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The expression of CB1R proteins in different auditory nuclei was demonstrated by fluorescence signals. CN, cochlear nucleus; SOC, superior olivary complex; LL, lateral lemniscus; IC, inferior colliculus; MGB, medial geniculate body; AC, auditory cortex. CN (n=10), SOC (n=7), LL (n=5), IC (n=8), AC (n=3), and MGB (n=3). The scale-bar for all the micrographs is 100 μm.(b)(c) The number of CB1R-expressing neurons and the average fluorescence intensities of CB1R proteins were quantified across different auditory nuclei. The number of CB1R-expressing neurons in distinct nuclei was manually counted using the unbiased stereological method, and the average fluorescence intensity (IntDen/Area) was analyzed for each nucleus.
Figure 2. Expression patterns of CB1Rs in subregions of central auditory nuclei. (a) Immunostaining of CB1Rs in subregions of auditory nuclei from Cnr1flox/flox mice. The auditory nuclei of the CN, SOC, and IC, along with their anatomical subregions, are shown in detail. DCN, dorsal cochlear nucleus; AVCN, anterior ventral cochlear nucleus; PVCN, posterior ventral cochlear nucleus; MSO, medial superior olive; LSO, lateral superior olive; MNTB, medial nucleus of the tapezoid body; ECIC, external cortex inferior colliculus; DCIC, dorsal cortex inferior colliculus; CNIC, central nucleus inferior colliculus. Numbers of mice used for analysis were: CN (n=10), SOC (n=7), LL (n=5), IC (n=8), AC (n=3), and MGB (n=3). (b) Number of CB1Rs expressing neurons. The number of CB1R-expressing neurons in subregions was manually counted using the unbiased stereological method (For CN, F (2, 28) = 59.40; DCN vs. PVCN, P=0.0018; DCN vs. AVCN,P<0.0001; PVCN vs. AVCN, P<0.0001. For SOC, F (2, 36) = 80.65; MNTB vs. LSO, P= 0.0101; MNTB vs. MSO, P<0.0001; LSO vs. MSO, P<0.0001. For IC, F (2, 24) = 180.9; ECIC vs. CIC, P<0.0001; ECIC vs. DCIC, P<0.0001; CIC vs. DCIC,P<0.0001). (c) Average fluorescence intensity of CB1R proteins. The average fluorescence signal intensity was analyzed for each subregion of the CN, SOC, and IC. (For CN, F (2, 27) = 26.53; DCN vs. PVCN, P=0.0146; DCN vs. AVCN, P=0.0007; PVCN vs. AVCN,P<0.0001.For SOC, F (2, 18) = 2.559; MNTB vs. LSO,P= 0.1049; MNTB vs. MSO, P=0.2317; LSO vs. MSO,P<0.8893, For IC, F (2, 21) = 3.7; ECIC vs. CIC,P=16.94; ECIC vs. DCIC, P=15.76; CIC vs. DCIC,P=1.185).The data are presented as means ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ****p < 0.0001.
Figure 3. CB1R knockout decreases the distribution of CB1R+ PV-neurons. (a) Double staining of PV and CB1Rs. The immunoreactivity for PV is shown in red, while CB1Rs are shown in green. Co-localization of the two markers in the CN and SOC regions is depicted in yellow (merged). The number of mice used for the double-staining analysis was: non-cKO: non-cKO (n=5) and Cnr1-cKO (n=5). (b) Comparison of PV and CB1Rs expression. The number of CB1R+ neurons in the CN and SOC was manually counted for each subregion, and the results were compared between PV-Cre;Cnr1flox/flox and control groups.
Similarly, the analysis of PV+ neurons was performed. The average fluorescence intensity of PV was also quantified for each subregion of the CN and SOC. The data are presented as means ± standard error of the mean (SEM) (For the average fluorescence intensity of PV, the interaction between non-cKO and Cnr1-cKO was significant: F (5, 62) = 8.066; main effect of Cnr1-cKO: F (1, 62) = 18.45). For total numbers of CB1R+ neurons, the interaction between non-cKO and Cnr1-cKO: F (5, 61) = 50.83; main effect of Cnr1-cKO: F (1, 61) = 569.1. DCN, P<0.0001; PVCN, P=0.0155; AVCN,P<0.0001; MNTB, P<0.0001; MSO,P<0.0001; LSO, P<0.0001). *p< 0.05, **p < 0.01, ****p < 0.0001. (c) Co-localization analysis of PV and CB1Rs in the CN and SOC. The ratios of PV+ neurons expressing CB1Rs were calculated and compared with those lacking CB1Rs.