Monitoring activity-dependent bulk endocytosis with the genetically-encoded reporter VAMP4-pHluorin

J Neurosci Methods. 2016 Jun 15:266:1-10. doi: 10.1016/j.jneumeth.2016.03.011. Epub 2016 Mar 23.


Background: Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during intense neuronal activity, implicating it as a major contributor to presynaptic plasticity under these stimulation conditions. However methods to monitor this endocytosis mode have been limited to either morphological or optical observation of the uptake of large fluid phase markers.

New method: We present here a method to monitor ADBE using the genetically-encoded reporter VAMP4-pHluorin in primary neuronal cultures.

Results: Individual nerve terminals expressing VAMP4-pHluorin display either an increase or decrease in fluorescence after stimulation terminates. The decrease in fluorescence reflects the slow acidification of large bulk endosomes to which VAMP4-pHluorin is selectively recruited. Use of VAMP4-pHluorin during sequential high frequency stimuli revealed that all nerve terminals perform ADBE, but not all do so in response to a single stimulus. VAMP4-pHluorin also displays a rapid activity-dependent decrease in fluorescence during high frequency stimulation, a response which is particularly prominent when expressed in hippocampal neurons. The molecular mechanism responsible for this decrease is still unclear, but is not due to loss of VAMP4-pHluorin from the nerve terminal.

Comparison with existing methods: This method allows the selective reporting of ADBE for the first time, when compared to previous approaches using markers of fluid phase uptake.

Conclusions: The development of VAMP4-pHluorin as a selective genetically-encoded reporter of ADBE increases the palette of approaches used to monitor this endocytosis mode both in vitro and in vivo.

Keywords: Clathrin; Endocytosis; Presynapse; VAMP4; Vesicle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Cerebellum / cytology
  • Cerebellum / physiology
  • Endocytosis / physiology*
  • Female
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Hippocampus / cytology
  • Hippocampus / physiology
  • Male
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence* / methods
  • Neurons / cytology
  • Neurons / physiology
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • Rats, Sprague-Dawley
  • Synaptic Transmission / physiology
  • Synaptic Vesicles / physiology*
  • Synaptophysin / genetics
  • Synaptophysin / metabolism
  • Transfection


  • PHluorin
  • R-SNARE Proteins
  • Synaptophysin
  • Green Fluorescent Proteins