Characterisation of putative retrotrapezoid nucleus (RTN) chemoreceptor neurons in the adult human brainstem

Abstract The retrotrapezoid nucleus (RTN) of rodents is located ventral to the facial motor nucleus (7N) and consists of acid-sensitive neurons that activate breathing and mediate the central component of the ventilatory response to hypercapnia. In rodents, RTN neurons can be histologically identified by the presence of paired-like homeobox 2B positive nuclei (Phox2b+) and the absence of cytoplasmic choline acetyltransferase (ChAT−) and tyrosine hydroxylase (TH−). Up to 50% of rodent RTN neurons synthesise galanin, and 88% express pituitary adenylate cyclase activating polypeptide (PACAP). The human RTN (hRTN) has not been mapped to date. This study aimed to map the location and cytoarchitecture of the adult hRTN and compare the findings to the homologies of rodents, macaques and human infants. Formalin-fixed, paraffin-embedded tissue blocks from two adult cases, spanning the medulla-pons, were serially sectioned (10µm thick) and every four in thirty sections was assayed for immunohistochemistry for ChAT, or double-labelled Phox2b/TH, Phox2b/galanin and Phox2b/PACAP, followed by analysis using QuPath software. hRTN neurons, identified as Phox2b+/TH−/ChAT−, were located ventral to 7N and lateral to the superior olive, overlapped with the C1 or A5 catecholaminergic population and extended rostrocaudally from Obex +13 to +17 mm. In the parafacial area, 90% of Phox2b immunoreactive (−ir) neurons are hRTN neurons, totaling around 5000 bilaterally, and were surrounded by numerous TH-ir fibers. Galanin- and PACAP-ir was identified in 43% and 39% of Phox2b-ir parafacial neurons, respectively. This is the first study to characterise and quantitatively map the adult human RTN using a series of neurochemical markers. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵* Co-senior authors Manuscript text revised for clarity and reduced by 2000 words Abbreviations - 4V - 4th ventricle - 6N - abducens nucleus - 6n - abducens nerve - 7N - facial motor nucleus - 7n - facial nerve - 8n - vestibulocochlear nerve - A5 - A5 catecholaminergic neurons - Biotin-SP-conjugated - biotin-succinimidyl ester conjugated - C1 - C1 adrenergic neurons - CCHS - congenital hypoventilation syndrome - ChAT - choline acetyltransferase - CO₂ - carbon dioxide - D - dorsal - DAB - 3,3’-diaminobenzidine - DPX - dibutylphthalate polystyrene xylene - FFPE - formalin-fixed, paraffin-embedded - H₂O₂ - hydrogen peroxide - H&E - hematoxylin & eosin - hRTN - human retrotrapezoid nucleus - HIER - heat-induced epitope retrieval - icp - inferior cerebellar peduncle - IC - inferior colliculus - IF - immunofluorescence - IgG - immunoglobulin G - IHC - immunohistochemistry - IO - inferior olive - IOP - inferior olive, principal nucleus - -ir - immunoreactivity - IRt - intermediate reticular nucleus - IS - inferior salivatory nucleus - ISH - in situ hybridisation - mcp - medial cerebellar peduncle - ml - medial lemniscus - MSO - medial superior olive - NDS - normal donkey serum - NK1R - neurokinin-1 receptor - NMB - neuromedin B - NTS - nucleus of the solitary tract - PACAP - pituitary adenylate cyclase activating polypeptide - PBS - phosphate-buffered saline - PCRt - parvocellular reticular nucleus - PCRtA - parvocellular reticular nucleus, alpha - Phox2b - paired-like homeobox 2B - PPGAL - preprogalanin - Pn - pontine nuclei - py - pyramidal tract - R - rostral - Rn - Red nucleus - RNA - ribonucleic acid - ROI - regions of interest - RRID - Research Resource Identifier - RTN - retrotrapezoid nucleus - SC - superior colliculus - SLC17A6 - solute carrier family 17 member A6 (vesicular glutamate transporter 2 gene) - SO - superior olive - Sp5O - spinal trigeminal nucleus, oral part - sp5 - spinal trigeminal tract, nerve - TBS - Tris-buffered saline - TH - tyrosine hydroxylase - Thal - thalamus - V - ventral - vGlut2 - vesicular glutamate transporter 2 - VRC - ventral respiratory column - WB - Western blotting.