Embryonic Rhombomere Hindbrain Patterning Delineates Distinct Functional Neuron Populations in the Adult Central Noradrenergic System
Authors: Sun Jenny Jia , Key Megan , Martinez Vena K , Morales Fatima Saldana , Zhu Ping , Ray Russell S 
Published: November 2023

Central noradrenergic (NA) neurons are a critical participant in the brainstem networks that maintain respiratory homeostasis. However, limitations in previous approaches have provided conflicting results and it is still not entirely clear how and which NA neurons are integrated into central respiratory circuits and how they may play a role in congenital respiratory pathophysiologies including Congenital Central Hypoventilation Syndrome and Sudden Infant Death Syndrome (SIDS). Studies suggest that proper partitioning of the developing hindbrain into transient, genetically‐defined segments called rhombomeres (r) is required for normal respiratory function. Thus, we hypothesize that the genetic programs within individual rhombomeres play a role in functionally patterning the adult NA system into distinct circuit modules with unique respiratory functions. To test our hypothesis, we used acute pharmaco‐genetic DREADD receptors to either perturb (hM4D) or stimulate (hM3D) genetically‐defined rhombomere‐specific NA subpopulations (derived from r1, r2, r3&5, r4, and r7&8) in the adult brainstem. Whole‐body barometric plethysmography with conscious and unrestrained mice was used to determine the effect of hM4D inhibition and hM3D stimulation in the whole NA population and rhombomeric NA subtypes under room air, hypercapnic (5% CO2), and hypoxic (10% O2) challenges that parallel conditions thought to frequently occur in SIDS.

Our data demonstrate that perturbation of the whole NA system results in no significant change under room air conditions (n=24) and a reduced hypercapnic ventilatory response (n=12, −12.99 in VE/VO2, p=0.0078) primarily mediated by a reduction in VE (−0.6111 mL/min/g, p=0.0446). Stimulation of the whole NA system results in significant changes in room air conditions (n=29) where we see increased Vf (+57.15 breaths/min, p=0.00000089), VT (+0.0035 mL/breath/g, p=0.0000029), VE (+1.0922 mL/min/g, 0.00000000029), VO2 (+0.3822 mL/min/g, p=0.0000000042), and VE/VO2 (+5.1, p=0.0094); and under hypercapnic conditions (n=12) where we see increased VO2 (+0.0178 mL/min/g, p=0.0015) and a reduced VE/VO2 (−17.913, p=0.0179). Finally, we have identified a critical subpopulation of NA neurons derived from rhombomeres 3&5 involved in the hypercapnic ventilatory reflex. Under 5% CO2, perturbation of r3&5 derived NA neurons (n=12) results in a decrease in VE/VO2 (−16.3899, p=0.03) primarily mediated by a reduction in VE (−0.7993 mL/breath/g, p=0.02), while preliminary data suggests that stimulation of r3&5 derived NA neurons (n=8) results in an increase in VE/VO2 (+20.5821, p=0.058) mediated by increases in VT (+0.0063 mL/breath/g, p=0.006) and VE (+2.4095 mL/min/g, p=0.036). Preliminary data suggests that no significant changes are seen after perturbation of r1 or r2 NA subpopulations under room air or hypercapnic ventilatory conditions. We will also present hypoxic mapping data suggesting that r1 NA neurons are required for the full hypoxic ventilatory response. Cumulatively, our data supports the contribution of early embryonic patterning in defining the functional organization of the adult respiratory network and provides candidate populations for further study that may be perturbed in congenital respiratory pathophysiologies and sets the stage for molecular profiling experiments and small molecule screens toward potential therapeutics that target key NA neurons without disrupting other critical NA functions.

Support or Funding Information

NHLBI R01 HL130249, BCM McNair Scholar Program, March of Dimes Basil O'Connor Research Award, Parker B. Francis Fellowship, Dunn Collaborative Research Award, CJ Foundation for SIDS, American Heart Association