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 āVā-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.