Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter

Abstract

Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

Fig 1. Reduced quantal content in VAChT KD^HOM neuromuscular junctions

A. Representative end-plate potentials from a wild-type and an age-matched VAChT KD^HOM end plate. Solid lines are the uncorrected recordings, broken lines are corrected for resting potential and non-linear summation. B. Amplitude distribution of MEPPs obtained from the same endplates used for Panel A. Points are individual MEPP amplitudes. Smooth lines are Gaussian distributions with mean and variance matched to the data. C. Average values of end-plate potentials and MEPP amplitude (WT) and VAChT KD^HOM (KD). D. Quantal content, calculated by the direct method for each synapse. [Ca²⁺]=1.2 mM. [Mg²⁺]=1.3 mM. Error bars in C and D are 95% confidence limits. Differences are significant with P<0.05.

Fig 2

Paired pulse facilitation. Paired-pulse interval was 10 ms. WT: wild-type. KD: VAChT KD^HOM. [Ca²⁺]=1.2 mM. [Mg²⁺] as indicated. Error bars are 95% confidence intervals. Differences are not statistically significantly different.

Fig 3. Post-tetanic potentiation of EPP amplitude

A. EPP amplitude before, during, and after 30 Hz stimulation, indicated by the black bar. Initial stimulation frequency was 0.3 Hz. EPP amplitudes were normalized to the last 20 EPPs prior to the onset of high-frequency stimulation. For clarity, only every tenth EPP is shown during the high frequency stimulation. Solid lines are exponential fits to EPP amplitude during potentiation. [Ca²⁺]=2.4 mM. [Mg²⁺]=1.3 mM. B. Degree of depression at the end of high-frequency stimulation for three conditions of Ca²⁺/Mg²⁺. C. Amount of post-tetanic potentiation (amplitude of exponential component shown in A) for the three conditions in shown in part B. Error bars are 95% confidence intervals. Differences are significant with P<0.05.

Fig 4. Post-tetanic potentiation of MEPP frequency

A. MEPP amplitudes prior to, and during three time bins after high-frequency stimulation. B. MEPP frequency before and after tetanic stimulation. Error bars are 95% confidence intervals. Differences are significant with p<0.05.

Fig 5. Quantal composition of EPPs in 0.6 mM Ca²⁺ / 7 mM Mg²⁺

A-D MEPPs and EPPs recorded from a representative WT synapse. A. Average MEPP waveform. Scale bars are 0.1 mV and 5 ms. B. Distributions of MEPP amplitudes and time integrals. Solid lines are data, broken lines are Gaussian distributions with mean and variance matched to the data. C. Average presumed uniquantal and diquantal EPP waveforms from the same synapse as above. D. Distributions of EPP amplitudes and areas. Solid lines are data. Broken lines are simulations as described in text. E. Summary data for WT (black) and VAChT KD^HOM (gray) synapses. At each synapse, quantal content was measured as -ln(F_Failure) and by the direct method (EPP/MEPP). Open symbols used EPP and MEPP amplitudes whereas filled symbols used areas for the direct method calculations. F. Summary of quantal content estimates using the three methods (from left to right, -ln(F_Failure), direct method based on EPP and MEPP amplitudes, direct method based on EPP and MEPP areas). Error bars are 95% confidence limits. Difference between WT and VAChT KD was significant (P<0.05).