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.

Figure 1. Genetically encoded pH-indicators (GEpHIs) report changes in pH_cyto in presynaptic motor neuron (MN) termini in situ

A, fluorescence images of termini of two MNs (MN13-Ib and MNSNb/d-Is) expressing different GEpHIs innervating larval body-wall muscle 13. The excitation and emission wavelength combination used for ratiometric imaging is indicated for each image. Fluorescence images were collected sequentially (PtGFP, RA-pHluorin) or simultaneously (SE-pHluorin 2EM, SE-pHluorin/DsRed). B, representative plots of changes in fluorescence intensity (ΔF/F) for each indicator in response to a 20 mm NH₄Cl pulse (grey column). C, representative plots of the change in fluorescence ratio (R) in response to a 20 mm NH₄Cl pulse. Fluorescence image pairs were collected at 1.66 Hz. Downward deflections represent cytosolic acidification.

Figure 2. Fluorescent pH-indicators reveal activity-dependent cytosolic acidification of presynaptic motor neuron (MN) termini

A, representative plots of changes in fluorescence intensity (ΔF/F) for each pH-indicator in response to nerve stimulation (grey bar). Fluorescence images were collected sequentially (PtGFP, RA-pHluorin, BCECF-dextran) or simultaneously (SE-pHluorin 2EM, SE-pHluorin/DsRed). B, plots of the average change in fluorescence ratio (R) during ratiometric imaging in response to stimulation (n= 6 preparations), except for the bottom panel where an average plot of SE-pHluorin/DsRed ΔR/R is presented (n= 6 preparations). Fluorescence image pairs were collected at 1.66 Hz. Downward deflections represent cytosolic acidification.

Figure 3. In situ calibration of fluorescent pH-indicators

A–C, calibration curves were constructed by measuring cytosolic fluorescence levels in nigericin-permeabilized MN13-Ib termini. For SE-pHluorin/DsRed and BCECF-dextran, each preparation was first exposed to saline titrated to pH ∼7, followed by a pseudo-random sequence of solutions titrated to various pH values before being returned to pH ∼7 (representative data from single preparations are shown in the left panels of A and B). For SE-pHluorin 2EM (C), a different preparation was used for each measurement at different pH values. Data traces from SE-pHluorin/DsRed were normalized to the maximum fluorescence (pH 9) for each preparation. D, averaged plots of pH_cyto shift reported by each pH-indicator in response to 40 Hz nerve stimulation for 4 s (grey bar) (n= 6 per GEpHI, same data as Fig. 2B). The SE-pHluorin/DsRed ΔR/R, SE-pHluorin 2EM emission ratio and BCECF-dextran emission ratio were converted to pH_cyto using the curve fits in A–C. E, pH_cyto changes as reported by each pH-indicator. Each pair of empty circles represents the resting pH_cyto and pH_cyto immediately after stimulation in a single preparation. Filled circles are mean values. Error bars (SEM). The resting pH_cyto of SE-pHluorin/DsRed was assumed to be 7.30. All pH-indicators revealed significant acidification upon stimulation (P < 0.05). F, decrease in pH_cyto calculated as the difference between resting value and peak response revealed by each pH-indicator. Error bars (SEM).

Figure 4. Bicarbonate-derived buffering does not buffer the rapid acidification phase of brief activity-induced acid transients but can buffer slow activity-induced acid loading

A, representative trace of pH_cyto changes in MN13-Ib termini of a larva expressing SE-pHluorin/DsRed induced by stepwise NH₄Cl pulses. pH_e was lowered to 6.35 to reduce pH_cyto to ∼7.15 so as to maximize the linear range of SE-pHluorin/DsRed (pK_a= 7.16). The extracellular solution was modified bicarbonate-free HL6 buffered with 19 mm BES and bubbled with 100% O₂ to calculate intrinsic buffering power (β_i). B, plot of β_i of motor neuron (MN) termini as a function of pH_cyto. Data are from 6 larvae, each exposed to the NH₄Cl pulse protocol in A. Data are grouped by each NH₄Cl withdrawal step. The mean pH_cyto during the acidification phase of each NH₄Cl withdrawal step was used as the corresponding pH_cyto value for the β_i calculated during the same step. Error bars (SEM). C, average traces of pH_cyto changes induced by long, low-frequency stimulation (black bar; 5 Hz, 60 s) in the presence of 200 μm ETZ and vehicle control. The bar graph shows the recovery decay constant and the change in pH_cyto in both conditions. Error bars (SEM). *P≤ 0.05 (paired two-way Student's t test, n= 6). D, average traces of pH_cyto changes induced by short, high-frequency stimulation trains (black bar; 40 Hz, 4 s) in the presence of 200 μm ETZ and vehicle (DMSO) control. Error bars (SEM). *Time point at which the values of the ETZ and control traces become significantly different (P≤ 0.05, two-way ANOVA, n= 6).

Figure 5. The presence of HCO₃⁻/CO₂-derived buffering decreases the time course of recovery from activity-induced acid loads, but does not change the molar flux of acid equivalents during the rapid acidification phase

A, activity-induced pH_cyto shifts in MN13-Ib termini of SE-pHluorin/DsRed larvae as measured in HL6 with different buffering conditions. Stock HL6 contained 5 mm BES and 10 mm HCO₃⁻, and was not bubbled with a gas mixture. Traces are averages of 6 preparations. Stimulation (black bar) was 40 Hz, 4 s in all cases. B, the decay constant (τ, top panel) of the recovery phase (grey portion of trace in A following stimulation) and the molar flux during the stimulation rapid acidification phase (indicated in black in A, not statistically different between groups, P≥ 0.20 by one-way ANOVA, n= 6) are compared between the different buffering conditions. Error bars (SEM). *P≤ 0.05 (one-way ANOVA). All data in A and B were collected at 1.66 Hz.

Figure 6. Activity-induced acid transients correlate with cytosolic Ca²⁺ increase and arise due to proton influx through the plasma membrane Ca²⁺-ATPase (PMCA)

A, representative traces of stimulation-induced pH_cyto and normalized [Ca²⁺]_cyto changes in MN13-Ib termini in a single preparation. Larval motor neurons (MNs) expressing SE-pHluorin were forward-filled with rhod-dextran and stimulated (black bars) in stock HL6 (pH_e 7.3) containing 0.5, 1.0 and 2.0 mm[Ca²⁺]_e. The rapid acidification phase of each trace is displayed in black. B, representative traces of stimulation-induced pH_cyto and normalized [Ca²⁺]_cyto changes in a single preparation in stock HL6 containing 0.6 mm[Ca²⁺]_e buffered to pH_e 7.3 (first stimulation) and then 8.8 (second stimulation). Rhod-dextran traces are normalized to the maximum fluorescence observed in a control stimulation in stock HL6 (2 mm Ca²⁺, pH_e buffered to 7.3, not shown). Resting pH_cyto was assumed to be 7.3 and 7.65 in stock HL6 buffered to 7.3 and 8.8, respectively. C, net molar acid flux (mm s⁻¹) plotted as a function of normalized rhod-dextran fluorescence during stimulation. Molar flux was calculated from the rapid acidification phase of the traces in A and B. Data are from 3 preparations, with each preparation stimulated once in each value of [Ca²⁺]_e. Half-filled circles represent the same 3 preparations presented in B. The line is a linear fit (R²= 0.99) to data from preparations in HL6 with [Ca²⁺]_e= 0.5, 1.0 and 2.0 mm. D, top panel, net molar acid fluxes from SE-pHluorin larvae forward-filled with rhod-dextran in response to stimulation (40 Hz, 4 s, [Ca²⁺]_e= 0.6 mm) in stock HL6 buffered to pH_e 7.3 (‘control’) and then 8.8 (same data from C, 10 min between stimulations). *P < 0.05. D, bottom panel, net molar acid fluxes from SE-pHluorin/DsRed larvae in response to stimulation (40 Hz, 4 s, [Ca²⁺]_e= 0.6 mm) in stock HL6 before (‘control’) and after 30 min incubation in 100 μm CEDA-SE (n= 5 preparations). *P < 0.05. Error bars in D are SEM. A–D collected at 4.3 Hz.

Figure 7. Endogenous motor activity causes substantial presynaptic acidification in situ and in vivo

A, traces representing simultaneous changes in [Ca²⁺]_cyto and pH_ctyo of a motor neuron (MN) terminal as reported by rhod-dextran and SE-pHluorin, in response to nerve stimulus trains (black bars, 40 Hz, 2 s each). B, traces representing spontaneous simultaneous changes in [Ca²⁺]_cyto and pH_ctyo as reported by rhod-2 AM and SE-pHluorin in a MN terminal still attached to the CNS. No external stimuli were applied. In A and B, rhod-dextran or rhod-2AM fluorescence change was normalized to a value of 1 (maximum response). The rhod-2 AM trace was corrected for cytosolic dye loss and bleaching using a single exponential curve fit. No data averaging or smoothing in A or B. C, averaged plots of pH_cyto shift in MN13-Ib termini reported by SE-pHluorin/DsRed in response to nerve stimulation (black bar) in preparations in which the axons had been cut (black trace, n= 6, data acquisition rate of 1.66 Hz) and left intact (grey trace, n= 6). The value of τ was calculated by fitting a single exponential to the recovery phase of each trace in each condition (R²_cut= 0.93, R²_intact= 0.97). D, a trace demonstrating spontaneous changes in pH_ctyo in a MN terminal in a restrained intact larva (in vivo). Fluorescence intensity data were acquired from presynaptic SE-pHluorin and DsRed through the cuticle. The relative change in the ratio of these fluorophores (ΔR/R) is plotted over a period of 120 s. In A–D, data were acquired at a frame rate of 4.3 Hz unless noted otherwise. Resting pH_cyto of SE-pHluorin/DsRed was assumed to be 7.30 in C and 7.15 in D, as this was the value observed in the presence of a CO₂/HCO₃⁻-based buffering system in situ.