Quantification was performed blind to condition and animal number by random coding of slides as previously described (6). Autoradiographic film images of GAD67, CB1R, and DAGLα mRNA were captured using a Microcomputer Imaging Device (MCID) (5.1 μm/pixel resolution) and digitized. All images for slides processed in an experimental run were acquired in the same session under identical room illumination and with the same gain and black levels and flatfield correction. Three sections evenly spaced at ~144 μm intervals containing the medial prefrontal cortex (mPFC; +1.98 to +1.54 bregma (16)), including the cingulate and prelimbic cortices, were selected from each mouse for quantification. For each section, optical density (OD) levels of GAD67, CB1R, and DAGLα mRNA were measured bilaterally from the pial surface to the white matter in the mPFC and expressed as nanocuries per gram of tissue (nCi/g) by reference to carbon-14 standards (ARC Inc., St. Louis, MO) exposed on the same film. All cortical density measures were corrected by subtracting background measured in the white matter.
