title: Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo.
abstract: All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pH(cyto)) 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 pH(cyto) 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 pH(cyto). 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 Ca(2+) increase upon stimulation, and partial inhibition of the plasma membrane Ca(2+)-ATPase, a Ca(2+)/H(+) exchanger, attenuated pH(cyto) shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (~0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pH(cyto) shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pH(cyto) shifts cannot be dismissed as artifacts of ex vivo preparations.
authors: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors: Adam J
authors: AJ
authors: Rossano
authors: Amit K
authors: AK
authors: Chouhan
authors: Gregory T
authors: GT
authors: Macleod
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D003600:Cytosol / Q000737:chemistry*; D004330:Drosophila / Q000235:genetics / Q000502:physiology*; D005260:Female; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009043:Motor Activity / Q000502:physiology*; D009046:Motor Neurons / Q000502:physiology*; D009411:Nerve Endings / Q000502:physiology*
references: J Physiol. 1996 Jun 1;493 ( Pt 2):457-70
references: 8782109
references: Ukr Biokhim Zh (1978). 1996 Nov-Dec;68(6):50-61
references: 9273745
references: Prog Neurobiol. 1990;34(5):401-27
references: 2192394
references: J Biol Chem. 1992 Jan 25;267(3):1761-8
references: 1309796
references: Biochemistry. 1979 May 29;18(11):2210-8
references: 36128
references: Curr Neurovasc Res. 2007 Aug;4(3):205-15
references: 17691974
references: J Physiol. 1977 Dec;273(1):295-316
references: 23428
references: J Physiol. 1998 Jun 1;509 ( Pt 2):471-85
references: 9575296
references: Nat Neurosci. 2011 Aug 28;14(10):1285-92
references: 21874016
references: Neurosci Lett. 1986 Dec 3;72(1):54-8
references: 2880318
references: J Physiol. 2002 Oct 15;544(2):487-99
references: 12381821
references: J Neurophysiol. 2005 Nov;94(5):3563-72
references: 16093328
references: J Comp Physiol A. 1994 Aug;175(2):179-91
references: 8071894
references: Neuron. 2002 Feb 14;33(4):545-58
references: 11856529
references: Am J Physiol Cell Physiol. 2011 Jan;300(1):C187-97
references: 20881233
references: J Biol Chem. 1989 May 15;264(14):8171-8
references: 2498308
references: Nature. 1998 Jul 9;394(6689):192-5
references: 9671304
references: J Neurosci. 1990 Nov;10(11):3754-68
references: 1700086
references: J Neurosci. 2006 Nov 22;26(47):12283-93
references: 17122054
references: J Biol Chem. 1982 Mar 10;257(5):2350-6
references: 6460758
references: Physiol Rev. 2003 Oct;83(4):1183-221
references: 14506304
references: Physiol Rev. 1981 Apr;61(2):296-434
references: 7012859
references: J Physiol. 1990 Jun;425:429-48
references: 2170631
references: J Physiol. 1976 Oct;262(1):189-214
references: 11339
references: Mol Membr Biol. 2000 Apr-Jun;17(2):101-8
references: 10989460
references: J Neurobiol. 2001 Jul;48(1):58-73
references: 11391649
references: Adv Physiol Educ. 2004 Dec;28(1-4):160-79
references: 15545345
references: Am J Physiol Cell Physiol. 2000 May;278(5):C965-72
references: 10794670
references: EMBO J. 1998 Aug 10;17(5):1297-303
references: 9482727
references: J Negat Results Biomed. 2010 Jun 23;9:5
references: 20573195
references: Neuroreport. 1996 Aug 12;7(12):2000-4
references: 8905712
references: J Neurosci. 2009 Mar 11;29(10):3252-8
references: 19279262
references: J Vis Exp. 2007;(6):250
references: 18997898
references: J Neurochem. 2003 Oct;87(1):30-43
references: 12969250
references: Plant Methods. 2006 Apr 06;2:7
references: 16600023
references: J Neurophysiol. 2010 Feb;103(2):667-76
references: 19939954
references: Nat Rev Mol Cell Biol. 2010 Jan;11(1):50-61
references: 19997129
references: J Biol Chem. 2000 Aug 11;275(32):24552-9
references: 10827195
references: Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984-9
references: 11050229
references: J Physiol. 2002 Jan 15;538(Pt 2):371-82
references: 11790806
references: J Neurosci. 2010 Feb 3;30(5):1869-81
references: 20130196
references: Chem Rev. 2008 May;108(5):1550-64
references: 18447377
references: J Neurophysiol. 1988 Jan;59(1):248-58
references: 2449521
references: J Physiol. 2009 Jan 15;587(2):315-27
references: 19064619
references: J Neurophysiol. 2002 Nov;88(5):2659-63
references: 12424301
references: J Neurosci. 2011 May 11;31(19):6997-7004
references: 21562261
references: Physiol Rev. 1967 Oct;47(4):595-781
references: 4964060
references: Neuron. 2010 Dec 22;68(6):1097-108
references: 21172612
references: J Gen Physiol. 1993 Nov;102(5):797-816
references: 8301258
references: J Neurophysiol. 1997 Feb;77(2):639-53
references: 9065837