Study On The Relationship Between The Expression Of CRHR2 And CRHBP And The Abnormal Behavior Of Rats With Single Prolonged Stress In Adolescence And Adulthood

Objective: Post-traumatic stress disorder(PTSD)is a mental illness that develops from exposure to severe trauma or life-threatening events,which may affect the daily life and social functions of patients.Exposure to early stressful events increases the risk of PTSD in adolescents and also increases the susceptibility of adolescents to PTSD in adulthood.Animal studies have found that rodents that experience early stressful events exhibit anxiety-like behavior,cognitive impairment,and fear during adolescence or later adulthood.The main pathogenesis of PTSD is hypothalamic-pituitary-adrenal axis(HPA)dysfunction.Adolescence is a critical period of brain development and a sensitive period of HPA axis,so it is necessary to give teenagers enough attention and support.Trauma in adolescence can lead to disorders of the HPA axis,causing anxiety or depression in the adolescent or in adulthood.Corticotropin-releasing hormone(CRH)is an initiating hormone of HPA axis and a neurotransmitter,which can coordinate the response of neuroendocrine to stress and enhance signal transduction and induce wakening-like response during stress.CRH is expressed in the amygdala and hippocampus,and plays a crucial role in the regulation of emotion and the function of cognitive basic circuits.CRH in amygdala is thought to be related to fear and anxiety,and CRH in hippocampus can cause stress-related memory changes.CRH exerts its biological effects by acting on two receptors(CRHR1 and CRHR2),while corticotropin releasing hormone binding protein(CRHBP)can compete with CRH receptors to bind CRH,thus blocking CRH from exerting its effects.The role of CRHR2 and CRHBP in abnormal behaviors such as anxiety and depression during stress events is rarely reported.In this study,Single prolonged stress(SPS)animal model was used to simulate PTSD.We studied the behavior of adolescent SPS rats and adult rats who experienced SPS in adolescence,and then explored the relationship between the expression of CRHrelated factors and behavior abnormalities of rats.Methods: 1.Grouping: 28-day-old rats were divided into adolescent control group(TC),adolescent model group(TS)and adult group for modeling in adolescence(SA).70-day-old rats were divided into adult control group(AC)and adult model group(AS).All rats were sampled after the end of behavior test.2.Establish SPS model: The animals were first restrained for two hours,then subjected to forced swim 20 minutes,followed by a 15-minute rest and ether anesthesia.3.Behavior: The rats’ anxiety was gauged by open-field and elevated plus-maze test,the Morris water maze test to assess spatial learning and memory,and environment test and extinction training after conditioned fear training to evaluate short-term fear memory and extinction ability,respectively.4.ELISA was employed to quantify plasma CRH and corticosterone levels5.Immunohistochemical staining was utilized to evaluate the staining intensity of CRHR1 and CRHBP.Immunofluorescence was then employed to detect the staining intensity of CRHR2.6.The mRNA levels of CRH,CRHR1,CRHR2 and CRHBP genes in amygdala and hippocampus were detected by qPCR.7.Western blotting revealed the protein levels of CRHR1,CRHR2,CRHBP,ERK1/2 and p-ERK1/2 in both amygdala and hippocampus.8.The interaction between CRHBP and CRHR2 in rat hippocampus was detected by immunoprecipitation.Results: 1.The ability of spatial learning and memory was impaired in adolescent rats after SPS(TS group),and the anxiety-like behavior was increased in adult rats after SPS and in adult rats with SPS experience in adolescence(AS and SA group).2.Adolescent and adult rats with SPS(TS and AS groups)and adult rats with SPS experience in adolescence(SA group)had enhanced fear memory and impaired fear extinction.3.Basal plasma CRH and corticosterone concentrations increased in adult rats with SPS experience in adolescence and in adult rats after SPS.(SA and AS group).4.No alteration in the amount of CRH mRNA in the hippocampus was observed,yet the amygdala’s CRH mRNA augmented in both adolescent and adult rats with SPS(TS and AS groups).5.No alteration in CRHR1 was observed in the hippocampus and amygdala of adolescent rats after SPS(TS group).CRHR1 expression in the hippocampus of adult rats with SPS experience in adolescence and those after SPS(SA and AS group)decreased,while the expression of CRHR1 in the amygdala increased.The intensity of CRHR1 staining in the hippocampal CA1 region of adult rats was found to be reduced after SPS,as evidenced by immunohistochemical staining.6.In the hippocampus,CRHR2 expression was augmented in both adolescent and adult rats with SPS(TS and AS groups)and adult rats with SPS experience in adolescence(SA group),yet not in the amygdala.Immunofluorescence showed that CRHR2 staining intensity increased in hippocampal CA1 region of SA and AS group 7.The expression of CRHBP in hippocampus was increased in adolescents rats after SPS(TS group)and decreased in adult rats with SPS experience in adolescence and in adult rats after SPS.(SA and AS group).CRHBP was unchanged in amygdala in TS and AS group but increased in SA group.The intensity of CRHBP staining in the hippocampal CA1 region of TS group was observed to be augmented,while the intensity of CRHBP staining in the hippocampal CA1 region of AS and SA group was seen to diminish,as revealed by immunohistochemical staining.8.The phosphorylation of ERK1/2 in hippocampus was increased in adolescent and adult rats after SPS(TS and AS groups)and adult rats with SPS experience in adolescence(SA group).The phosphorylation of ERK1/2 in amygdala was increased in TS group,but not in AS and SA group.9.There is an interaction between CRHBP and CRHR2 in the hippocampus of rats,and the interaction between them is more significant after SPS in adolescent rats.Conclusions: 1.Adolescent rats did not show anxiety-like behavior after SPS stress,but showed impaired spatial learning and memory ability.2.CRHBP in hippocampus may inhibit CRH-mediated anxiety-like behavior and enhance CRHR2-mediated spatial cognitive and learning disabilities.3.The increased expression of CRHR1 in amygdala may be related to anxiety-like behavior in adult rats with adolescent SPS experience.