Nucleus Tractus Solitarius Neurons Activated by Hypercapnia and Hypoxia Lack Mu Opioid Receptor Expression

Figures

• 

  Figure 1. Morphine suppresses breathing during control and hypercapnic conditions in wild-type mice. Respiration was measured during control conditions (standard room air) or hypercapnia challenge (7% CO2) using whole-body plethysmography following an injection of saline (black symbols) or morphine (30 mg/kg; gray symbols). (A) Representative plethysmography traces during each of the four conditions. Scale bar applies to all traces. Inspiration is downward. (B–D) Saline control n = 11, morphine control n = 11, saline hypercapnia n = 10, morphine hypercapnia n = 10. Data are graphed as mean ± SEM. *p < 0.05, ***p < 0.0001, ns p > 0.05 by two-way ANOVA and Holm-Sidak multiple comparison's test.

• 

  Figure 2. Hypercapnia and morphine activate non-overlapping populations of NTS neurons. Wild-type mice were exposed to standard room air (Control) or hypercapnia (7% CO2) following an injection of saline or morphine (30 mg/kg). (A–D) Example images of cFos immunolabeling (green) in NTS sections from mice exposed to each of the four treatment groups. Scale bar = 100 μm. AP, area postrema; cc, central canal; 10N, dorsal motor nucleus of vagus. (E,F) Summary data of the average number of cFos+ NTS cells per section (n = 6–9 mice/group, 2–6 sections/mouse). Hypercapnia increased the number of cFos+ cells in saline (E) and morphine (F) treated mice. Each data point represents the average # of cFos+ cells per section for an individual mouse. Bar and error are group mean ± SEM. *p < 0.05 by unpaired t-test.

• 

  Figure 3. Hypercapnia activates MOR-negative neurons. Identification of cFos immunolabeling and tdTomato expression, as an indicator of MOR expression, in the NTS of Oprm1Cre/tdT mice that were exposed to standard air (Control) or hypercapnic air (7% CO2). (A–C) Example images of cFos immunostaining (green) and MOR+ tdTomato expression (red) from a mouse exposed to hypercapnia. Very few cells co-expressed cFos and tdTomato (indicated by yellow vertical arrows). White diagonal arrows are pointing at example cells that are cFos+, but do not express MORs. (A1–C1) are zoomed in views of the boxed region in (A–C). Scale bar = 100 μm. (D,E) Summary data of the average number of cFos+ NTS cells per section (D), or the average number of co-labeled cFos+/MOR+ NTS cells per section (E) (n = 7–8 mice/group, 2–6 sections/mouse). Each data point represents the average # of cFos+ cells per section for an individual mouse. Bar and error are group mean ± SEM. *p < 0.05 by unpaired t-test.

• 

  Figure 4. Hypoxia activates MOR-negative neurons. Identification of cFos immunolabeling and tdTomato expression, as an indicator of MOR expression, in the NTS of Oprm1Cre/tdT mice that were exposed to standard air (Control) or hypoxic air (10% O2). (A–C) Example images of cFos immunostaining (green) and MOR+ tdTomato expression (red) from a mouse exposed to hypoxia. Very few cells co-expressed cFos and tdTomato (indicated by yellow vertical arrows). White diagonal arrows are pointing at example cells that are cFos+, but do not express MORs. (A1–C1) are zoomed in views of the boxed region in (A–C). Scale bar = 100 μm. (D,E) Summary data of the average number of cFos+ NTS cells per section (D), or the average number of co-labeled cFos+/MOR+ NTS cells per section (E) (n = 6 mice/group, 4–6 sections/mouse). Each data point represents the average # of cFos+ cells per section for an individual mouse. Bar and error are group mean ± SEM. *p < 0.05; ns, p > 0.05 by unpaired t-test.

• 

  Figure 5. Morphine induces cFos expression in MOR-positive and MOR-negative NTS neurons. Identification of cFos immunolabeling and tdTomato expression, as an indicator of MOR expression, in the NTS of Oprm1Cre/tdT mice that were injected with morphine (30 mg/kg). (A–C) Example images of cFos immunostaining (green) and MOR+ tdTomato expression (red). Scale bar = 100 μm. Cells that co-expressed cFos and tdTomato are indicated by yellow vertical arrows.