3.4 PV-Cre;Cnr1flox/flox mice exhibit abnormal auditory brainstem responses
Among the tone-burst stimuli with five frequencies ranging from 4 kHz to 32 kHz, the hearing thresholds in the mice presented a ā€˜V’-shaped curve, with the lowest thresholds observed at 16 kHz, indicating that the mice had the best hearing sensitivity in this frequency range (Fig. 4b). To further evaluate the auditory function, the ABR waveforms recorded at 90 dB SPL were superimposed for both Cnr1-cKO and non-cKO mice under click and 16 kHz stimuli (with 16 kHz serving as the representative frequency). The comparison of the waveforms between the two groups revealed notable differences, especially in terms of wave amplitude and latency (Fig. 4c). Surprisingly, there were significant differences in the ABR wave characteristics between Cnr1-cKO and non-cKO groups. These differences were observed in wave amplitude, latency, and waveform morphology (Fig. 4c-e). In response to click stimuli, the amplitude of wave II in Cnr1-cKO mice decreased dramatically when compared to non-cKO mice, while the amplitudes of waves I, III, IV, and V showed only a slight decrease in Cnr1-cKO mice (Fig. 4c, 4e). Both the latencies and amplitudes of the waveforms I-V in Cnr1-cKO mice were significantly changed when compared to non-cKO mice in response to 16 kHz pure tones (Fig. 4c-e, Fig. S5-S7). Especially the wave II and wave III, which are typically linked with the physiological functions of the CN and the SOC, respectively, were drastically changed in both amplitudes and latencies in Cnr1-cKO mice (Fig. 4c-e, Fig. S5-S7). As expected, the changes in ABR wave II and wave III were consistent with the immunostaining results from the CN and SOC in Cnr1-cKO mice, which were demonstrated previously (Fig. 3). Except for the data collected under 32 kHz, which was too ambiguous to analyze, the amplitudes of waves I-IV derived from ABR waveforms under click stimuli or other frequencies of tone burst were decreased in Cnr1-cKO mice (Fig. S6 and Fig. S7). The longer latencies and lower amplitudes observed in the ABR waveforms reflect a decrease in transduction velocity and strength of the auditory signal.
Taken together, these findings strongly suggest that the conditional knockout of Cnr1 in PV interneurons caused auditory dysfunction in mice. Specifically, the prolonged latencies indicate slower neural transmission in the auditory pathways, while the attenuated amplitudes point to a weakened neural response. This disruption in both the timing and magnitude of auditory signals in PV-Cre;Cnr1flox/flox mice points to the critical role of CB1Rs signaling in maintaining normal auditory processing, particularly in PV interneurons within the central auditory system.