Invited:
TWO-PHOTON COMPATIBLE LIGHT-GATED NMDARS

The intricate connections between neurons in the brain (i.e., connectome) is the substrate that allows us to sense the outside world, coordinate activity, store and retrieve memories, as well as instigate disease. Understanding the connectome requires knowledge from the system`s level, through cellular, all the way down to the molecular architecture of synapses. Addressing these questions requires methodologies that allow selective and reversible modulation of diverse classes of synaptic functions in vivo. Synthetic optogenetics is precisely suited for this task. This approach employs genetically-modified receptors to which synthetic light-sensitive molecules—or photoswitches—are appended. Photon-absorption by the molecules causes change in their geometry, which is then leveraged to modulate the activity of the protein they are immobilize onto. The change in geometry is completely reversible, therefore providing the user with the ability to toggle between the active and inactive states of the photoswitches, correspondingly triggering or stopping the protein’s activity. This approach provides remote and reversible means to control select receptors of the synapse, as well as their signaling mechanisms, by light. Using this technology, we have previously created a family of light-gated NMDARs (LiGluNRs), explicitly light-blocked Glu1a and -2A; as well as light-activated GluN2A and -B. We now expand this palette and introduce light-gated GluN2C and –D. To explore the role of receptors’ activities in disease requires means to control these using better tissue-penetrating wavelengths, namely two-photon excitation (2PE). Whereas 2PE can activate various optogenetic tools, as well as photoswitches, so far LiGluNs could not be amended by 2PE. With the use of second generation red-shifted photoswitches, and new LiGluN variants, we succeeded in achieving this in hippocampal neurons. We now present new 2PE-compatible LiGluNs with which we intend to explore whether select spatiotemopral activation or block of GluN2A and -B can modulate disease phenotypes.