The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.

␣-Synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, ␣-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, ␣-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of ␣-synuclein with cell membranes, we find that ␣-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of ␣-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of ␣-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute ␣-synuclein away from synapses, indicating an important role for raft association in the normal function of ␣-synuclein and its role in the pathogenesis of PD.

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

The presynaptic protein ␣-synuclein has a central role in Parkinson's disease (PD). However, the mechanism by which the protein contributes to neurodegeneration and its normal function remain unknown. ␣-Synuclein localizes to the nerve terminal and interacts with artificial membranes in vitro but binds weakly to native brain membranes. To characterize the membrane association of ␣-synuclein in living neurons, we used fluorescence recovery after photobleaching. Despite its enrichment at the synapse, ␣-synuclein is highly mobile, with rapid exchange between adjacent synapses. In addition, we find that ␣-synuclein disperses from the nerve terminal in response to neural activity. Dispersion depends on exocytosis, but unlike other synaptic vesicle proteins, ␣-synuclein dissociates from the synaptic vesicle membrane after fusion. Furthermore, the dispersion of ␣-synuclein is graded with respect to stimulus intensity. Neural activity thus controls the normal function of ␣-synuclein at the nerve terminal and may influence its role in PD.