Figure 4. PV-Cre;Cnr1flox/flox mice exhibit hearing loss and abnormal auditory brainstem responses. (a) ABR Waveform Analysis. The ABR waveforms were recorded under click and 16 kHz tone burst stimuli. The black line represents the non-cKO mice (Cnr1flox/flox), while the red line represents the Cnr1-cKO mice (PV-Cre;Cnr1flox/flox). The numbers of mice used for the analysis were: non-cKO (n=5) and Cnr1-cKO (n=5). (b) Hearing Thresholds Obtained from ABR Waveforms. The hearing thresholds for each mouse were recorded under click and pure-tone stimuli across five frequencies (4-32 kHz) and analyzed from their ABR waveforms. Statistical analysis showed significant differences between the non-cKO and Cnr1-cKO groups (The interaction between non-cKO and Cnr1-cKO: F (4, 30) = 3.586; main effect of Cnr1-cKO: F (1, 30) = 26.97. 4 kHz, P=0.1450; 8 kHz, P=0.1450; 16 kHz, P=0.9995; 24 kHz, P=0.5990; 32 kHz, P<0.0001). (c) Superimposition of ABR Waveforms. The ABR waveforms at 90 dB SPL, recorded under click and 16 kHz tone burst stimuli, were superimposed to highlight the differences in amplitude and latency for waves I-V between the non-cKO (black line) and Cnr1-cKO (red line) transgenic mice. This comparison reveals discrepancies in the wave characteristics between the two groups. (d) The latency and (e) amplitude from ABR waveforms. Latency and Amplitude from ABR Waveforms. The latencies and amplitudes for each ABR wave (I-V) were calculated from the ABR data collected at 90 dB SPL under a 16 kHz tone burst. Additionally, the amplitudes obtained from ABR data collected at 90 dB SPL under click stimuli are also presented. These measurements reveal significant differences in the auditory brainstem responses between non-cKO and Cnr1-cKO mice. The data are presented as means ± SEM (For the latency, the interaction between non-cKO and Cnr1-cKO: F (4, 30) = 2.406; main effect of Cnr1-cKO: F (1, 30)=15.45. I Wave, P=0.9985; II Wave, P=0.9500; III Wave,P=0.0103; IV Wave, P=0.0274; V Wave, P=0.2366. For the amplitude under 16 kHz, the interaction between non-cKO and Cnr1-cKO: F (4, 26)=2.359; main effect of Cnr1-cKO: F (1, 26)=16.50. I Wave, P=0.5682; II Wave, P=0.0004; III Wave,P=0.7907; IV Wave, P=0.8171; V Wave, P=0.8606. For the amplitude under click, the interaction between non-cKO and Cnr1-cKO: F (4, 28) = 28.68; main effect of Cnr1-cKO: F (1, 28)=13.46. I Wave,P=0.0023; II Wave, P<0.0001; III Wave,P<0.0001; IV Wave, P=0.0519; V Wave,P<0.0001). *p < 0.05, **p< 0.01, ****p < 0.0001.