Exocytosis of neurotransmitter from a synaptic vesicle is followed by efficient retrieval of its constituent membrane and proteins. Several different mechanisms of endocytosis occur at synapses, but their relative importance has been unclear (Royle & Lagnado, 2003). We have investigated the role of clathrin-mediated endocytosis (CME) by using RNA interference (RNAi) to selectively knock-down the expression of clathrin heavy chain (CHC) in hippocampal neurons.

Primary cultures of rat hippocampal neurons were prepared from E19 pups and all animals were humanely killed. Exocytosis and endocytosis were measured by confocal imaging of the optical probe synapto-pHluorin (SpH), which is quenched at the acidic pH within synaptic vesicles (Sankaranarayanan et al. 2000) and the styryl dye FM4-64. RNAi was achieved by transfection of short interfering RNAs (siRNAs).

At 72 h post-transfection the expression of CHC was reduced to 10.6 ± 0.8 % compared to control neurons (mean ± S.E.M.) and transferrin uptake by CME was reduced to 38.6 ± 0.9 %. Depolarization for 60 s by local perfusion of hyperkalaemic saline caused an increase in SpH fluorescence, reflecting exocytosis. SpH fluorescence then decayed back to resting levels, reflecting retrieval of the probe from the membrane surface into acidified organelles (Sankaranarayanan et al. 2000). In control neurons transfected with scrambled siRNA, SpH fluorescence increased 4-fold on stimulation and recovery was complete within 3-4 mins (220 boutons in 7 neurons). In neurons in which CHC was knocked-down, the exocytic response was inhibited in 83 % of boutons and there was little or no recovery over the 5 mins following exocytosis (210 boutons in 12 neurons). In separate experiments, FM4-64 (15 µM) was applied in the hyperkalaemic saline and then washed off to stain any vesicles that were internalised. Synaptic boutons in control neurons stained brightly, but the dye was not internalized by boutons of neurons in which CHC was knocked down.

These findings suggest that CME plays an important role in the retrieval of synaptic vesicles at the synapse. Inhibition of CME also leads to a reduction in the number of vesicles available for exocytosis.

This work was supported by the Human Frontiers Science Program (HFSP)