The recent advent of optogenetics and photopharmacology has made the dream of remotely controlling neurons with light come true. Azobenzene photoswitches are at the core of some of the main strategies explored to reach this goal and, as such, an increasing number of azo derivatives are being developed to photomodulate neural activity. However, to make these molecular tools viable for real application, optimization of their light-induced response is required. In particular, a key challenge that must be addressed is multiphoton excitation of azoaromatic switches with near-infrared light, an essential feature to accomplish neuron manipulation with sub-micrometric resolution in three dimensions, deeper penetration into tissue, lower photodamage and patterned illumination.
Prompted by this need, back in 2009 we started a collaboration with Pau Gorostiza’s group at IBEC, which led us to report in 2014 the first case of manipulation of neural activity using two-photon excitation of azo compounds with NIR light. Unfortunately, the practical use of this technology to photostimulate individual cells in neuronal tissue has so far been hampered by two main factors: (a) the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and (b) the short lifetime of the cis state produced upon excitation of the two-photon responsive azo switches.
In view of this situation, we recently decided to take a step back and conduct the rational design of optimized azobenzenes for two-photon operation based on theoretical calculations (Nature Communications 10, 907 (2019)). This allowed us to identify the structural motifs in azo switches that increase their two-photon excitability without compromising their cis state lifetime, an information that was then exploited for the synthesis of new compounds capable to phototrigger ionotropic glutamate receptors regulating excitatory neurotransmission. Optimized and sustained two-photon neuronal stimulation was achieved with these photoswitches both in light-scattering brain tissue and in Caenorhabditis elegans nematodes (in collaboration with Michael Krieg's group at ICFO), obtaining photoresponses that are comparable to those registered under one-photon excitation. These results constitute a proof of concept that opens the way to use azobenzene switches to dissect intact neuronal circuits in three dimensions.
If you want to read more about our work, check out this link: https://www.nature.com/articles/s41467-019-08796-9