Individual cortical afferents (black reaction product) branch to contact both nitrergic interneurons (blue reaction product) and SC output neurons (brown reaction product). (A) Low-powered photomicrograph through SGI showing BDA-labeled fibers (asterisk) from AES cortex and their relationship with NADPH-labeled interneurons (blue-black) and SMI-32 immunostained output neurons (brown). The region highlighted by the dotted lines is shown at higher magnification in B. (B) An individual fiber apposes the soma and dendritic processes of NADPH interneurons (black arrowheads) and the axon of an SMI-32 output neuron (red arrowheads). (C) Note that a cortical fiber (asterisk) selectively may appose an SMI-32 immunostained neuron (yellow arrowhead) that also receives nitrergic inputs (white arrowheads). Individual cortical fibers commonly had multiple appositions on both nitrergic neurons (black arrows in D-F) and output neurons (yellow arrows in D-F). Additionally, an SMI-32 immunostained neuron could receive appositions (yellow arrows in E and F) from the same nitrergic neurons (white arrows in E and F) that have AES appositions (black arrows in E and F). Appositions are indicated by arrows. Yellow arrows=AES-SMI-32 appositions; black arrows=AES-nitrergic appositions; white arrows= nitregic-SMI-32 appositions. Red arrows indicate a cortical fiber apposing SMI-32 axons. The drawing on the top left corner in (A) indicates the approximate location of the tissue magnified in A. Abbreviations: stratum griseum superficiale (SGS); stratum opticum (SO), stratum griseum intermediale (SGI), stratum griseum profundum (SGP); periaqueductal gray (PAG). Scale bar = 50μm in A, B and E; 25μm in C, D and F.