Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for adult breathing
Authors: Chang Yuan , Ray Russell 
Published: May 2023

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders such as Sudden Infant Death Syndrome (SIDS) and Rett Syndrome. Previous studies suggest that glutamate is co-transmitted in subsets of NA neurons and may play a role in breathing control. However, the extent of glutamate co-transmission in central NA system remains uncharacterized. Furthermore, no direct evidence exists that NA-derived glutamate is required for respiratory homeostasis. Thus, our aim is to fully characterize the expression profiles of all three Vesicular Glutamate Transporters (Vglut1, Vglut2, and Vglut3) in NA neurons and to determine if Vglut2-based glutamatergic signaling in NA neurons is required for adult respiratory homeostasis. We utilized intersectional genetics to characterize the cumulative fate maps of Vglut1/2/3 co-expressing NA neurons. For each Vglut1/2/3 transporter , a corresponding Cre knock-in recombinase line was combined with DBH-p2a-Flpo mice . These Vglut1, 2, or 3-Cre; DBH-p2a-Flpo compound lines were crossed with the RC::F_TdTomato_P_GFP intersectional reporter mice. In each of the three intersectional reporter crosses, NA neurons co-expressing either Vglut1, Vglut2, or Vglut3 are labeled by green fluorescent protein (GFP) while NA neurons without any Vglut1, 2, or 3 expression are labelled by red fluorescent protein (tdTomato). We found 84.6 ± 3.75% of NA neurons express Vglut2 and 26.9 ± 3.16% posterior medullary NA neurons express Vglut3 over the lifetime of the mouse but no Vglut1 expression was observed (n=3).Functionally, to determine the requirement of NA-based Vglut2 signaling in adult breathing, we conditionally ablated Vglut2 in central NA neurons by crossing DBH-Cre/+ with Vglut2flox/flox mice. Mutants and sibling controls (6-8 weeks, n=16 per group) were assessed for respiratory function by whole-body barometric plethysmography. Ventilation and VO2 were assessed under room air (21%O2,79%N2), hypercapnia (21%O2, 74/72/69%N2, 5/7/10%CO2) and hypoxia (10%O2, 90%N2). Surprisingly, Vglut2 removal from NA neurons failed to significantly alter breathing under room air, hypercapnia and hypoxia. (A linear mixed-effects regression model was used to compare each respiratory parameter between mutants and controls.)In conclusion, anatomically, our study confirmed Vglut2 co-expression in NA neurons and identified a novel posterior NA population co-expressing Vglut3. Functionally, our data demonstrates that Vglut2-based glutamatergic signaling within the central NA system is not required for normal baseline breathing and hypercapnic, and hypoxic chemosensory reflexes, in contrast to prior studies. Our work suggests that glutamate may be not a critical target to understand NA neuron dysfunction in respiratory diseases. R01HL130249 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.