Activity-Dependent Bulk Endocytosis and Clathrin-Dependent Endocytosis Replenish Specific Synaptic Vesicle Pools in Central Nerve Terminals

Abstract

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activitydependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high-and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

“…Our 30 Hz 2 s stimuli evokes an identical extent of RRP unloading to hypertonic sucrose 8 . Since the size of the RRP is defined by hypertonic sucrose unloading 15 , we can state that this protocol unloads all RRP SVs, in agreement with studies in hippocampal neurons a paradigm (2 stimuli with 50 mM KCl) that depletes 95% of all dye-labelled SVs 8,17 .…”

“…2. Optional -If the effect of a drug on endocytosis is to be tested, perfuse neurons with drug solution during this period (Figure 2b) 3,8 .…”

“…FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals [3][4][5][6][7][8] . They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes.…”

“…As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation 1,2 . It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrindependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals [3][4][5][6][7][8] . They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes.…”

Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis

“…Exocytosis has to be followed by endocytosis to recapture the granule membrane that has been added to the plasma membrane of the cell in order to maintain cellular homeostasis and integrity (Rizzoli & Jahn 2007). The classification of the endocytotic mechanisms includes conventional endocytosis involving recruitment of clathrin and a group of adaptor proteins; rapid endocytosis, for example, by 'kiss and run'; bulk endocytosis (Ales et al 1999, Jahn et al 2003, Harata et al 2006, Rizzoli & Jahn 2007, Cheung et al 2010; and excess endocytosis (Eliasson et al 1996, Smith & Neher 1997. Endocytosis is Ca 2+ -dependent as has been shown in synaptic systems and neuroendocrine cells (Eliasson et al 1996, Photowala et al 2006, Rizzoli & Jahn 2007, Cheung et al 2010, including β-cells (Jarousse & Kelly 2001, Murthy & De Camilli 2003, MacDonald et al 2005, He et al 2008).…”