Distribution And Role Of Receptors In Vestibular Hair Cells Of Rat

Part IExpression of nicotinic acetylcholine receptor subunit α 9 in typeII vestibular hair cells of ratObjective: ACh was considered as the predominant efferent transmitter in peripheral vestibular system in vertebrates. It was suggested that nicotinic acetylcholine receptor subunit α 9 is one of the important mediators of efferent cholinergic signaling in peripheral vestibular system. The innervation pattern of the ChAT-like immunostained efferents in the human is similar to that of other mammals including the rat, while there were many differences between the efferent vestibular system of birds and that of mammals. This study was aimed to explore the cell specific existence of α 9 AChR in the vestibular type II hair cells (VHCs II) of rat. This study will help to know the signaling transduction of human vestibular system and the mechenism of disease of vestibular system.Methods: To detect the expression of α 9 AChR messenger RNA(mRNA) in vestibular endorgans and single VHC II of rat by using reverse transcription polymerase chain reaction (RT-PCR) technique and single cell RT-PCR technique respectively.Results: It was shown that α 9 AChR mRNA was detected and sequenced from vestibular endorgans by RT-PCR. Further by using single-cell RT-PCR, mRNA encoding α 9 AChR was detected and sequenced from VHCs II of rat.Conclusion: This study established a method which could effectively detect the cell specific expression of mRNA in an individual VHC. Present data confirm that α 9 AChR mRNA express in the VHC II of rat and lend further support for the role of α 9 AChR as a mediator of efferent cholinergic signaling in mammalian vestibular hair cells. Part IIExpression of GABA_B receptor GB1 subunit in the vestibularendorgans of ratObjective: To investigate the expression of GABA_B receptor GB1 subunit in vestibular endorgans of rat and to discuss the distribution of GABA_B receptor in vestibular endorgans of rat.Methods: Gene specific primers used were based upon the rat GABA_B receptor GB1 subunit sequence. Reverse transcription polymerase chain reaction was carried out in the vestibular endorgans. Subsequently, the PCR products were sequenced.Results: After the amplification, the PCR products can be seen at the predicted size. Sequence analysis of the band confirmed identity to rat GB1 subunit cDNA sequence in the predicted region.Conclusion: These results indicate that there is the expression of the GB1 subunit in the vestibular endorgans, giving the direct evidence to date for that GABA as a vestibular transmitter and lending support for the role of GABA_B receptor as a mediator of GABA-ergic signaling in vestibular hair cells. Part IIIEvidence of the expression of GABA_B receptors on some type Ivestibular hair cells of ratObjective: GABA is considered as one of the efferent transmitters in peripheral vestibular system of rat. There is general consensus that the mammalian type I vestibular hair cells (VHCs I) are surrounded by an afferent calyx which effectively prevent efferent terminals from directly contacting this hair cells. However, Wackym et al. (1991) noted that the efferent CGRPi terminals occasionally passed through the primary afferent calyx surrounding the VHCs I and directly contacted a few of VHCs I of rat. It remains unknown now that weather there is GABA-ergic innervation on single rat VHC I . This study was aimed to explore the possible existence of GABA receptors on the VHCs I of rat.Methods: To detect the expression of GABA_B receptor GABA_B1 subunit messenger RNA(mRNA) in single VHC II of rat by using single cell RT-PCR technique. Patch clamp and calcium imaging were applied to measure the ion current and intracellular free calcium change induced by GABA.Results: It was shown that mRNA encoding GABA_B receptor GABA_B1 subunit was detected and sequenced from a few of VHCs II (6/94) of rat. In electrophysiological experiments, it was shown GABA induced a decrease in outward current elicited by depolarizing voltage steps. CGP35348, a GABA_B antagonist can completely stop the effect, while Picrotoxin, a GABA_A antagonist didn't affect this effect. It was also found that application of Baclofen, a GABA_B agonist, can inhibit the calcium current isolated by TEA and TTX in a few of VHCs I (9/128). This effect was completely blocked by CGP35348, while was not affected by Picrotoxin. In calcium imaging experiments using Fura-2 to measure the intracellular free-calcium, GABA induced a decrease of free-calcium concentration in some of VHCs I . CGP35348 can completely stop the change of free-calcium concentration induced by GABA, while Picrotoxin didn't affect this effect induced by GABA.Conclusion: The results suggest GABA_B receptors exist on some VHCs I of rat, and indicate that the GABA-ergic vestibular efferent system might be capable of directly modulating, at least in part, the function of VHCs I of rat.