Role of acid sensing ion channels on mice neuromuscular transmission

C. GONZALEZ-INCHAUSPE.; F. J. URBANO.; A. WUNSCH.; J. WEMMIE.; O. D. UCHITEL.

Chicago., USA.

Congreso; 39th Annual Meeting of the Society for Neuroscience; 2009

Society for Neuroscience

We studied the effects of extracellular low pH during intracellular recordings of neuromuscular transmission in wild type and ASIC1-/- transgenic juvenile mice (1-2 months old). We optically chose superficial neuromuscular junctions from the thin levator auris longus muscle and locally pressure injected pH 6.0 (MES based, 1-2 seconds long) modified extracellular solution using a Picrospritzer. Continuous replacement of O2 aerated Hepes based external solution was achieved with a peristaltic pump (pH 7.4).During intracellular recording of the postsynaptic muscle cell, nerve paired stimuli at 20 Hz were delivered in the presence of μ-conotoxin GIIIB (1 μM, to prevent muscle contraction). At pH 7.4, characteristic endplate potentials (EPPs) were observed for wild type and ASIC1 -/- terminals with a small facilitation (i.e., EPP2/EPP1 amplitude ratio >1). The transient extracellular reduction of pH to 6.0 drastically reduced the amplitude of both first and second EPPs, in a reversible way. Interestingly, pH 6.0 did increase significantly average paired-pulse ratios for ASIC1 -/- neuromuscular synapses but not for wild types. During 75 Hz (5 seconds long) trains stimulation Epps amplitudes decreased more during the train in wild type terminals compare to the KO ones, supporting the idea that ASIC1 channels might be reducing ACh release during repetitive stimulation. Finally, we used a recently described ASIC1-blocking toxin, Psalmotoxin-1 (5 mg/ml) in wild type terminals during both 20Hz (300 ms long) and 50 Hz (250 ms long) train stimulation. Blocking ASIC1 channels did increase, in a frequency dependent fashion (i.e., smaller des-inhibition at 20Hz than at 50 Hz), transmitter release in wild types.Our results suggested the presence of ASIC1 channels at neuromuscular junction from adult mice, which would be inhibiting ACh release during repetitive stimulation at high frequency stimulation.Support: This work was supported by grants from Wellcome Trust, grant# 068941/Z/02/Z; ANCyT; grant#6220; UBACYT; grant# X171 & X223; FONCYT-BID 1728 OC.AR. PICT2005 #32113 and #13367; and BID 1728 OC.AR. PICT 2006 #199 (to Dr. Uchitel), VA merit review (JAW) (to Dr. Wemmie) and FONCYT-BID 1728 OC.AR. PICT 2007-01009 (to Dr. Urbano).