Yumei Wu; Eileen T O'Toole; Martine Girard; Brigitte Ritter; Mirko Messa; Xinran Liu; Peter S McPherson; Shawn M Ferguson; Pietro De Camilli

“ Once a neuron receives an electrical stimulus, the membranes that enclose the synaptic vesicles fuse with the plasma membrane that encloses the neuron. This releases the neurotransmitters, which then trigger an electrical signal in the neighboring cell. ”

“ Bulk endosomes could be the direct source of SVs, or they may first fuse back to the plasma membrane from which new SVs could then bud by CME. To discriminate between these possibilities, cultures of all four genotypes were exposed to CTX-HRP only during the recovery phase, following a 90 s stimulus, and then analyzed at the 10 and 30 min time points (Figure 1B, Figure 2E–H) . In these samples, only very few large vacuoles were HRP labeled (Figure 2G,H) , indicating that bulk endocytosis occurs nearly exclusively during the high K+ stimulation. ”

“ Our results demonstrate that SV reformation following a strong, acute, secretory stimulus (either high K+ depolarization or high frequency electrical activity) occurs predominantly via the budding of SVs from endosomal intermediates generated by bulk endocytosis (bulk endosomes) . ”

“ To assess the contribution to bulk endocytosis of the other neuronal dynamin, dynamin 3, which is encoded by the DNM3 gene, and to monitor reformation of SVs from bulk endosomes, we performed experiments with primary cultures of WT, dynamin 1 KO (Dyn1 KO) , dynamin 3 KO (Dyn3 KO) and dynamin 1 and 3 double KO (Dyn1/3 DKO) mice. ”

“ Interestingly, dynamin 1 is constitutively phosphorylated in resting nerve terminals and its Ca2+ triggered dephosphorylation upon synaptic stimulation results in its binding to the F-BAR domain containing protein syndapin/pacsin ( [33] ; [29] ; [34] ) , which has also been implicated in bulk endocytosis ( [35] ) . However, studies of dynamin 1 knock-out (KO) neurons have indicated the occurrence of robust bulk endocytosis even in the absence of dynamin 1 ( [17] ) , in spite of a strong impairment of CME ( [36] ; [37] ) . ”

“ SVs reformed from bulk endosomes can undergo exocytosis upon stimulation. (A) Control (Dyn3 KO) and Dyn1/3 DKO neurons were stimulated by a 90 s high K+ stimulation in the presence of HRP, rested for 30 min in the absence of HRP and then subjected to a second round of high K+ stimulation and recovery, always in the absence of HRP. Loss of HRP-labeled SVs during the second stimulus, and the subsequent recovery of unlabeled SVs, demonstrates that vesicles generated from bulk endosomes are functional SVs. ”

“ High K+ stimulation induces bulk endocytosis, as assessed by CTX-HRP, at both WT and dynamin mutant synapses.Following incubation in resting buffer (R) , neurons were stimulated with 90 mM K+ for 90 s, then incubated in resting buffer for 10 and 30 min. CTX-HRP, added either during or after the stimulus (A) , or selectively after the stimulus (B) , was used as an endocytic tracer. In all genotypes, loss of SVs during the stimulus is compensated by the appearance of numerous bulk endosomes. ”

“ The HRP-positive CCPs in the resting (R) and stimulated (K+) samples reflect accessibility to the extracellular medium of arrested plasma membrane CCPs in dynamin KO synapses. (E) Electron micrograph of a Dyn1 KO synapse incubated for 5 min with CTX-HRP selectively during recovery (as for field A) . Most CCPs are HRP reaction product positive (red arrows) . (F) Dyn1 KO synapse incubated with soluble HRP during stimulation and then washed and examined at 3 min (as for field C) . All CCPs are HRP reaction product negative. Scale bar = 100 nm. ”

“ Pie charts summarizing results of fields A and B concerning the reformation of SVs at different times after recovery. Entire pie: total SVs before stimulation; white sector: unlabeled SVs; black sector: labeled SVs after stimulation; striped section: total labeled SVs subtracted by the SVs formed during stimulation. ****, ***, **, * indicate p-values of <0.0001, <0.001, <0.01, and <0.05, respectively. N.D., ‘not determinated’. Black asterisks refer to comparisons between total vesicles, colored asterisks to comparisons between HRP-labeled vesicles. ”

“ All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#07422-2012) of Yale university. All animals were euthanized with CO2 and embryos were used for primary neuron culture. ”