mGluR1/IP3/ERK signaling pathway regulates vestibular compensation in ON UBCs of the cerebellar flocculus

Abstract

Aims: To investigate the role of mGluR1α in cerebellar unipolar brush cells (UBC) in mediating vestibular compensation (VC), using mGluR1α agonist and antagonist to modulate ON UBC neurons, and explore the mGluR1/IP3/extracellular signal-regulated kinase (ERK) signaling pathway.

Methods: First, AAV virus that knockdown ON UBC (mGluR1α) were injected into cerebellar UBC by stereotactic, and verified by immunofluorescence and western blot. The effect on VC was evaluated after unilateral labyrinthectomy (UL). Second, saline, (RS)-3,5-dihydroxyphenylglycine (DHPG), and LY367385 were injected into tubes implanted in rats at different time points after UL separately. The effect on ON UBC neuron activity was evaluated by immunofluorescence. Then, Phosphoinositide (PI) and p-ERK1/2 levels of mGluR1α were analyzed by ELISA after UL. The protein levels of p-ERK and total ERK were verified by western blot. In addition, the effect of mGluR1α activation or inhibition on VC-related behavior was observed.

Results: mGluR1α knockdown induced VC phenotypes. DHPG increased ON UBC activity, while LY367385 reduced ON UBC activity. DHPG group showed an increase in PI and p-ERK1/2 levels, while LY367385 group showed a decrease in PI and p-ERK1/2 levels in cerebellar UBC of rats. The western blot results of p-ERK and total ERK confirm and support the observations. DHPG alleviated VC-related behavior phenotypes, while LY367385 exacerbated vestibular decompensation-like behavior induced by UL.

Conclusion: mGluR1α activity in cerebellar ON UBC is crucial for mediating VC through the mGluR1/IP3/ERK signaling pathway, which affects ON UBC neuron activity and contributes to the pathogenesis of VC.

Keywords: mGluR1α; unipolar brush cells; vestibular compensation.

FIGURE 1

mGluR1α was knockdown in ipsilateral flocculus of UL. (A) Experimental design. AAV‐NC or AAV‐mGluR1α‐shRNA were stereotaxically injected into the ipsilateral cerebellar flocculus of UL. After 3 weeks, confirmed knockdown by immunofluorescence and western blot. Conducted behavioral tests to analyze phenotypes: spontaneous nystagmus, postural asymmetry, head tilt, and tail suspension. (B, C) Stereotactic injection schematic (relative to bragma coordinates: AP = −4.5 mm, ML = −10 mm, DV = 7.8 mm). (D) Cerebellar flocculus injection coordinates were confirmed by methylene blue. (E, F) Fluorescence images of GFP in flocculus after virus infection. The number of GFP‐positive cells were similar in both groups. (G, H) The expression of mGluR1α was significantly reduced in the flocculus after AAV‐mGluR1α‐shRNA injection, confirmed by western blot. UL, unilateral labyrinthectomy; NC, normal control. ns, indicates no statistical difference; ***p < 0.001.

FIGURE 2

mGluR1α downregulation in ipsilateral cerebellar flocculus impairs VC 3 days after UL. Downregulation of mGluR1α expression affected spontaneous nystagmus (A), postural asymmetry (B), head tilt (C), and tail‐hang test (D), compared to the NC group. Downregulation of mGluR1α expression showed no significant difference in the tail‐hanging test compared to the NC group. UL, unilateral labyrinthectomy; NC, normal control; VC, vestibular compensation. ns, indicates no statistical difference; *p < 0.05; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Immunofluorescence staining investigated the effects of drug intervention on the activity of ON UBC neurons. (A) Experimental design of pharmacological interventions. Rats were implanted with catheters and injected with saline, DHPG, or LY367385 at different time points after undergoing UL. Behavioral assessments were conducted daily and tissues were collected after drug administration. (B) Immunofluorescence staining on the activity of ON UBC neurons. LY367385 decreased co‐localized positive neurons for mGluR1α and c‐fos, while DHPG increased them, as shown by immunofluorescence staining in the cerebellar flocculus of rats. (C) Quantitative analysis of the number of co‐localized positive neurons for mGluR1α and c‐fos. UL, unilateral labyrinthectomy; UBC, unipolar brush cells; DHPG, mGluR1α agonist; LY367385, mGluR1α antagonist. **p < 0.01; ***p < 0.001. Scale bar = 50 μm.

FIGURE 4

Effects of mGluR1α agonists and antagonists on vestibular compensation behavior. (A) SN of rats was recorded by electrooculography. DHPG reduced SN in rats after UL, with significant effects observed at 4, 8 h, and 1 day. In contrast, LY367385 increased SN in rats at 3 and 7 days compared to the Saline group. (B) Posture asymmetry of rats was assessed. DHPG reduced posture asymmetry in rats at 4, 8 h, 1, and 3 days compared to Saline group, while LY367385 increased posture asymmetry in rats at 3 and 7 days compared to Saline group. (C) The head tilt angle of rats was evaluated. The DHPG group suppressed UL‐induced SN in rats compared to the Saline group, with significant results at 4, 8 h, and 1 day. In contrast, the LY367385 group increased the head tilt of rats at 1, 3, and 7 days compared to the Saline group. (D) The tail hanging test score of rats was assessed. DHPG significantly reduced the tail hanging test score compared to Saline at 4, 8 h, 1, and 3 days, while no significant difference was observed between LY367385 and Saline at any time points (4, 8 h, 1, 3, and 7 days). UL, unilateral labyrinthectomy; UBC, unipolar brush cells; DHPG, mGluR1α agonist; LY367385, mGluR1α antagonist; Control: UL + saline group; DHPG: UL + DHPG group; LY367385: UL + LY367385 group. ns, indicates no statistical difference; *p < 0.05; **p < 0.01; ****p < 0.0001.

FIGURE 5

Effects of drug stimulations on PI/p‐ERK1/2 levels at various time points post‐UL. (A) DHPG increased while LY367385 decreased the PI level in the cerebellar flocculus of rats, compared to the UL + Saline group. Two‐way ANOVA showed a significant effect of drug stimulation (F (8,45) = 5.838, p < 0.0001). Bonferroni post‐hoc tests showed significant differences between the UL + Saline and UL + DHPG groups at 4, 8 h, 1, and 7 days (4 h, p < 0.0001; 8 h, p < 0.0001; 1 day, p < 0.0001; 7 days, p = 0.0019), but no significant difference at 3 days after UL (3 days, p = 0.9808). There were significant differences between the UL + Saline and UL + LY367385 groups at 4 h and 7 days (4 h, p < 0.0001; 7 days, p = 0.0041), but no significant difference at 8 h, 1, or 3 days after UL (8 h, p = 0.8851; 1 day, p = 0.0741; 3 days, p = 0.0629). (B) Compared to the Saline group, the p‐ERK1/2 level was increased in the cerebellar flocculus of rats in the DHPG group, while it was decreased in the LY367385 group. Two‐way ANOVA indicated a significant effect of DHPG drug stimulation (F (8,45) = 4.166, p = 0.0009). Bonferroni post‐hoc analysis showed significant differences between the UL + Saline group and the UL + DHPG group at 8 h, 1, and 7 days (8 h, p < 0.0001; 1 day, p = 0.000; 7 days, p = 0.0001), but no significant differences at 4 h and 3 days after UL (4 h, p = 0.0858; 3 days, p = 0.7964). Significant differences were observed between the UL + Saline group and the UL + LY367385 group at 4 h, 1, 3, and 7 days after UL (4 h, p < 0.0001; 1 day, p = 0.0002; 3 days, p = 0.0001, 7 days, p < 0.0005), but not at 8 h after UL (8 h, p = 0.9917). (C) The protein levels of ERK and p‐ERK in the cerebellar flocculus of rats, ERK were no change and p‐ERK were increased in DHPG‐injected Rat. (D) The quantification of ERK levels (n = 4 rat/group, two‐way ANOVA, F (4, 30) = 0.8065, p = 0.538). (E) The quantification of p‐ERK levels (n = 4 rat/group, two‐way ANOVA, F (4, 30) = 4.647, p = 0.005). (F) The protein levels of ERK and p‐ERK in the cerebellar flocculus of rats, ERK showed no change and p‐ERK was increased in LY367385‐injected Rat. (G) The quantification of ERK levels (n = 4 rat/group, two‐way ANOVA, F (4, 30) = 2.579, p = 0.058). (H) The quantification of p‐ERK levels (n = 4 rat/group, two‐way ANOVA, F (4, 30) = 74.300, p < 0.0001). UL, unilateral labyrinthectomy; UBC, unipolar brush cells; DHPG, mGluR1α agonist; LY367385, mGluR1α antagonist; Control: UL+ Saline group; DHPG: UL+ DHPG group; LY367385: UL+ LY367385 group; C: control group; D: DHPG group; L: LY367385 group. ns, indicates no statistical difference, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Algorithm of the mGluR1/IP3/ERK signaling pathway in cerebellar ON UBCs. Mossy fibers transmit signals to UBCs, which in turn relay them to granule cells or other UBCs and ultimately to Purkinje cells via parallel fibers. Purkinje cells, as the sole output neurons in the cerebellar cortex, inhibit the MVN in the vestibular‐cerebellar pathway. ON UBCs exhibit prolonged depolarization response to mossy fiber input, primarily mediated by mGluR1α receptors. Activation of mGluR1α triggers intracellular signaling through G proteins, PKC, IP3, and ERK, resulting in increased calcium efflux. The mGluR1α activity in cerebellar ON UBCs may crucial for mediating VC via the mGluR1/IP3/ERK pathway. UL, unilateral labyrinthectomy; UBC, unipolar brush cells; DHPG, mGluR1α agonist; MVN, medial vestibular nuclei; PKC, protein kinase C; ERK, extracellular signal‐regulated kinase; IP3, inositol 1,4,5‐triphosphate; IP3R, inositol 1,4,5‐triphosphate receptor, GC, granular cell; Glu, glutamic acid; PF, parallel fibers; ER, endoplasmic reticulum; VC, vestibular compensation.