Tridimensional mapping of Phox2b expressing neurons in the brainstem of adult Macaca fascicularis and identification of the retrotrapezoid nucleus

Chemosensitivity is a key mechanism for the regulation of breathing in vertebrates. The retrotrapezoid nucleus is a crucial hub for respiratory chemoreception within the brainstem. It integrates chemosensory information that are both peripheral from the carotid bodies (via the nucleus of the solitary tract) and central through the direct sensing of extracellular protons. To date, the location of a genetically defined RTN has only been ascertained in rodents. We first demonstrated that Phox2b, a key determinant for the development of the visceral nervous system and branchiomotor nuclei in the brainstem including the RTN, had a similar distribution in the brainstem of adult macaques compared to adult rats. Second, based on previous description of a specific molecular signature for the RTN in rats, and on an innovative technique for duplex in situ hybridization, we identified parafacial neurons which coexpressed Phox2b and ppGal mRNAs. They were located ventrally to the nucleus of the facial nerve and extended from the caudal part of the nucleus of the superior olive to the rostral tip of the inferior olive. Using the previously described blockface technique, deformations were corrected to allow the proper alignment and stacking of digitized sections, hence providing for the first time a 3D reconstruction of the macaque brainstem, Phox2b distribution and the primate retrotrapezoid nucleus. This description should help bridging the gap between rodents and humans for the description of key respiratory structures in the brainstem. From a whole macaque (Macaca fascicularis) brain, serial transversal sections of the brainstem were made using a blockface setup that takes high‐definition pictures of the organ surface, before microtome slicing. Different histological techniques were applied to series of sections, which allowed us to identify RTN neurons among Phox2b immunoreactive cells within the macaque brainstem. Our findings rely on the specific signature previously assessed in rats for chemosensitive neurons of the parafacial region: Phox2b/VGluT2/Galanin. Blockface photographs allowed us to correct histology‐induced deformations on digitized sections. Series of sections were virtually stacked and ppGal/Phox2b positive cells identified to provide with the first 3D description of the main chemosensitive brainstem region in nonhuman primates.