Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin

Abstract

Synaptic vesicles are recycled locally within presynaptic specializations. We examined how vesicles are reused after endocytosis, using transgenic mice expressing the genetically encoded fluorescent indicator synaptopHluorin in subsets of neurons. At both excitatory and inhibitory synapses in cultured hippocampal neurons, newly endocytosed vesicles did not preferentially enter the releasable pool of vesicles. Rather, they entered the reserve pool first and subsequently the readily releasable pool over a period of several minutes. All vesicles in the recycling pool could be accessed by spaced stimuli, arguing against preferential local reuse of the readily releasable vesicles. Interestingly, nearly half the vesicles at excitatory synapses, and a third at inhibitory synapses, could not be recruited for release even by sustained stimuli. We conclude that, at presynaptic terminals in the hippocampus, most vesicles vacate release sites after exocytosis and are replaced by existing vesicles from the reserve pool, placing constraints on kiss-and-run recycling.endocytosis ͉ exocytosis ͉ hippocampus ͉ kiss and run S ynaptic vesicles are recycled locally within the presynaptic terminal for reuse (1, 2). Several mechanisms of recycling have been proposed, including a conventional pathway involving clathrin coat formation (1, 2) and a clathrin-independent pathway involving kiss-and-run exocytosis (3-9) that might explain rapid endocytosis observed at some synapses (5, 10-12). Although recent attention has centered on modes of endocytosis, several key questions can also be raised regarding postendocytic traffic of synaptic vesicles. Where do vesicles go after endocytosis? Are recently recaptured vesicles preferentially reused? Rapid reuse of recently recaptured vesicles (13, 14) might be advantageous to synapses if it allows vesicles to become release-competent more rapidly than would be possible by recruitment from the reserve pool.Studies in hippocampal and cortical cultures generally have focused on glutamatergic synapses or assumed that the synapses under investigation are glutamatergic synapses because they are more abundant (15). It is unclear whether inhibitory presynaptic terminals have the same mechanisms of recycling as excitatory synapses. Some differences might be anticipated based on previous studies establishing differences in the molecular composition (16, 17) and short-term synaptic dynamics (16,18,19). Additionally, the loss of specific proteins, for example, synapsin I (20), appears to have different consequences in glutamatergic and GABAergic synapses. Therefore, it is important to understand the properties of and mechanisms in vesicle recycling at inhibitory synapses.Genetically encoded probes such as synaptopHluorin (spH), which is a fusion protein of the vesicle protein VAMP2 and a pH-sensitive EGFP (21), offer a method to target tracers to selected populations of neurons. We have generated lines of transgenic mice that express spH in subsets of neurons in the brain. We first show that exocytosis and en...

Heterogeneity in Synaptic Vesicle Release at Neuromuscular Synapses of Mice Expressing SynaptopHluorin

“…As has been shown in previous studies using the thy1.2 promoter to drive transgene expression in mice (Feng et al, 2000;Li et al, 2005), we found that the pattern of spH expression varied between the different lines (Fig. 1).…”

“…To circumvent these limitations we generated transgenic lines of mice in which synaptopHluorin (spH), a pH-sensitive variant of green fluorescent protein (GFP) tethered to the vesicular membrane protein vesicle-associated membrane protein 2 (VAMP2) (Miesenbock et al 1998), is expressed in motor nerve terminals using the thy1.2 promoter (Feng et al, 2000;Li et al, 2005). This construct has been extensively used to investigate synaptic vesicle properties within hippocampal neurons in vitro Ryan, 2000, 2001;Fernandez-Alfonso et al, 2006) and at Drosophila (Poskanzer et al, 2003;Reiff et al, 2005) and mouse (Tabares et al, 2007) neuromuscular junctions in vivo.…”

Visualizing circuits and systems using transgenic reporters of neural activity

“…Both ratiometric and ecliptic pHlourins have been engineered [26], although the superecliptic synaptopHluorin has been most widely employed to address important biological questions in mammalian systems. For example, synaptic vesicle recycling dynamics in neurons from Thy1-driven synaptopHluorin transgenic mice have been examined, providing results that challenged current models about constraints on vesicle reuse [27].…”

Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis

“…We compared the fluorescence response of spH synapses to a stimulus of 300 APs at 10 Hz in the presence of bafilomycin with the response in the presence of dynasore. Fluorescence changes were normalized to the total pool of vesicles, which was estimated by neutralizing all vesicles with ammonium chloride (34). As shown in Fig.…”

“…We also used the method of alkaline trapping at spH-expressing synapses (33,34) to rule out a direct effect of dynasore on exocytosis. Alkaline trapping of vesicles by bafilomycin allows for the estimation of cumulative exocytosis throughout the stimulation; endocytosed vesicles do not contribute to additional changes in fluorescence because they cannot be reacidified.…”