Figure 4. Neuronal CB₂R mRNA Expression in the Hippocampus by RNAscope ISH and FACS-qPCR Assays.

(A) Hippocampal image (DAPI staining) illustrates the target region (CA3) in (C).

(B) The CB₂R mRNA structure in CB₂R-floxed mice and the target gene region of a CB₂R RNAscope probe (CB₂-O2, 506–934 bp) used to detect CB₂R mRNA. The CB₂-O2 probe targets the floxed region of mouse CB₂R mRNA (NM_009924.4) in CB₂R-floxed mice. CDS, (CB₂R)-coding DNA sequence (478–1,521 bp).

(C) CB₂R mRNA staining illustrates significant CB₂R (Cnr2, green) and NeuN (Rbfox, red) mRNA co-localization in WT hippocampal CA3 neurons (upper panels), while such co-localization is substantially diminished in Syn-CB₂R^−/− (lower panels).

(D) A representative image shows FACS-sorted NeuN+ neurons and NeuN− non-neuronal cells in the hippocampus.

(E) The qPCR assays show that CB₂R mRNA is detected mainly in NeuN+ hippocampal cells of WT mice, while the CB₂R mRNA in NeuN+ hippocampal cells in Syn-CB₂R^−/− mice was substantially reduced (~70% reduction), and abolished in the CB₂R^−/− mice.

(F) The qPCR assays for CB₁R mRNA (as controls) in the same samples demonstrate similar levels of CB₁R mRNA expression in NeuN+ neurons and NeuN− cells in WT, Syn-CB₂R^−/−, and const. CB₂R^−/− mice.

(G) The qPCR assay results of neuronal and glial markers in two cell populations to examine the purity of sorted cells, illustrating that Rbfox3 was detected mainly in FACS-sorted NeuN+ neurons (a), but not in NeuN− cells (b). In contrast, the glial marker genes Itgam, Cspg4, and Aldh1l1 were mainly detected in NeuN− nonneuronal cells (b), but not in NeuN+ hippocampal neurons (a). Data shown in (a) were normalized to Rbfox3 expression in the NeuN+ population, which was defined as 1. Data shown in (b) were normalized to each respective marker gene level in NeuN+ (Rbfox3) and NeuN− cells (all three glial markers).