Kleinhans Christian, Kafitz Karl W, Rose Christine R
Institute of Neurobiology, Heinrich Heine University Düsseldorf.
Institute of Neurobiology, Heinrich Heine University Düsseldorf;
J Vis Exp. 2014 Oct 8(92):e52038. doi: 10.3791/52038.
Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.
多光子荧光显微镜能够以高空间和时间分辨率分析完整组织中脑细胞的形态和生理参数。与电生理学相结合，它被广泛用于研究诸如树突和树突棘等小亚细胞区室中与活动相关的钙信号。除了钙瞬变外，突触活动还会诱导突触后钠信号，但其特性目前了解甚少。在这里，我们描述了一种在中枢神经元的细胞微区室中结合全细胞膜片钳和多光子钠成像的方法。此外，我们介绍了一种用于紫外线（UV）诱导的谷氨酸光解笼锁的改良程序，该程序允许在组织中可靠且局部地激活谷氨酸受体。为此，在小鼠海马体急性组织切片中的海马1区（CA1）锥体神经元上进行了全细胞记录。通过膜片吸管将钠敏感荧光染料SBFI注入神经元，对SBFI的多光子激发使得能够可视化树突和相邻的树突棘。为了建立紫外线诱导的局部光解笼锁，对包括光强度、受紫外线光解笼锁光束影响的体积、光束的定位以及笼锁化合物的浓度等几个参数进行了测试和优化。我们的结果表明，用笼锁谷氨酸（MNI-谷氨酸）进行局部灌注及其局部紫外线光解笼锁会导致树突和树突棘中的内向电流和钠瞬变。内向电流和钠信号的时间进程和幅度与光解笼锁脉冲的持续时间相关。此外，我们的结果表明，在存在离子型谷氨酸受体阻滞剂的情况下，细胞内钠信号被阻断，这表明它们是由通过该途径的钠内流介导的。总之，我们的方法为研究完整脑组织中局部受体激活诱导的细胞内钠信号提供了一个可靠的工具。
