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).