A dark quencher genetically encodable voltage indicator (dqGEVI) for high fidelity recordings in murine and human in vitro systems

Abstract

To understand neurons and neural circuits it is necessary to visualize their electrical activity. <br /> In contrast to traditional electrophysiological techniques, optical methods are very promising as they tend to be less invasive, and enable high throughput visualization of the activity of neuronal ensembles. Genetically encoded optical indicators can enable the targeting of specific neuronal subpopulations. The most widely used such indicators are genetically encoded calcium indicators (GECIs). These, however, are inherently unable to resolve hyperpolarizations and subthreshold events, have a poor temporal resolution and act as calcium buffers, thereby interfering with physiological calcium signaling cascades. <br /> Genetically encoded voltage indicators (GEVIs) are ideally suited to directly monitor the electrical changes at the plasma membrane. However, voltage imaging at the plasma membrane is challenging for several reasons: the space for positioning the fluorophores is minimal as it is restricted to the thinness of the plasma membrane. The resulting higher illumination intensities then lead to rapid photobleaching and phototoxic effects which impede long-term recordings of neuronal activity. Likewise, the resulting low signal-to-noise ratios require averaging of the fluorescent signals. Furthermore, until now, GEVIs have been based on voltage-sensitive domains from potassium channels or phosphatases or on rhodopsins, inserted in multiple copies into the plasma membrane. This in turn leads to increases in membrane capacitance, which has a direct impact on neuronal excitability. <br /> The work described in this thesis aimed to identify a GEVI that does not increase membrane capacitance, is slow to bleach on constant illumination, has a high temporal resolution for both action potential and subthreshold events, and does not require averaging to resolve the fluorescence. Based on a promising alternative approach (Chanda et al., 2005), a GEVI was developed that satisfies these requirements. This hybrid genetically encoded voltage sensor consists of a Förster resonance electron transfer (FRET) pair composed of a fluorescent enhanced green fluorescent protein (eGFP) anchored to the extracellular leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor and an azo-benzene quenching molecule, Disperse Orange 3 (D3), which is not fluorescent itself and therefore referred to as dark quencher (dq). D3 moves in the membrane in a voltage-dependent fashion and thereby quenches and unquenches the fluorescence and modifies the fluorescent signal. In murine cells, dqGEVI is shown to be capable of performing high-fidelity recordings of changes in membrane potential. It outperforms previous hybrid and non-hybrid GEVI approaches by leaving the intrinsic membrane properties of the cell unaffected, and is exceptionally photostable and fast. <br /> This thesis also reports on the applicability of dqGEVI for detecting neuronal activity in human iPSC-derived sensory and cortical neurons. dqGEVI enabled action potential firing patterns to be visualized. In a cell line derived from a patient suffering from inherited erythromelalgia (EM) and a healthy control. Using dqGEVI it was possible to detect significant differences in action potential burst firing, which could not be shown with other methods. <br /> This novel sensor is a highly applicable tool for the non-invasive, fast, and detailed monitoring of iPSC-derived neuronal activity.