Development of new voltage imaging tools for studying mammalian neuronal plasticity in-vivo
1) Robert Prevedel (PI; EMBL Heidelberg), coworker:
2) Paul Heppenstall (PI; SISSA, Italy), coworker:
There is currently a major demand in neuroscience for indicators and instrumentation to optically monitor voltage in the nervous system. While there has been much progress in developing technologies for voltage imaging, no single system is yet able to combine the high sensitivity, temporal precision and signal to noise ratio needed for robust in vivo recordings. For example, in probe development, the two types of indicator which are most commonly used, synthetic voltage sensitive dyes (VSDs) and genetically encoded voltage indicators (GEVIs), are hampered by unspecific background (in the case of VSDs), or low sensitivity and slow kinetics (GEVIs). Instrumentation also has certain trade-offs in that efforts to increase imaging speed in order to temporally resolve fast voltage dynamics often lead to significant reductions in image volume and vice versa. Thus in this proposal we wish to address these shortcomings by 1), generating a novel hybrid probe that combines the superior optical properties of VSDs with the genetic targeting ability of GEVIs. 2), developing a two-photon imaging system for high-speed microscopy in living mice. And 3), using this technology to address a key question in sensory biology as to how innocuous touch becomes painful under neuropathic pain conditions. To develop a new hybrid voltage indicator we will build upon previous work in the Heppenstall lab in which they demonstrated that the fluorescent dye Nile Red is voltage sensitive when inserted in the membrane electric field. Utilizing a peptide/protein tag system, we will covalently tether Nile Red to the extracellular surface of cells and determine the optimal conditions for voltage sensitivity. We will then use this information to design strategies to directly tether Nile Red to exposed surfaces of the opsin-based GEVI, Ace2N. Through electrochromic FRET, we expect that the resultant hybrid probe will display higher sensitivity and faster kinetics than current state of the art indicators, with substantially higher signal to noise. On the instrumentation side, the Prevedel lab will build upon previous work and develop a novel, high-speed two-photon microscope capable of resolving millisecond voltage dynamics over large volumes. For this, spatio-temporal multiplexing will be used to upgrade the recently demonstrated light-sculpting approach in order to achieve kHz frame rates over 500x500?m large neuronal tissues. Finally, to validate and benchmark our new technology in vivo, we will test it in complex tissue of the mouse spinal cord. We will investigate how neuronal plasticity in the dorsal horn underlies hypersensitivity to light touch under neuropathic pain conditions. By optogenetically activating mechanoreceptors in the skin and monitoring evoked neuronal activity in the dorsal horn, we aim to understand how spinal circuits are recruited under pathological conditions to drive the transition from innocuous touch to noxious pain after injury.