Persistence of quantal synaptic vesicle recycling following dynamin depletion

Olusoji A.T. Afuwape, Natali L. Chanaday, Merve Kasap, Lisa M. Monteggia, Ege T. Kavalali

Preprint posted on 12 June 2020

Pinch me— I'm dreaming of dynamin-free vesicle recovery

Selected by Julia Bleier

Categories: neuroscience, physiology

Background and Approach:

During chemical neurotransmission, synaptic vesicles fuse with the presynaptic plasma membrane and release neurotransmitters into the cleft, but the membrane belonging to the vesicles must also be retrieved to reform vesicles. While mechanisms of endocytosis of synaptic vesicles are somewhat contentious, it is generally accepted that the GTPase dynamin plays an essential role in the process by forming rings that pinch off and recover endocytic vesicles from the plasma membrane. Because the triple KO mouse of all three dynamin isoforms is not viable, and dynamin inhibitors have been found to be nonspecific (1), it has been difficult to fully assess the role of dynamin in synaptic vesicle endocytosis.

To directly address this question, the authors used hippocampal neuronal cultures from mice with constitutive and conditional loss of dynamin isoforms. One of the key methods the authors use to examine vesicle release and endocytosis is the pH-sensitive fluorescent reporter vGluT1-pHluorin (2). This reporter is quenched when vesicles are intact due to their acidic interior and becomes fluorescent upon fusion with the plasma membrane due to the neutral extracellular environment. This allows Afuwape et al. to determine the time course of fusion and vesicle recovery following fusion. They examine dynamin’s function in neurotransmission and synaptic vesicle recycling during both strong stimulation and during single synaptic vesicle endocytosis.

Key Findings:

While synapses lacking all three dynamin isoforms (Dnm TKO) showed no deficits in morphology, these synapses exhibited a decrease in the number of both total and docked synaptic vesicles, compared to synapses lacking only dynamin 3. Accordingly, Dnm TKO neurons, but not Dnm 2 KOs, demonstrated impaired excitatory neurotransmission: evoked excitatory postsynaptic currents were decreased and synapses demonstrated facilitation to trains of stimuli, a common indicator of decreased evoked release probability.

Though the expectation might be to find arrested synaptic vesicle recycling following high frequency stimulation, the authors find that different synapses respond distinctly to the absence of all dynamins. Only 40% of boutons showed a deficit in endocytosis, the rest either showing no, or only partial deficit compared to boutons lacking only dynamin 3.

Figure 1, taken directly from Figure 2B of Afuwape et al. under a Creative Commons CC BY 4.0 license. 60% of synapses demonstrated no deficit in vGlut1-pHluorin recovery during 20Hz stimulation in the absence of all dynamins, suggesting that endocytosis persists in its absence.


This significant result implies that dynamin-independent endocytosis of vesicles occurs at central synapses. It also suggests that dynamin-dependent and independent endocytosis may have unequal importance across synapses. Even more strikingly, single synaptic vesicle fusion events show a similar distribution of vGlut1-pHluorin amplitudes dwell times in the absence of just dynamin 3 or all three dynamins, suggesting that dynamins are not required for single vesicle endocytosis.

Figure 2, taken directly from Figure 2K and 2L of Afuwape et al. under a Creative Commons CC BY 4.0 license. vGlut1-pHluorin amplitude and dwell time of single synaptic vesicles is nearly identical in Dnm TKO neurons compared to Dnm3 KO controls. This suggests that dynamins are not essential for single synaptic vesicle endocytosis.


To provide further support for the idea that dynamins are only required for multi-vesicular endocytosis, the authors more closely examined the fraction of events which decayed rapidly (<1s), with a short dwell time (1-10s), or with a longer dwell time (>10s). For synapses that demonstrated slowed endocytosis after high frequency stimulation, a very small fraction had long dwell times compared to all synapses from TKO or control neurons, supporting the idea that dynamin is most critical for multi-vesicle endocytosis.

These results imply that there must be other proteins which can carry out single vesicle recycling in addition to dynamin. The authors pharmacologically tested the involvement of actin, DRP-1 and Arp2/3 complex, which have previously been implicated in synaptic vesicle endocytosis (3, 4), finding that they do not play a major role in the recycling of spontaneously fused synaptic vesicles.


Though other studies have suggested dynamin independent vesicle endocytosis (5, 6), this study elegantly shows that single synaptic vesicles can be recycled in the absence of dynamin. Determining the proteins responsible for scission during endocytosis of synaptic vesicles in its absence is an exciting new direction for the field. This work also points to the possibility that different synapses operate with distinct endocytosis mechanisms, both within the population of excitatory synapses and between excitatory and inhibitory synapses, which is a potential mechanism for physiological diversity.


Questions for authors and open questions for future study:

  1. Under basal conditions, are the three dynamin isoforms likely to function at each synapse or does their distribution vary across synapses? What is the current understanding of the degree to which they are unique or redundant in hippocampal synapses?
  2. The authors find that depleting all three dynamins reduces release probability in both excitatory and inhibitory synapses, mentioning reduced vesicle availability and limited viable release sites as two potential mechanisms. How might one distinguish between these two possibilities? Is it also possible that dynamins are involved more directly in exocytosis?
  3. The authors justify the use of dynamin 3 KOs as controls by stating that this provides the most consistent comparisons with littermates and because previous studies showed no significant functional or structural deficits. I’m wondering if dynamin 1 and dynamin 2 are upregulated at the mRNA or protein level in dynamin 3 KO neurons and what effect this might have over developmental time.
  4. Is it possible that some of the rapid decay events correspond to kiss-and-run type release events in which the fusion pore opens and closes without fully fusing with the plasma membrane?
  5. If observed events all correspond to full fusion of vesicles in which all neurotransmitter is released, are individual events with different dwell times likely to be different in terms of the physiological effect on the post-synapse?



  1. Park, R.J., Shen, H., Liu, L., Liu, X., Ferguson, S.M., and De Camilli, P. (2013). Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors. Journal of cell science 126, 5305-5312.
  2. Voglmaier, S.M., Kam, K., Yang, H., Fortin, D.L., Hua, Z., Nicoll, R.A., and Edwards, R.H. (2006). Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51, 71-84.
  3. Lee, E., and De Camilli, P. (2002). Dynamin at actin tails. Proc Natl Acad Sci U S A, 161-166
  4. Orth, J.D., Krueger, E.W., Cao, H., and McNiven, M.A. (2002). The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc Natl Acad Sci U S A 99, 167- 594 172.
  5. Xu, J., McNeil, B., Wu, W., Nees, D., Bai, L., and Wu, L.G. (2008). GTP-independent rapid and slow endocytosis at a central synapse. Nat Neurosci 11, 45-53.
  6. J. Van Hook, W.B. Thoreson Rapid synaptic vesicle endocytosis in cone photoreceptors of salamander retina J. Neurosci., 32 (2012), pp. 18112-18123



Tags: dynamin, endocytosis, hippocampal culture, synapses, synaptic vesicles

Posted on: 30 June 2020 , updated on: 14 July 2020


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Author's response

The author team shared

1. We currently do not know if the relative levels of each dynamin isoform are different among
individual synapses. To our knowledge, available dynamin antibodies are not sufficiently
specific to discern expression of distinct isoforms at the synaptic level using immunostaining.
However, we agree that this is an important point. We will address this question as soon as we
can identify the necessary tools.

2. There is some evidence that dynamins may play a direct role in exocytosis. In synapses,
dynamins may impact fusion by facilitating clearance of previously fused vesicles from the
plasma membrane. Our results suggest that fundamental aspects of the fusion process — as
detected using electrophysiology and pHluorin based imaging — are unaffected while fusion
propensity is decreased.

3. The dynamin 3 mouse KO line was generated in the laboratory of Dr. Pietro de Camilli. In
their publication (see Raimondi et al., Neuron 2011; specifically Figure 1C) they show by
immunoblot that protein levels of dynamins 1 and 2 are not changed in several tissues
(including brain) of the dynamin 3 KO mice, compared to wild-type littermates. So other
isoforms do not seem to be upregulated in dynamin 3 KO animals. In agreement with these
earlier results, in our hands dynamin 3 KO mice and synapses appear completely normal.

4. This is a possibility. The kinetics of fast events, with very short dwell times (i.e. extremely
fast endocytosis) are consistent with kiss-and-run. However, our measurements do not provide
a direct assessment of fusion pore dynamics, therefore, we cannot rule out if another cellular
mechanism with the same time course is responsible for these events.

5. This is an intriguing possibility. As indicated in our manuscript, we only detect a difference in
the propensity (or probability) of fusion events but not in their kinetics as measured by AMPA
receptor mediated responses. In our optical recordings, even the fastest events we detect are
in the order of 100 ms, which is at least two-orders of magnitude slower than expected time
course of a synaptic vesicle fusion pore. That said, we cannot exclude the possibility that a more
in-depth analysis of synaptic events at a fast time scale may reveal differences.

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