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.