Intersectional targeting with AAV.Con/Fon.GFP

Four female CR^Cre;VGAT^Flp mice received injections of an AAV that expressed GFP in the presence of both Cre and Flp recombinases under transcriptional control of the human elongation factor 1 alpha promoter fragment hEF1α (AAV1.C^on/F^on.GFP) into the right side of the spinal cord as described previously^27,84. Briefly, the mice were anaesthetised with isoflurane and received injections in the spaces between the T12/T13 and T13/L1 vertebrae, to allow targeting of the L3 and L5 spinal segments. In some cases an additional injection was made through a small hole drilled in the lamina of T13 to allow targeting of the L4 segment. At each site, 300 nl of virus containing 8.1 × 10⁸ GC was injected 400 μm lateral to the midline and 300 μm below the pial surface. The wound was closed and the mice received perioperative analgesia (0.5 mg/kg buprenorphine and 5 mg/kg carprofen). After a 13–23 day survival period the mice were reanaesthetised and perfused transcardially with a fixative that contained 4% formaldehyde. In two cases (used for electron microscopy) the fixative also contained 0.2% glutaraldehyde.

Tissue from the mice fixed with formaldehyde/glutaraldehyde was processed for immunoperoxidase labelling. The L4 segments were fixed overnight at 4 °C and cut into 60 μm thick transverse sections with a vibrating blade microtome (Leica VT1200). The sections were incubated for 3 days in rabbit anti-GFP (see Table ^S2), followed by overnight incubations in biotinylated donkey anti-rabbit IgG and avidin-HRP. All of these reagents were diluted in PBS. The sections were then reacted with DAB, and processed for electron microscopy. We used the same procedure as described above, except that post-fixation was performed with a "reduced osmium" protocol as described by Zhang et al.⁶⁸. Ultrathin sections were cut onto Formvar-coated slot grids and stained with lead citrate, before being viewed on the EM. Tissue from the other 2 mice was cut into transverse or sagittal sections, which were reacted to reveal GFP with an immunofluorescence method, as described above.

Two male Tac1^Cre;VGAT^Flp mice received intraspinal injections of AAV1.C^on/F^on.GFP into the L3 and L5 segments on the right side as described above. In this case 1.6 × 10⁹ GC in 300 nl was injected at each site. After a 7 day survival time, mice were perfused with fixative containing 4% formaldehyde. Spinal cord regions that included the injection sites was cut into transverse or sagittal sections, which were immunoreacted with antibody against GFP.