Neuroplastin65敲除通过调控中枢5-HT影响小鼠的焦虑样行为

Neuroplastin65 knockout influences anxiety-behavior by mediating central 5-HT expression in mice

目的:研究neuroplastin65(NP65)敲除对小鼠焦虑样行为的影响并初步探讨其机制。方法:成年NP65敲除(NP65^(-/-))小鼠和同基因型背景的野生型(wild type,WT)小鼠,通过Western Blot鉴定NP65^(-/-)小鼠;用旷场实验、探洞实验和埋珠实验检测小鼠的焦虑水平;ELISA检测小鼠海马、前额叶皮质和纹状体谷氨酸和5-羟色胺(5-hydroxytryptamine,5-HT)的含量;5-HT合成酶色氨酸羟化酶2(tryptophan hydroxylase 2,TPH2)免疫荧光染色观察小鼠脑干中缝核5-HT能神经元;Western Blot检测小鼠脑干TPH2的蛋白表达。结果:与WT小鼠相比,在旷场实验中,NP65^(-/-)小鼠进入中心区的次数显著减少(P<0.001),中心区停留时间显著降低(P<0.001),运动总距离和平均运动速度无显著变化。探洞实验中,NP65^(-/-)小鼠探洞次数显著增加(P<0.001)、探洞时间增多(P<0.05)、后肢竖立次数减少(P<0.01)和排便次数增多(P<0.001)。埋珠实验中,NP65^(-/-)小鼠掩埋潜伏期延长(P<0.05),埋珠个数显著减少(P<0.05)。神经递质检测结果显示:NP65^(-/-)小鼠海马、前额叶皮质和纹状体谷氨酸的含量不变,海马5-HT的含量显著减少(P<0.05),但前额叶皮质和纹状体5-HT的含量没有显著性差异。免疫荧光染色观察到NP65^(-/-)小鼠脑干中缝核TPH2阳性细胞数目显著减少(P<0.05)。Western Blot分析显示:NP65^(-/-)小鼠脑干TPH2的蛋白表达量显著降低(P<0.05)。结论:NP65敲除导致小鼠表现出显著的焦虑样行为,可能与脑干中缝核TPH2表达减少而导致海马5-HT含量降低有关。

Objective： To study the effect of neuroplatin65 （NP65） knockout on the anxiety-like behaviors in mice and related mechanisms. Methods： Anxiety-like behaviors of adult NP65 knockout （NP65-/-） mice and wild type （WT） mice were measured by open field test, hole-board test and marble burying test. The concentrations of glutamate and 5-hydroxytryptamine （5-HT） in the hippocampus, prefrontal cortex and striatum of mice were determined by ELISA. Se- rotonergic neurons in the raphe nuclei of mice were stained by immunofluorescence detection of tryptophan hydroxylase 2 （ TPH2 ）. The expression of TPH2 in the brainstem of mice was also detected by Western Blot. Results： In the open field test, NP65 -/- mice exhibited decreased number of entries into center zone （ P 〈 0.001 ） and spent significantly less time in center （ P 〈 0.001）. No differences in total distance and mean velocity were found between two groups. In the hole-board test, NP65-/- mice showed increased number in head dipping （P 〈 0. 001 ）, spent more time in head dipping （P 〈 0.05 ）, reduced numbers of rearing （ P 〈 0.01 ） and increased defecation （ P 〈 0. 001 ）. In the marble burying test, NP65 -/- mice exhibited a longer latency in digging （ P 〈 0.05 ） and fewer numbers of marble buried （ P 〈 0.05 ）. Neuro-transmitter analysis revealed that ablation of NP65 has no effect on the concentrations of glutamate, but a significant reduction of the concentration of 5-HT in the hippocampus were observed in the NP65 -/- mice （P 〈 O. 05 ）, has no effect on the concentrations of 5-HT in the prefrontal cortex and striatum. Subsequently, the decreased number of TPH2 positive cells in NP65 -/- mice was confirmed （P 〈 0.05 ）. Western Blot analysis showed that the expression of TPH2 was signifi- cantly reduced in the brainstem of NP65 -/- mice compared to WT mice （ P 〈 0. 05 ）. Conclusion： The low 5-HT levels in hippocampus and decreased TPH2 expression in raphe nuclei may be involved in ablation of NP65 induced anxiety-like behaviors in mice.