Fig. 5: Descending projections of the PB^FoxP2 neurons.

Bilateral injections of Cre-dependent AAV-ChR2 (magenta) into the PBcl in (a) FoxP2-Cre mouse (a₁) are shown with immuno-labeled for FoxP2 (green); nearly all of the ChR2-labeled cell bodies are doubly labeled (white) in the PBcl area along the dorsal margin of the PBel (which is unlabeled) (a, magnified view of injection site on right). The bottom right inset a is a magnified view of the area marked by the dashed rectangle. Descending fibers and terminals in the medulla are shown in magenta in (b, c) on the right side of the brain. The magnified views (3×) of the areas in the dashed boxes in both (b, c) are shown as b₁–c₂ to demonstrate that ChR2-mCherry labels only the fibers and does not label any cell bodies in the area. Note that inferior olive neurons (green in b, d) also express FoxP2. Scale in a = 100 µm (inset = 50 µm), in c = 500 µm and c₂ = 150 µm. Data shown in micrographs a–c were consistently observed in n = 6 mice. The photomicrograph in d shows the injection site of the retrograde tracer CTb (0.2%, magenta) in the pre-Bötzinger area (PBZ) (n = 3; scale = 500 µm). This injection retrogradely labeled many FoxP2 neurons in the KF (e₁–e₄) and the PBcl and lateral crescent areas that surround the PBel subnucleus (f₁–g₃). The arrowheads in e₄, f₃, and g₃ mark the cells doubly labeled for FoxP2 and CTb (white), while the arrows mark cells labeled only with CTb (magenta). The insets in f₃ and g₃ are 2× magnified views of the areas encompassed by dashed rectangles, highlighting the double-labeled cells. Scale in e₁ = 100 µm, in e₃ = 60 µm, and in e₄ = 30 µm. Scale in g₁ and g₃ = 100 µm. CVLM caudal ventrolateral medulla, KF Kölliker-Fuse PB subnucleus, NTS nucleus tractus solitarii, PBZ pre-Bötzinger area, PBcl central lateral PB subnucleus, PBel external lateral PB subnucleus, RVLM rostral ventrolateral medulla, scp superior cerebellar peduncle, vsct ventral spinocerebellar tract, XIIn hypoglossal nucleus.