Noronha-Matos_2020_J.Neurochem__e14975

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g. neostigmine). Nicotinic alpha7 receptors (alpha7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (i) the outflow of [(3) H]ACh, (ii) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (iii) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective alpha7 nAChR agonist, PNU 282,987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that alpha7 nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca(2+) ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The alpha7nAChR agonist, PNU 282,987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [(3) H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of alpha7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca(2+) ]i transients triggered by PNU 282,987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that alpha7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.