Functional Characterization of Developmental Whole Rhombomere Brainstem Populations in Adult Respiratory Physiology

Abstract

The perpetual rhythm of breathing is essential for survival and emerges through the interactions of a highly redundant and anatomically complex array of brainstem neuronal networks. Previous studies suggest that proper partitioning of the hindbrain into transient, genetically‐defined segments called rhombomeres (r) is required for normal respiratory development. However, because most studies of rhombomeric mispatterning result in embryonic or neonatal lethality, it remains unclear if rhombomeres ultimately play a role in adult circuit modules that regulate unique aspects of respiration. Further, gross loss or perturbation of a whole rhombomeric domain during development likely results in cell non‐autonomous effects such as the mis‐patterning of neighboring rhombomeric segments or neuronal populations. To bypass embryonic lethality and avoid non‐autonomous developmental perturbations, we used conditional mouse lines crossed to rhombomeric specific Cre drivers to express pharmaco‐genetic DREADD receptors in adult cell populations derived from one or more rhombomeric domains.We expressed two different DREADD receptors for acute and non‐invasive perturbation (RC::FP_hM4D) and stimulation (RC::FP_hM3D) of targeted adult brainstem neurons in the conscious and unrestrained mouse. In combination with whole‐body plethysmography and ECG, we are able to accurately measure respiratory parameters under room air, hypercapnic (5% CO2), and hypoxic (10% O2) conditions in conscious and unrestrained mice while observing heart rate in a subset of experiments. Our data demonstrate that distinct adult rhombomere derived populations (r1, r2, r3&5, r4, and r7&8) differentially affect respiratory rate, tidal volume, minute ventilation, oxygen consumption, and waveform patterns when they are perturbed or stimulated under various respiratory conditions. For example, perturbation of r2 neurons resulted in no significant changes under room air (n=24) or 5% CO2 (n=12) conditions, but under 10% O2 after CNO administration, experimental animals (n=12) showed a reduced Vf (−36.54 breaths/min, p=0.000035), VT (−0.0012 mL/breath/g, p=0.0134), VE (−0.6435 mL/min/g, p=0.00043), and VE/VO2 (−10.6826, p=0.0212) as compared to sibling controls (n=12). On the other hand, perturbation of r3&5 neurons resulted in significant changes under all three ventilatory conditions: under room air (n=24) we saw reduced VT (−0.0011 mL/breath/g, p=0.0016), VE (−0.2639 mL/min/g, p=0.0043), and VE/VO2 (−6.3370, p=0.003); under hypercapnic conditions (n=12) we saw lower Vf (−58.046 breaths/min, p=0.00000073), VT (−0.1110 mL/breath/g, p=0.00000059), VE (−1.9847 mL/min/g, p=0.000000013), VO2 (−0.0030 mL/min/g, p=0.0355), and VE/VO2 (−40.8103, p=0.00000017); and under hypoxic conditions (n=12) we saw reduced Vf (−38.5233 breaths/min, p=0.0018), VT (−0.0010 mL/breath/g, p=0.048), VE (−0.5992 mL/min/g, p=0.0000056), and VE/VO2 (−12.5255, p=0.0054).Finally, DREADD mediated excitation of targeted rhombomeric domains all resulted in rapid death, with unique differences in the patterns of cardio‐respiratory failure. These data show the contribution of early embryonic patterning in defining the functional organization of the adult respiratory network, and set the stage to use intersectional genetics to further identify neuron subtypes within each rhombomere that are involved in specific aspects of respiratory homeostasis.Support or Funding InformationNHLBI 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