We found NMDAR1s present throughout the cerebellar cortex. NMDAR1s in Purkinje cell somas, dendrites of the molecular layer, and granular cell layer (Figures 1A-D). We found CB1Rs expression on pre-synaptic terminals around Purkinje cell somas, consistent with their presence in the pinceau (Figures 1E-H). There was also diffuse expression in the molecular layer, corresponding to parallel fibers (20). In the granule cell layer the expression corresponded to granule cells somas and dendrites (21).
We performed a multi-way ANOVA test on the expression patterns of each receptor. The test compared genetic background (C57 vs BTBR), sex (male vs female), environmental condition (standard vs enriched caging), and layer in the cerebellar cortex (molecular, Purkinje, and granular). In the case of NMDAR1/DAPI, this test showed that sex was the only significant effect (\(F\left(\text{1,32}\right) = 56.95, p=1.71 x{10}^{-10}\), and the effect size was \({Ƞ}^{2}= 0.44\) ). The same test for the expression of CB1R showed that sex (sex:\(F\left(\text{1,32}\right)= 11.09, p=14.00 x{10}^{-4}, {Ƞ}^{2}= 0.11\)) and environmental condition (\(F\left(\text{1,32}\right) = 16.75, p=1.00 x{10}^{-4}, {Ƞ}^{2}= 0.17\)) had a significant effect.
Since sex has a strong influence on the expression of both, NMDAR1 and CB1R, we performed another multi-way ANOVA separating the groups by sex. For NMDAR1 this shows that, for both males and females, the genetic background had a significant effect on the expression of this receptor (males: \(F\left(\text{1,32}\right) = 13.73, p=8.00 x{10}^{-4}, {Ƞ}^{2}=0.22\); females: \(F\left(\text{1,32}\right) = 26.69, p = 1.33 x{10}^{-5}, {Ƞ}^{2}=0.22\)). This was also the case for the environmental condition (males: \(F\left(\text{1,32}\right) = 15.98, p = 3.00 x{10}^{-4}, {Ƞ}^{2}=0.25\); females: \(F\left(\text{1,32}\right) = 56.94, p = 1.66 x{10}^{-8}, {Ƞ}^{2}=0.48\)). We obtained the same result when performing the test for the CB1R images (Genetic background: males, \(F\left(\text{1,32}\right) = 14.91, p= 5.00x{10}^{-4},{Ƞ}^{2}=0.23\); females, \(F\left(\text{1,32}\right) = 14.40, p = 6.00 x{10}^{-4}, {Ƞ}^{2} = 0.27\); Environmental condition: males, \(F\left(\text{1,32}\right) = 18.21, p = 1.00 x{10}^{-4}, {Ƞ}^{2} = 0.28\); females, \(F\left(\text{1,32}\right) = 6.27, p = 0.01, {Ƞ}^{2}=0.11\)).
Next, we performed a post-hoc multi-compare analysis of the multi-way ANOVA tests (groups separated by sex) to determine differences in the effect of environmental enrichment and genetic background in the expression of each receptor. In males, the expression of NMDAR1/DAPI in the BTBR enriched environment group was more than 5 times larger (and significantly different, mean 6.42) than for all the other experimental groups (C57 standard and enriched environment groups, and the BTBR standard environment group, Figure 2A). The same analysis for expression of NMDAR1 in females shows that the effect of environmental enrichment is similar for the C57 and BTBR groups. In both cases, the enriched environment had a higher expression of the receptor than the corresponding standard environment group. Thus, suggesting that the genetic background does not modify the effect of environmental enrichment in females (Figure 2B).
We also performed a post-hoc analysis for the CB1R data. This shows that the expression of CB1R in males is identical to the pattern we found for NMDAR1 males (Figure 2C). The expression in females was different from CB1R males and from NMDAR1 males or females. The BTBR groups showed a lower ratio of CB1R/DAPI than the C57 groups. Environmental enrichment had no effect in changing the expression of CB1R (Figure 2D).
Since we used tissue from the same animals to stain for CB1R or NMDAR1 we studied the relative expression of these receptors. We calculated the correlation coefficient between these values across all animals. We found that the correlation coefficient for all males was large and significant (\(r = 0.89, p = 1.10x{10}^{-4}\)). In contrast, the correlation coefficient for females was not significant (\(r = 0.10, p = 0.77\)). This analysis suggests a differential regulation in the co-expression of NMDAR1 and CB1R between males and females.