Figure 3. P2Y1 inhibition abolishes spontaneous currents in inner supporting cells and inner hair cells.

(A) Schematic of whole-cell recording configuration from ISCs. (B) Spontaneous inward currents recorded from an inner supporting cell before and during application of MRS2500 (1 μM). Recordings were performed at room temperature (~25°C). (C) Plot of event frequency, amplitude, and integral (charge transfer) before and after application of MRS2500. Measurement periods were five minutes long. n = 9 ISCs, 9 cochleae from 8 P6-8 mice (two-tailed paired Student’s t test; ****p<5e-5, ***p<0.0005) (D) Intrinsic optical imaging performed before and after application of the P2RY1 antagonist, MRS2500 (1 μM). Detected crenations are outlined in colors based on time of occurrence as indicated by timeline below image. Imaging was performed at room temperature (~25°C). (E) Plot of crenation frequency and area before and after application of MRS2500. n = 8 videos, 8 cochleae from 8 P6-8 mice (two-tailed paired Student's t test; **p<0.005) for frequency calculation and n = 5 cochleae (two-tailed paired Student's t test; *p<0.05) for area calculation. Cochleae that did not crenate after MRS2500 were excluded from the area calculation. (F) Schematic of whole-cell recording configuration from IHCs. (right) Whole-cell voltage clamp recording from an IHC before and during application of MRS2500. (G) Plots of event frequency and amplitude before and after application of MRS2500. n = 6 IHCs, 6 cochleae from 6 P6-8 mice (two-tailed paired Student's t test with Bonferroni correction; **p<0.005, *p<0.05). (H) Whole-cell voltage clamp recording of an ISC with application of MRS2500 following pre-incubation in aCSF containing CdCl₂ (100 μM), TTX (1 μM), ouabain (10 μM), and bumetanide (50 μM) to limit potassium release into the extracellular space. (I) Plot of the change in holding current, defined as the 95% percentile current value for each period. n = 6 ISCs, 6 cochleae from 6 P6-8 mice for each condition (two-tailed Student’s t test; ***p<0.0005). (J) Whole-cell voltage clamp recording of an IHC with a Cs-TEA internal solution (to inhibit K+ channels) before and after MRS2500 application. (K) Plot of the change in IHC holding current following control (superfusion of aCSF only) and MRS2500 with K-MeS and Cs-TEA internal. n = 5 IHCs, 4 cochleae from 4 P6-8 mice for aCSF, n = 6 IHCs, 6 cochleae from six mice for MRS2500, n = 4 IHCs, 3 cochleae from three mice for aCSF with Cs-TEA internal, and n = 6 IHCs, 6 cochleae from six mice for MRS2500 with Cs-TEA internal (one-way ANOVA; ***p<0.005, **p<0.005, ns, not significant). See Figure 3—source data 1 for plotted values and statistics.

Figure 3—figure supplement 1. P2ry1 inhibition abolishes spontaneous inward currents near physiological temperature.

(A) Schematic of whole-cell recording configuration from ISCs. (B) Spontaneous inward currents recorded from an ISC before and after application of MRS2500 (1 μM) and subsequent broad spectrum purinergic antagonists suramin (100 μM) and PPADS (50 μM). Recordings were performed near physiological temperature 32–34°C. (C) Quantification of event frequency. Each window measured was 5 min in length, washout was taken 20 min after superfusion of aCSF. n = 5 ISCs, 5 cochleae from 5 P7-8 mice (one-way ANOVA; ****p<5e-5, ***p<0.0005, ns, not significant). (D) Quantification of event amplitude. n = 5 ISCs, 5 cochleae from 5 P7-8 mice (one-way ANOVA; ****p<5e-5, ns, not significant). (E) Quantification of average charge transfer (integral). n = 5 ISCs, 5 cochleae from 5 P7-8 mice (one-way ANOVA; ****p<5e-5, ns, not significant). (F) Intrinsic optical imaging performed before and after application of the P2Y1 antagonist, MRS2500 (1 μM). Detected crenations are outlined in colors based on time of occurrence as indicated by timeline below image. Imaging was performed near physiological temperature 32–34°C. (E) Plot of crenation frequency before and after application of MRS2500. n = 6 videos, 6 cochleae from 3 P6-8 mice (two-tailed paired Student's t test; **p<0.005). See Figure 3—figure supplement 1—source data 1 for plotted values and statistics. (G).

Figure 3—figure supplement 2. Spontaneous inward currents and crenations are dramatically reduced in P2ry1 KO mice.

(A) Spontaneous inward currents recorded from ISCs in wildtype and P2ry1 KO mice. Recordings were performed near physiological temperature 32–34°C. (B) Plots of event frequency, amplitude, and integral (charge transfer). n = 17 ISCs, 17 cochleae from 17 P6-8 wildtype mice and 14 ISCs, 14 cochleae from 9 P2ry1 KO mice (two-tailed Student's t-test with Bonferroni correction; ****p<0.0005, ns, not significant). (C) Intrinsic optical imaging performed in wildtype and P2ry1 KO mice. Detected crenations are outlined in colors based on time of occurrence as indicated by the timeline below image. Imaging was performed at room temperature. (D) Plots of crenation frequency and area in wildtype and P2ry1 KO mice. n = 8 videos, 8 cochleae from 6 P6-8 wildtype mice and six videos, 6 cochleae from 5 P2ry1 KO mice (two-tailed paired Student's t test with Bonferroni correction; *p<0.05, ns, not significant). (E) Spontaneous inward currents recorded from an inner supporting cell in P2ry1 KO mice before and during application of MRS2500 (1 μM) and subsequent broad spectrum purinergic antagonists suramin (100 μM) and PPADS (50 μM). (F) Plots of event frequency, amplitude, and charge transfer. n = 5 ISCs, 5 cochleae from 4 P2ry1 KO mice (one-way ANOVA; ns, not significant). See Figure 3—figure supplement 2—source data 1 for plotted values and statistics.