Design and Synthesis of Chemical Tools for Imaging Neuronal Activity
The ultimate goal of neuroscience is to correlate the activity of neurons and neuronal networks to higher order behaviors and cognition. This colossal task necessitates a variety of optical tools that can both measure and modulate neuronal activity. This dissertation describes the design, synthesis and characterization of a variety of such tools. In order to interrogate the electrical activity of neuronal circuits directly, we synthesized tetramethylrhodamine-based voltage reporters, or RhoVRs, that possess excitation and emission profiles in the green to orange region of the visible spectrum and use photoinduced electron transfer (PeT) as a trigger for voltage sensing. Two RhoVRs with particularly attractive photophysical properties were developed: RhoVR 1, which possessed the highest voltage sensitivity of any RhoVR (47% ΔF/F per 100 mV), and RhoVR(Me), a less sensitive (13% ΔF/F per 100 mV) but far brighter RhoVR (4-fold brighter than RhoVR 1). In order to facilitate the use of RhoVRs in complex tissues such as brain slice or in vivo, we developed genetically targetable RhoVRs using the HaloTag system (RhoVR-Halos). RhoVR-Halos maintained the fast kinetics and high sensitivities typical of RhoVRs (up to 34% ΔF/F per 100 mV) while labeling HaloTag expressing cells with high selectivity in culture, brain slice and in vivo. In addition, RhoVRs possess high two-photon cross sections (up to 190 GM), making them excellent candidates for two-photon voltage imaging. While voltage indicators allow for the study of fast voltage dynamics in real time, they necessitate very fast imaging speeds that are not well suited for mapping activity across large neuronal networks. In order to facilitate neuronal activity mapping, we developed a Ca2+ integrator MethylAzid-1 (MA-1). MA-1 acts as a coincidence detector for both light and Ca2+, allowing a temporally precise “snapshot” of [Ca2+] to be taken. MA-1 undergoes a 25 nm shift in absorbance upon chelation of Ca2+ (Kd = 270 nM) that allows for the selective photolysis of Ca2+-free MA-1 with 400 nm light. The extent of photolysis can be visualized post hoc through either “click chemistry” or the fluorogenic reduction of remaining MA-1. In addition to tools which report neuronal activity, we synthesized a new class of photocages based on 6-aminobenzofurans (BFCs) that can be used to selectively release caged compounds, such as neurotransmitters, upon irradiation with UV light. BFCs operate through a photo-induced elimination reaction that generates an extended azaquinone-methide from the excited state. Initial studies revealed BFCs are strong absorbers of UV/Violet light (λmax = 365 nm, ε = 30,000 M-1 cm-1) that readily photolyze (Φphotolysis up to 0.18) to release their caged cargo. Together, this work demonstrates the power of optical tools to study neuronal systems ranging from sub-cellular domains to large networks of neurons and lays the groundwork for their application in complex tissues such as brain slice or in vivo.