Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells
Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells
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DOI：
10.1152/jn.1997.78.3.1320
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发表时间：
1997-09-01
影响因子：
2.5
通讯作者：
Slater, NT
中科院分区：
文献类型：
--
作者：
Kinney, GA;Overstreet, LS;Slater, NT
采用全细胞和切除膜片钳记录方法，研究了大鼠前庭小脑凌乱纤维(MF)-单极刷细胞（UBC）突触中α -氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体介导的长时间兴奋性突触后电流（EPSCs）发生的细胞机制。MFs的激活会引起全或无AMPA受体介导的双相突触电流，其后期成分在100-800 ms达到峰值，这被认为是源于谷氨酸在MF-UBC突触间隙中的捕获，并由AMPA受体的稳态激活产生。环噻嗪可阻断AMPA受体的脱敏作用，可使突触电流振幅呈剂量依赖性增强（中位有效剂量为30 μ M），并减缓快速EPSC的上升时间。环噻嗪（100 μ M）对ubc中n -甲基- d -天冬氨酸受体介导的EPSCs在振幅和时间上没有增强作用。谷氨酸作用于从UBC和颗粒体膜切除的外部斑块的AMPA受体所引起的稳态电流的剂量-反应关系是双相的，在50 μ M时达到峰值，在1 mM谷氨酸时下降到该值的50-70%。当将谷氨酸从斑块中缓慢冲洗以模拟突触中谷氨酸的逐渐下降时，两种细胞类型的斑块中都观察到跨膜电流的晚期峰。在慢速EPSC的峰值处给予第二次MF刺激，诱发了一个振幅降低的快速EPSC，随后是一个慢速电流的下冲，这与慢速EPSC的峰值反映了两相稳态剂量-反应曲线的峰值的假设一致。根据快速EPSC振幅的比值，以及配对脉冲实验中初始稳态电流的振幅和极性，对受体占用率和谷氨酸浓度的估计，预测谷氨酸在800 ms的时间常数下缓慢下降，在5.4 s内下降到无效浓度。通过降低细胞外钙或提供简短刺激来控制谷氨酸浓度，分别消除了缓慢的EPSC，并恢复了配对刺激的不足，这与谷氨酸在谷氨酸中延长寿命的方式一致。谷氨酸再摄取抑制剂l -反式吡咯烷-2,4-二羧酸盐延长了EPSC的慢速组分的持续时间，这表明谷氨酸转运有助于ubc突触电流的时间过程。这些数据支持这样的观点，即MF- ubc突触代表了一种超微结构的特化，可以在MF末端释放谷氨酸后的异常长时间内有效地捕获谷氨酸。突触后受体的特性以及突触超微结构和谷氨酸转运体对谷氨酸扩散逃逸的限制共同决定了由此产生的慢速EPSC的时间过程。这反过来又驱动了对单一突触前刺激的长期动作电位反应。
The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) al the messy fiber(MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 mu M) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 mu M). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 mu M and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations al 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime-of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting slow EPSC. This in turn drives a long-lasting train of action potentials in response to single presynaptic stimuli.