Abstract

This thesis examines the efficiency of neurotransmitter vesicle recycling in two contexts, first, in the previously uncharacterized synapses of dopamine-releasing neurons, and second, in excitatory and inhibitory mouse cortical neurons lacking the inositol phosphatase Synaptojanin1.

Dopamine-releasing neurons in the ventral midbrain have well-documented roles in neurological function and mental health. Dopamine signaling is also distinguished from excitatory or inhibitory synaptic transmission by its slower timescale and greater spatial range. This study therefore characterizes the process of neurotransmitter vesicle recycling in dopaminergic neurons, using the synaptopHluorin and FM-dye based assays used to examine other model systems. In dopaminergic neurons, unlike hippocampal neurons, a six-fold increase in stimulus frequency failed to significantly deplete the recycling pool of vesicles. The rate of vesicle exocytosis and endocytosis were both modulated by stimulus frequency, but their efficiencies were coupled so as to limit vesicle pool depletion. Finally, the rate constant for endocytic retrieval after synaptic activity was slower than that during synaptic activity, suggesting that different endocytic modes were operational in these regimes.

The second part of this thesis examines the vesicle cycle in neurons from Synaptojanin1 knockout animals. Synaptojanin1 is a multifunctional protein conserved from yeast to humans, and contains two phospho-inositol phosphatase domains and a proline-rich domain. Genetic ablation of Synaptojanin1 leads to pleiotropic defects in presynaptic function, including synaptic depression, accumulation of free clathrin-coated vesicles and delayed post-endocytic vesicle re-availability. Our studies show that Synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and post-endocytic vesicle re-availability can be fully restored upon reintroduction of Synaptojanin1. Finally, 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 re-availability.