Synapses transform neurotransmitter signals derived from other neurons into intracellular electrical and biochemical responses. In turn, this transformation affects the response of the parent neuron and is modified by the state of the neuron and its environment. In this thesis, we investigate three separate mechanisms regulating synapse structure and function.

PSD-95 is a multi-domain scaffolding protein of the postsynaptic density (PSD) that regulates both basal synaptic transmission and plasticity. Through a series of multivalent interactions with itself and other proteins, PSD-95 is believed to form a stable protein lattice within the PSD. To elucidate molecular mechanisms underlying PSD-95 stabilization, we photoactivate PAGFP-tagged PSD-95 within dendritic spines and directly measure its turnover and exchange. Using this technique, we confirm that PSD-95 is more stable than other PSD proteins and identify distinct sets of domains within PSD-95 mediating PSD formation, PSD-95 stabilization and activity-dependent modifications of the PSD.

Synaptic transmission is metabolically expensive due to the costs of reversing large ion fluxes across neuronal membranes against substantial concentration gradients. We test the hypothesis that AMP-activated protein kinase (AMPK) helps coordinate synaptic transmission and neuronal metabolism. We determine that AMPK is basally active in neurons and that its catalytic activity is driven by ongoing neuronal activity. As the regulation of synaptic transmission might serve as a mechanism to limit metabolic demand, we examine whether AMPK regulates synaptic transmission on both acute and chronic time-scales. We do not find evidence for acute effects of AMPK but we identify a role for AMPK in the regulation of spine morphology over chronic time scales.

In order to determine whether asymmetries in phosphoinositide distribution exist across dendrites and spines, we utilize fluorescent reporters harboring phosphoinositide-specific binding domains. To enable measurement of phosphoinositide distribution in dendrites and spines, we develop two separate methods for detecting changes in phosphoinositide distribution in small structures. In the first, changes in the relative distribution of reporter between spines and dendrites are used to infer changes in phosphoinositide levels at the plasma membrane within these structures. In the second, we measure the effect of membrane binding on reporter diffusion.