Presynaptic pH and vesicle fusion in Drosophila larvae neurones

Abstract

Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca(2+) -sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca(2+) i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca(2+) i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca(2+) -free solution. Acidification by propionate superfusion, NH(4)(+) withdrawal, or the inhibition of acid extrusion on the Na(+)/H(+) exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH(4)(+) removal. In the presence of NH(4)(+), 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca(2+) -dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering.

Keywords: Na+/H+ exchanger; exocytosis; intracellular pH; neuromuscular junction; synaptic transmission.

Fig 1

Fluorescence imaging of Drosophila larva neuromuscular junctions in 0.5 mM Ca²⁺. a 488 nm excited fluorescence image of motoneurone terminals forward-filled with HPTS (shown in yellow, average of 214 frames) superimposed on the transmitted reference image of body-wall muscle fibres 7 and 6 (red, average of 74 frames). b Motoneurone boutons forward-filled with HPTS (shown in green) and extracellular plasmalemmal surfaces stained with Sulforhodamine B (SRB, 565 nm excitation, >586 nm emission shown in red). The SRB staining surrounding boutons was 1.39±0.24 μm thick (n=2, 4 NMJs, pinhole 1.2 airy units) corresponding to the infoldings of the postsynaptic target muscle membrane (Lnenicka et al., 2006). c Aligned F/F₀ OGB-1 (Ca²⁺_i, blue trace) and HPTS (pH_i, red trace) fluorescence intensities during 80Hz 2 s long stimulus trains from ROIs placed over distal nerve bouton regions. The pH transient is overlaid with an exponential fit from which pH_i τ_recovery was extracted. d Aligned Ca²⁺_i (blue) and pH_i (red) transients at higher temporal resolution. The plots show exponential fits to the Ca²⁺ recovery and acidification, from which [Ca²⁺]_i τ_recovery and pH_i τ_{acidification} were extracted.

Fig 2

Effect of 5 μM carboxyeosin on Ca²⁺ and pH_i transients. a 488 nm excited HPTS fluorescence reference image of NMJ boutons overlaid with an ROI covering 3 boutons (average of 27 frames) and superimposed plots of F/F₀ fluorescence intensity during stimulus trains. The red trace is in control and the black trace is after 5 μM CE has been removed. Intervals between frames with no laser illumination were altered to allow fast changes to be captured whilst minimizing dye photodamage. b Ca²⁺_i transient ΔF/F_o and τ_recovery prior to CE application and 20 min after 5 μM CE was removed. c pH_i transient ΔF/F_o and τ_{acidification} prior to CE application and after CE removal.

Fig 3

Postsynaptic membrane potential and mEPPs. a Traces of E_m, low pass differential of E_m (used for counting), the derived mEPP markers, and the AC component of E_m trace used to aid visualization of mEPP count accuracy. b E_m and mEPP frequency during exposure to alkaline and acidic pH_o.

Fig 4

Effect of 50 μM EIPA on 20 mM propionate-induced mEPP frequency transients. a E_m and mEPP frequency during 10 min application of 20 mM propionate. b E_m and mEPP frequency during 50 μM EIPA and 20 mM propionate application. c mEPPs at high time resolution from (b).

Fig 5

The effect of 50 μM EIPA on 20 mM NH₄⁺ prepulse-induced mEPP frequency transients. a E_m and mEPP frequency upon NH₄⁺ removal in control solution. b E_m and mEPP frequency upon NH₄⁺ removal in the presence of EIPA.

Fig 6

Effect of 20 mM NH₄⁺ exposure on mEPP frequency. a E_m and mEPP frequency during the superfusion of NH₄⁺ showing mEPP frequency oscillations. b E_m and mEPP frequency from another preparation during NH₄⁺ exposure. The microelectrode became displaced from the muscle fibre, following 20 mM NH₄⁺ removal, upon repositioning the recordings were unaffected. c mEPPs at high time resolution from b as indicated. d Steady-state baseline mEPP frequency prior to oscillations occurring during 10 min exposures to 20 mM NH₄⁺ plotted against mean number of oscillations in resting mEPP frequency bins of 0.4 Hz width (n=1,3,3,1,2 respectively). Only preparations exhibiting oscillations were considered. e Successive oscillation amplitudes relative to that of the first NH₄⁺-induced oscillation plotted against the time from NH₄⁺ application (n=4, each preparation represented by a different symbol).