Membrane Lipids and Synaptic Vesicle Trafficking in the CNS

Abstract

Most vesicles within a synapse are dormant. The rest participate in synaptic neurotransmission, with a portion of these preferentially fusing first. Moreover, all synapses experience spontaneous neurotransmitter release which may originate from the random exocytosis of vesicles prepared to fuse immediately upon calcium influx; however, spontaneously fusing vesicles may be independent because they prefer spontaneous fusion. The functional separation argues that the compositions the synaptic vesicle membranes are somehow unique between pools.

The first three chapters explore the role of cholesterol in synaptic transmission. We treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed augmented spontaneous neurotransmission. In contrast, the same treatments severely impaired responses evoked by action potentials and hypertonicity. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. Chapter five examines the role of sphingosine on neurotransmitter release. By adding sphingosine to hippocampal cultures, we found that sphingosine enhances neurotransmission in a synaptobrevin-2-dependent manner. Chapter six investigates the stability of actively recycling synaptic vesicles. We employed several approaches (fluorescent and ultrastructural imaging) to monitor not only the fate recycling vesicles, but also the origin and reuse of spontaneously fusing vesicles. We conclude that at rest, the total recycling pool remains active and resists spontaneous fusion up to at least six hours; while spontaneous fusion of spontaneously fusing vesicles is much faster. This argues that vesicles fusing spontaneously do not originate from the recycling pool. In chapter seven, we observe how modifying synaptic vesicle membranes might affect neurotransmitter release. By the uptake of horseradish peroxidase into vesicles followed by hydrogen peroxide perfusion, we induced free radical modification of vesicle membranes and found that modifying recycling pool vesicles increased spontaneous fusion and attenuated evoked release. Taken together, the results of each chapter appear to suggest that the fusion of action potential-dependent and-independent vesicles are regulated by different mechanisms, supporting the theory that some vesicles may be unique within a synapse.