Title: Development of Fluorescent Probes for Imaging Synaptic Activity at Individual Presynaptic Terminals
Description: This thesis describes the design, synthesis and development of fluorescent probes to monitor synaptic transmission at individual presynaptic terminals in the mouse brain. Two distinct approaches to accomplish this are discussed. The first approach seeks to monitor synaptic activity by using pH-sensitive endocytic membrane probes to label active presynaptic terminals. The second approach seeks to monitor synaptic activity by loading small fluorescent molecules into presynaptic vesicles and studying their evoked release upon stimulation. The first chapter of this thesis describes currently available techniques that are used to study synaptic transmission in the brain. The use of electrochemical techniques is discussed and the use of fluorescent reporters is introduced as a means to image single synapses with high resolution. Chapter II of this thesis describes the rational design of pH-sensitive membrane probes for labeling recycling vesicles. The synthesis, photophysical properties and biological characterization of these probes are described.

Although these probes proved to be too lipophilic to work well in the brain tissue and neuronal culture, their use on the cell surface is demonstrated. Furthermore, the structure activity relationship established by this library of probes can be used to direct the future development of pH-sensing endocytic dyes. Chapter III and IV of this thesis describe the development of new generations of Fluorescent False Neurotransmitters (FFNs) for imaging vesicular content release from individual presynaptic terminals in the brain. Chapter III introduces a novel imaging agent, FFN200, for monitoring and quantifying dopamine release from individual synaptic terminals in the mouse brain. Chapter IV describes the exploration and screening of small fluorescent molecules in the mouse brain for the purpose of developing FFNs at synaptic terminals other than dopamine. FFN7122 is introduced as the first FFN to be developed for terminals outside of dopamine. FFN7122 is shown to be a marker for glutamatergic terminals in the hippocampus, dorsal striatum, and motor cortex of the mouse brain.

The evoked release of this probe from presynaptic vesicles is demonstrated and two hypotheses for its uptake mechanism are proposed.