Research Center of Nanjing Medical University.They had free access to food and water before and after all procedures.2.Drugs treatment on days 24-28 of the experimental procedure:NMD Ar agonist NMDA was given to EW mice,NR2B inhibitor Ro25-6981 and Src inhibitor dasatinib were given to HSP mice and control mice,all drugs were intraperitoneally(IP)injected daily.Intracerebroventricular(ICV)injection of TrkB inhibitor K252a was given to HSP mice and control mice in right lateral ventricle,control mice or HSP mice were given the injection(ICV)of vehicle(1.0%DMSO)at the same volume and time course.3.Emotional behaviors tests were performed at 3-4 h after the injection of drugs on days 26-28 of the experimental procedure:(1)EPM was used to evaluate the anxiety-like behaviors by the time spent in open arms.(2)FST was used to evaluate the depression-like behaviors by the immobility time.(3)TST was used to evaluate the depressive-like behaviors by the immobility time.4.The survival and neurite growth of newborn neurons in the DG of hippocampus on day 28 of the experimental procedure:(1)The mice were given three IP injections of BrdU(50 mg/kg)with an interval of 8 h on day 1 of the experimental procedure.The BrdU+ cells were examined on day 28 of the experimental procedure.(2)DCX immunostaining was used to examine the density of DCX+ fibers per DCX+cell.5.Western Blot Analysis:Proteins were extracted from the hippocampal regions to examine the levels of p-NR2B,p-Src,and BDNF on day 28 of the experimental procedure.6.Reverse transcription-polymerase chain reaction(RT-PCR):Total RNA was isolated from the hippocampus to detect the BDNF mRNA on day 28 of the experimental procedure.7.To test whether the decline of BDNF level by EB withdrawal affects the behavioral tests and the survival and neurite growth of newborn neurons,the HSP mice and control mice were treated with the repeated ICV injection of TrkB receptor inhibitor K252a or vehicle for 5 days.Results and Summary1.Levels of hippocampal NR2B phosphorylation(p-NR2B)and Src phosphorylation(p-Src):(1)Compared with the controls,the levels of p-NR2B and p-Src were significantly reduced in the EW mice,elevated both in the HSP mice and OVX/EB mice,but were not affected in the OVX mice.(2)Administration of the Src inhibitor dasatinib for 5 days could suppress the increase in p-NR2B levels in the HSP mice and OVX/EB mice.The above results indicate that the level of p-NR2B can be increased after EB use in HSP mice and OVX/EB mice and that the EB-induced NR2B phosphorvlation in the HSP mice and OVX/EB mice depends on the activation of Src.2.Examination of affective disorders:(1)The use of NMDA in EW mice reversed the reduced time spent in the open arms of the EPM,the prolonged immobility time in the FST and TST.(2)The administration of Ro25-6981 reduced the time that the control mice and the HSP mice spent in the open arms of the EPM,and increased their immobility time in the FST and TST.(3)The administration of dasatinib reduced the time in the open arms of the EPM and increased the immobility time in the FST and TST in HSP mice.The above results indicate that an NMDAr aeonist reverses the EB withdrawal-induced affective disorder.while the NMDAr antagonist and the Src inhibitor block the protective effects of EB on affective disorders.3.Examination of the BrdU+ cells and the density of DCX+ fibers:(1)The use of NMDA in EW mice reversed the loss of the 28-day-old BrdU+cells;however,it had no effect on the decreased density of DCX+ fibers.(2)The administration of Ro25-6981 decreased the number of 28-day-old BrdU+cells in the control mice and the HSP mice and also decreased the density of DCX+ fibers in the control mice.(3)The administration of dasatinib decreased the number of 28-day-old BrdU+cells,but had no influence on the density of DCX+ fibers in the control mice and the HSP mice.The above results indicate that an NMDAr agonist reverses the EB withdrawal-induced hippocampal neurogenesis deficits.while the NMDAr antagonist and the Src inhibitor block the protective effects of EB on hippocampal neurogenesis.4.Levels of the hippocampal BDNF levels examined using RT-PCR and Western blot analysis:Compared with the controls,the levels of the BDNF mRNA and BDNF protein were decreased in the EW mice and OVX mice,while they were not altered in the HSP mice and OVX/EB mice.The above results indicate that EB withdrawal after HSP reduces the levels of BDNF.5.The effects of K252a ICV administration on the survival and neurite growth of hippocampal newborn neurons:(1)The density of the DCX+ fibers in the control mice and the HSP mice were decreased after ICV injection of K252a,but the number of 28-day-old BrdU+cells was not affected.(2)Neither the time in the open arms of the EPM nor the immobility time of FST and TST in the HSP mice was affected by the administration of K252a compared to the HSP mice injected with vehicle.(3)Neither the time in the open arms of the EPM nor the immobility time of FST and TST in the control mice was affected by the administration of K252a compared to the control mice injected with vehicle.The above results indicate that the decrease in the BDNF levels following EB withdrawal causes the neurites of hippocampal newborn neuronal cells to become hvpoplastic,but this seems to be insufficient to impair the survival of the mature newborn neurons.ConclusionThe postpartum estrogen withdrawal leads to deficits of the hippocampal neurogenesis and the development of depression-and anxiety-like behaviors,which are associated with the down-regulated levels of NMDAr via the Src signal pathway.The present study indicates that an NMDAr agonist may have a novel therapeutic effect in postpartum depression and protect the capacity of hippocampal neuronal replacement.