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

Abstract

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis 1,2 . The recycling pool can generally be subdivided into two distinct pools -the readily releasable pool (RRP) and the reserve pool (RP). 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. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane 9 . Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal 10 , the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable 11 .Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs) 12,13 .

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

“…In this study, Ara-C was used to minimize the growth of glial cells, although there was still about a 1 -5% presence of glial cells in the CGN culture. This problem is also presented in other groups' studies 10 . In CGN culture, the sizes and shapes of glial cells are largely different from neurons.…”

IGF-II promotes neuroprotection and neuroplasticity recovery in a long-lasting model of oxidative damage induced by glucocorticoids

“…The fluorescent probe FM®1-43 has been extensively used as a direct index of presynaptic function to study vesicle turnover for live-cell imaging [91], [92], allowing the study of the processes of exocytosis in addition to endocytosis. Glucocorticoids have been reported to impair neurotransmitter release, synaptic plasticity and cognition [52], [93], [94].…”

Analysis of shape and spatial interaction of synaptic vesicles using data from focused ion beam scanning electron microscopy (FIB-SEM)

“…Upon arrival of an action potential at the nerve terminal, the pre-synaptic plasma membrane depolarizes and voltage-gated Ca 2+ channels open; the initial rapid rise in intracellular Ca 2+ triggers exocytosis of readily releasable synaptic vesicles at the active zone of the pre-synaptic membrane and release of their neurotransmitter into the synaptic cleft (Südhof, 1995 ; Rosenmund and Stevens, 1996 ). The reserve pool of synaptic vesicles becomes available for neurotransmitter release upon strong stimulation (Rizzoli and Betz, 2005 ); This pool may be mobilized to replenish the pool of readily releasable vesicles (Prekeris and Terrian, 1997 ; Cheung and Cousin, 2011 ). The synaptic vesicles movement have been observed in different synaptic preparations (Llinás et al, 1989 ; Koenig et al, 1993 ; Ryan and Smith, 1995 ; Henkel et al, 1996 ).…”