小鼠前额叶皮层出生后发育过程中篮状细胞的电生理特性和基因表达变化

Characterization of electrophysiological properties and changes in gene expression in basket cells during the postnatal development of mouse prefrontal cortex

篮状细胞是一类表达小清蛋白(parvalbumin,PV)的抑制性中间神经元。本文利用增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记篮状细胞的G42转基因小鼠,研究小鼠前额叶皮层出生后发育过程中篮状细胞的电生理特性和基因表达变化。麻醉下取出生后第7天(postnatal day 7,P7)、14天(P14)和21天(P21)G42小鼠的前额叶皮层脑组织,制作脑片,人工脑脊液中孵育1 h后利用全细胞膜片钳技术记录篮状细胞的电生理特性;另外,消化上述时间点的脑组织,用流式细胞术分选EGFP标记的篮状细胞,建库后进行转录组测序。从P7到P21,篮状细胞的静息膜电位(resting membrane potential,RMP)超极化程度越来越大,膜输入阻抗(membrane input resistance,Rm)逐渐降低;动作电位(action potential,AP)幅度逐步增大,AP持续时间逐渐缩短,而AP阈电位无显著变化;自发兴奋性突触后电流(spontaneous excitatory postsynaptic currents,sEPSCs)的频率逐渐变大,但幅度无显著变化。转录组测序发现,P14相对于P7的篮状细胞有682个基因的表达水平发生显著改变(22个基因上调,660个基因下调),而P21相对于P14的篮状细胞仅有176个基因的表达水平发生显著改变(107个基因上调,69个基因下调)。这些发育过程中的差异表达基因主要富集在神经凋亡、RNA剪接、高尔基体囊泡转运和轴突导向生长等生物学通路。以上结果揭示了篮状细胞成熟过程中的电生理特性和基因表达变化规律,为进一步研究调控篮状细胞发育的分子机制提供了新的线索。

This study aims to explore the electrophysiological properties and changes in gene expression of basket cells,a unique population of GABAergic interneurons expressing parvalbumin(PV),during the postnatal development of mouse prefrontal cortex(PFC).Toward this goal,we took use of the G42 transgenic mouse line which specifically expresses enhanced green fluorescent protein(EGFP)in basket cells.The brain slices of PFC were prepared from the postnatal 7(P7),14(P14)and 21 days(P42)G42 mice and whole-cell patch clamp recording was performed in basket cells.In addition,we sorted the basket cells by flow cytometry and analyzed their transcription profiling on P7,P14,and P21 using RNA-seq technology.The results showed that the resting membrane potential and membrane input resistance decreased gradually from P7 to P21.The amplitude and duration of action potential of basket cells increased and decreased from P7 to P21,respectively.In contrast,the threshold of action potential of basket cells did not have a significant change from P7 to P21.The frequency of spontaneous excitatory postsynaptic currents(sEPSCs)of basket cells increased gradually,while the amplitudes of sEPSCs of basket cells remained constant from P7 to P21.RNA sequencing from basket cells revealed that the expression of 22 and 660 genes was upregulated and downregulated from P7 to P14,respectively.By contrast,the expression of 107 and 69 genes was upregulated and downregulated from P14 to P21,respectively.The differentially expressed genes in basket cells from P7 to P21 were significantly enriched in pathways such as neuron apoptotic process,mRNA processing,Golgi vesicle transport and axon guidance.Altogether,we characterized electrophysiological properties and changes in gene expression of basket cells during the postnatal development in mouse PFC.These results provide insight into the mechanisms underlying the development of basket cells in mouse cortex.