The Changes Of μ Opioid Receptors In Acute Stress And The Construction Of Conditional Gene Knockout Mice

Stress induces the body to produce a series of complex physiological and psychological reactions.Excessively strong or persistent stress response often leads to cognitive dysfunction,changes in emotional behavior,and even induces severe neuropsychiatric problems such as anxiety,depression,and post-traumatic stress disorder.A large number of studies have shown that the endogenous opioid system plays an important role in the process of learning and memory.However,it is not clear whether this system participates in the learning and memory impairment caused by acute stress.Previous studies in our laboratory using the Morris water maze test demonstrated that elevated platform stress impaired the spatial reference memory retrieval,which could be reversed by pre-injection of specific μ-opioid receptor antagonist.These results suggest that endogenous opioid system may play a crucial role in the formation of the stress-induced memory impairment,but the changes in p,opioid receptor and its ligand during acute stress are not clear.Furthermore,to address the exact mechanism of μ-opioid receptor mediated stress-induced memory impairment,the specific μ-opioid receptor gene conditional knockout mice are constructed,including the mice specifically lacking μ-opioid receptors in GABAergic neurons(μRGABA),glutamatergic neurons(μRGlut)or astroglial cells(μRAstro).The main results are as following:Part 1 The changes of μ-opioid receptor and endogenous opioid peptide in acute stress.1.After 50 min of stress,the concentration of enkephalin was detected by ELISA,and the results showed that the content of enkephalin in the hippocampus was significantly increased after acute stress.It indicates that acute stress causes memory impairment by enhancing enkephalin release.2.The level of μ-opioid receptor expression and phosphorylation in the hippocampus was detected immidiately after acute stress.The results showed that the acute stress increased the phosphorylation of p,-opioid receptors.The cell surface μ-opioid receptor protein were decreased significantly after 50 min stress but not after 25-min stress,although the total μ-opioid receptor protein content was unchanged.It shows that acute stress activates μ opioid receptor3.The corticosterone level in mice increased after acute elevated platform stress,and pretreatment with μ-opioid receptor antagonist did not change the stress elevated corticosterone level.The results of this study indicate that acute elevated platform stress indeed activates μ opioid receptors and suggest that endogenous opioid system may play a crucial role in the formation of the stress-induced memory impairment.Part 2 Construction and identification of specific μ opioid receptor gene conditional knockout miceThe cell-specific μ-opioid receptor gene knockout mice were constructed based on the Cre/Loxp gene recombination system,and the genotype of mutant mice and their littermates were selected by PCR.The expression of opioid receptors in mutant mice was identified by western blot.The results showed there was decreased expression of μ-opioid receptors in μRGABA-/-andμRAstro-/-mice.There was no significant change of μ-opioid receptor expression in the hippocampus of μRGiut-/-mice,compared to wildtype.The expression of opioid receptors in mutant mice was further identified by west Immunofluorescence.The results showed that the specific μ-opioid receptor gene conditional knockout mice are constructed successfully.The expression of opioid receptors in mutant mice was identified by immunofluorescence.The results showed that the specific p,opioid receptor gene conditional knockout mice are constructed successfully.