Voltage imaging with ANNINE dyes and two-photon microscopy of Purkinje dendrites in awake mice

Voltage imaging is the next generation of functional imaging in neuroscience. It promises to resolve neuronal activity 10 to 100-times faster than calcium imaging and to report not only supra but also subthreshold activity on a single cell or even subcellular level. Lately, several different voltage sensors and imaging techniques were published which can achieve this. Here, we focus on a technique based on the synthetic pure electrochromic voltage-sensitive dyes ANNINE-6 and ANNINE-6plus and the excitation of this dye at the red spectral edge of absorption to maximize voltage sensitivity and minimize phototoxicity and bleaching. Importantly, voltage imaging with ANNINE dyes can be done with one and two-photon excitation. Two-photon microscopy allows in vivo, depth resolved imaging and line-scan recordings with sub-millisecond temporal resolution. Interestingly for many future applications, the spectral characteristics of ANNINE dyes allows simultaneous imaging with green indicators, like the genetically encoded calcium indicator GCaMP6. We used this method to study supra and subthreshold dendritic voltage changes in Purkinje neurons of awake mice. Simultaneously, we imaged dendritic calcium and recorded electrical activity from the soma or locally applied drugs to show the full potential of the technique to study dendritic integration in awake animals.