背景 人类及哺乳动物的视觉发育主要是在生后关键期内完成的,但此期并非是哺乳动物接受视觉经验刺激的最早期.小鼠等哺乳动物视觉发育的关键期前还存在前关键期.目前,前关键期视皮层神经元的反应特性及突触可塑性研究仍处于探索阶段. 目的 探讨小鼠前关键期视皮层神经元的反应特性及突触可塑性特点. 方法 选择生后13～17 d的C57BL/6J小鼠48只,分别采用在体膜片钳全细胞记录及离体脑片膜片钳全细胞记录法记录小鼠视皮层第Ⅳ层神经元的电生理反应.在体记录在小鼠麻醉下进行,在电流钳模式下给予步阶电流刺激,测量其在体膜反应特性.给予最优刺激参数的移动光棒刺激,测量其视觉诱发反应特性.完成在体实验后行离体实验,分别测量神经元离体膜反应特性及白质-第Ⅳ层通路刺激条件下的诱发反应特性.采用随机数字表法将实验动物随机分成4个组,各组雌雄比例分配均匀.每组测定12个细胞,按照刺激频率的不同分别行低频刺激(LFS)和高频刺激[θ波脉冲刺激(TBS)]模式训练,按照刺激时序的不同进行突触前-后(pre-post)模式和突触后-前(post-pre)模式训练,在-70 mV电压钳制下分别记录训练前后兴奋性突触后电流(EPSCs).采用pClmap 10软件对原始数据进行预处理,采用Matlab 2008a软件进行统计分析.结果 在体成功记录的细胞数为39个,离体记录48个.在体和离体条件下视皮层第Ⅳ层神经元稳态平均发放动作电位(AP)个数分别为1.01±0.03和1.01±0.05,AP阈值分别为(-40.2±3.2)mV和(-39.6±2.0)mV,阈电流水平分别为(126.7± 17.4) pA和(129.6±17.5)pA,差异均无统计学意义(AP数:t=0.512,P=0.610;AP阈值:t=-1.074,P=0.286;阈电流:=-0.776,P=0.440).在体最优视觉刺激条件下平均膜电位峰值幅度为(7.3±4.3)mV,鲜见AP;离体最强通路刺激条件下平均膜电位峰值反应幅度为(6.4±2.8)mV,未见AP,在体与离体记录的平均膜电位峰值幅度差异无统计学意义(t=1.234,P=0.221).离体条件下,LFS训练前后EPSCs幅度分别为(138.1±51.9)pA和(76.1±34.8) pA,差异有统计学意义(t=4.437,P=0.001),而TBS训练前后EPSCs幅度差异无统计学意义(t=-0.756,P=0.466),pre-post训练前后EPSCs幅度分别为(122.4±62.2)pA和(78.5±46.7)pA,post-pre训练前后分别为(131.9±48.0)pA和(74.3±30.7)pA,差异均有统计学意义(pre-post:t=3.558,P=0.004;post-pre:t=4.283,P=0.001).结论 前关键期小鼠视皮层第Ⅳ层已完成神经回路的基本构建,但神经元的膜反应性以及突触连接仍未成熟.在低频或高频突触前后时序差异性输入条件下,突触功能受到抑制,而在高频输入条件下突触功能得到继续保持.前关键期小鼠视觉神经系统的发育具有不同于关键期的特征.%Background The visual development is completed during the critical period in human and mammals.However,the critical period is not the initial of receiving visual experience.It is known that before the onset of critical period in mammals,such as mouse,there is an earlier stage for visual development,the pre-critical period.The research of response characteristics of the visual cortical neurons and the synaptic plasticity in the pre-critical period is still in the exploratory stage.Objective The study aimed to preliminarily investigate the response properties of neurons and synaptic plasticity in mouse visual cortex during the pre-critical period.Methods Fortyeight postnatal day 13-17 C57BL/6J mice were used for in vivo whole-cell recordings and in vitro brain slice wholecell recordings.In vivo whole-cell recordings were done in anesthetized mice.Moving bars in different directions were produced and controlled by a Matlab program.Cell recordings were obtained at the depth of layer Ⅳ of visual cortex.Step current stimuli under current clamp were given to measure the membrane response properties of neurons.Optimal visual stimuli were given to measure the in vivo largest responses of membrane potentials.In vitro experiments were performed after in vivo experiments.All cells were given current step stimuli to measure the membrane response properties of neurons.Different intensities of white-matter-to-layer-Ⅳpathway stimulation were given to measure the evoked response properties.All cells from 48 mice were randomized into 4 groups according to different stimulus training modes,including low frequency stimulation (LFS),high frequency theta-burst stimulation (TBS),pre-post synaptic timing stimulation (pre-post TS) and post-pre synaptic timing stimulation (post-pre TS).Under the voltage clamp of-70 mV,excitatory postsynaptic currents (EPSCs) before and after training were recorded to measure the plastic changes of excitatory synaptic connections.pClamp 10 was used for the pre-analysis of data and Matlab 2008a was used for statistical analysis.The use and care of the animals followed the Statement for the Use of Animals in Ophthalmic and Vision Research.Results Thirty-nine cells and 48 cells were successfully recorded in the in vivo and in vitro experiments,respectively.The steady-state average number of action potentials (APs) were (1.01 ± 0.03)/sweep and (1.01 ±0.05)/sweep,the AP thresholds were (-40.2 ± 3.2) mV and (-39.6 ±2.0) mV,and the threshold step current levels were (126.7 ± 17.4) pA and (129.6 ± 17.5) pA in the in vivo and in vitro recordings,respectively,with no significant differences between them (APs:t =0.512,P =0.610;AP thresholds:t =-1.074,P =0.286;current levels:t =-0.776,P =0.440).Under the optimal visual or pathway stimulation,the average peak response of membrane potentials was (7.3 ±4.3)mV and (6.4±2.8)mV with rarely evoked APs in the in vivo and in vitro experiments,respectively,with no significant difference between them (t =1.234,P =0.221).Under the in vitro recording,the EPSCs before LFS were [(138.1 ±51.9)pA],which was significantly higher than that after LFS [(76.1 ± 34.8)pA] (t=4.437,P=0.001),but no significant differences were seen in EPSCs before and after TBS (t=-0.756,P=0.466).The EPSCs before and after pre-post TS were (122.4±62.2)pA and (78.5±46.7)pA,and those before and after post-pre TS were (131.9 ±48.0) pA and (74.3 ± 30.7) pA,showing significant differences between them (pre-post TS:t =3.558,P =0.004;post-pre TS:t =4.283,P =0.001).Conclusions The construction of fundamental neural circuits in layer Ⅳ of mouse visual cortex is completed during pre-critical period.However,the membrane responsive capability of neurons and the synaptic connections are in an immature state,and the evoked responses to visual pathway inputs are basically subthreshold.The strength of synaptic connections is depressed with low frequency stimulation or pre-post/post-pre synaptic timing stimulation,and kept unchanged with high frequency stimulation.The development of visual neural system of PSP in mouse presents different characteristics from CP.
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