Lighting-up neurons: characterization of new membrane-targeted photoswitches for geneless neural stimulation

Doctoral Program in Neuroscience Curriculum in Neuroscience and Brain Technologies XXXVI cycle   Summary In the last decades, light-sensitive nanotools have emerged as a powerful approach for modulating neural circuits’ activity with exquisite precision. Among the light-sensitive approaches, membrane-targeted photoswitches hold particular promise in enabling light-induced control of neuronal activity without directly interfering with physiological mechanisms and avoiding genetic manipulation. This doctoral thesis aims to unravel the intricate dynamics driven by light in the context of four distinct newly-synthetized membrane-targeted photochromic molecules. The foundation of this research lies in the characterization of Ziapin2, a newly synthetized photoswitch that demonstrated remarkable success in inducing firing activity in response to light, both in vitro and in vivo. Ziapin2 showed a distinct affinity for lipid rafts, membrane domains characterized by high concentrations of diverse proteins, including ion channels and receptors. Building upon this knowledge, our investigation focused on the light-dependent modulation of synaptic neurotransmission in primary neurons mediated by Ziapin2, recognizing the pivotal role of lipid rafts in neurotransmitter release. This exploration involved distinguishing between excitatory and inhibitory neuronal subpopulations, considering their inherent physiological differences in protein expression and synaptic machinery. The results revealed an opposite modulation pattern in light-triggered modulation of synaptic transmission, providing insights on its potential applications in controlling neural networks. Moreover, we conducted a comprehensive characterization of 2Pyr-2Pyr, a photoswitch closely related to Ziapin2 but featuring a structural distinction that could potentially enhance its functionality. Our findings indicate that 2Pyr-2Pyr shares similar properties in modulating neuronal activity, offering the prospect of exploiting its structural difference for further engineering. Specifically, it could enable prolonged cellular membrane permanence, addressing a crucial challenge faced by Ziapin2, which is internalization through membrane recycling mechanisms over time. In addition, this thesis studies newly synthetized Push-Pull molecules, employing a distinct donor-acceptor mechanism for membrane voltage modulation, unlike Ziapin2's capacitance modulation. The characterization of these molecules not only offered insights into alternative strategies for light-induced neuronal stimulation but also enhanced our understanding of the essential patterns of membrane voltage modulation required to initiate light-dependent firing activity. Lastly, we present BV-1, a donor-acceptor photoswitch without the azobenzene core, in contrast to the previously studied photoswitches. BV-1 not only demonstrated efficient membrane voltage modulation but also exhibited the unique ability to induce light-triggered membrane poration, expanding the potential repertoire of light-sensitive tools for neuroscience research. In conclusion, this Ph.D. thesis characterizes four membrane-targeted photoswitches with a focus on their potential applications in modulating membrane voltage, neuronal activity, and cell viability, while exploring the underlying mechanisms. The findings highlight the diverse functional properties of these photoswitches and their potential applications in neuroscience. This research contributes to the growing body of knowledge in the field of optostimulation and paves the way for the development of more efficient and precise tools for neuroscience experimentation.