(A) Schematic of anterograde transsynaptic tracing from the FN to identify fastigial disynaptic input to forebrain areas. AAV1.hSyn.Cre injected in the FN is transsynaptically transported to postsynaptic neurons in which Cre recombinase drives reporter (tdTomato) expression in Ai14 mice, thereby axonal projections of those postsynaptic neurons to the forebrain areas are robustly labeled. (B–D) Disynaptic projections from the FN to the cortical areas. Panels show example coronal sections of the forebrain in which labeled axons (black) of the fastigial postsynaptic neurons show a characteristic distribution. Fastigial disynaptic input to layer one was widely distributed over the M2 (B–D), frontal association cortex (FrA) (B), lateral/ventral orbital cortex (LO/VO) (B), prelimbic cortex (PrL) (B and C), S1 (C and D), insular cortex (C and D), cingulate cortex (Cg) (C and D), and M1 (D). Inset in (B) is high magnification of layer 1 of the M2 (region circumscribe by rectangle in upper image), showing dense terminal arborization at layer 1. Fastigial disynaptic input also terminates at deeper layers of the FrA (B), LO/VO (B), M2 (B–D), and M1 (D). Note no labeled axons in all layers of medial orbital cortex (MO) (B) and infralimbic cortex (IL) (C), and deeper layers of insular cortex (C), S1 (C and D), and Cg (D). (E–G) Color-coded illustration of the cortical areas that receive disynaptic input from the FN to layer 1 (pink) and layers 3–5 (purple). Darker colors indicate denser input. Dorsal (E) and lateral (F and G) views are shown. Fastigial disynaptic input to layer one broadly covers the cortical areas including the FrA, PrL, LO/VO, M1, M2, S1, barrel field (S1BF), S2, insular cortex, parietal cortex (Ptl), and visual cortices (V1 and V2), but excluding MO, IL, retrosplenial cortex (Rsp), auditory cortex (Aud), and rhinal/temporal cortex (Rhi/Tem) (gray). Input to layers 3–5 (purple) is restricted in the FrA, LO/VO, M2, M1, S1BF, and S2. In (E), approximate levels of the sections in (B–D) are also shown. Results were consistent across n = 4 male and n = 4 female mice. (H–K) Disynaptic fastigial projection targets in subcortical forebrain areas, revealed by axonal terminals transsynaptically labeled with AAV1.hSyn.Cre (tsAAV). (H) The axonal terminal arborization of fastigial postsynaptic neurons (cyan) at proximity of neurons of caudate putamen (red, Nissl). (I) The labeled axonal terminals at the accumbens nucleus. (J) The labeled axonal terminals and tertiary infected neuron at the nucleus of diagonal band, a basal forebrain structure. (K) Sparse but consistent labeling at the medial septum. (L) Schematics summarizing disynaptic fastigial input to the cortex from F2 and F4 neurons. These circuits are likely to be comprised of two separate pathways, as the F2 and F4 neurons target different thalamic neurons that give rise discrete thalamocortical projections. F4 neurons provide synaptic input to the ventromedial (VM) and mediodorsal (MD) thalamic neurons. Axons of postsynaptic VM neurons target layer 1 of the widespread areas of the cortex (Kyuhou and Kawaguchi, 1985; Noda and Oka, 1985; Kuramoto et al., 2015). Axons of postsynaptic MD neurons target prelimbic, cingulate, and orbitofrontal cortices (Kuramoto et al., 2017). F2 neurons provide synaptic input to the ventrolateral (VL) thalamic neurons which then project to the deeper layers of the sensorimotor cortex (Jones, 2009; Kuramoto et al., 2009). (M) Schematics summarizing disynaptic fastigial input to the striatum from F2 and F4 neurons. These circuits are also likely to be comprised of two separate pathways, as the F2 and F4 neurons target different thalamic neurons that give rise discrete thalamostriatal projections. F2 neurons provide synaptic input to the parafascicular (PF) thalamic neurons that project to both the dorsal (caudate putamen; CPu) and ventral striatum (accumbens nucleus; NAcc) (Sadikot and Rymar, 2009). F4 neurons provide synaptic input to the centrolateral (CL) thalamic neurons that project to the dorsal striatum. Scale bar in D applies to low magnification images in B-D. Abbreviations, Aud, auditory cortex; Cg, cingulate cortex; CL, centrolateral thalamic nucleus; CPu, caudate putamen; FrA, frontal association cortex; IL, infralimbic cortex; LO, lateral orbital cortex; M1 and M2, primary and secondary motor cortex; MD, mediodorsal thalamic nucleus; MO, medial orbital cortex;; PF, parafascicular thalamic nucleus; PrL, prelimbic cortex; Ptl, parietal association cortex; Rhi, ecto-/peri-/ento-entorhinal cortex; Rsp, retrosplenial cortex; S1 and S2, primary and secondary sensory cortex; S1BF, barrel field of primary sensory cortex; Tem, temporal association cortex; V1 and V2, primary and secondary visual cortex; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VO, ventral orbital cortex.