Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

哺乳动物脑片中具有细胞分辨率的电压成像仍然是一项艰巨的任务。在这里，我们描述并验证了一种将压敏染料ANNINE-6plus（A6 +）输送到组织中以进行电压成像的方法，该方法比传统的镀浴方法具有更高的信噪比（SNR）。使用立体定向手术在急性切片制备之前0、1或7天将未完全溶解的染料注入到下橄榄（IO）中。我们发现潜伏期延长后电压成像会改善体内就标记的体积而言，均具有显着标记的躯体的均质Neuropil标记和SNR。染料注入后7天准备急性切片，其SNR足够高，可以使用宽场（网络级）以及高倍率（单细胞级）电压成像对IO亚阈值振荡进行单次试验记录。 CMOS相机。这种方法很容易适应其他难以使用基因编码电压传感器且需要超快速，纯电致变色传感器（例如A6 +）的大脑区域。由于此处展示了持久的染色效果，因此该方法可以与例如使用可植入GRIN透镜的深脑成像相结合。