PERFORATED PATCH-CLAMP ANALYSIS OF THE PASSIVE MEMBRANE-PROPERTIES OF 3 CLASSES OF HIPPOCAMPAL-NEURONS
PERFORATED PATCH-CLAMP ANALYSIS OF THE PASSIVE MEMBRANE-PROPERTIES OF 3 CLASSES OF HIPPOCAMPAL-NEURONS
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DOI：
10.1152/jn.1992.67.3.508
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发表时间：
1992-03-01
影响因子：
2.5
通讯作者：
JOHNSTON, D
中科院分区：
文献类型：
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作者：
SPRUSTON, N;JOHNSTON, D
1.穿孔膜片钳记录从三个主要类别的海马神经元在常规的体外切片制备的成年豚鼠。该技术提供了被动膜特性（输入电阻R（N）和膜时间常数tau（m））的实验估计，所述被动膜特性在不存在与常规全细胞记录相关的细胞质成分的冲洗的微电极穿刺相关的泄漏电导的情况下确定。为了便于我们的数据与以前的结果进行比较，并确定被动膜性能的条件下，尽可能的生理，记录在静息电位，在生理盐水中，没有任何添加的电压依赖性电导的阻断剂。分析了膜电位对电流阶跃的响应，并使用四个标准来确定受电压依赖性电导激活影响最小的电压响应。根据这些标准认为被动的响应的多指数拟合的最慢分量（tau（0））估计Tau（m）。R（N）由电压-电流关系的超极化方向的线性区域的斜率估计.这是不可能的，以衡量纯粹的被动膜特性是完全独立的膜电位在任何三类海马神经元。通过恒定电流注入改变膜电位导致R（N）和tau（0）的变化;对于所有三类海马神经元，阈值下去极化产生R（N）和tau（0）的增加，超极化降低。三类海马神经元中的每一类在较大的超极化电压瞬变期间也显示出去极化“下垂”。为了评估导致这种下垂的电导对被动膜性质的影响，将2-5 mM Cs+加入生理盐水中。细胞外Cs+有效地阻断了所有三类海马神经元的下垂，但Cs+对R（N）、tau（0）以及这些参数的电压依赖性的影响对于每类神经元是独特的.在静息电位为-64 +/- 2 mV时，CA 1锥体神经元的R（N）为104 +/- 10（SE）M-OMEGA，tau（0）为28 +/- 2 ms（n = 12）。在这些神经元中，R（N）和tau（0）在更高的去极化电位下更大，但生理盐水中加入Cs+逆转了这种电压依赖性. CA 3锥体神经元的R（N）为135 +/- 8 M-OMEGA，tau（0）为66 +/- 4 ms，静息电位为-64 +/- 1 mV（n = 14）。在这些神经元中，R（N）和tau（0）在更多的去极化电位下更大，并且向生理盐水中添加Cs+导致在所有电位下R（N）和tau（0）的增加，而不逆转或消除这些参数的电压依赖性。齿状颗粒神经元的R（N）为446 +/- 87 M-OMEGA，tau（0）为43 +/- 4 ms，静息电位为-73 +/- 2 mV（n = 12）。在这些神经元中，R（N）和tau（0）在更高的去极化电位下更大，并且在生理盐水中加入Cs+对这些参数几乎没有影响。通过使用穿孔膜片钳记录确定的被动膜特性与先前微电极记录的估计值的比较显示，我们对R（N）的估计值相对于tau（0）的估计值增加不成比例地大。为了确定理论模型是否预测这一结果，我们研究了在具有被动膜的神经元的分析和数值模型中引入体细胞泄漏的影响。与我们的实验数据一致，这些模型预测体细胞漏导对R（N）的影响大于tau（0）。这些结果支持的解释，即通过使用膜片钳和微电极记录获得的被动膜特性之间的差异是在微电极记录的冲击诱导的电导的结果。此外，体细胞泄漏对R（N）和tau（0）的影响差异对于具有电紧张性较长树突电缆的模型更大。因此，通过使用膜片钳记录确定的R（N）和tau（0）的增加估计值的差异幅度表明，海马神经元即使在稳态下也不是等电位的。我们的结论是，对于海马神经元的三个主要类别，R（N）和tau（0）都大于微电极记录提供的先前估计。此外，我们的数据表明，传统上被认为是被动的膜特性至少部分地受到海马神经元静息电位附近的电压依赖性电导的影响。Cs+对R（N）和tau（0）的影响的差异以及这些参数的电压依赖性表明，各类海马神经元的整合膜特性可能受到在静息膜电位处或附近开放的电压门控离子通道的独特类型、密度和分布的影响。
1. Perforated patch-clamp recordings were made from the three major classes of hippocampal neurons in conventional in vitro slices prepared from adult guinea pigs. This technique provided experimental estimates of passive membrane properties (input resistance, R(N), and membrane time constant, tau(m)) determined in the absence of the leak conductance associated with microelectrode impalement of the washout of cytoplasmic constituents associated with conventional whole-cell recordings.2. To facilitate comparison of our data with previous results and to determine the passive membrane properties under conditions as physiological as possible, recordings were made at the resting potential, in physiological saline, and without any added blockers of voltage-dependent conductances.3. Membrane-potential responses to current steps were analyzed, and four criteria were used to identify voltage responses that were the least affected by activation of voltage-dependent conductances. Tau(m) was estimated from the slowest component (tau(0)) of multiexponential fits of responses deemed passive by these criteria. R(N) was estimated from the slope of the linear region in the hyperpolarizing direction of the voltage-current relation.4. It was not possible to measure purely passive membrane properties that were completely independent of membrane potential in any of the three classes of hippocampal neurons. Changing the membrane potential by constant current injection resulted in changes in R(N) and tau(0); subthreshold depolarization produced an increase, and hyperpolarization a decrease, in both R(N) and tau(0) for all three classes of hippocampal neurons.5. Each of the three classes of hippocampal neurons also displayed a depolarizing "sag" during larger hyperpolarizing voltage transients. To evaluate the effect of the conductances underlying this sag on passive membrane properties, 2-5 mM Cs+ was added to the physiological saline. Extracellular Cs+ effectively blocked the sag in all three classes of hippocampal neurons, but the effect of Cs+ on R(N), tau(0), and the voltage dependence of these parameters was unique for each class of neurons.6. CA1 pyramidal neurons had an R(N) of 104 +/- 10 (SE) M-OMEGA and tau(0) of 28 +/- 2 ms at a resting potential of -64 +/- 2 mV (n = 12). R(N) and tau(0) were larger at more depolarized potentials in these neurons, but the addition of Cs+ to the physiological saline reversed this voltage dependence.7. CA3 pyramidal neurons had an R(N) of 135 +/- 8 M-OMEGA and tau(0) of 66 +/- 4 ms at a resting potential of -64 +/- 1 mV (n = 14). R(N) and tau(0) were larger at more depolarized potentials in these neurons, and addition of Cs+ to the physiological saline resulted in an increase in R(N) and tau(0) at all potentials without reversing or eliminating the voltage dependence of these parameters.8. Dentate granule neurons had an R(N) of 446 +/- 87 M-OMEGA and tau(0) of 43 +/- 4 ms at a resting potential of -73 +/- 2 mV (n = 12). R(N) and tau(0) were larger at more depolarized potentials in these neurons, and addition of Cs+ to the physiological saline had little or no effect on these parameters.9. Comparison of the passive membrane properties determined by the use of perforated patch-clamp recording to previous estimates from microelectrode recordings revealed that our estimates of R(N) are disproportionately large relative to the increased estimate of tau(0). To determine whether theoretical models predict this result, we investigated the effect of introducing a somatic leak in analytic and numerical models of neurons with passive membranes. In agreement with our experimental data, these models predict that a somatic leak conductance has a greater effect on R(N) than tau(0). These results support the interpretation that differences between the passive membrane properties obtained by the use of patch-clamp and microelectrode recordings are the result of an impalement-induced conductance in the microelectrode recordings. Furthermore, the difference in the effect of a somatic leak on R(N) and tau(0) was greater for models having electrotonically longer dendritic cables. The magnitude of the discrepancy in the increased estimates of R(N) and tau(0) determined by the use of patch-clamp recordings therefore suggests that hippocampal neurons are not isopotential, even in the steady state.10. We conclude that for the three major classes of hippocampal neurons, both R(N) and tau(0) are larger than previous estimates provided by microelectrode recordings. In addition, our data indicate that membrane properties traditionally considered passive are at least partially influenced by voltage-dependent conductances active near the resting potential in hippocampal neurons. Differences in the effects of Cs+ on R(N) and tau(0) and the voltage dependence of these parameters suggest that the integrative membrane properties of each class of hippocampal neurons are likely to be influenced by unique types, densities, and distributions of voltage-gated ion channels open at or near the resting membrane protential.