Using pHluorins to Investigate Synaptic Function

Abstract

Genetically encoded probes are becoming increasingly powerful for measuring neuronal activity in vitro and in vivo. pHluorins, such as synaptopHluorin (spH), are genetically encoded reporters whose fluorescence depends on the pH of their biological environment. Because there are large differences in pH between synaptic vesicles and the extracellular medium, spH can be used to monitor presynaptic vesicle cycling, with various experimental manipulations allowing the study of specific exo‐ or endocytosis dynamics. pHluorins can also be used to study the turnover of neurotransmitter receptors on the postsynaptic membrane, and are being used with great effect to study the activity of neurons in a variety of in vivo systems.

Key concepts

  • pH‐sensitive probes can be used to measure important aspects of synaptic function, including presynaptic vesicle cycling and postsynaptic receptor turnover.

  • SynaptopHluorin (spH) can be co mbined with other experimental manipulations to provide detailed information about specific presynaptic vesicle cycling processes.

  • Whereas synaptopHluorin possesses many advantages as an indicator of presynaptic vesicle cycling, it also possesses numerous drawbacks. These must be considered when interpreting spH studies.

  • The sensitivity of spH is not particularly good. However, presynaptic vesicle cycling can be followed with greater sensitivity using different pHluorin‐based probes.

  • pHluorins can provide useful information about neuronal activity in vivo.

Keywords: synaptopHluorin; vesicle cycling; neurotransmitter release; presynaptic; functional imaging

Figure 1.

pHluorins and synaptopHluorin. (a) Two types of pHluorin exhibit pH‐dependent changes in fluorescence. The arrow at 488 nm shows the often‐used excitation wavelength for ecliptic pHluorin where the ratio is maximal between pH 5.5 and 7.5 fluorescence signals. (b) Neuronal organelles exhibit different internal pH levels. (c) Synaptophluorin is formed from superecliptic pHluorin (SEP) fused to the lumenal end of the synaptic vesicle protein VAMP2. At rest, the vesicle lumen is at pH 5.5 and the SEP fluorescence is quenched. Upon vesicle exocytosis, the SEP is exposed to an extracellular pH of approximately 7.4, and approximately 20‐fold increase in spH fluorescence occurs. Following endocytosis and subsequent reacidification of the vesicle, spH returns to its quenched baseline state. (d) Sample images of spH‐expressing synaptic terminals from a cultured hippocampal neuron before (1), during (2) and after (3) electrical stimulation of the axon. (e) Typical fluorescence response profile of spH, averaged over the synaptic terminals shown in (d).

Figure 2.

Tools to exploit spH. (a) Bafilomycin (baf) blocks reacidification of endocytosed synaptic vesicles, making spH fluorescence indicative of exocytosis only. Subtraction of the control trace from the bafilomycin trace produces a read‐out of endocytotic activity. (b) NH4Cl diffuses through organelle membranes, unquenching all spH molecules. The increase in spH fluorescence after NH4Cl addition can be used to calculate the fraction of spH that normally resides on the surface of the synaptic terminal. (c) Different stimulation protocols, with bafilomycin and NH4Cl, can reveal the relative sizes of different presynaptic vesicle pools. A short series of 40 action potentials (APs) at 20 Hz mobilizes the readily releasable pool (RRP), whereas 900 APs at 20 Hz exhausts the reserve pool, and a final NH4Cl wash reveals the resting pool (see Burrone et al., ).

Figure 3.

In vivospH responses. (a) Mice expressing spH in all olfactory receptor neurons within the main olfactory endothelium (MOE) project their axons through the olfactory tract (OT) and terminate at specialized sites called glomeruli in the olfactory bulb (OB). Each glomerulus receives inputs from neurons expressing the same olfactory receptor. (b) Top view of mice expressing spH in the OB. Note the glomeruli seen as round clusters of green fluorescence. (c, d) spH responses to two different odours (butyraldehyde and hexanone) shows different sets of glomeruli ‘light’ up. The responses are given as changes in fluorescence (ΔF) normalized to the maximum response for a given trial. Reproduced from Bozza et al., by permission of Cell Press.

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How to Cite close
Grubb, Matthew S, and Burrone, Juan(Mar 2009) Using pHluorins to Investigate Synaptic Function. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021387]