ABSTRACT
Objective To investigate the effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice.Methods Twelve healthy C57BL/6 male mice were randomly divided into experimental group and control group,with 6 mice in each group.The mice in the two groups underwent contextual fear conditioning training in the contextual fear response box to establish the contextual fear models,and the freezing time of mice after each footshock was recorded.After 7 days,the mice in the experimental group were fed with food containing Pexidartinib formulation PLX3397,while the mice in the control group were fed with regular food until the end of behavioral experiment.On the 16th day after contextual fear conditioning training,the mice were put back into the contextual fear box for recalling the fear memory without any stimulation.The mice were taken out after 5 minutes of free exploration,and the freezing time of mice during this period was recorded.At 24 h after the fear memory was recalled,the mice were again placed in the contextual fear box,allowing them to explore freely for 3 minutes,and the freezing time of mice during this period was recorded;the fear response of mice was indicated by the percentage of freezing time.After the behavioral experiment,the mice were anesthetized by intraperitoneal injection of pentobarbital sodium,and three mice from the two groups were taken and rapidly opened the chest to expose their hearts,the perfusion needle was inserted into the left ventricle from the tip of heart and then was perfuse with 40 g·L-1 paraformaldehyde(pH=7.4)which precooled at 4 ℃ until the involuntary convulsion disappeared and the body limbs of mice were stiff.The brain tissue of mice was taken and fixed with paraformaldehyde solution,and then placed in sucrose solution for dehydration.The brain tissue of mice was coated with tissue embedding agent.The number of microglial cells in the hippocampus of mice in the two groups was detected by immunohistochemistry.The remaining mice in the two groups were taken and quickly decapitated to obtain brain tissues,and the hippocampus tissues of two sides were separated on ice.The expressions of phosphorylated bromodomain-containing protein 4(pBRD4),gasdermin D(GSDMD)and mixed lineage kinase domain-like protein(MLKL)in the hippocampus of mice were detected by Western blot.Results In the stage of contextual fear conditioning training,the percentage of freezing time of mice in two groups increased with the increase of the number of footshock(P＜0.05),but there was no significant difference in the percentage of freezing time of mice after the first,second,third,fourth and fifth footshock between the two groups(P＞0.05).There was no significant difference in the percentage of freezing time of mice between the two groups during recall period(P＞0.05).The percentage of freezing time of mice in the experimental group was significantly lower than that in the control group at the phase of memory test(P＜0.05).The number of microglia in CA1,CA3 and DG regions of hippocampus of mice in the experimental group was significantly lower than that in the control group(P＜0.05).The relative expressions of pBRD4,GSDMD and MLKL in hippocampus of mice in the experimental group were significantly lower than those in the control group(P＜0.05).Conclusion Microglia inhibitor Pexidartinib can injury the reconsolidation of remote contextual fear memory,which may be related to its inhibition of microglial cell activation and the down-regulation of the expressions of pBRD4,GSDMD and MLKL.
ABSTRACT
【Objective】 To explore the role of environmental enrichment (EE) in paternal stress-induced anxiety and depression-like behaviors in offspring and its potential mechanisms. 【Methods】 Male C57BL/6 mice (F0) were treated with unpredictable chronic mild stress (UCMS) and subsequently mated with normal females to obtain F1 offspring mice. The standard environment (SE) and enriched environment (EE) were administered to F1-UCMS offspring mice during their early life (3-5 weeks of age). Anxiety-like behaviors were detected by open field test (OFT) and elevated plus-maze test (EPM); depression-like behaviors were detected via forced swimming test (FST) and sucrose preference test (SPT) at the age of 8 weeks. The expressions of LIM and SH3 domain protein 1 (LASP1) in the hippocampus of adult F1 offspring mice were detected by Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. 【Results】 Compared to F1 offspring of normal paternal (F1-Nor), F1 offspring mice of the stressed paternal (F1-UCMS) showed significantly anxiety-like behavior with reduced percentage of time spent in the central region of OFT and in the open arm of EPM (P<0.05); mice from the F1-UCMS group showed a significantly increased percentage of immobility in FST and a reduced percentage of sugar consumption in SPT (P<0.01), which demonstrated significant depression-like behaviors. Compared to the SE group, mice in the EE group had an increased percentage of time spent in the central region of the OFT [males: (7.44±0.75)% vs. (14.93±1.74)%, P<0.01; females: (8.89±1.06)% vs. (15.10±1.82)%, P<0.05] and an increased percentage of time in the open arm of EPM [males: (8.09±1.05)% vs. (14.15±1.88)%, P<0.05; females: (9.13±1.14)% vs. (14.04±1.37)%, P<0.05]. This indicated that EE ameliorated anxiety-like behavior in F1-UCMS mice with paternal stress. Compared to the SE group, mice in the EE group had an decreased percentage of immobility in FST [males: (58.63±4.51)% vs. (42.15±3.81)%, P<0.05; females: (57.96±4.19)% vs. (43.25±4.22)%, P<0.05] and an increased percentage of sugar consumption in SPT [males: (50.38±3.47)% vs. (70.39±3.12)%, P<0.01; females: (52.42±2.84)% vs. (69.99±3.55)%, P<0.01]. This indicated that EE ameliorated depression-like behavior in F1-UCMS mice with paternal stress. Hippocampal LASP1 expression was reduced in SE group compared to F1-Nor group (males: P<0.01; females: P<0.05), while LASP1 was increased in EE group compared to SE group (P<0.05) detected by RT-qPCR and Western blotting. 【Conclusion】 EE ameliorates paternal stress-induced anxiety and depression-like behaviors in F1-UCMS mice, and the mechanism may be associated with increased hippocampal LASP1 expression in F1-UCMS mice.