Fig. 1

DREADD design and stereotaxic delivery results in high ChAT-DREADD+ neuronal expression within the rat PPTg. a Schematic showing the Cre-dependent AAV containing mutant hM3Dq. Upon expression, hM3Dq-mCherry and ChR2-eYFP is inverted to enable transcription from the EF-1a promoter. b The location for unilateral stereotaxic injection of either constructs into the PPTg of Chat::Cre-transgenic rats, along with either lactacystin or lactacystin vehicle into the unilateral SNc. c High magnification confocal photomicrographs show strong ChAT- immunofluorescence signaling for individual cell bodies that overlap considerably with mCherry-tagged hM3Dq, expressed in the plasma membrane. The intensity of the immunofluorescence signal for ChAT (green) significantly surpassed the intrinsic brightness of expressed hM3Dq (red), the latter that was induced through viral vector-mediated transduction. Co-expressing neurons appear mainly green, due to the strong immunosignal of the applied secondary antibody for ChAT, but with some yellow labelling. d A low magnification image shows the PPTg resident cholinergic neurons (green), interspersed with cholinergic neurons co-expressing (yellow) mCherry-tagged hM3Dq. e High magnification confocal images show red labelled ChAT+ neurons co-expressing (yellow/orange) eYFP-tagged hChR2. f A low power confocal image shows the high degree of co-expressing ChAT+ (red) and yellow labelled hChR2 PPTg neurons. In the case of both hM3Dq and hChR2, viral vector-mediated expression was limited to PPTg ChAT+ neurons. However, some variance in viral vector-mediated expression was seen, especially in the case of ChAT-hChR2, with some individual ChAT+ cells that expressed the gene construct to a lesser extent (visible as a lower fluorescence intensity, such neurons being indicated by a red asterisk in (e), left panel) than other transduced cholinergic cells. High power images were taken with a 20×, air-immersion objective lens, while low power images were captured with a 63× oil-immersion objective lens. Arrowheads point to ChAT+ neurons that did (yellow) and did not (white) express the respective virally transduced constructs. Scale bars: 50 μm (c, e); 100 μm (d, f). g The degree of overlap between ChAT+ (green) and hM3Dq + (red) neurons for the V + D (n = 12) and L + D rats (n = 12) and the overlap between ChAT+ (red) and hChR2+ (yellow) neurons for V + CV (n = 12) and L + CV rats (n = 12) revealed that dual expression of the viral vector-ChAT+ PPTg neurons exceeded 70 %, relative to all ChAT+ PPTg neurons. This demonstrates that our approach using Cre-mediated recombination to selectively express DREADD within PPN cholinergic neurons resulted in high transduction efficiency