The first developing "mixed" synapses between vestibular sensory neurons mediate glutamate chemical transmission

In the present study, the nature of the synaptic transmission responsible for a monophasic potential generated by vestibular nerve stimulation of the principal cells in the chick tangential nucleus was established. This work was performed in slice preparations at the critical embryonic age of 15-16 days, the time of first observation of morphologically mixed (chemical and electrical) synapses at the axosomatic endings called spoon endings. The spoon endings are formed by the primary vestibular fibers with the largest diameters, the colossal vestibular fibers. This monophasic potential fits the criteria for chemical rather than electrical transmission due to the following responses in most cases: (i) the absence of collision between a direct spike initiated by depolarization in the principal cell and a vestibular-evoked action potential; (ii) failure to follow high frequency stimulation (up to 50 Hz); (iii) sensitivity to low calcium solution (0.1 mM). These tests indicate that strong electrical coupling between spoon endings and principal cells does not prevail at this stage. The recordings were obtained from principal cells injected intracellularly with biocytin, allowing their identification by morphological criteria. The lack of tracer coupling between the stained principal cells and their innervating vestibular fibers (n = 17) is consistent with the absence of electrical coupling. Identification of the neurotransmitter involved in this vestibular response was achieved by bath application of glutamate receptor antagonists, DL-2-amino-5-phosphonovaleric acid (40 microM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM), which blocked transmission reversibly. These results suggest that at the onset of formation of these "mixed" vestibular synapses, the gap junctions identified morphologically are likely not functional, and that the main response of the principal cells to vestibular nerve stimulation is mediated by glutamate.