Neuroscience

Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for normal breathing and chemosensory reflexes

Yuan Chang
Savannah Lusk
Russell Ray author has email address

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston TX
Department of Neuroscience, Baylor College of Medicine, Houston TX
McNair Medical Institute, Houston TX

https://doi.org/10.7554/eLife.88673.1

Open access
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Figures and data

Cumulative fate maps of glutamate co-expressing NA neurons characterized by intersectional genetics.
(A) Breeding schematic. Vglut1-Cre/+, Vglut2-Cre/+ and Vglut3-Cre/+ were each bred with DBH-p2a-Flpo/+ mice respectively. These Vglut1, Vglut2, or Vglut3-Cre/+; DBH-p2a-Flpo/+ compound lines were then crossed with the RC::FLTG 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 (eGFP) while NA neurons without any Vglut1,2, or 3 expression are labelled by red fluorescent protein (tdTomato). Shown as an example is the Vglut2-Cre/+; DBH-p2a-Flpo/+; RC::FLTG/+ intersectional reporter line. (B) Quantification of the percentage of Vglut2-coexpressing NA neurons among NA neurons in each brainstem NA nuclei including A7, LC, A5, sub CD/CV, anterior C1/A1 and C2/A2, posterior C1/A1 and C2/A2. (C) Quantification of the percentage of Vglut3-coexpressing NA neurons among NA neurons in each NA nuclei. (D) Fluorescent expression of tdTomato (red) and eGFP (green) in coronal sections of Vglut1, Vglut2, or Vglut3 intersectional reporter lines in brainstem NA nuclei. Nucleus is labeled by DAPI (blue). Scale bar 50μm.

Cumulative fate maps of glutamate co-expressing NA neurons characterized by intersectional genetics.
(A) Breeding schematic. Vglut1-Cre/+, Vglut2-Cre/+ and Vglut3-Cre/+ were each bred with DBH-p2a-Flpo/+ mice respectively. These Vglut1, Vglut2, or Vglut3-Cre/+; DBH-p2a-Flpo/+ compound lines were then crossed with the RC::FLTG 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 (eGFP) while NA neurons without any Vglut1,2, or 3 expression are labelled by red fluorescent protein (tdTomato). Shown as an example is the Vglut2-Cre/+; DBH-p2a-Flpo/+; RC::FLTG/+ intersectional reporter line. (B) Quantification of the percentage of Vglut2-coexpressing NA neurons among NA neurons in each brainstem NA nuclei including A7, LC, A5, sub CD/CV, anterior C1/A1 and C2/A2, posterior C1/A1 and C2/A2. (C) Quantification of the percentage of Vglut3-coexpressing NA neurons among NA neurons in each NA nuclei. (D) Fluorescent expression of tdTomato (red) and eGFP (green) in coronal sections of Vglut1, Vglut2, or Vglut3 intersectional reporter lines in brainstem NA nuclei. Nucleus is labeled by DAPI (blue). Scale bar 50μm.

Characterization of real time co-expression of Vglut1, Vglut2 and Vglut3 in brainstem NA neurons in adult mice by fluorescent in situ hybridization.
(A) Representative images of Vglut1, Vglut2, or Vglut3 and DBH double ISH in coronal sections of WT mice in all of brainstem NA nuclei in adult mice. DBH (green), Vglut1/2/3 (red), DAPI (cyan). Scale bar 50μm. (B) Quantification of the percentage of Vglut2 co-expression in NA neurons in each NA nuclei. (C) Quantification of the percentage of Vglut3 co-expression in NA neurons in each NA nuclei.

DBH with Vglut2 co-staining by fluorescent in situ hybridization confirms that Vglut2 expression is disrupted in the whole central NA system in DBH-Cre; Vglut2 cKO mice.
(A) Representative images of Vglut2 and DBH double ISH in anterior C1/A1, posterior C1/A1, anterior C2/A2 and posterior C2/A2 in control and DBH-Cre; Vglut2 cKO mice. DBH (green), Vglut2 (red), DAPI (cyan). Scale bar 20μm.(B) Quantification of intensity of Vglut2 signal in control and DBH-Cre; Vglut2 cKO mice.

Vglut2 conditional knockout in central noradrenergic neurons fails to alter baseline breathing and the hypercapnic ventilatory reflex (5% CO₂).
(A) Mouse model schematic and experimental protocol including habituation and hypercapnia protocol. (B) Under both room air and hypercapnia (5% CO₂), DBH-Cre; Vglut2 cKO mice didn’t show significant changes in respiratory output (V_E/V_O2), minute ventilation (V_E), respiratory rate (V_f), tidal volume (V_T), and metabolism demand (V_O2). Linear mixed-effects regression model, *p<0.05.

Respiratory and metabolic values under room air conditions and the statistical tests.

Respiratory and metabolic values under 5% CO₂ conditions and the statistical tests.

Vglut2 conditional knockout in central noradrenergic neurons fails to alter the majority of hypercapnic ventilation parameters and metabolism under 7% CO₂.
(A) Mouse model schematic and experimental protocol including habituation and hypercapnia protocol. (B) Knocking out Vglut2 in the whole NA system failed to alter ventilation (V_E/V_O2, V_f, V_E) and metabolism (V_O2) but only showed a significantly reduce in V_T under 7% CO₂ condition. Linear mixed-effects regression model, *p<0.05.

Respiratory and metabolic values under 7% CO₂ conditions and the statistical tests.

Vglut2 conditional knockout in central noradrenergic neurons fails to alter the hypercapnic ventilatory reflex (10% CO₂).
(A) Mouse model schematic and experimental protocol including habituation and hypercapnia protocol. (B) Knocking out Vglut2 in the whole NA system didn’t significantly alter ventilation (V_E/V_O2, V_f, V_T, V_E) and metabolism (V_O2) under 10% CO₂ condition. Linear mixed-effects regression model, *p<0.05.

Respiratory and metabolic values under 10% CO₂ conditions and the statistical tests.

Vglut2 conditional knockout in central noradrenergic neurons fails to alter the hypoxic ventilatory reflex (10% O₂).
(A) Mouse model schematic and experimental protocol including habituation and hypoxia protocol. (B) Knocking out Vglut2 in the whole NA system didn’t show significant breathing changes including V_E/V_O2, V_E, V_f, V_T, and V_O2 in either of the three five-minute time periods under hypoxia (10% O₂). Linear mixed-effects regression model, *p<0.05.

Respiratory and metabolic values under 10% O₂ conditions and the statistical tests.

Virus injection of Cre-dependent tdTomato into LC of Vglut2-Cre mice.

Vglut2 positive innervations from central noradrenergic neurons to the brain nuclei important in breathing control.

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