Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft

Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca2+ extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca2+ release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle expressed GCaMP3 to monitor the contribution of postsynaptic Ca2+. Activity-induced liberation of intracellular Ca2+ in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca2+ liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates.

Automated summary

Neurotransmission is influenced by extracellular pH. Alkalization enhances transmitter release and receptor activation, while acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain unclear. In this study, researchers investigated the extracellular pH transients at the cholinergic synapse of the mouse NMJ under near physiological conditions and identified their sources. They found that activity-induced intracellular Ca2+ release in the muscle plays a role in cleft alkalization, whereas PMCA inhibition decreases alkalization. This suggests that cholinergic synapses at the mouse NMJ typically alkalize due to cytosolic Ca2+ liberation during activity, unless under highly strenuous conditions where acidification predominates.