Effect And Mechanism Of DRN-PFC Pathway On Depression-Like Behavior In Hypothyroidism Rats

Background:Hypothyroidism can be caused by a variety of reasons,such as lack of iodine uptake in the living environment and autoimmune thyroiditis(Hashimoto’s thyroiditis).Especially in recent years,with the popularity of physical examination,hypothyroidism in patients with thyroid-related diseases after thyroidectomy has become more frequent.Numerous studies have shown that patients with hypothyroidism are prone to apathy,social dysfunction and other depressive behaviors,which made the quality of life of patients decreased and difficult to integrate into social life.In addition,hypothyroidism also caused changes in brain areas,especially decreased blood flow in brain areas associated with emotion such as the prefrontal cortex(PFC).We have previously demonstrated that thyroidectomy(TX)-induced hypothyroidism rats exhibit depression-like behaviors.These behaviors may be achieved by inhibiting the synthesis and release of 5-hydroxytryptamine(5-HT)within the dorsal raphe nucleus(DRN).As a neural pathway that played an important role in the regulation of depression,DRN-PFC pathway may be the direction of future research in the treatment of hypothyroidism and depression.Objectives:Based on our previous study that depression-like behavior in hypothyroidism may be caused by inhibition of 5-HT synthesis and release in DRN,we continued to observe depression-like behavior in TX rats and changes in activity of DRN and PFC neurons.We intended to investigate whether TX rats caused depression-like behavior through DRN-PFC pathway and explore the mechanism,so as to provide clinical therapeutic targets for depression-related directions caused by hypothyroidism.Methods:(1)Behavioral changes in TX rats: Male Wistar rats were randomly divided into Sham group and TX group.The body weight of each group was measured 6 weeks after operation.We used the open-field test(OFT),the forced swim test(FST),the sucrose preference test(SPT)and Morris water maze test(MWM)to measure the depressionlike behavior of rats in both groups.(2)Depressive behavior and changes in DRN and PFC activity in TX rats after intraperitoneal injection of thyroid hormone(TH): Experiment was divided into three groups,respectively for the Sham,TX and TX + TH group,including TX + TH group for 2 weeks intraperitoneal injection of thyroid hormone(6mg/100g),Sham and intraperitoneal injection of TX group equal volume saline solution.The behavioral changes of OFT,FST and SPT in three groups were observed.The rats in the three groups were put to death under deep anesthesia after the behavioral test.Half of the rats were taken DRN and PFC,and the expressions of c-fos and BDNF in the DRN and PFC were detected by western blot(WB).The other half was made into paraffin sections while c-fos positive neurons in DRN and PFC were assessed by immunohistochemical staining(IHC).(3)Depression-like behavior and PFC activity changes in TX rats after local chemical activation of DRN: The experiment was divided into three groups,namely Sham+m Cherry,TX+m Cherry and TX+h M3 Dq group.In the m Cherry group,the virus r AAV-h Syn-m Cherry-WPRE-h GH p A was given.The h M3 Dq group was given r AAV-h Syn-h M3D(Gq)-m Cherry-WPREs virus.After 3 weeks of virus infection,the rats in the three groups were intritoneally injected with Clozapine N-oxide(CNO,5mg/kg)for 1 hour.The behavioral changes of OFT and FST in the three groups were observed.The activation of neurons in DRN was observed under fluorescence microscope in frozen sections.The expressions of c-fos and BDNF in rat PFC were detected by WB,and the number of c-fos positive neurons in PFC was calculated by IHC.Results:(1)The TX rats showed depressed-like behavior,which was shown as follows: the weight of the TX group decreased compared with the Sham group after surgery,the total travel distance of the TX group decreased in OFT,the immobility time of the TX group increased in FST,the sucrose preference and sugar water intake decreased in SPT,the escape time increased in MWM,and the number of platform crossing decreased.(2)Intraperitoneal injection of TH can improve the depression-like behavior of TX rats,and reverse the decline in DRN and PFC activity caused by hypothyroidism.Specifically,in OFT,the total travel distance of TX group was reduced compared with that of Sham group or TX+TH group,while the total travel distance of Sham group and TX+TH group was not significantly different.In FST,the immobility time of TX group was increased compared with that of Sham group or TX+TH group,but there was no significant difference in immobility time between Sham group and TX+TH group.In SPT,sugar preference and sugar water intake decreased in TX group compared with Sham group or TX+TH group,but there was no significant difference in sugar preference and sugar water intake between Sham and TX+TH groups.WB results showed that the expression levels of c-fos and BDNF in DRN and PFC in TX group were lower than those in Sham group or TX+TH group,and the difference was statistically significant,while the expression levels of c-fos and BDNF in DRN and PFC in Sham group and TX+TH group were not significantly different.IHC showed that the number of c-fos positive neurons in DRN and PFC decreased in TX group compared with Sham group or TX+TH group.Similarly,there was no significant difference in the number of c-fos positive neurons in DRN and PFC between Sham group and TX+TH group.(3)Local chemical activation of DRN can improve the depression-like behavior and increase the activity of PFC in TX rats,which s shown as follows: Compared with Sham+m Cherry or TX+h M3 Dq,TX+m Cherry and TX+h M3 Dq had less total travel distance in OFT,while there was no significant difference in total travel distance between Sham+m Cherry and TX+h M3 Dq.TX+m Cherry group showed an increased immobility time in FST compared with Sham+m Cherry group or TX+h M3 Dq group,while there was no significant difference in immobility time between Sham+m Cherry group and TX+h M3 Dq group.Activation of DRN neurons was observed under fluorescence microscopy.WB results showed that the expression levels of c-fos and BDNF in PFC in TX+m Cherry group were lower than those in Sham+m Cherry or TX+h M3 Dq group,and the difference was statistically significant.There was no significant difference in the expression levels of c-fos and BDNF between Sham+m Cherry group and TX+h M3 Dq group.IHC showed that the number of c-fos positive neurons in PFC decreased in TX+m Cherry group compared with Sham+m Cherry group or TX+h M3 Dq group.Similarly,there was no significant difference in the number of c-fos positive neurons in PFC between Sham+m Cherry group and TX+h M3 Dq group.Conclusions:(1)Hypothyroidism can lead to depression-like behavior in rats.(2)TH supplementation can improve depression-like behavior in hypothyroidism rats and reverse the decline in DRN and PFC activity caused by hypothyroidism.(3)Local chemical activation of DRN can improve depression-like behavior and increase PFC activity in hypothyroidism rats.(4)Depression-like behavior in hypothyroidism may be caused by decreased PFC activity due to DRN inhibition.