3.1 CB1Rs are highly expressed in auditory brainstem
In the current study, we first analyzed the distribution and expression
of CB1Rs in the central auditory system of mice. The distribution and
expression level of CB1Rs in various regions of central auditory system
were both qualitatively and quantitatively evaluated using
immunostaining through fluorescence imaging, and coronal sections of
auditory nuclei were subjected to immunohistochemical analysis.To investigate the characteristics of CB1Rs expression in
central auditory system of Cnr1flox/flox mice, CB1R-immunostaining was detected in several nuclei,
including the cochlear nucleus (CN), superior olivary complex (SOC),
inferior colliculus (IC), the lateral lemniscus (LL) , medial geniculate
body (MGB) and auditory cortex (AC).
The expression of CB1Rs proteins in different auditory nuclei
was quantitatively analyzed using average fluorescence intensity
(fluorescence intensity per unit area) (Fig. 1 and Fig. 2), and total
cell counting was used to address both the density of
expression and the distribution of CB1R-expressing cells contained in
different nuclei (Fig. 1 and Fig. 2). For each nucleus, three
to five coronal sections at different distances from the bregma were
used for analysis to ensure a comprehensive analysis of each nucleus.
As expected, we found that the CB1Rs are widely distributed
throughout the central auditory system (Fig. 1a), particularly
abundantly enriched in CN, SOC and LL. In the CN, we observed the
differential expression of CB1Rs within the subregions of the CN. The
VCN showed higher CB1Rs expression when compared to the DCN. Within the VCN, the anteroventral cochlear nucleus (AVCN) has
significantly higher CB1Rs expression than the posteroventral cochlear
nucleus (PVCN) (Fig. 2a). In SOC, CB1Rs proteins are expressed stably
with similar strength across the medial superior olive (MSO), lateral
superior olive (LSO), and medial nucleus of the trapezoid body (MNTB)
(Fig. 2a). We observed that CB1R proteins are also detected in the MGB
at a moderate expression level relative to the CN, SOC, and LL (Fig.
1a). In contrast, CB1Rs expression in the IC) and AC was poorly
detected, with almost no visible positive immunoactivity in the AC (Fig.
1a). Intriguingly, the IC showed weak expression of CB1Rs, with sporadic
fluorescence signals in the dorsal central nucleus of the inferior
colliculus (DCIC) and the central nucleus of the inferior colliculus
(CNIC) (Fig. 2a). Moreover, our finding that the total number of
CB1R+ neurons was significantly higher in the SOC and
LL compared to other regions is consistent with a megascopic observation
(Fig. 1b). The CN ranks second in terms of the number of
CB1R+ neurons among all auditory nuclei, following the
SOC and LL, while the IC has fewer CB1R+ neurons than
SOC, LL, and CN. Much fewer CB1R+ neurons were
identified in the MGB and AC, with relatively lower expression levels of
CB1Rs proteins in these regions (Fig. 1b).