Lou Xuelin
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.
Methods Mol Biol. 2018;1847:95-108. doi: 10.1007/978-1-4939-8719-1_8.
Endocytosis is fundamental to cell function. It can be monitored by capacitance measurements under patch-clamp recordings. Membrane capacitance recording measures the cell membrane surface area and its changes at high temporal-resolution and sensitivity, and it is a powerful biophysical approach in the field of exocytosis and endocytosis. A popular one is the frequency domain method that entails processing passive sinusoidal membrane currents induced by a sinusoidal voltage. This technique requires a phase-sensitive detector or "lock-in amplifier" implemented in hardware or software during patch-clamp recordings. It has been widely used in many secretory cells, but its application directly at central presynaptic terminals is technically challenging. We have applied this technique to study synaptic endocytosis in the calyx of Held, a large glutamatergic synaptic terminal, as well as mouse pancreatic β-cells. The presynaptic capacitance measurements provide a unique alternative to measuring transmitter release and presynaptic endocytosis. Here, we describe this method at the calyx of Held in acute brain slices and provide a practical guide to obtaining high quality capacitance measurements at presynaptic terminals.
内吞作用对于细胞功能至关重要。它可以通过膜片钳记录下的电容测量来监测。膜电容记录能够以高时间分辨率和灵敏度测量细胞膜表面积及其变化，是胞吐作用和内吞作用领域一种强大的生物物理方法。一种常用的方法是频域法，该方法需要处理由正弦电压诱导产生的被动正弦膜电流。这种技术在膜片钳记录过程中需要硬件或软件中实现的相敏探测器或“锁相放大器”。它已在许多分泌细胞中广泛应用，但其在中枢突触前终末的直接应用在技术上具有挑战性。我们已将此技术应用于研究Held壶腹（一种大型谷氨酸能突触终末）以及小鼠胰腺β细胞中的突触内吞作用。突触前电容测量为测量递质释放和突触前内吞作用提供了一种独特的替代方法。在此，我们描述在急性脑片的Held壶腹中的这种方法，并提供在突触前终末获得高质量电容测量的实用指南。
