UC San Diego

The brain teems with bursts of fleeting activity. To deduce the meaning of its electrochemical flashes, an accurate record of their presence must first be captured. The acquisition of precise patterns of synaptic activity has been limited by the lack of tools to directly observe neurotransmitter release and propagation. This thesis demonstrates how a new class of experimental tools, genetically-encoded fluorescent reporters, provides an experimental modality well-suited to solve this problem. Novel reporters for optical recording of neurotransmitter release were developed. These were constructed by genetic fusion of fluorescent proteins to environmentally sensitive elements. These reporters rapidly changed their fluorescence in response to changes in neurotransmitter concentration or pH. The most successful reporter was a sensor for the excitatory neurotransmitter glutamate. The dynamic range this reporter was strongly enhanced by a comprehensive screen of linker mutations and its affinity was tuned for measurement of synaptically released glutamate by mutagenesis of its ligand-binding pocket. This optimized reporter was genetically targeted to the extracellular surface of neurons. The release, propagation, and recycling of synaptically released glutamate in response to electrical stimulation was quantitatively determined by recording the changes in the color of the reporter's fluorescence. Functionally relevant levels of glutamate were found to spill beyond the synaptic cleft to extrasynaptic regions in an activity-dependent manner, implying that the independence of synaptic signaling is determined by the degree of neuronal excitation. These glutamate reporters were also used as a measurement of presynaptic strength in a model system of long-term potentiation. Prototype reporters for inhibitory neurotransmission and a spectrally distinct reporter of synaptic vesicle fusion were also designed and tested, but had limited functionality. Besides the creation of a powerful new method of recording synaptic activity, the successes and failures of sensor development provide an illustrated primer to the design and optimization of future genetically-encoded fluorescent reporters