The Effects Of CGRP On The Glutamate-elicited Excitotoxicity In Spiral Ganglion Neurons

Spiral ganglion neurons (SGNs) are primary afferent fibers of auditory pathway, which accept the stimulation of glutamate released by hair cells and conduct the excitatory action potentials to the nucleus of cochlear nerve. Under certain pathological conditions, such as noise, ischemia, hypoxia, infection and ototoxic drugs, the over accumulated glutamate could cause swelling, even apoptosis of spiral ganglion neurons and result in irreversibly hearing loss. The efferent systems of cochlear play an important role in the protection against noise through neuronal circuit by negative feedback to the hair cells and primary afferent neurons. Of note, CGRP, one of the neurotransmitters of the efferent system, is found both in the efferent systems and the autonomic nerve endings innervating the spiral modiolar artery. What is more, it is locally synthesized during the regeneration of injured axon of peripheral sural sensory nerve in rats. Herein, we are to observe the effects of exogenous CGRP on protection of the SGN from the over accumulated glutamate and detect whether the CGRP type I receptor is existed on the SGN or not.Methods:In this work,3～5 days postnatal mouse were used. The SGNs were dissected and cultured for 24 hours in the DMEM containing 10% fetal bovine serum. Then,3 groups of the cells were treated with 500μM glutamate for 6,12 and 24 hours separately. Another 3 groups, which were pretreated with 1 nM,10 nM and 100 nM CGRP respectively, were subsequently treated with 500μM glutamate for 24 hours. Total cell proteins were abstracted and csapase-3 protein were detected by western blot. The total RNA and proteins of normally cultured cells for 24 hours were also abstracted to detect the existence of CGRP receptor by RT-PCR and western blot.Results:1.6 hours after being treated with 500 u M glutamate, the activation of csapase-3 was enhanced in SGNs, when it comes to 24 hours the level of activated caspase-3 dropped to below control.2. Pretreated with low dose of CGRP caused the down-regulation of activated caspase-3 level. However, when the concentration of CGRP added up to 10 nM, the activation of caspase-3 began to up-regulate in a dose-dependent manner.3. There was no CGRP type I receptor existed in the SGNs as evidenced by PCR or western blot.4.Conclusions:1. At the early stage of exposure to high level glutamate, there is a detectable activation of caspase-3 resulting in apoptosis of neurons. 2. CGRP has dual effects on SGNs. CGRP at low concentration could reduce the activation of caspase-3, and protect the spiral ganglions from apoptosis. However, high concentrations of CGRP cause a dose-dependent increase in caspase-3 activation.3. We assume that the effects of CGRP on the SGNs are not via the known type I receptors.