小鸡脑片突触反应长时程增强的细胞内记录和多参数分析

Intracellular recordings and multi-parameter analysis of long-term potentiation of synaptic responses in chick brain slices

本文旨在建立日龄小鸡脑片制备及其神经元细胞内记录技术,观察中间腹内侧原皮质（intermediate medialmesopallium,IMM）神经元的电学特性、突触反应及其长时程增强（long-term potentiation,LTP）现象。取210日龄小鸡制备左侧前脑切片（500μm厚）,对69个IMM神经元进行细胞内记录,测得静息电位（-59.4±5.3）mV、膜斜率电阻（70.8±27.2）MΩ、时间常数（10.2±4.3）ms,动作电位幅度（85.2±9.4）mV、阈电位（-38.7±7.6）mV、超射（25.6±8.9）mV、半幅时程（2.1±0.5）ms、最大上升斜率（150.5±41.2）mV/ms、最大下降斜率（-64.3±14.0）mV/ms。其中32个神经元的放电频率随刺激电流强度增大而升高[直线回归斜率为（21.5±10.9）Hz/nA,均P〈0.05]。在所记录IMM神经元的背侧（n=25）、腹侧（n=62）局部电刺激（0.1Hz）均可诱发具有强度、膜电位依赖性的去极化突触反应（即EPSP）。对12个测试细胞给予腹侧局部强直刺激（5Hz,300脉冲/串,2串,串间隔10min）,在6个细胞诱导出EPSP的LTP,呈现为EPSP的幅度、曲线下面积、时程和最大上升斜率均增大（强直刺激后45min与强直刺激前相比,P〈0.05,n=5）,但相应的膜电学特性无明显变化（P〉0.05）。以上结果表明,在日龄小鸡脑片上建立的IMM神经元细胞内记录技术稳定可靠,可用于突触反应LTP的多参数分析。

Day-old chick is unique animal model in brain development and behavior study. The intermediate medial mesopallium （IMM）, a region of the chick forebrain, is intimately involved in the early learning processes, which offers the ideal opportunity to study the neural changes that underlie behavioral plasticity. In this paper, the intracellular recordings were conducted from IMM neurons in chick forebrain slices, in which electrophysiological properties, synaptic responses and long-term potentiation （LTP） were observed. Coronal sections of left forebrains （500 mum thick）, containing IMM, were prepared from domestic chicks, aged 2-10 days. In 69 IMM neurons, the resting membrane potential was measured to be （-59.4＋/-5.3） mV, slope membrane resistance （70.8＋/-27.2） MOmega, and time constant （10.2＋/-4.3） ms. The amplitude, threshold, overshoot, half-width, max rise slope and max decay slope of action potential evoked by intracellular current injection were （85.2＋/-9.4） mV, （-38.7＋/-7.6） mV, （25.6＋/-8.9） mV, （2.1＋/-0.5） ms, （150.5＋/-41.2） mV/ms and （-64.3＋/-14.0） mV/ms, respectively. Spike-firing frequency was increased with depolarizing current intensity in 32 of 69 tested cells [linear regression slope was （21.5＋/-10.9） Hz/nA, P〈0.05 in all cells]. The depolarizing synaptic responses （i.e. EPSPs）, with stimulus intensity- and membrane potential-dependent properties, were elicited by dorsal （n=25） or ventral （n=62） focal electrical stimuli at 0.1 Hz in all tested IMM neurons and could be nullified reversibly by perfusion with 100 mumol/L AP5 （NMDA receptor antagonist） and 3 mumol/L DNQX （non-NMDA receptor antagonist）, but enlarged by 6 mumol/L bicuculline （GABA（A） receptor antagonist）. The EPSPs evoked by ventral stimulation were persistently increased after tetanic stimulation （5 Hz, 300 pulses/train, 2 trains, train interval 10 min） in 6 of 12 tested IMM neurons. The amplitude of EPSPs was potentiated to more than 120% of control level （when analyzed at 45 min of enhancement, P〈0.05, n=5）, which lasted at least 30 min and then could be referred to as LTP. Moreover, area under curve, duration and max rise slope of EPSPs were also enhanced （P〈0.05）, while no significant changes were observed in the electrophysiological parameters of IMM neurons following induction of LTP （P〉0.05）. These results suggest that the intracellular recording techniques in the chick brain slices can be used to perform multi-parameter analysis of synaptic responses and their LTP.