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

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.