Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo
Article first published online: 11 FEB 2013
© 2013 The Authors. The Journal of Physiology © 2013 The Physiological Society
The Journal of Physiology
Volume 591, Issue 7, pages 1691–1706, April 2013
How to Cite
Rossano, A. J., Chouhan, A. K. and Macleod, G. T. (2013), Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo. The Journal of Physiology, 591: 1691–1706. doi: 10.1113/jphysiol.2012.248377
- Issue published online: 27 MAR 2013
- Article first published online: 11 FEB 2013
- Accepted manuscript online: 9 JAN 2013 07:40AM EST
- (Received 14 November 2012; accepted after revision 4 January 2013; first published online 7 January 2013)
- • Changes in pH occur within neurons during nerve activity and in response to hypoxic insult.
- • Many aspects of neurophysiology are potentially influenced by intracellular pH changes.
- • At the fruit fly larval neuromuscular junction, fluorescent genetically encoded pH-indicators (GEpHIs) revealed significant cytosolic acidification of presynaptic termini during nerve activity.
- • GEpHIs revealed that presynaptic pH changes occur in live intact larvae, indicating for the first time that such pH changes are not an artifact of experimental conditions.
- • The pH changes in presynaptic termini are substantial and are likely to influence synaptic function.
Abstract All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pHcyto) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pHcyto shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pHcyto. Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤4 s) trains of action potentials but did buffer slow (∼60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca2+ increase upon stimulation, and partial inhibition of the plasma membrane Ca2+-ATPase, a Ca2+/H+ exchanger, attenuated pHcyto shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (∼0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pHcyto shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pHcyto shifts cannot be dismissed as artifacts of ex vivo preparations.