The firing activity of striatal cholinergic interneurons (CINs) is a neural correlate of conditioned learning. However, its circuitry is not entirely described. Here we report that midbrain GABA and Glutamate neurons discriminate auditory cues and encode the association of a predictive stimulus with a footshock. Through their mono- and di-synaptic inputs via the thalamic-parafasicular sub-nucleus onto CINs, they contribute to processing fear learning.