Live imaging of synaptic vesicle release and retrieval in dopaminergic neurons.

Dopaminergic (DA) neurons represent <0.01% of neurons in the human brain, but are essential for normal neurological and psychiatric function. The majority of these neurons reside in the ventral midbrain, but they exert their profound influences on brain function through projections to both the cortex and the basal ganglia. These projections secrete dopamine from small clear synaptic vesicles (SVs) in axonal varicosities. DA signaling has unique spatial and temporal characteristics as compared to the fast, focal synaptic transmission of excitatory and inhibitory neurons. However, as with fast-acting neurotransmitters, DA SVs must be locally recycled for use following exocytosis. Little is known about these DA SV recycling properties and how they might impact efficacy of DA neurotransmission. Here we used the pH-sensitive fluorescent probe synaptopHluorin to investigate SV recycling in DA neurons and compared their properties to prototypical fast neurotransmitter synapses of the hippocampus. These measurements showed that DA SVs, like hippocampal SVs, have a resting pH of approximately 5.6. However, compared to hippocampal neurons, DA neurons show limited depletion of the recycling pool of vesicles as the stimulus frequency is increased from 5 to 30 Hz. Additional measurements show that exocytosis rates at this frequency are comparable between hippocampal and DA neurons. Thus, limited vesicle depletion likely arises from a stimulus frequency-dependent acceleration of DA SV endocytosis or re-acidification. Our observations imply differential regulation of endocytic-exocytic balance in DA neurons. Finally, our assay can also be used to investigate the effects of genetic and chemical modulation of the SV cycle.

The dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals.

Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phosphoinositol phosphatase domains and a proline-rich domain. Genetic ablation of synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle reavailability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1(-/-) neurons. Our studies show that synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and postendocytic vesicle reavailability can be fully restored upon reintroduction of synaptojanin1. However, expression of synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and reavailability.