Budde T, White J A
Otto-von-Guericke Universität, Institut für Physiologie, Magdeburg, Germany.
Eur J Neurosci. 1998 Jul;10(7):2309-21. doi: 10.1046/j.1460-9568.1998.00242.x.
Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).
采用全细胞膜片钳技术研究成年大鼠皮质I层急性分离神经元中电压依赖性钠（Na+）、钙（Ca2+）和钾（K+）电导。通过γ-氨基丁酸（GABA）免疫细胞化学鉴定I层细胞。一部分细胞中钙结合蛋白钙视网膜蛋白呈阳性染色，表明存在Cajal-Retzius（C-R）细胞。所有研究的细胞均表现出相当均匀的电压依赖性膜电流特征。一种快速Na+电流在约-45 mV时激活，在-66.6 mV时达到半数最大稳态失活，失活后的恢复遵循双指数过程（tau1 = 8.4 ms，tau2 = 858.8 ms）。Na+电流以两个电压依赖性时间常数迅速衰减，几十毫秒后达到基线电流。在一部分细胞（<50%）中，90 ms电压阶跃结束时恒定电流水平<65 pA。一种瞬时外向电流（Ifast）在约-40 mV时激活，以电压不敏感的时间常数（tau约350 ms）迅速衰减，对四乙铵（TEA，20 mM）相对不敏感。根据对4-氨基吡啶（4-AP）的敏感性，Ifast可分为两个成分：一个被低浓度（40 μM）阻断，另一个被高浓度（6 mM）阻断。通过预处理脉冲（50 ms至-50 mV）消除Ifast后，可单独研究慢K+电流（I(KV)）。I(KV)仅受到4-AP（6 mM）的中度影响，而TEA（20 mM）阻断了大部分（>80%）电流。I(KV)在约-40 mV时激活，以电压依赖性方式单指数衰减（在-30 mV时tau约850 ms），并表现出不完全的稳态失活。除了Ifast和I(KV)外，还发现了钙依赖性外向电流成分的迹象。当Na+电流、Ifast和I(KV)被河豚毒素（TTX，1 μM）、4-AP（6 mM）和TEA（20 mM）阻断时，发现了由Ca2+携带的内向电流。Ca2+电流在约-30 mV的去极化电位下激活，被50 μM镉（Cd2+）完全阻断，对维拉帕米敏感（10 μM时约40%阻断），不受镍（50 μM）影响。在电流钳记录期间，分离的I层神经元表现出快速放电行为，动作电位短（约2 ms，在半数最大幅度处测量），幅度相对较小（约83 mV，从动作电位阈值测量）。
