Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice

Figures

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  Figure 1.. Expression of genes relevant to carotid body (CB) function in TH-Olfr78 KO and Epas1 KO mice. (A) Scheme of the generation of the TH-Olfr78 KO mouse model. (B) and (C) Levels of Olfr78 mRNA, relative to wild type (WT), in CB samples (B) and in superior cervical ganglion (SCG) samples (C) of WT mice and TH-Olfr78 KO mice. Data are expressed as mean ± SEM with individual values superimposed. Values are CB (WT: 1 ± 0.10; KO: 0.00046 ± 0.00008, n = 3 replicates/group); SCG (WT: 1 ± 0.14; KO: 0.0009 ± 0.0001, n = 4 replicates/group). Statistically significant P-values (<0.05) calculated by unpaired two-tailed t-test are indicated. (D) and (E) Levels of mRNA, relative to WT, of genes relevant to glomus cell function: Olfr78, Epas1, Cox8b, Higd1C, and Rgs5 (D) and Cox4i2, Pcx, and Th (E) in CB samples of WT mice and TH-Olfr78 KO mice. Data with normal distribution are presented by bar diagrams (D) and expressed as mean ± SEM with data values superimposed. Values that do not follow a normal distribution are represented as boxplots (E) indicating median (middle line), 25th, 75th percentile (box), and largest and smallest values (whiskers). All data are plotted individually. P-values…

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  Figure 2.. Hypoxic and hypercapnic ventilatory responses in wild-type (WT) mice and TH-Olfr78 KO mice. (A), (B), and (E) Time course of the increase in respiratory frequency (breaths/min, left ordinate axis) induced by hypoxia (10% O2, A and 12% O2, B) and hypercapnia (5% CO2, E) in WT mice (10% O2, n = 10, 12% O2, n = 10, and 5% CO2, n = 10) and TH-Olfr78 KO mice (10% O2, n = 11, 12% O2, n = 10, and 5% CO2, n = 11). Changes in % O2 and % CO2 over time are presented related to the right ordinate axis. (C), (D), and (F) Respiratory frequency (in breaths/min) during normoxia (basal, 21% O2), hypoxia (10% O2, C and 12% O2, D) and hypercapnia (20% CO2, F) measured by plethysmography in WT mice and TH-Olfr78 KO mice. Respiratory frequency during hypoxia was calculated at the peak (box and “p” indicated in panels A and B) and during the whole stimuli (box and “a” in panel A and B). Data points are presented as bar diagrams indicating mean ± SEM with all data points superimposed. Values (in breaths/min) are WT mice (basal: WT, 194.2 ± 8; KO, 189.3 ± 4; average 10% O2: WT, 223.4 ± 4; KO, 223.2 ± 4; peak 10% O2: WT, 235.3 ± 7; KO, 241.1 ± 3; average 12% O2: WT, 196.2 ± 10; KO, 202.3 ± 7; peak 12%…

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  Figure 3.. Changes in carotid body (CB) structure upon chronic hypoxia. (A) Scheme of the chronic hypoxia protocol. (B) Immunohistochemical analyses of carotid bifurcations in wild-type (WT) mice (left) and TH-Olfr78 KO mice (right) exposed to chronic hypoxia (14 d, 10% O2). The typical growth of the TH + glomus cell mass under hypoxia (upper panel) is also observed in TH-Olfr78 KO mice. Proliferating cells were labeled with Ki67 (middle panel). Colocalization of TH and Ki67 is represented in the lower panel, where nuclei were labeled with DAPI. CB, carotid body; SCG, superior cervical ganglion. (C) Average number of TH + cells, Ki67 + cells, and double TH/Ki67 + cells in CB of WT mice and TH-Olfr78 KO mice. Data are expressed as mean ± SEM. Values are TH+ (WT, 3062 ± 626, n = 3; KO, 2200 ± 511, n = 3), Ki67+ (WT, 282 ± 75, n = 3; KO, 284 ± 54, n = 3), and double TH/Ki67+ (WT, 102 ± 35, n = 3; KO, 124 ± 35, n = 3). (D) Quantification of CB volume of WT mice and TH-Olfr78 KO mice. Data (in μm3) are presented as mean ± SEM. WT, 6.3 × 108 ± 1.2 × 108; KO, 5.6 × 108 ± 1.1 × 108). (E) Average hematocrit values measured in WT mice and TH-Olfr78 KO mice in normoxia (Nx) and after exposure to…

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  Figure 4.. Passive electrical parameters and voltage-dependent K+ currents of dispersed glomus cells of wild-type (WT) mice and TH-Olfr78 KO mice. (A) and (B) Average cell capacitance (in pF, A) and input resistance (in GOhm, B) measured in glomus cells of WT mice and TH-Olfr78 KO mice. Data are presented as boxplots indicating median (middle line), 25th, 75th percentile (box), and largest and smallest values range (whiskers), and with all data values superimposed. Mean ± SEM are: capacitance, WT, 3.955 ± 0.245 (n = 61); KO, 3.589 ± 0.0.251 (n = 59); input resistance, WT, 3.298 ± 0.259 (n = 48); KO, 3.726 ± 0.293 (n = 44). (C) Top, representation of the depolarizing pulse protocol (50-ms depolarizing pulses reaching membrane potentials between −30 to +50 mV in steps of 20 mV from a holding potential of −70 mV). Families of representative macroscopic K+ currents recorded from dispersed glomus cells of WT mice (middle) and TH-Olfr78 KO mice (bottom) illustrating the variability in the time course of the current in both types of mice. (D) Average maximal K+ current density (in pA/pF, ordinate) versus voltage (in mV, abscissa) relationship in glomus cells of WT mice and TH-Olfr78 KO mice. Each…

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  Figure 5.. Voltage-dependent Na+ and Ca2+ currents of dispersed glomus cells of wild-type (WT) mice and TH-Olfr78 KO mice. (A) Macroscopic Na+ and Ca2+ currents elicited by membrane depolarization in a patch clamped glomus cell in the presence (left) and absence (right) of extracellular Na+. Complete replacement of Na+ was achieved with the impermeant cation N-methyl glucamine. Na+ current (INa) and Ca2+ current (ICa) were measured at the indicated time. (B) and (C) Representative examples of macroscopic inward currents with variable components of Na+ and Ca2+ current recorded from glomus cells of WT mice (B) and TH-Olfr78 KO mice (C). Standard (full Na+) external solution and pulse protocol are as illustrated in panel A (left). (D) and (F) Average Na+ (D) and Ca2+ (F) current density (in pA/pF, ordinate) versus voltage (in mV, abscissa) relationship in glomus cells of WT mice and TH-Olfr78 KO mice. Each point is the average of 18 (WT) and 21 (KO) different experiments. (E) and (G) Average Na+ (E) and Ca2+ (G) current density (in pA/pF) recorded in response to depolarizing pulses to +10 mV from glomus cells of WT mice and TH-Olfr78 KO mice. Values, expressed as mean ± SEM, are Na+ current…

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  Figure 6.. Components of Ca2+ currents of dispersed glomus cells of wild-type (WT) mice and TH-Olfr78 KO mice. (A) Representative Ca2+ tail current recorded from a glomus cell at the end of a 10-ms depolarizing pulse to +10 mV upon repolarization to −70 mV. Deactivation time course is fitted by a double exponential function (left) with fast and slow components (right). (B) and (C) Average deactivation time constants (in ms) for the fast (τf, B) and slow (τs, C) component of the tail Ca2+ currents recorded from glomus cells of WT mice and TH-Olfr78 KO mice. Pulse protocol as indicated in (A). Data are expressed as mean ± SEM. Values are τf (WT, 0.1373 ± 0.010, n = 16; KO, 0.1194 ± 0.006, n = 16); τs (WT, 1.264 ± 0.177, n = 16; KO, 1.017 ± 0.120, n = 16). (D) and (E) Average % of tail current amplitude for the fast (D) and slow (E) component of the tail Ca2+ current recorded from glomus cells of WT mice and TH-Olfr78 KO mice. Data are expressed as mean ± SEM. Values are Af (WT, 0.1373 ± 0.010, n = 16; KO, 0.1194 ± 0.006, n = 16); As (WT, 69.55 ± 3.86, n = 13; KO, 72.6 ± 3.47, n = 16).

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  Figure 7.. Changes in cytosolic [Ca2+] elicited by high extracellular K+ and hypoxia in glomus cells of wild-type (WT) mice and TH-Olfr78 KO mice. (A) Representative recordings of the changes in cytosolic Ca2+ (measured as the increase in the 340/360 fluorescence ratio) recorded in single Fura-2-loaded dispersed glomus cells from WT mice (left) and TH-Olfr78 KO mice (right) in response to depolarization with high KCl (40 m m). (B) Increase in cytosolic [Ca2+] elicited by 40 m m K+ in dispersed glomus cells of WT mice mice and TH-Olfr78 KO mice. Data are presented as mean ± SEM. Values (in fluorescence ratio) are (WT, 0.597 ± 0.047, n = 18; KO, 0.555 ± 0.058, n = 25). (C) and (D) Representative recordings of nonspiking (C) and spiking (D) cytosolic [Ca2+] signals recorded in dispersed FURA-2-loaded glomus cells of WT mice (C) and TH-Olfr78 KO mice (D) in response to different levels of hypoxia (external solutions bubbled with either 0% O2 ∼15 mmHg or 3% O2 ∼30 mmHg). (E) Percentage of spiking (S) and nonspiking (NS) hypoxia-induced Ca2+ responses recorded in WT mice (n = 44/6 cells/mice) and TH-Olfr78 KO mice (n = 41/5 cells/mice). (F) Quantification of the cytosolic Ca2+ changes (area under…

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  Figure 8.. Secretory response to hypoxia of glomus cells in carotid body (CB) slices from wild-type (WT) mice and TH-Olfr78 KO mice. (A) Scheme illustrating the amperometric recording of quantal dopamine release from a single glomus cell (modified from Torres-Torrelo et al. 2021). (B) Representative amperometric recordings of the secretory activity recorded from glomus cells of WT mice (left) and TH-Olfr78 KO mice (right) under normoxia (21% O2, 150 mmHg) and during exposure to hypoxia (6% O2 ∼50 mmHg; 3% O2 ∼30 mmHg; 0% O2, ∼15 mmHg) and depolarization with potassium (40 m m K+). (C) Average secretion rate (ordinate) elicited by different levels of hypoxia (abscissa) in glomus cells of WT mice and TH-Olfr78 KO mice. Values in picocoulombs/min are (mean ± SEM): WT (21% O2, 0.43 ± 0.15, n = 13; 6% O2, 2.1 ± 0.4, n = 6; 3% O2, 3.52 ± 0.5, n = 6; 0% O2, 6 ± 0.7, n = 10); KO (21% O2, 0.2 ± 0.03, n = 13; 6% O2, 1.3 ± 0.24, n = 7; 3% O2, 2 ± 0.4, n = 7; 0% O2, 3.3 ± 0.4, n = 14). Lines have been drawn by eye. Discontinuous line was obtained by scaling the red line. (D-G) Boxplots representing average secretion rate in basal conditions 21% O2 (D), 6% O2 (E), 3% O2 (F), and 0% O2 (G). The boxplots…

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  Figure 9.. Secretory response to hypercapnia, K+, and hypoglycemia of glomus cells of wild-type (WT) mice and TH-Olfr78 KO mice. (A) Amperometric recordings of catecholamine release in response to hypercapnia (20% CO2) of glomus cells of WT mice (left) and TH-Olfr78 KO mice (right). (B) and (C) Average secretion rates (in pC/min) during exposure of glomus cells of WT mice and TH-Olfr78 KO mice to hypercapnia (20% CO2, B) and depolarization with 40 m m K+ (C). Data are presented as bar diagrams indicating mean ± SEM. Values are hypercapnia (WT: 6.09 ± 3.3 pC/min, n = 4 cells exp; KO, 4.91 ± 0.9, n = 6 cells); 40 m m K+ (WT: 24.43 ± 3.4 pC/min, n = 13 cells; KO, 19.15 ± 4.3, n = 14 cells). (D) Representative amperometric signal showing catecholamine release from glomus cells of WT mice (top) and TH-Olfr78 KO mice (bottom) in response to hypoglycemia (0.5 and 0 m m glucose). (E) Average secretion rates (in pC/min) during normoglycemia (5 m m glucose, basal) and in response to hypoglycemia (0.5 and 0 m m glucose) in WT mice and TH-Olfr78 KO mice. Data are represented as boxplots indicating median (middle line), 25th, 75th percentile (box), and largest and smallest values range (whiskers) with…

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  Figure 10.. Secretory responses of glomus cells to short-chain lipid compounds. (A-B) Representative amperometric recordings illustrating glomus cell secretory responses to acetate and β-hydroxybutyrate in carotid body slices of TH-Olfr78 KO mice. (C) Quantification of the secretory responses of glomus cells to acetate (5 m m) and 3β-hydroxybutyrate (2-2.5 m m) in wild-type (WT) mice and TH-Olfr78 mice. Data are presented as bar diagrams indicating mean ± SEM. P-values (<0.05) calculated with parametric Dunnett’s multiple comparison test. Values in pC/min are WT (basal, 0.19 ± 0.15, n = 5; acetate, 1.7 ± 1, n = 3; 3β-hydroxybutyrate, 1.4 ± 0.1, n = 5); KO (basal, 0.24 ± 0.1, n = 5; acetate, 1.8 ± 0.4, n = 5; 3β-hydroxybutyrate, 2 ± 0.4, n = 5).