Two-photon voltage imaging using a genetically encoded voltage indicator

Abstract

Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2–3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.

Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators

“…With appropriate speed, it is also possible to deconvolve the images to obtain an indication of spiking (Vogelstein et al, 2010). There is also the possibility of detecting decreases in fluorescence (Cheng, Krishnan, & Jesuthasan, 2016), which occurs when a cell with ongoing tonic activity is inhibited. Calcium imaging has led to a number of discoveries, such as the existence of discrete attractor states in the olfactory bulb (Niessing & Friedrich, 2010), and of compartmentalized activity within single neurons (Hendricks, Ha, Maffey, & Zhang, 2012).…”

“…This was first done with organic dyes, whose fluorescence or absorbance changes with membrane potential (Cohen et al, 1974) and which have millisecond resolution (reviewed in Chemla & Chavane, 2010). Dyes have been used in vivo, for example in the characterization of functional connectivity in the rat cortex (Yuste, Tank, & Kleinfeld, 1997) or of odour-evoked oscillation in the olfactory bulb (Friedrich et al, 2004). However, labelling neurons with a dye requires an invasive procedure, and the label does not persist.…”

“…This state can be changed as an integral part of information processing. In the olfactory system, for example, neuronal activity in the bulb not only affects downstream neurons but also changes the immediate network by triggering local oscillations (Friedrich, Habermann, & Laurent, 2004). Nutritional status can further change activity in the bulb, through the action of modulators (reviewed in Palouzier-Paulignan et al, 2012).…”

“…One approach has been the use of multi-electrode arrays (Spira & Hai, 2013). An alternative approach is to use imaging, rather than electrodes, to characterize electrical signals in the brain (Broussard, Liang, & Tian, 2014). One advantage of imaging is that regions of a cell, e.g.…”

Genetically encoded voltage indicators for large scale cortical imaging come of age

“…GEVIs with response dynamics that are compatible with recordings of fast action potentials in cultured cells are already available [20,21 ,23 ,26], but robust resolution of individual action potentials from multiple neurons in living rodents has not yet been demonstrated with any GEVI. While proof-ofprinciple experiments reaching this milestone can be expected in the very near future, further challenges will include the necessary advances in optical instrumentation and analysis methods of activity-map data [37,38]. For cellular resolution functional imaging, novel two-photon microscopes are required that provide activity maps updated at approximately each millisecond with large field-of-views ()1 mm 2 ).…”