Function Regulation Of Cerebellar Flocculus Unipolar Brush Cells During Vestibular Compensation

PART I Expression changes and distribution characteristics of different subtypes of UBCs in rat cerebellar flocculus after unilateral labyrinthectomyObjective: To explore the expression changes and distribution characteristics of different subtypes of unipolar brush cells（UBCs）in rat cerebellar flocculus during different periods after unilateral labyrinthectomy（UL）.Methods: 1.SD rats were randomly divided into the operation group（UL）and the shamoperated group.The rats were tested for quantitative vestibular function at 4 h,8 h,1 day,3 days and 7 days after UL.2.The mRNA and protein of mGluR1α and calretinin in rat cerebellar flocculus were tested by RT-PCR and Western blot at different time points（4 h,8 h,1 day,3 day,7 day）after UL,and the contralateral and sham operation groups were used as reference.3.Immunofluorescence staining was used to study the localization of mGluR1α and calretinin in the cerebellar flocculus of normal rats.The number,size and distribution of positive cells were observed,and the changes of mGluR1α and calretinin in the cerebellar flocculus of rats before and after UL were observed.Results: 1.According to the results of quantitative vestibular function tests,UL-induced behavioral symptoms gradually recovered within 7 days.2.The RT-q PCR showed that mGluR1α in the rat cerebellar flocculus increased at 4 h after UL and disappeared at 8 h.The calretinin in the rat cerebellar flocculus decreased at 4 h and 8 h after UL and returned to normal at 1 d after UL.Western blot showed that mGluR1α in the rat cerebellar flocculus increased at 4 h after UL and calretinin decreased at 8 h after UL.3.Immunofluorescence showed the presence of different subtypes of UBCs in normal rat cerebellar flocculus,labeled by mGluR1α and calretinin,respectively.mGluR1α was mostly expressed in the dendrites of smaller ON UBCs,accounting for about 60%,and calretinin was mostly expressed in the cytosol and dendrites of slightly larger OFF UBCs,accounting for about 40%.There was no significant difference in the number of positive cells before and after UL.Conclusion: In the early stage of vestibular compensation,different subtypes of UBCs in the cerebellar flocculus of rats are involved in the vestibular center plasticity,while ON UBC and OFF UBC may play the opposite role.PART II Construction of ON UBC model of targeted knockdown in cerebellar flocculus and the effect on vestibular compensatory behavior phenotypeObjective: To observe the effect of specific knockdown of mGluR1α（ON UBC）on the behavior phenotype of vestibular compensatory in rats.Methods: 1.The SD male rats were randomly divided into the negative control（NC）group and mGluR1α-shRNA group.The AAV-NC virus and AAV-mGluR1α-shRNA virus were injected into the cerebellar flocculus of rats by stereotaxic injection.After 3 weeks the UL was performed.Then immunofluorescence was used to observe the infection effect and western blot was used to detect the expression of mGluR1α to verify the knockdown efficiency.2.The behavioral phenotype of the rats was analyzed to evaluate spontaneous nystagmus,postural asymmetry,head tilt and tail-hanging test.To observe the effect of the mGluR1α knockdown model on vestibular compensatory behavior phenotype in rats.Results: 1.Both AAV-NC and AAV-mGluR1α-shRNA viruses were infected effective in rat cerebellar flocculus by immunofluorescence.The expression of mGluR1α（ON UBC）was significantly downregulated in the mGluR1α-shRNA group compared with the AAV-NC group,as shown by Western blot,verifying the knockdown efficiency of mGluR1α.2.The results of vestibular functional behavior showed that the down-regulation of mGluR1α expression in cerebellar flocculus induced vestibular compensatory phenotypes,including spontaneous nystagmus,postural asymmetry,head tilt and tail-hanging test.Conclusion: It is further proved that the expression changes of mGluR1α in the cerebellar flocculus may be a key factor affecting the vestibular compensatory phenotype.PART III Regulation and mechanism of mGluR1/IP₃/ERK signaling pathway in vestibular compensation in ON UBC of cerebellar flocculusObjective: The regulation and mechanism of mGluR1/IP₃/ERK signaling pathway in vestibular compensation were investigated by activating and inhibiting ON UBC neurons by mGluR1α agonist DHPG and antagonist LY367385,respectively.Methods: 1.SD rats（180～200 g）were randomly divided into three groups,including UL + saline groups,UL + DHPG groups,UL + LY367385 groups.Firstly,the rats were embeded with a cannula.After the rats were recovered and UL model was established,mGluR1α agonist DHPG or antagonist LY367385 was injected into the cannula at 4 hours,8 hours,1 day,3 days and 7 days after UL.The vestibular function tests were detected 30 min after administration,including spontaneous nystagmus,postural asymmetry,head tilt and tailhanging test.2.The levels of phosphatidylinositol（PI）and phosphorylated extracellular signal-regulated kinases（p-ERK1/2）in the downstream of mGluR1α at different time points after UL were analyzed by ELISA.3.By c-fos co-localization with mGluR1α immunofluorescence,the effects of DHPG or LY367385 on the activity of cerebellar flocculus neurons were evaluated,and the possible mechanism of mGluR1α in vestibular compensation was further explored.Results: 1.The mGluR1α agonist DHPG alleviated UL-induced spontaneous nystagmus,postural asymmetry,head tilt,and tail-hanging test,whereas the mGluR1α antagonist LY367385 aggravated UL-induced vestibular decompensation behaviors.In the 4-hour,8-hour,and 1-day groups,the DHPG injected into the flocculus reduced the sensitivity and severity of the UL state,in contrast,the LY367385 increased the severity of the UL state at 3 and 7 days.2.Compared with the UL + saline group,the levels of PI and p-ERK1/2 mediated by mGluR1α in the cerebellar flocculus of UL + DHPG group were increased.The levels of PI and p-ERK1/2 mediated by mGluR1α in the cerebellar flocculus of UL + LY367385 group were decreased.3.Immunofluorescence showed that compared with the UL + saline group,the UL + DHPG group significantly enhanced the activity of cerebellar flocculus neurons,which was manifested by a significant increase in the number of mGluR1α and c-fos co-localized positive neurons.Compared with the UL + saline group,the UL + LY367385 group reduced the activity of cerebellar flocculus ON UBC neurons,which was manifested by a decrease in the number of mGluR1α and c-fos co-localized positive neurons.Conclusion: The activity of m GluR1α in the cerebellar flocculus may be a key factor in mediating the vestibular compensation,and may play a positive role in the vestibular compensation through the mGluR1/IP₃/ERK signal transduction pathway,affecting the ON UBC neuron in the cerebellar flocculus and participating in the pathogenesis of vestibular compensation.