Supplementary Figure 1: Cholinergic neurons projecting to the VTA are concentrated in the caudal mesopontine region.

(a) Schematic showing the sites of retrograde tracer injections in the VTA: cholera toxin b (CTb) was injected in the rostral VTA and red retrobeads (RB-R) in the caudal VTA to identify the topography of the projections from the PPN and LDT. (b) ChAT-immunopositive neurons contained CTb and/or RB-R in both PPN and LDT (arrows; the PPN neuron is triple-labeled and the LDT neuron in double-labeled). (c) Pattern of retrograde labeling in relation to the expression of ChAT at three mediolateral levels of the PPN/LDT in a representative animal. (d) Similar numbers of VTA-projecting cholinergic neurons were detected at different mediolateral levels. A larger proportion of non-cholinergic VTA-projecting neurons was observed in the most lateral sections of the PPN. (e) The number of cholinergic neurons containing one of the tracers was normalized against the total number of cholinergic neurons and expressed as a percentage of the total. This was quantified at three rostrocaudal 900 μm-segments of the PPN and in the LDT. The number of cholinergic neurons projecting to the VTA is higher in the caudal PPN segments and the LDT. This contrasts with the low number of ChAT+/RB-R+ neurons projecting to the caudal VTA located in the rostral PPN. Bars represent SEM (n = 4 rats).