(A) Mouse breeding scheme of the Cre-recombinase Kiaa1107 line crossed to Chatflox/flox mice to generate ChAT-cKO mice with conditional gene deletion of Chat in habenular neurons. (B) Western blot analysis with CHAT and α-tubulin antibodies in MHb and IPN extracts from wt and ChAT-cKO mouse brains. (C) Angled sections of the midbrain immunostained for CHAT (red). In wt mice (left panel), CHAT is highly expressed in MHb neurons, along their axons in the fasciculus retroflexus (fr) and in their axonal terminals in the IPN. In ChAT-cKO mice (right panel), CHAT immunoreactivity is no longer detected in the MHb-fr-IPN tract. Scale bars: 300 µm. (D) Sagittal sections of the midbrain immunostained for CHAT (red) and neurofilament (green). CHAT immunoreactivity is strong in the IPN (arrowhead) of wt mice (upper panel), and absent in ChAT-cKO brains (lower panel) while the laterodorsal tegmental nucleus (LDTg) - an efferent target of the IPN - and adjacent third cranial nerves (3N) show similar CHAT expression in wt and ChAT-cKO mice. Scale bars: 300 µm. (E) Immunostaining analyses in wt and ChAT-cKO show that CHAT immunoreactivity (red) is no longer detected in the MHb of ChAT-cKO mice. Wt and ChAT-cKO mice show similar CHAT immunoreactivity in striatum and LDTg. Scale bars: MHb and striatum 100 µm, LDTg 200 µm. (F) Quantification of CHAT positive neurons in the indicated cholinergic areas. ChAT-cKO mice do not express CHAT in the MHb, while striatum, PPTg and LDTg show comparable number of neurons positive for CHAT (percentage of the number of cells counted per section normalized to wt is shown in bars. MHb wt: 100.1 ± 3.676, number of sections=22; MHb ChAT-cKO: 0.3326 ± 0.1380, n=22; unpaired t-test p<0.0001; Striatum wt: 100.0 ± 7.233, n=22; Striatum ChAT-cKO: 92.27 ± 7.112, n=22; unpaired t-test p=0.45; PPTg wt: 100.1 ± 11.70, n=14; PPTg ChAT-cKO: 115.8 ± 10.78, n=14; unpaired t-test=0.33; LDTg wt: 100.0 ± 11.50, n=13; LDTg ChAT-cKO: 122.0 ± 16.86, n=13; unpaired t-test=0.29. The total number of neurons counted per brain area is shown above the bars; n=3 mice per genotype). (G) Sagittal brain sections immunostained for CHAT (red) and neurofilament (green). The axonal projections and general anatomy of the fr, IPN and surrounding tegmental areas are comparable between wt (left panels) and ChAT-cKO (right panels). ChAT-cKO mice lack CHAT-immunoreactivity in the MHb-fr-IPN projection but show similar CHAT expression as wt mice in other brain areas, such as the LDTg, third cranial nerve (3N), striatum (Str) and BF. Scale bar: 800 µm. BF: Basal forebrain; cKO: Conditional knockout; IPN: Interpeduncular nucleus; LDTg: Laterodorsal tegmental nucleus; MHb: Medial habenula.