Identification of Neurotransmitter released from hair cells

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In order to determine the neurotransmitter released from auditory hair cells, we have invented an artificial synapse formed between isolated hair cells and cultured cerebellar granule cells. Both hair cells and granule cells were whole cell voltage clamped, and the current detected in the granule cells in response to the hair cell membrane depolarization were analyzed. Outward current was detected in the granule cells voltaged clamped at positive membrane potentials. This current was blocked reversibly by the bath applied DL-APV,therefore, concluded as due to the activation of NMDA type glutamate receptors on the granule cell. From detailed investigations of the dose-dependencies of several excitatory amino acids for NMDA and non-NMDA receptors expressed on the granule cells and on the Purkinje cells, respectively, glutamate was concluded as the most likely neurotransmitter released from hair cells. When glutamate was released from hair cells, it could mobilized up to 860 synaptic vesicles in 0.5 sec and such vesicles are likely fused with the presynaptic membrane at a rate of 500 Hz at each active zones in the hair cell.