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Open Access Methodology article

Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique

Serguei S Khiroug1, Evgeny Pryazhnikov1, Sarah K Coleman2, Andreas Jeromin3, Kari Keinänen2 and Leonard Khiroug1*

Author Affiliations

1 Neuroscience Center, University of Helsinki, FIN-00014, Helsinki, Finland

2 Department of Biological and Environmental Sciences, Division of Biochemistry, University of Helsinki, FIN-00014, Helsinki, Finland

3 Allen Institute for Brain Science, Seattle, WA 98103, USA

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BMC Neuroscience 2009, 10:141  doi:10.1186/1471-2202-10-141

Published: 30 November 2009

Abstract

Background

Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.

Results

We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.

Conclusion

The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.