We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: co-transmitted acetylcholine (ACh)/GABA, GABA only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA co-transmission. In response to repeated stimulation the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA co-transmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by co-transmitted ACh/GABA was more effective in eliciting an action potential compared to current, glutamate, or ACh/GABA alone. Th...