Coexistence of glutamate and acetylcholine in the developing motoneurons

Glutamate receptors mediate excitatory neurotransmission in the central nervous systems and are important in neuronal development, acquisition of memory, and are related to some neurodegenerative disorders. In the current study, co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryo. Spontaneous synaptic currents were recorded from innervated myocytes using whole-cell recording. Local perfusion of glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and non-NMDA, at synaptic regions with another micropipette all increased the frequency of spontaneous synaptic currents. Whole-cell recording was also made in the nerve growth cone of cultured spinal neurons. Local application of glutamate, NMDA or kainate all induced an inward current, indicating the coexistence of NMDA and non-NMDA receptors in the nerve terminals of developing motoneurons. Some innervated myocytes contracted spontaneously in the cultures, which were processed for glutamate immunocytochemistry after the recording of spontaneous synaptic currents. Glutamate immunoreactivity appeared in neuronal varicosities and neuromuscular junctions, indicating that glutamate is co-stored with acetylcholine in motoneurons. Double staining for glutamate and choline acetyltransferase further provides the evidence of the colocalization of glutamate and acetylcholine in developing motoneurons. These results suggest that both NMDA and non-NMDA receptors exist in the nerve terminals of developing motoneurons. Furthermore, glutamate and acetylcholine coexist in the motoneurons. The presynaptic glutamate receptors may thus have a physiological role in neuromuscular synaptogenesis in early embryonic stages.