Dittert Ivan, Benedikt Jan, Vyklický Ladislav, Zimmermann Katharina, Reeh Peter W, Vlachová Viktorie
Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
J Neurosci Methods. 2006 Mar 15;151(2):178-85. doi: 10.1016/j.jneumeth.2005.07.005. Epub 2005 Aug 29.
We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.
我们开发了一种改进技术，用于在膜片钳或钙成像条件下对灌注分离细胞的溶液进行快速冷却和加热。该系统满足研究各种离子通道温度依赖性的要求，特别是温度门控离子通道。它允许在5 - 60摄氏度范围内以最大-40摄氏度/秒至60摄氏度/秒的速率施加温度变化。装有不同溶液的桶连接到一个由七个硅胶毛细管（内径320微米）组成的歧管上。一个公共出口由一根玻璃毛细管组成，溶液通过该毛细管施加到细胞表面。该毛细管的上部嵌入由微型珀尔帖装置驱动的温度交换器中，该装置对通过的溶液进行温度预处理。毛细管的下部带有一根绝缘铜线，在7毫米的长度上紧密缠绕，并连接到直流电流源以进行电阻加热。珀尔帖装置和加热元件电连接到头级探头，该探头固定在微操纵器上以定位歧管。流动溶液的温度通过插入公共出口毛细管靠近其孔口处的微型热电偶测量，该孔口距离被检查细胞的表面小于100微米。温度可以通过电压命令手动控制，也可以通过传统数据采集接口的数模转换器进行调节。文中给出了在异源表达的TRPV1、TRPM8以及培养的大鼠感觉神经元的膜片钳研究中使用该装置的示例。
