Reply to Wilkinson et al.: Concerning the use of pHluorin-tagged GluA2 as a reporter for NMDA-induced AMPA receptor recycling

As pointed out in the letter by Wilkinson et al. (1), we question in our recent paper by Rathje et al. (2) the use of pHluorin-GluA2 (pH-GluA2) as a reporter of NMDA- and AMPA-induced endocytosis because application of the agonists NMDA and AMPA results in intracellular acidification of hippocampal neurons. This conclusion rests on two important key observations: (i) the pHluorin response to NMDA treatment was almost intact after removal of surface-exposed pHluorin by thrombin cleavage (figure 1 of Rathje et al.) (2) and (ii) NMDA stimulation reduces fluorescence of both ER-retained pH-GluA2 and cytosolic pHluorin, in agreement with intracellular acidification, which was further supported by measurements using the pH-sensitive dye SNARF-1 (figures 2 and 3 of Rathje et al.) (2).