Figure 1. Generation and validation of Cnr1/Gad1 transgenic mouse line.

(A) Schematic representation of the construct used to generate the Cnr1/Gad1 transgenic mouse line. A centrally located minigene, containing a truncated part of the first exon, intervening intron, and second exon of the β-globin gene were the non-coding, but transcribed backbone which served as a ‘carrier’ for a synthetic miRNA directed against the Gad1 mRNA. The transcribed-translated part of the construct was the Gfp sequence inserted in frame with the start codon of the BAC-encoded Cnr1 gene, marking the cells in which the construct was activated. (B–C–D) Immunohistochemical assessment of Tg mice. (B) Triple immunohistochemistry showing the near-perfect co-localization of GFP with CNR1 in the hippocampus, stratum, substantia nigra, amygdala and cerebellum. Larger images on the left denote merged CNR1-GFP-DAPI triple-labeled images, small image stripes on the right denote enlarged part of the image on the left with signge-channel label (CNR1, GFP, DAPI and combined). DAPI co-labeling was performed to reveal overall tissue structure. Note that the GFP (produced from our transgene) and the co-expressed CNR1 (from the endogenous source) showed a staining pattern that is consistent with the previously described CNR1 expression across the rodent brain(Pettit et al., 1998; Tsou et al., 1998), suggesting that our transgene achieved the right spatial targeting. (C–D) To examine the cell-type specificity of the construct effects, we performed GFP-GAD67-PV triple-labeling of neocortical (C) and hippocampal (D) tissue sections. Larger pictures represent merged triple-labeled images, smaller monochrome images denote single channel fluorescence, higher magnification images (PV, GAD67 and GFP) originating from the larger picture (denoted by the white rectangle). As expected, the GFP expressing neurons (thus CNR1-expressing neurons) lacked GAD67 expression (yellow circles), suggesting that we achieved our goal of eliminating GAD67 production in the CNR1+ neuronal population. In contrast, GAD67 expression in the parvalbumin+ interneurons (blue circles) was not affected (retained both GAD67-PV immunostaining), suggesting that our construct specifically affected the CNR1-GAD67 subpopulation of interneurons. Abbreviations. Neocortex: Roman numerals denote neocortical laminae; WM - white matter; Hippocampus: so – stratum oriens, sp - stratum pyramidale, sr - stratum radiatum, slm - stratum lacunosum moleculare; Striatum: LGP - lateral globus pallidus ; CPu - caudate putamen; Substantia nigra: SNR - pars reticulata, SNC - pars compacta ; Amygdala: La – lateral amygdaloid nucleus, BLA - basolateral amygdaloid nucleus, ec - external capsule, En – endopiriform nucleus; Cerebellum: ml – molecular layer, plc – Purkinje cell layer, igl - internal granule layer. Calibration bar=100μm.