Brainstem locations of naloxone microinjection. (A) Phrenic neurogram tracings illustrate the functional identification of the parabrachial nucleus, Kölliker–Fuse nucleus, and pre-Bötzinger complex through typical responses to injection of the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; vertical arrows). (B) Dorsal view of the brainstem. Superimposed are the approximate distribution areas of the naloxone injections into the parabrachial nucleus (blue), Kölliker–Fuse nucleus (green), and pre-Bötzinger complex (red). We estimate an effective spherical diffusion radius for our injection volume of 1 to 1.2 mm.²²  There is little overlap between the parabrachial nucleus and Kölliker–Fuse nucleus injections in the brainstem, because the Kölliker–Fuse nucleus is located 2 mm ventral to the parabrachial nucleus. (C) Stereotaxic coordinates of the naloxone injection sites for parabrachial nucleus injections (blue squares) and Kölliker–Fuse nucleus injections (green squares), projected over coronal slices of the rostral pons. Coordinates for the bilateral injections were averaged for each animal. For clarity, injection sites less than 1.5 mm caudal to the inferior collicle are summarized in the slice labeled “-1.0mm”; coordinates between 1.5 and 2.5 mm caudal to the inferior collicle are summarized in the slice labeled “-2.0mm”; and injection sites 2.5 mm or more caudal to the inferior collicle are summarized in the slice labeled “-3.0mm.” To account for residual cerebellar tissue covering the dorsal brainstem in our preparation, we subtracted 5 mm from the measured stereotaxic depth coordinate in all animals (i.e., the depicted depth of injection is an approximation). (D) Stereotaxic coordinates of the naloxone injections into the pre-Bötzinger complex after injection into the parabrachial nucleus and Kölliker–Fuse nucleus (yellow squares, cohort A) or solely into the pre-Bötzinger complex (red squares, cohort B), projected over coronal slices of the caudal medulla oblongata. Coordinates for the bilateral injections were averaged for each animal. Injection sites less than 1.5 mm rostral to obex are summarized in the slice labeled “+1.0mm.” Injection sites 1.5 mm or more rostral to obex are summarized in the slice labeled “+2.0mm.” Please see “Identification of the Parabrachial Nucleus, Kölliker–Fuse Nucleus, and Pre-Bötzinger Complex” for the average stereotaxic coordinates. The outlines of the maps are redrawn from histologic sections obtained for our previous studies in adult rabbits.^8,22  The atlas of Meessen and Olszewski⁴⁶  was used for comparison. Please note the different scale for (D).