(A) Left, coronal sections showing GCaMP6m labelled neurons throughout the auditory thalamus of a CR-IRES-cre mouse injected with AAV1.Syn.GCaMP6m.WPRE.SV40 and AAV1.CAG.Flex.tdTomato.WPRE.bGH. Right, coronal sections showing GCaMP6m labelled thalamic axons in auditory cortex, amygdala and striatum. (B) Left, same coronal sections as in left panels of A, showing tdtomato labelled neurons exclusively in non-lemniscal and paralaminar nuclei. Right, same coronal sections as in right panel of A, showing tdtomato labelled thalamic axons almost exclusively in secondary auditory cortex, amygdala and striatum. (C) Bottom, overlay of GCaMP6m (all thalamic input) and tdtomato (CR+ input) labelled axons in transition area from secondary to primary auditory cortex indicated by white rectangle in A and B. Top, cortical depth profile of labelling from non-lemniscal thalamic axons (red, CR+) versus all thalamic axons (green) within primary auditory cortex (average across area within white bracket in bottom panel). The red and green lines were normalized to have same peak height in layer I. MGBd, dorsal division of medial geniculate body; MGBm, medial division of medial geniculate body; MGBv, ventral division of medial geniculate body; SG, suprageniculate nucleus; PIN, posterior intralaminar nucleus; PP, peripeduncular nucleus; LGNd, dorsal division of lateral geniculate nucleus; LGNv, ventral division of lateral geniculate nucleus; IGL, intergeniculate leaf; br, bregma; CR+, calretinin-positive; RF, rhinal fissure. Scale bars, 200 µm. Locations of thalamic subdivisions adopted from Lu et al., 2009. CR+ boutons typically responded very poorly to acoustic stimulation. See Figure 4—figure supplement 1 for In vivo two-photon image of CR+ thalamic axons of CR-IRES-cre mouse injected with AAV1.Syn.Flex.GCaMP6m.WPRE.SV40 and example FRAs.