Effects Of AC₃ In The Main Olfactory Epithelium On Depression,Anxiety,and Learning And Memory In Mice

With the increasing pressure of work and life and the growth of the elderly population,the incidence of anxiety,depression and learning and memory impairment has increased year by year,but the clinical treatment was not satisfactory.Therefore,the research on its pathogenesis has become one of the focuses of brain science research.Some studies have suggested that depression,anxiety and learning and memory impairment are closely related to the dysfunction of the hippocampus（HIP）.Meanwhile,olfactory dysfunction is also related to these diseases.It has been reported that type III adenylate cyclase（AC₃）is expressed in the main olfactory epithelium（MOE）and HIP,and plays an important role in olfactory signal transduction and olfactory nerve development.At the same time,there is a neuroanatomical and functional relationship between MOE and HIP.Literatures and our previous work have shown that AC₃ knockout mice exhibit depression-like behavior and learning and memory impairment.However,it is unclear whether AC₃ in MOE is involved in the pathophysiology of these diseases.In this paper,we used the AC₃ gene knockout mice（mutant,MUT mice）and the wild-type mice（WT mice）to evaluated their anxiety-like and depression-like behaviors by a series of behavioral paradigms.Then,5%zinc sulfate were infused to bilateral nasal of the wild type of C57 mice（ZnSO₄ mice）to destroy the MOE of mice,and control mice were infused normal saline to bilateral nasal（NaCl mice）.After the MOE of mice were destroyed evaluating with HE staining,the behaviors of anxiety,depression and learning memory were investigated by corresponding behavioral experiments.The whole MOE was damaged by intranasal injection of ZnSO₄,but which protein damage is responsible for the abnormal behaviors of ZnSO₄ mice?Does AC₃ in MOE plays a key role in anxiety,depression and learning and memory impairment?In this paper,CRISPR/cas9-AAV nasal perfusion technology was used to specifically knock down AC3 in C57 mouse(AC_3KD/MOE mice),and C57 mice perfused with empty vector virus in the nasal cavity as the control mice（NC mice）.Western blotting was used to evaluate the expression of AC₃ in MOE and HIP after two months of virus perfusion.The above behavioral experiments were carried out to evaluate the effect of specific knock-down of AC₃ in MOE on related behaviors after confirming that AC₃in the MOE was significantly knocked down.In order to explore the mechanism of the above diseases caused by knock-down or deficengcy of AC₃ in MOE,the content of monoamine neurotransmitter,dopamine receptor（Drds）,tyrosine hydroxylase（TH）and N-methyl-D-aspartate receptor 2B subunit（GluN2B）in the hippocampus was evaluated.The results and conclusions are as follows:1.Compared with WT mice,AC₃ gene knockout had no significant effect on the spontaneous activity of mice;the struggling time of MUT mice were decreased significantly in the tail suspension test（TST）and the forced swimming test（FST）;the immobility time was prolonged significantly in FST;the time spent in international zone was shortened significantly in the social interaction test（SIT）;the numbers of interaction with unfamiliar mouse was decreased significantly;the distance travelled in the dark box was prolonged and the distance travelled in the light box was significantly shortened significantly in the light/dark transition test（L/DTT）;the latency to find the food was significantly prolonged and the food consumption in 5 min was significantly decreased in the novelty-suppressed feeding test（NSFT）;the content of neurotransmitter dopamine（DA）in hippocampus was significantly decreased;the mRNA levels of TH,Drd2,Drd3,Drd4 and GluN2B were significantly decreased;the protein levels of TH,Drd1 and Drd3 were significantly decreased.The results showed that AC₃ gene knockout not only led to depression-like behavior but also anxiety-like behavior in mice.The decrease of TH,Drds and GluN2B expression in the hippocampus was one of the causes of depression and anxiety caused by AC₃ gene knockout.2.Compared with NaCl mice,the structure of MOE in ZnSO₄ mice was obviously damaged,the expression of AC₃ in MOE was significantly decreased;the latency to find the buried cookies in ZnSO₄ mice was significantly prolonged,the struggling time were significantly shortened in TST and FST;the time spent in international zone was shortened significantly in SIT,the numbers of interaction with unfamiliar mouse was decreased significantly in SIT;the distance travelled in the dark box of light/dark transition test（L/DTT）was prolonged significantly,the distance travelled in the light box was significantly shortened in L/DTT;the latency to find the food was significantly prolonged,the food consumption in 5min was significantly decreased in NSFT;the discrimination index of new objects was decreased significantly in the novel object recognition test（NORT）;In the 4th day of the Morris water maze test,the escape latency was significantly lower than that of the NaCl mice,the time of searching in the quadrant of the original platform was significantly shortened,and the numbers of crossing the original platform was significantly reduced.The content of neurotransmitter DA in the HIP was significantly reduced;the mRNA levels of TH,Drd3,Drd5 and GluN2B were significantly reduced in the HIP;the protein levels of Drd3 and GluN2B were significantly reduced in the HIP.The above results confirmed that ZnSO₄ could cause depression-like behavior and learning and memory impairment in mice after MOE destroyed.Injuried MOE may has effect on depression,anxiety and learning and memory related behaviors by down-regulating the dopaminergic nervous system function and the expression of downstream GluN2B in the HIP.3.Compared with NC mice,the expression of AC₃ was significant decreased in in MOE of AC_3KD/MOE mice,but there was no significant change in the expression of AC₃ in the HIP.The time of finding buried cookies was increased significantly in AC_3KD/MOE mice,the immobility time was increased significantly in TST,the struggle time was decreased significantly in FST,the numbers of interaction with unfamiliar mouse was decreased significantly in SIT.In addition,the entries to the open arms in the elevated plus maze test（EPMT）were significantly reduced,the distance travelled in the light box was significantly shortened and the numbers of transition were significantly reduced in L/DTT;the latency to find the food was significantly prolonged in the NSFT;the correct reaction rate was decreased in Y maze test（YMT）,the discrimination index of new objects was decreased significantly in the NORT;the latency to step down was shortened and the number of errors was increased in SDPTA;the escape latency was significantly lower than that of the NC mice on the 4th day,the time of searching in the quadrant of the original platform was significantly shortened,and the numbers of crossing the original platform was significantly reduced in Morris water maze.The protein expressions of TH,Drd3 and GluN2B were significantly decreased in the HIP.The results showed that the down-regulation of AC₃ in MOE can lead to anxiety,depression and learning and memory impairment in mice,and the mechanism is related with the down-regulation of TH,Drd3 and GluN2B protein expression in the MOE of the AC_3KD/MOE mice.In conclusion,we used AC₃ gene knockout mice,ZnSO₄ damage MOE mice and specific AC₃ gene knockdown in MOE mice to evaluate the anxiety,depression,learning and memory related behaviors.The content of neurotransmitter in mice HIP and the expression levels of TH,Drds and GluN2B were estimated.The results showed that AC₃ in MOE plays a vital role in the pathogenesis of anxiety,depression and learning and memory impairment and we discussed preliminarily the mechanism of the dopaminergic nervous system and GluN2B in the pathophysiology of these diseases.The results of this study promote people’s understanding of the pathogenesis of diseases related with the abnormal behaviours（such as Alzheimer’s disease,depression,anxiety）,and provide theoretical basis for the design of new treatment programs for these diseases.