(A) Right half of the rhythmic slice preparation after PFA fixation and optical clearing using the Scale method. White arrow points to the soma of the inspiratory-modulated Dbx1 neuron, which is further characterized in panels B-E. (B) From left to right, IR-DIC image of the recorded neuron and epifluorescence images showing tdTomato, Alexa 488 dialyzed from the intracellular solution, and a composite of tdTomato and Alexa 488 labeling. (C) Whole cell recording of imaged neuron showing rhythmic inspiratory firing (top trace) and integrated inspiratory XII nerve output (bottom trace). (D) Membrane potential (upper black traces), whole cell stimulation (0.8 mA, 1 ms duration, middle blue traces), and XII stimulation time (0.3 mA, 0.2 ms duration, bottom black trace, stimulation time is at vertical line); Left panel: antidromic action potentials activated from XII nucleus stimulation, 5 traces overlaid; Middle: evoked orthodromic action potentials followed 46 ms later by XII nucleus stimulation, 4 traces overlaid; Right: evoked orthodromic action potential followed 16 ms later by XII stimulation resulted in the extinction of the antidromic action potential (i.e., a successful collision test, 7 traces overlaid). (E) Composite diagram showing biocytin-filled, FITC-labeled, reconstructed neuron; arrowheads point to the axon. Orientation is the same as the image in A (images adjusted for brightness and contrast). (F) Morphologic reconstruction of all neurons that only projected ipsilaterally, illustrating axon trajectory and dendritic tree organization (n = 2). (G) Morphologic reconstruction of all commissurally-projecting neurons illustrating axon trajectory and dendritic tree organization (n = 5). Reconstructed neurons on the left-hand side were also antidromically activated. Arrowhead indicates axon bifurcation. (H) Image showing location for 24 of 34 recorded inspiratory Dbx1 IRt neurons. Open squares: antidromic activation not tested; open circles: negative antidromic activation; filled circles: positive antidromic activation; open triangles: commissural projection, negative antidromic activation; filled triangles: commissural projection, positive antidromic activation; open diamonds: ipsilateral projection, negative antidromic activation; filled diamond: ipsilateral projection, positive antidromic activation. XII – hypoglossal nucleus, IO – inferior olive, SP5 – trigeminal nucleus, scNA – semicompact division of nucleus ambiguus.