Using pHluorins to Investigate Synaptic Function


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 (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 (APs) at 20 Hz mobilizes the (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 vivo spH responses. (a) Mice expressing spH in all olfactory receptor neurons within the (MOE) project their axons through the (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.



Ashby MC, De La Rue SA, Ralph GS et al. (2004) Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. Journal of Neuroscience 24: 5172–5176.

Ashby MC, Maier SR, Nishimune A and Henley JM (2006) Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology. Journal of Neuroscience 26: 7046–7055.

Atluri PP and Ryan TA (2006) The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals. Journal of Neuroscience 26: 2313–2320.

Balaji J, Armbruster M and Ryan TA (2008) Calcium control of endocytic capacity at a CNS synapse. Journal of Neuroscience 28: 6742–6749.

Balaji J and Ryan TA (2007) Single‐vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode. Proceedings of the National Academy of Sciences of the USA 104: 20576–20581.

Bayazitov IT, Richardson RJ, Fricke RG and Zakharenko SS (2007) Slow presynaptic and fast postsynaptic components of compound long‐term potentiation. Journal of Neuroscience 27: 11510–11521.

Bozza T, McGann JP, Mombaerts P and Wachowiak M (2004) In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron 42: 9–21.

Burrone J, Li Z and Murthy VN (2006) Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin. Nature Protocols 1: 2970–2978.

Cantallops I and Cline HT (2008) Rapid activity‐dependent delivery of the neurotrophic protein CPG15 to the axon surface of neurons in intact Xenopus tadpoles. Developmental Neurobiology 68: 744–759.

Fernandez‐Alfonso T and Ryan TA (2004) The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals. Neuron 41: 943–953.

Gandhi SP and Stevens CF (2003) Three modes of synaptic vesicular recycling revealed by single‐vesicle imaging. Nature 423: 607–613.

Granseth B, Odermatt B, Royle SJ and Lagnado L (2006) Clathrin‐mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51: 773–786.

Hemmings HC Jr, Yan W, Westphalen RI and Ryan TA (2005) The general anesthetic isoflurane depresses synaptic vesicle exocytosis. Molecular Pharmacology 67: 1591–1599.

Jacob TC, Bogdanov YD, Magnus C et al. (2005) Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. Journal of Neuroscience 25: 10469–10478.

Kim JH, Udo H, Li HL et al. (2003) Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long‐term facilitation in Aplysia. Neuron 40: 151–165.

Kopec CD, Li B, Wei W, Boehm J and Malinow R (2006) Glutamate receptor exocytosis and spine enlargement during chemically induced long‐term potentiation. Journal of Neuroscience 26: 2000–2009.

Leonoudakis D, Zhao P and Beattie EC (2008) Rapid tumor necrosis factor alpha‐induced exocytosis of glutamate receptor 2‐lacking AMPA receptors to extrasynaptic plasma membrane potentiates excitotoxicity. Journal of Neuroscience 28: 2119–2130.

Li Z, Burrone J, Tyler WJ et al. (2005) Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proceedings of the National Academy of Sciences of the USA 102: 6131–6136.

Lin DT and Huganir RL (2007) PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor‐induced internalization. Journal of Neuroscience 27: 13903–13908.

McGann JP, Pirez N, Gainey MA et al. (2005) Odorant representations are modulated by intra‐ but not interglomerular presynaptic inhibition of olfactory sensory neurons. Neuron 48: 1039–1053.

Miesenbock G, De Angelis DA and Rothman JE (1998) Visualizing secretion and synaptic transmission with pH‐sensitive green fluorescent proteins. Nature 394: 192–195.

Moulder KL, Jiang X, Taylor AA et al. (2007) Vesicle pool heterogeneity at hippocampal glutamate and GABA synapses. Journal of Neuroscience 27: 9846–9854.

Ng M, Roorda RD, Lima SQ et al. (2002) Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36: 463–474.

Nicholson‐Tomishima K and Ryan TA (2004) Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I. Proceedings National Academy of Sciences of the USA 101: 16648–16652.

Petzold GC, Albeanu DF, Sato TF and Murthy VN (2008) Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways. Neuron 58: 897–910.

Poskanzer KE, Marek KW, Sweeney ST and Davis GW (2003) Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature 426: 559–563.

Reiff DF, Ihring A, Guerrero G et al. (2005) In vivo performance of genetically encoded indicators of neural activity in flies. Journal of Neuroscience 25: 4766–4778.

Samuel AD, Silva RA and Murthy VN (2003) Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory. Journal of Neuroscience 23: 373–376.

Sankaranarayanan S, De Angelis D, Rothman JE and Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophysics Journal 79: 2199–2208.

Sankaranarayanan S and Ryan TA (2000) Real‐time measurements of vesicle‐SNARE recycling in synapses of the central nervous system. Nature Cell Biology 2: 197–204.

Sankaranarayanan S and Ryan TA (2001) Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nature Neuroscience 4: 129–136.

Sano H and Yokoi M (2007) Striatal medium spiny neurons terminate in a distinct region in the lateral hypothalamic area and do not directly innervate orexin/hypocretin‐ or melanin‐concentrating hormone‐containing neurons. Journal of Neuroscience 27: 6948–6955.

Shang Y, Claridge‐Chang A, Sjulson L, Pypaert M and Miesenbock G (2007) Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe. Cell 128: 601–612.

Tabares L, Ruiz R, Linares‐Clemente P et al. (2007) Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptopHluorin. Journal of Neuroscience 27: 5422–5430.

Tretter V, Jacob TC, Mukherjee J et al. (2008) The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. Journal of Neuroscience 28: 1356–1365.

Voglmaier SM, Kam K, Yang H et al. (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51: 71–84.

Wienisch M and Klingauf J (2006) Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nature Neuroscience 9: 1019–1027.

Wilson NR, Kang J, Hueske EV et al. (2005) Presynaptic regulation of quantal size by the vesicular glutamate transporter VGLUT1. Journal of Neuroscience 25: 6221–6234.

Yu D, Ponomarev A and Davis RL (2004) Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment. Neuron 42: 437–449.

Yudowski GA, Puthenveedu MA and von Zastrow M (2006) Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nature Neuroscience 9: 622–627.

Further Reading

Burrone J (2005) Synaptic physiology: illuminating the road ahead. Current Biology 15: R876–R878.

LoGiudice L and Matthews G (2006) The synaptic vesicle cycle: is kissing overrated? Neuron 51: 676–677.

Miesenbock G and Kevrekidis IG (2005) Optical imaging and control of genetically designated neurons in functioning circuits. Annual Review of Neuroscience 28: 533–563.

Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547.

Wu (2004) Kinetic regulation of vesicle endocytosis at synapses. Trends in Neuroscience 9: 548–554.

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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. [doi: 10.1002/9780470015902.a0021387]