Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

Miesenböck, Gero; Angelis, Dino A. De; Rothman, James E.

doi:10.1038/28190

Cited by 2,397 publications

(2,440 citation statements)

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“…Synaptic vesicles undergo a dramatic change in pH upon vesicle fusion with the plasma membrane, a property that has been employed for the synaptic vesicle fusion sensor synaptopHluorin [26]. 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.…”

Section: Phluorins Ph-based Sensorsmentioning

confidence: 99%

“…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].…”

Section: Phluorins Ph-based Sensorsmentioning

confidence: 99%

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Visualizing circuits and systems using transgenic reporters of neural activity

Barth

2007

Current Opinion in Neurobiology

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Genetically encoded sensors of neural activity enable visualization of circuit-level function in the central nervous system. Although our understanding of the molecular events that regulate neuronal firing, synaptic function, and plasticity has expanded rapidly over the past fifteen years, an appreciation for how cellular changes are functionally integrated at the circuit level has lagged. A new generation of tools that employ fluorescent sensors of neural activity promises unique opportunities to bridge the gap between cellular and system-levels analysis.This review will focus on genetically-encoded sensors. A primary advantage of these indicators is that they can be non-selectively introduced to large populations of cells using either transgenic or viral-mediated approaches. This ability removes the non-trivial obstacles of how to get chemical indicators into cells of interest, a problem that has dogged investigators who have been interested in mapping neural function in the intact CNS. Five different types of approaches and their relative utility will be reviewed here: 1) reporters of immediate-early gene (IEG) activation using promoters such as c-fos and arc, 2) voltage-based sensors, such as GFPcoupled Na + and K + channels, 3) Cl − based sensors, 4) Ca ++ -based sensors, such as Camgaroo and the troponin-based TN-L15, and 5) pH-based sensors, which have been particularly useful for examining synaptic activity of highly convergent afferents in sensory systems in vivo. Particular attention will be paid to reporters of IEG expression, since these tools employ the built-in threshold function that occurs with activation of gene expression, provoking new experimental questions by expanding the timescale of analysis for circuit and system-level functional mapping. Fluorescent reporters of inducible gene expressionImmediate-early gene expression (IEG) can be a reliable marker of elevated neuronal firing, reporting activity over time scales that range from minutes to hours compared to ms and seconds for voltage-or ion-based sensors. In ion-based approaches, indicators are constitutively present in the cell, and the fluorescent protein must be engineered to be a sensor with rapid onset and offset kinetics to achieve temporal fidelity with the process being measured. In contrast, monitoring IEG expression can be directly coupled to transcription of the reporter gene. However, because maturation of the fluorophore is required for detection, a temporal delay may be introduced into reporter detection. This delay in reporting activity is advantageous in that it expands the types of experimental questions that can be addressed. For example, do IEGexpressing cells remain more excitable after stimulus cessation? Are cells activated by two temporally distinct stimuli (i.e. training and recall in a memory task) overlapping or distinct?

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Section: Phluorins Ph-based Sensorsmentioning

confidence: 99%

Section: Phluorins Ph-based Sensorsmentioning

confidence: 99%

Visualizing circuits and systems using transgenic reporters of neural activity

Barth

2007

Current Opinion in Neurobiology

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“…1E, Bozza et al, 2004). SpH corresponds to the fusion protein between the synaptic vesicle protein VAMP-2 and a green fluorescent protein pH-sensitive (Miesenbock et al, 1998). Upon odour stimulation, vesicular release at the terminal induces an observable increase of fluorescence that reveals the pattern of nervous inputs to the OB (Fig.…”

Section: In Vivo Imaging Of Odour Evoked Signalsmentioning

confidence: 99%

Wavelet-based multi-resolution statistics for optical imaging signals: Application to automated detection of odour activated glomeruli in the mouse olfactory bulb

Bathellier¹,

Ville²,

Blu³

et al. 2007

NeuroImage

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“…In parallel experiments, we observed that the fluorescence of syt-eGFP is consistently brighter than that of syb-eGFP. One plausible explanation is that the fluorescence of eGFP is dimmer at lower pH (Tsien, 1998), the lumen of vesicles is acidic (pH 5.6; Miesenbock et al, 1998), and therefore it quenches the fluorescence of eGFP, as the eGFP end of syb-GFP is inside the lumen (see Fig. 1 Legend).…”

Section: Figmentioning

confidence: 99%

Living synaptic vesicle marker: Synaptotagmin‐GFP

Zhang

Rodesch

Broadie

2002

Genesis

234

192

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Synapses are the site of chemical communication between neurons and between neurons and muscles. The synaptic vesicle (SV) is a prominent presynaptic organelle which contains chemical neurotransmitters and fuses with the plasma membrane to mediate neurotransmission. There are about 50 or so synaptic proteins which are either integral vesicle membrane proteins (e.g., synaptotagmin, syt; and synaptobrevin, syb) or vesicle-associated proteins (e.g., cysteine string protein, CSP; Fernandez-Chacon and Sudhof, 1999). We have transformed Drosophila with a novel syt-eGFP (enhanced GFP) fusion protein, the fluorescence pattern of which colocalizes with native SV proteins at synapses, suggesting that the syt-eGFP fusion protein is correctly localized as an integral SV protein and therefore a good SV marker in living synapses. We demonstrate that the syt-eGFP line can be used to study SV dynamics in vivo by fluorescence recovery after photobleach (FRAP).The syt-eGFP fusion was constructed as shown in Figure 1. The eGFP carries double substitution of Phe 64 to Leu and Ser 65 to Thr and fluoresces 35-fold more intensely than wild-type GFP when excited at 488 nm, based on spectral analysis of equal amounts of soluble protein (Cormack et al., 1996). Four syt-eGFP transgenic lines were generated; one with insertion on the X chromosome, two on the second chromosome, and one on the third chromosome. All of these lines produced clear fluorescence when crossed to a pan-neuronal GAL4 driver (elav-GAL4, see Fig. 2) or a subset neuronal GAL4 driver 4G-GAL4 (data not shown). 4G-GAL4 is identified from an enhancer trap screen for neuronal-specific genes; it starts expression at late embryogenesis panneuronally, but in a subset of motor neurons in the third instar larvae, and enriched in mushroom body in adult brain. The eGFP-positive animals, from embryos to adults, can be readily recognizable under a fluorescence dissecting scope. Stocks with expression of syt-eGFP in all neurons (recombinant chromosome carrying both elav-GAL4 and syt-eGFP on the X chromosome) or subset of neurons (recombinant chromosome carrying both 4G-GAL4 and syt-eGFP on the second chromosome) were established.Multiple lines of evidence indicate that syt-eGFP is present in SVs, with expression similar to the native syt (Fig. 2). First, syt-eGFP is highly enriched in the neuropil region of the ventral nerve cord (VNC) of the embryo (data not shown) and larva ( Fig. 2A, left), as well as in the axonal lobes of the larval mushroom body ( Fig. 2A, right). These neuropil regions are densely packed with neuronal synapses. Second, at neuromuscular junction (NMJ) synapses, where we have higher resolution of single synaptic boutons, the syt-eGFP pattern perfectly matches the staining pattern seen with antibodies against SV-associated proteins (Fig. 2B) FIG. 1.Map of syt-eGFP and syb-eGFP fusion constructs. Syt (accession number M55048) or syb (neuronal synaptobrevin, accession number S66686) coding region was fused to the N-terminal of eGFP (enhanced GFP, catalog number 6...

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Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

Cited by 2,397 publications

References 30 publications

Visualizing circuits and systems using transgenic reporters of neural activity

Visualizing circuits and systems using transgenic reporters of neural activity

Wavelet-based multi-resolution statistics for optical imaging signals: Application to automated detection of odour activated glomeruli in the mouse olfactory bulb

Living synaptic vesicle marker: Synaptotagmin‐GFP

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