Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging

Abstract

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode.

Introduction

Neurotransmitter release is dependent on the fusion of small synaptic vesicles (SVs) with the neuronal plasma membrane. The maintenance of neurotransmitter release is dependent on the subsequent retrieval and recycling of fused SVs. There are at least three modes by which a SV can be internalised. Both clathrin-dependent endocytosis and kiss-and-run modes of retrieval internalise single SVs (Edeling et al., 2006, Harata et al., 2006) and are the dominant modes of SV retrieval during low intensity stimulation (Granseth et al., 2006, Zhang et al., 2009, Zhu et al., 2009). However, during high intensity stimulation another SV endocytosis mode is triggered to increase the retrieval capacity within the nerve terminal, called activity-dependent bulk endocytosis (ADBE) (Cousin, 2009). ADBE is an activity-dependent fluid phase uptake mode that generates endosome-like structures direct from the plasma membrane. SVs can then bud from these endosomes to rejoin the recycling pool of SVs (Richards et al., 2000). Due to its large capacity, ADBE is the dominant SV retrieval mode in central nerve terminals during high intensity stimulation.

Fluorescence-based approaches have been predominantly employed to visualise SV recycling in neuronal culture, mainly due to the fact that it is difficult to directly measure either SV fusion or retrieval in a typical small central nerve terminal. The great majority of these methods utilise either the uptake of small fluorescent molecules (such as FM1-43, Cochilla et al., 1999, Cousin and Robinson, 1999) or the fusion of SV proteins to fluorescent proteins that report the pH of their immediate environment (Ryan, 2001). Unfortunately these methods do not differentiate between different SV retrieval modes such as clathrin-dependent endocytosis and ADBE. Therefore it is impossible to determine the contribution of either mode to SV retrieval during intense stimulation.

Because of the limitations in existing fluorescence approaches, we decided to establish a selective assay of ADBE, using dextran, a large inert fluid phase marker. Fluorescent-tagged dextrans are too large to be internalised within a single SV (Berthiaume et al., 1995, Araki et al., 1996, Holt et al., 2003, Teng et al., 2007). This means that any observed internalised fluorescence should be due to ADBE, since all other SV retrieval modes occur at the level of a single SV. We now report the development of a reliable, quantifiable and accurate method to monitor ADBE in a typical central nerve terminal in culture. The extent of ADBE was monitored by quantifying the number of nerve terminals loaded with dextran, rather than the fluorescence intensity of the nerve terminals themselves. This simple and efficient assay will allow the molecular mechanism of ADBE to be specifically monitored using both pharmacological and molecular technologies.