Chub N, O'Donovan M J
Section on Developmental Neurobiology, Laboratory of Neural Control, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurophysiol. 2001 May;85(5):2166-76. doi: 10.1152/jn.2001.85.5.2166.
Whole cell recordings were obtained from ventral horn neurons in spontaneously active spinal cords isolated from the chick embryo [embryonic days 10 to 11 (E10-E11)] to examine the post-episode depression of GABAergic transmission. Spontaneous activity occurred as recurrent, rhythmic episodes approximately 60 s in duration with 10- to 15-min quiescent inter-episode intervals. Current-clamp recording revealed that episodes were followed by a transient hyperpolarization (7 +/- 1.2 mV, mean +/- SE), which dissipated as a slow (0.5-1 mV/min) depolarization until the next episode. Local application of bicuculline 8 min after an episode hyperpolarized spinal neurons by 6 +/- 0.8 mV and increased their input resistance by 13%, suggesting the involvement of GABAergic transmission. Gramicidin perforated-patch recordings showed that the GABAa reversal potential was above rest potential (E(GABAa) = -29 +/- 3 mV) and allowed estimation of the physiological intracellular [Cl(-)] = 50 mM. In whole cell configuration (with physiological electrode [Cl(-)]), two distinct types of endogenous GABAergic currents (I(GABAa)) were found during the inter-episode interval. The first comprised TTX-resistant, asynchronous miniature postsynaptic currents (mPSCs), an indicator of quantal GABA release (up to 42% of total mPSCs). The second (tonic I(GABAa)) was complimentary to the slow membrane depolarization and may arise from persistent activation of extrasynaptic GABAa receptors. We estimate that approximately 10 postsynaptic channels are activated by a single quantum of GABA release during an mPSC and that about 30 extrasynaptic GABAa channels are required for generation of the tonic I(GABAa) in ventral horn neurons. We investigated the post-episode depression of I(GABAa) by local application of GABA or isoguvacine (100 microM, for 10-30 s) applied before and after an episode at holding potentials (V(hold)) -60 mV. The amplitude of the evoked I(GABA) was compared after clamping the cell during the episode at one of three different V(hold): -60 mV, below E(GABAa) resulting in Cl(-) efflux; -30 mV, close to E(GABAa) with minimal Cl(-) flux; and 0 mV, above E(GABAa) resulting in Cl(-) influx during the episode. The amplitude of the evoked I(GABA) changed according to the direction of Cl(-) flux during the episode: at -60 mV a 41% decrease, at -30 mV a 4% reduction, and at 0 mV a 19% increase. These post-episode changes were accompanied by shifts of E(GABAa) of -10, -1.2, and +7 mV, respectively. We conclude that redistribution of intracellular [Cl(-)] during spontaneous episodes is likely to be an important postsynaptic mechanism involved in the post-episode depression of GABAergic transmission in chick embryo spinal neurons.
从鸡胚[胚胎第10至11天（E10 - E11）]分离出的自发活动脊髓腹角神经元进行全细胞记录，以研究GABA能传递的发作后抑制。自发活动以持续约60秒的反复、有节律的发作形式出现，发作间期静息间隔为10至15分钟。电流钳记录显示，发作后紧接着出现短暂的超极化（7±1.2 mV，平均值±标准误），该超极化以缓慢（0.5 - 1 mV/分钟）的去极化形式消散，直至下一次发作。发作8分钟后局部应用荷包牡丹碱使脊髓神经元超极化6±0.8 mV，并使其输入电阻增加13%，提示GABA能传递参与其中。短杆菌肽穿孔膜片钳记录显示，GABAa逆转电位高于静息电位（E(GABAa)= - 29±3 mV），并可估算出生理细胞内[Cl⁻]=50 mM。在全细胞配置（使用生理电极[Cl⁻]）下，在发作间期发现了两种不同类型的内源性GABA能电流（I(GABAa)）。第一种包括对河豚毒素（TTX）不敏感的、异步微小突触后电流（mPSCs），这是量子化GABA释放的指标（占总mPSCs的42%）。第二种（持续性I(GABAa)）与缓慢的膜去极化互补，可能源于突触外GABAa受体的持续激活。我们估计，在一个mPSC期间，单个GABA释放量子可激活约10个突触后通道，而在腹角神经元中产生持续性I(GABAa)大约需要30个突触外GABAa通道。我们通过在发作前后于钳制电位（V(hold)）- 60 mV局部应用GABA或异鹅掌楸碱（100 microM，持续10 - 30秒）来研究I(GABAa)的发作后抑制。在发作期间将细胞钳制在三个不同的V(hold)之一：- 60 mV（低于E(GABAa)，导致Cl⁻外流）、- 30 mV（接近E(GABAa)，Cl⁻通量最小）和0 mV（高于E(GABAa)，发作期间导致Cl⁻内流）后，比较诱发的I(GABA)的幅度。诱发的I(GABA)的幅度根据发作期间Cl⁻通量的方向而变化：在- 60 mV时降低41%，在- 30 mV时降低4%，在0 mV时增加19%。这些发作后的变化分别伴随着E(GABAa)的- 10、- 1.2和+ 7 mV的偏移。我们得出结论，在鸡胚脊髓神经元中，自发发作期间细胞内[Cl⁻]的重新分布可能是参与GABA能传递发作后抑制的重要突触后机制。
