Colocalization of c-Fos-IR (rhodamine) and ChAT-IR (FITC) to demonstrate that c-Fos-positive neurons in the IML are cholinergic sympathetic preganglionic neurons (SPNs). Insets in (A, B) are magnified as indicated. (A) Low magnification photomicrograph of the dorsal quadrant of a transverse section of T3 spinal cord segment. A triangular cluster of IML cells showed intense ChAT-IR that embraced nuclei labeled with c-Fos-IR (Ai). Distribution of ChAT-positive cells extended dorsomedially toward the intermediate zone of lamina X (Aii), where localizations of ChAT-IR and c-Fos-IR were separate. In (Aii), open arrowheads on Top indicate c-Fos-positive ChAT-negative cells in the dorsal lamina X; filled arrowheads at Bottom indicate nuclei with mild c-Fos-IR surrounded by diffusive ChAT-IR in the ventral lamina X. Arrows indicate ChAT-positive c-Fos-negative cells in the intermediate zone. (B) Low magnification photomicrograph of the ventral quadrant of a transverse section of T5 spinal cord segment. ChAT-IR was present in the IML and the ventral horn; only in the IML, ChAT-IR was colocalized with c-Fos-IR (Bi). (C) High magnification photomicrograph of a horizontal section of T7-T8 spinal cord segments, showing c-Fos-positive nuclei in close proximity to cytoplasmic-like ChAT-IR in the IML. Scale bars = 100 μm in (A, B), 25 μm in (Ai-ii), and 20 μm in (Bi, C).