Figure 3. Transgenic HA-tagged CB1 receptor is expressed in hippocampal pyramidal neurons.

AAV-Stop-CB1 was injected bilaterally to the hippocampus of NEX-Cre transgenic (AAV-Glu-CB1) mice and their respective wild-type (AAV-WT) littermates. A, Cre-activated CB1 expression occurred in pyramidal neurons and appeared in a similar pattern compared to AAV-Stop-GFP confirming the integrity of the system. GC, granule cell layer; Hil, hilar region of dentate gyrus; LMol, stratum lacunosum-molecularis; Mol, stratum molecularis; Or, stratum oriens; Pyr, CA1/CA3 pyramidal cell layer; Rad, stratum radiatum. Bar: 250 µm. B-D, Higher magnification micrographs of dentate granule cells shown in A. Immunohistochemistry for the HA-tag reveals co-expression of CB1 and VGluT1 in the inner molecular layer of the dentate gyrus, validating the presynaptic localization of transgenic CB1 receptor. Bar in D: 25 µm. E, Western blot analysis of hippocampal homogenates of AAV-Glu-CB1 (n = 3) and AAV-WT (n = 3) mice. AAV-Glu-CB1 mice express significantly increased levels of CB1 receptor protein. Data are normalized for α-tubulin (p<0.001; unpaired t test analysis, two-tailed). F, Stimulation of [³⁵S]GTPγS binding in hippocampal homogenates of AAV-Glu-CB1 mice (n = 6) and AAV-WT littermates (n = 3) was determined by various concentrations of the CB1 agonist HU-210. Basal binding was measured in absence of HU-210 and defined as 0% in each experiment. Data are presented as percentage stimulation above basal [³⁵S]GTPγS binding and are the means ± SEM, all performed in duplicates. The non-linear regression curve illustrates that overexpression of the CB1 receptor in hippocampal pyramidal neurons resulted in significantly enhanced cannabinoids-induced G-protein activation and thus increased cannabinoid signaling. EC₅₀ = 3.26±0.08 nM (AAV-Glu-CB1), 2.97±0.13 nM (AAV-WT); E_max  = 224.4±7.39 (AAV-Glu-CB1), 93.77±4.99 (AAV-WT). Unpaired t test analysis, two-tailed: p<0.0001.