3.2 Cnr1 conditional knockout Cnr1 conditional knockout leads to a decreased distribution of CB1R+ PV-neurons
To elucidate how CB1Rs influence inhibitory circuits in central auditory system and ultimately auditory signal processing, we generated the conditional knockout mice with Cnr1 specifically deleted from PV interneuron, a major type of GABAergic interneurons in the central auditory system.
To confirm the specific deletion of CB1Rs from PV neurons, we performed double-staining for CB1Rs and PV in both the PV-Cre;Cnr1flox/flox and Cnr1flox/flox mice. This approach allows us to assess the co-localization of CB1Rs and PV, providing direct evidence for the selective loss of CB1Rs in PV-expressing neurons and confirming the specificity of the conditional knockout (Fig. 3a ). We found that average fluorescence intensity of PV (indicated by Alexa Fluor 594, red fluorescence) is nearly unchanged in Cnr1-cKO mice when compared with non-cKO controls suggests that the conditional knockout of Cnr1 does not affect the overall expression of parvalbumin in the PV+ interneurons (Fig. 3b, Fig. S2 and Fig. S3 ). PV proteins are abundantly distributed in the CN and SOC, and that CB1Rs and PV proteins co-localize to a certain proportion (Fig. 3a, 3c ).
It is not surprising that the knockout of Cnr1 specifically in PV+ neurons in the PV-Cre;Cnr1flox/flox mice might lead to a significant change in CB1Rs expression in the CN or SOC (Fig. 3b and Fig. 3c ). The number of CB1R+ cells was reduced in the central auditory nuclei of the Cnr1-cKO mice compared with the non-cKO controls (Fig. 3b ). The ratio of CB1R+ PV-neurons (represented by PV+/CB1R+ cells) was notably decreased in the CN and SOC in the Cnr1-cKO mice (Fig. 3c ). This suggests that the co-localization of CB1Rs and PV proteins is reduced, which could have important implications for synaptic signaling and inhibitory control in these regions
The combination of real-time quantitative PCR (RT-PCR) and Western blotting was used to confirm the expression change of Cnr gene and CB1Rs proteins in the specific auditory regions (CN and SOC) following Cnr1-cKO (Fig. S1 ). Given the marginal anatomical definition of the CN, the region was exacted scrupulously for both western-blot and RT-qPCR. Moreover, IC and hippocampus were also exacted for RT-qPCR(Fig. S1 ). In contrast to the non-cKO group, the protein expression level of CB1Rs in the CN from Cnr1-cKO mice was significantly decreased, indicating that the conditional knockout of Cnr1 in PV interneurons led to a reduction in CB1R+ neurons in this region (Fig. S1a ). Moreover, in CN, IC, and hippocampus, the gene transcription level of Cnr1, as measured by mRNA transcripts, was also decreased in Cnr1-cKO mice, when normalized to β-actin(Fig. S1b ).
Overall, our results indicated that deleting CB1Rs specifically from PV interneurons dramatically decreased the distribution of CB1R+ PV-neurons in both the CN and SOC. This reduction in CB1R+ PV-neurons was observed at both the protein and mRNA levels. The loss of CB1Rs in PV+neurons likely disrupts the GABAergic inhibition in the central auditory system, which may lead to altered auditory signal processing.
3.3 CB1Rs cKO in PV interneurons results in hearing loss at 32 kHz
The CB1Rs were presented in many different cell types, with their expression levels dynamically varied amongst distinct subcellular locations (Kendall and Yudowski, 2016b; Busquets-Garcia et al., 2018b; B et al., 2024b). CB1Rs are recognized to have the most abundant expression in GABAergic interneurons, particularly in regions of the central auditory system such as the CN and SOC. In contrast, glutamatergic, glycinergic, and other neuronal types exhibit relatively low-to-moderate expression levels of CB1Rs (Huang and Paul, 2019). This suggests that CB1R-mediated signaling may primarily influence inhibitory circuits, especially in GABAergic interneurons, which are critical for regulating the balance between excitation and inhibition in auditory processing. The diversity of GABAergic neurons in the CNS can be characterized by multiple factors, including their transcriptomes, morphology, and electrophysiological properties. Among these, PV interneurons stand out one of the most abundant types of GABAergic neurons in the central auditory system (Hu et al., 2014; Huang and Paul, 2019). Therefore, our results suggest that loss of CB1Rs in PV interneurons might trigger abnormal hearing in mice.
To assess the impact of CB1Rs deletion in PV interneurons on hearing function, we conducted ABR tests on both Cnr1-cKO and non-cKO mice (Fig. 4 and Fig. S4). To further assess the auditory function in Cnr1-cKO and non-cKO mice, we analyzed the waveforms, thresholds, latencies, and amplitudes of waves I-V of the ABR (Fig. 4 and Fig. S4-S7). Compared with non-cKO mice, the average hearing thresholds for both click and pure-tone stimuli at 32 Hz in Cnr1-cKO mice were significantly shifted (Fig. 4b). The increased hearing thresholds in Cnr1-cKO mice indicated that the conditional knockout of CB1Rs specifically from PV-interneurons resulted in hearing loss at high frequency.