Activation of Ionotropic Glutamate Receptors on Peripheral Axons of Primary Motoneurons Mediates Transmitter Release at the Zebrafish NMJ

Abstract

The development and function of the vertebrate neuromuscular junction (NMJ) is continually being redefined. Previous studies have indicated that glutamate may play a role in the development or function of the NMJ by associating with presynaptic receptors. We have used larval zebrafish ( Danio rerio) to investigate the presence of presynaptic ionotropic glutamate receptors (iGluRs) at the NMJ in vivo. In whole-mount zebrafish larvae, antibody staining directed to NR2A subunits colocalized with specific staining of motoneuron axon tracts. Whole cell voltage-clamp recordings of miniature endplate currents (mEPCs) from axial white muscle were performed during application of iGluR agonists and antagonists. Local perfusion of the NMJ with iGluR agonists resulted in significant increases in the frequency of spontaneous acetylcholine (ACh) release. These increases were blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (50 μM) and by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (50 μM). Further pharmacological investigation revealed no effect of the kainate receptor-specific antagonist (2S,4R)-4-methylglutamate (10 μM) on kainate-induced rises in the frequency of spontaneous ACh release. However, these were blocked with the AMPA receptor-specific antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (50 μM). Application of glutamate (1 mM) in the presence of the glutamate uptake inhibitor d-threo-β-benzyloxyaspartate(200 μM) resulted in a significant increase in the frequency of mEPCs. These results suggest the presence of AMPA and NMDA receptors in association with motoneuron axons of larval zebrafish.