Fig. 2

Effect of Y-27632 on the synapses in the inner hair cells after LISW exposure. a Timeline of the experiments in this study. b The effect of 10 mM Y-27632 in the LISW exposed cochleae on relative mRNA expression levels of RhoA, ROCK1, and ROCK2, 2 days after Y-27632 treatment. The relative mRNA expression levels were standardized by the expression levels before LISW exposure in each mRNA (n = 6). *indicates a significant difference (p < 0.05, two-tailed Mann–Whitney U test). c–e The effect of Y-27632 on the degenerated synapses of the inner hair cells at cochlear 16 kHz area 28 days after treatment with various concentrations of Y-27632 (c without Y-27632, d: 1 mM, and e: 10 mM). In c–e, presynaptic ribbons (CTBP2-immunoreactive puncta, red), postsynaptic densities (GluA2 immunoreactive puncta, green), and hair cells labeled with myosin 7a (blue) are shown. (c'–e') Enlarged images of the white inlets in (c–e) focusing on the synapses in the inner hair cells area. White dotted lines show the contour of the inner hair cells. White arrowhead indicates normal synapse formation with CtBP2-positive patch (red) accompanied with glutamate-receptor patch (green). Red arrow indicates orphan ribbons, which is a CtBP2-positive patch without apposed glutamate-receptor patches. In c’, there are seven normal synapses (white arrowheads) and two orphan synapses (red arrows); in d’, there are 12 normal synapses and one orphan synapse; and in e’, all 14 synapses have both CtBP2 and glutamate-receptor patch. f, g The quantification of the synaptic ribbons (f) and the glutamate receptor (g) observed in the 28 days after treatment and control from single IHC. The number of synapse components is lower in the sham surgery groups than in the control group at all frequencies tested. The number of synapse components is significantly larger in the 10 mM Y-27632-treated group at higher frequencies (asterisks) than those in the sham surgery group. Scale bar is 5 µm. *indicates significant differences (p < 0.05, two-way ANOVA, followed by Bonferroni correction for multiple comparisons). Values are represented as mean ± SEM