CHARACTERIZATION OF SYNAPTICALLY ELICITED GABA-B RESPONSES USING PATCH-CLAMP RECORDINGS IN RAT HIPPOCAMPAL SLICES
CHARACTERIZATION OF SYNAPTICALLY ELICITED GABA-B RESPONSES USING PATCH-CLAMP RECORDINGS IN RAT HIPPOCAMPAL SLICES
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DOI：
10.1113/jphysiol.1993.sp019600
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发表时间：
1993-04-01
影响因子：
5.5
通讯作者：
MODY, I
中科院分区：
文献类型：
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作者：
OTIS, TS;DE KONINCK, Y;MODY, I
1. 采用紧密密封的全细胞电压钳记录技术来表征维持在 34-35 摄氏度的成年大鼠脑切片中单突触诱发的 GABA(B) 电流。阻断6-氰基-7-硝基喹喔啉-2,3-二酮(CNQX)、D-2-氨基-5-磷酸戊酸(D-AP5)和印防己毒素敏感的快速突触传递后，记录齿状回颗粒细胞的反应，以便可以单独研究剩余的突触电流。 2．在这些条件下，分子层中的刺激引起缓慢的外向电流，该电流被选择性 GABA(B) 拮抗剂 CGP 35348 以浓度依赖性方式 (200-800 muM) 阻断。在使用含有 10-15 mm 利多卡因衍生物 QX-314 的移液器进行的记录中或当用铯代替 K+.3 时，不存在该电流。正如能斯特方程所预测的，增加 [K+]o e 倍（从 2.5 到 6.8 mm）将 GABA(B) 电流的反转电位从 -97.9 mV 移动到 -73.2 mV。峰值电导恒定，但在 6.8 mm [K+]o 电压超极化至 E(K)（钾的平衡电位）时，有明显的小向外整流。4．电流的时间进程可以通过具有双指数失活的四次方指数激活动力学来描述。在34-35℃下，平均激活时间常数（tau（m））为45.2 ms，而两个失活时间常数（tau（h1）和tau（h2））分别为110.2和516.2 ms，相应的权重因子（w（h1）和w（h2））分别为0.84和0.16。这些时间常数的 Q10（温度系数）值在 1.82 和 2.31 之间。 tau(m)、tau(h)1 和 tau(hg) 在 -45 至 -95 mV 范围内均不依赖于电压。5。通过在宽范围（50-5000 ms）的刺激间隔（ISI）内提供相同的调节和测试刺激，研究了 GABA（B）电流的成对脉冲抑制。最大抑制（48％）发生在200ms ISI时，并且抑制持续超过5s。成对脉冲抑制的幅度不依赖于突触后膜电位。6．应用竞争性拮抗剂 CGP 35348 使峰值电流减少约 50%，对电流的激活或失活动力学没有影响。类似地，在成对脉冲抑制期间，测试电流的动力学与调节电流的动力学相同。这些发现支持这样的假设：导致配对脉冲抑制的机制涉及神经递质释放的减少，而 K+ 通道激活/失活动力学没有突触后改变。7。突触后 GABA(B) 电流的时间进程与成对脉冲抑制的时间进程的比较表明，与突触后 GABA(B) K+ 电导相同的突触前电导不足以单独通过超极化突触前末端来抑制神经递质释放。
1. Tight-seal, whole-cell voltage clamp recording techniques were used to characterize monosynaptically evoked GABA(B) currents in adult rat brain slices maintained at 34-35-degrees-C. Responses were recorded from granule cells of the dentate gyrus following the blockade of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-, D-2-amino-5-phosphonovaleric acid (D-AP5)- and picrotoxin-sensitive fast synaptic transmission, so that the remaining synaptic currents could be studied in isolation.2. Under these conditions, stimulation in the molecular layer elicited a slow outward current which was blocked by the selective GABA(B) antagonist CGP 35348 in a concentration-dependent manner (200-800 muM). This current was absent in recordings made with pipettes containing 10-15 mm of the lidocaine derivative QX-314 or when caesium was substituted for K+.3. Increasing the [K+]o e-fold (from 2.5 to 6.8 mm) shifted the reversal potential of the GABA(B) current from -97.9 to -73.2 mV, as predicted by the Nernst equation. Peak conductance was constant, but in 6.8 mm [K+]o at voltages hyperpolarized to E(K) (equilibrium potential for potassium), a small outward rectification was evident.4. The time course of the current could be described by fourth-power exponential activation kinetics with double exponential inactivation. At 34-35-degrees-C, the average activation time constant (tau(m)) was 45.2 ms, while the two inactivation time constants (tau(h1) and tau(h2)) were 110.2 and 516.2 ms, with corresponding weighting factors (w(h1) and w(h2)) of 0.84 and 0.16, respectively. The Q10 (temperature coefficient) values for these time constants were between 1.82 and 2.31. Neither tau(m), nor tau(h)1 and tau(hg) were voltage dependent in the range from -45 to -95 mV.5. Paired-pulse depression of the GABA(B) current was studied by giving identical conditioning and test stimuli over a wide range (50-5000 ms) of interstimulus intervals (ISIs). The maximal depression (48 %) occurred at 200 ms ISI, and the depression lasted for over 5 s. The magnitude of paired-pulse depression was not dependent on the postsynaptic membrane potential.6. Application of the competitive antagonist CGP 35348 such that the peak current was diminished by approximately 50% had no effect on the activation or inactivation kinetics of the current. Similarly, during paired-pulse depression the kinetics of test currents were identical to those of conditioning currents. These findings support the hypothesis that the mechanism responsible for paired-pulse depression involves a reduction in neurotransmitter release without postsynaptic alterations in K+ channel activation/inactivation kinetics.7. Comparison of the time course of the postsynaptic GABA(B) current with that of the paired-pulse depression indicates that a presynaptic conductance identical to the postsynaptic GABA(B) K+ conductance is not sufficient alone to depress neurotransmitter release by hyperpolarizing the presynaptic terminal.