Quantification of CB1R mRNA at the cellular level was performed as previously described (13) for the 5 pairs of GAD67 WT and heterozygous mice with available emulsion-dipped, Nissl-counterstained sections. Using the MCID software and a Nikon microscope with a motorized stage, sampling boxes (120 × 170 μm) were systematically tiled from the pial surface to the layer 6 - white matter border in both hemispheres of the mPFC. Sampling circles with a fixed diameter of 16 μm (15) were placed over CB1R silver grain clusters, and the number of silver grains per circle was quantified. Background signal, determined for each tissue section by quantifying grains in a 120 × 170 μm sampling box placed in the white matter, was subtracted from each grain cluster before analysis. Examination of individual grain cluster counts revealed 13 clusters that were ≥ 2SD away from the mean, and these clusters were excluded from analyses as outliers. A total of 119 and 118 CB1R grain clusters were analyzed for GAD67 WT and heterozygous mice, respectively.
