A rhodopsin-based two-photon voltage indicator for all-optical mapping of synaptic connectivity in vivo
Biophysics
Final Report Abstract
Unraveling functional synaptic connectivity -ideally in awake animals- remains one of the indispensable goals of modern neuroscience on the way to understand how the mammalian brain computes. To date, most approaches still fundamentally rely on electrical recordings as the gold-standard method to resolve (subthreshold) changes in membrane potential. The invasive, low-throughput nature of electrical approaches could be overcome by performing activity monitoring and manipulation with optical tools instead of electrodes. Hence, this project aimed at the establishment of a genetically-encoded, rhodopsin-based tool for the non-invasive interrogation and manipulation of neuronal activity under two-photon illumination, which could ultimately be used for the all-optical mapping of synaptic connectivity. Additionally, it was aspired to realize the activity interrogation with a voltage sensor to be able to detect subthreshold changes or hyperpolarization of the membrane potential, which is impossible with calcium indicators and essential to identify synaptic connections. In the first part of the project we were aiming at establishing a method for the all-optical mapping of local connectivity using two-photon illumination. We were able to demonstrate that we can probe hundreds of potential pre-synaptic cells in a short time using two-photon holographic multi-cell stimulation to non-invasively induce action potentials while reading out the post-synaptic response electrically to indentify connections. More importantly, we showed that this pre-synaptic stimulation can be complemented with simoultaneous two-photon voltage imaging to obtain an all-optical induction and read out of the pre-synaptic action potential including the exact spike timing, which is a major advance towards high-throughput connectivity mapping with optical approaches. In the second part of the project we succesfully engineered a FRET-opsin voltage indicator using the brightest reported GFP-homologe to date. The sensor showed robust expression in cultured cells and hippocampal slices and displayed reliable voltage responses using 1-photon widefield illumination. In contrast to the non-rhodpsin GEVIs ASAP3 and JEDI-2P, the sensor is characterized by a linear sensitivity curve, which could be an advantage for the detection of subthreshold voltage changes, and additionally -despite a lower ΔF/F0- the sensor displayed a higher SNR at the same imaging conditions. Lastly, we were able to show that the sensor can be used under temporally-focused two-photon holographic illumination to detect action potentials in a single trial at kHz acquisition rates, paving the way for the use of rhodopsin-based voltage indicators under two-photon illumination.
