Evidence for L-glutamate release in frog vestibular organs

The present study was devised in order to ascertain whether L-glutamate (Glu) is the neurotransmitter at the primary afferent synapse in frog vestibular organs. To this end different groups of frog isolated semicircular canals were stimulated by means of solutions slightly enriched in K+ (5 mM K(+)-rich solutions are sufficient to produce a strong, long-lasting, transmitter release from the basal pole of sensory cells) both in normal conditions and after low-Ca(2+)-high-Mg2+ impairment of the synaptic transmission. The concentration of Glu in the surrounding medium, determined by means of a bioluminescence-enzymatic method, was evaluated in two different experimental conditions: a) when the canals (5 canals placed inside little net bags) were immersed in a 5 mM K(+)-stimulating solution; b) during the superfusion of the canals (25 canals placed into a little perfusion chamber) with a 5 mM K(+)-stimulating solution. The net bag experiments demonstrated that K(+)-rich solutions can provoke an outflow of Glu from canal organs only if the crista ampullaris is present and functioning. Glu fluctuations were in fact suppressed by employing canals deprived of the ampulla or after low-Ca2(+)-high-Mg2+ synaptic blockade. The superfusion experiments demonstrated that the time course of 5 mM K(+)-induced release of Glu from the sensory organ strictly parallels the time course of 5 mM K(+)-induced EPSPs and spike discharge in afferent axons. These results strongly support the hypothesis that Glu is, or is released with, the afferent transmitter in frog inner ear sensory organs.