Fig. 2

Injection of CNO induces c-fos immunoreactivity in PPTg ChAT+ neurons of rats expressing Cre-dependent AAV-hM3Dq-mCherry. In ChAT::Cre rats where hM3Dq (red) was expressed unilaterally in the PPTg and when brains were obtained within 90 min of administering CNO, c-Fos immunoreactivity (green nucleic stain) overlapped with the hM3Dq signal, confirming that CNO stimulates hM3Dq-expressing PPN neurons. a c-Fos-expressing cell counts revealed relatively high expression in the ipsilateral but not the contralateral PPTg of V + D and L + D rats (n = 12/group). However, c-Fos signalling levels in rats that underwent a CNO wash-out phase lasting ~48 h, prior to sacrifice and brain collection, detected minimal hM3D+ neurons co-expressing c-Fos. b In both PPTg-CV rat groups (n = 12/group), ChAT-c-Fos neuronal overlap was nearly undetectable, when measured either at + CNO or -CNO. c Representative fluorescent microscopy images of contralateral and ipsilateral PPTg c-Fos fluorescence seen in the same brain section but in the different experimental groups. Merged images are shown in the right panels, with yellow indicating co-localisation. Magnification: ×20, scale bar: 25 μm. Mean ± SEM, *p <0.05