Knockdown of PHOX2B in the retrotrapezoid nucleus reduces the central CO2 chemoreflex in rats
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Canada
- Women and Children’s Health Research Institute, University of Alberta, Canada
- Neuroscience and Mental Health Institute, University of Alberta, Canada
Figures
Figure 1 with 1 supplement
Respiratory data 2 weeks post viral PHOX2B shRNA injection.
(A) Breathing frequency (ƒR,), (B) tidal volume (VT), (C) allometric minute ventilation (VE ALLO), (D) oxygen consumption (VO2 ALLO), (E) convective requirement ratio (VE/VO2 ALLO), (F) hypercapnic ventilatory response (HCVR absolute change in VE ALLO vs. corresponding room air) of baseline (pre-surgery, grey filled box), naive (black n=8), non-target control shRNA (NT-shRNA, grey n=17) and PHOX2B shRNA (PHOX2B-shRNA, red n=17) rats 2 weeks post injection during room air, hypercapnia 5% and 7.2% CO2. ƒR was equally reduced in all experimental groups compared to baseline but no treatment effect was observed (A). VT was significantly impaired following RTN injection in PHOX2B-shRNA group compared to naive animals (p=0.022) and baseline (p<0.001) at 7.2% CO2 (B). VE ALLO was increased in naive rats compared to the other treatment groups (p=0.005) in room air (C). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. Black*, different from naive; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.
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Figure 1—source data 1
Respiratory data 2 weeks post viral PHOX2B shRNA injection.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Respiratory and anatomical data 4 weeks post large viral PHOX2B shRNA injection.
(A) Allometric VE is equally impaired following RTN injection of non-target control (NT-shRNA n=6) and PHOX2B-shRNA (n=8) in 5% CO2 (p<0.001) and 7.2% CO2 (p<0.001). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. Asterisks, different from baseline. (B) The number of Nmb+/PHOX2B+ cells comprising the RTN are reduced in NT-shRNA (n=5; p=0.002), and shRNA rats (n=5; p=0.001) as compared to naive controls (n=4; Asterisks, different from baseline). The number of Nmb+/PHOX2B- cells was unchanged in both surgical treatments compared to baseline (One-way ANOVA p=0.8).
Figure 2
PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats two weeks post viral shRNA injection.
(A) Schematic and representative image of a transverse brainstem section at the level of the RTN (–11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in RTN Nmb+/PHOX2B+ and Nmb+/PHOX2B- neurons in naive (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view inserts below). Arrowheads indicate absence of PHOX2B protein. Scale bar = 400 μm (top figures), 150 μm (inserts below). (C) The number of total cells (Nmb+/PHOX2B+ plus Nmb+/PHOX2B-) comprising the RTN were reduced in PHOX2B-shRNA rats (n=4) as compared to naive (n=4) and NT-shRNA (n=4), and in NT-shRNA rats as compared to naive rats (black#, One-way ANOVA, p<0.001). The number of Nmb+/PHOX2B+ cells were reduced in PHOX2B-shRNA rats as compared to naive and NT-shRNA (blue*, One-way ANOVA, p<0 0.001). The number of Nmb+/PHOX2B- cells was unchanged across groups. (D,E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb+/PHOX2B+ (D) and Nmb+/PHOX2B- (E) neurons along the RTN.
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Figure 2—source data 1
PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats two weeks post viral shRNA injection.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig2-data1-v1.xlsx
Figure 3
PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.
(A) Schematic and representative image of a transverse brainstem section at the level of the RTN (–11.5 mm distance from Bregma) showing the area of investigation containing RTN neurons. (B) Expression of Phox2b mRNA (red), PHOX2B protein (white) and Nmb mRNA (green) in naive (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats (magnified view insert). Arrowheads indicate absence of PHOX2B protein. Scale bar = 400 μm (top figures), 150 μm (inserts below). (C) The number of total cells (Nmb+/PHOX2B+ + Nmb+/PHOX2B-) comprising the RTN were reduced in PHOX2B-shRNA rats (n=6) as compared to naive (n=4) (Black#, One-way ANOVA, p=0.0087) but not to NT-shRNA (n=10). The number of Nmb+/PHOX2B+ cells were reduced in PHOX2B-shRNA rats as compared to naive and NT-shRNA (Blue*, one-way ANOVA, p<0.001). The number of Nmb+/PHOX2B- cells were increased in PHOX2B-shRNA rats as compared to both naive and NT-shRNA (Green*, one-way ANOVA p<0.001). (D,E) Rostral-caudal distribution (distance from the caudal tip of the facial nucleus, 7Mn) of Nmb+/PHOX2B+ (D) and Nmb+/PHOX2B- (E) neurons along the RTN.
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Figure 3—source data 1
PHOX2B and Nmb expression and total cell count within the RTN area in naive, NT-shRNA and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig3-data1-v1.xlsx
Figure 4
Respiratory data following 4 weeks post viral shRNA injection.
(A) Breathing frequency (ƒR,), (B) tidal volume (VT), (C) allometric minute ventilation (VE ALLO), (D) convective requirement ratio (VE/VO2 ALLO), (E) hypercapnic ventilatory response (HCVR, absolute change in VE ALLO vs. corresponding room air), (F) HCVR at baseline, week 2 and week 4 post-viral injections in naive (black n=8), non-target control shRNA (NT-shRNA, grey n=10) and PHOX2B-shRNA (PHOX2B-shRNA, red n=6). ƒR was equally impaired in all experimental group compared to baseline but no treatment effect was observed (A). VT was significantly impaired following RTN injection in PHOX2B-shRNA group compared to baseline pre-surgery (p<0.001), naive rats (p<0.001), and NT-shRNA rats (p=0.002) at 7.2% CO2. (B). VE ALLO was impaired in PHOX2B-shRNA rats during exposure to hypercapnia (7.2% CO2) compared to baseline (p=0.0025), naive rats (p=0.007), and NT-shRNA rats (p=0.002). (C). VE/VO2 ALLO was reduced in PHOX2B-shRNA animals compared to NT-shRNA rats both at 5% (p=0.023) and 7.2% (p=0.004) CO2 (D). HCVR during 7.2% CO2 was lower in PHOX2B-shRNA rats compared to baseline (p=0.007), naive rats (p=0.001), and NT-shRNA rats (p=0.016) (E). Boxplots: median, 1st – 3rd quartiles and 10th – 90th percentiles, outliers = dots, ‘+’ indicates arithmetic mean. Bonferroni post-hoc as indicated. HCVR was significantly impaired only in PHOX2B-shRNA rats 4 weeks post-surgery (One-way ANOVA p=0.007) (F). Black*, different from naive; Grey*, different from NT-shRNA; Red*, different from PHOX2B- shRNA.
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Figure 4—source data 1
Respiratory data following 4 weeks post viral shRNA injection.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig4-data1-v1.xlsx
Figure 5
PHOX2B knockdown in RTN neurons does not alter TH or Nmb expression but impairs the hypercapnic ventilatory response.
(A) PHOX2B protein (white) and TH (red) expression in C1 neurons of nave (left), NT-shRNA (middle), and PHOX2B-shRNA (right) rats. Magnified view at the bottom. Arrowheads indicate the colocalization of PHOX2B and TH protein in C1 neurones cells. Scale bar = 400 μm (top figures), 150 μm (bottom figures). (B) No differences were observed in the rostral-caudal distribution of TH+/PHOX2B+ catecholaminergic C1 neurons cells caudal to the RTN between naive (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rats. (C) PHOX2B protein (white) and Nmb mRNA (green) expression in NT-shRNA rat at the level of NTS and RTN regions. Magnified view on the left. Arrowheads indicate cells with Nmb mRNA expression. Scale bar = 400 μm (right figures), 150 μm (left figures). (D) Quantification of single cells Nmb mRNA fluorescence intensity along the rostro-caudal extension of RTN in naive (black, n=4), NT-shRNA (grey, n=10) and PHOX2B-shRNA (red, n=6) rat calculated as average ratio between RTN and NTS cells showed no difference between treatment groups. Data are shown as average cell fluorescence value at different rostro-caudal levels. (E-F) X-Y plot of HCVR during 7.2% CO2 exposure relative to the number of Nmb+/PHOX2B+ (E, slope is different from ‘0’ at p<0.001; r2=0.739) and Nmb+/PHOX2B- (F, p<0.001 for difference between slopes; r2=0.482) in the RTN.
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Figure 5—source data 1
PHOX2B knockdown in RTN neurons does not alter TH or Nmb expression but impairs the hypercapnic ventilatory response.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig5-data1-v1.xlsx
Figure 6
Gpr4 and Task2 mRNA expression within the RTN area in naive, and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.
(A, D) Nmb (green), Gpr4, Task2 mRNA (red) and PHOX2B protein (grey) expression in RTN neurons in naive (top) and PHOX2B-shRNA (bottom) rats. Scale bar = 150 μm. Arrowheads indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb+/PHOX2B+ neurons. Asterisks indicate colocalization of Gpr4 (A) and Task2 (D) with Nmb+/PHOX2B- neurons. (B, E) Quantification of single cells Gpr4 and Task2 mRNA fluorescence intensity along the rostro-caudal extension of RTN in naive (black), NT-shRNA (grey) and PHOX2B-shRNA (red) rats. Black*, different from naive, p<0.001. Grey*, different from NT-shRNA, p<0.001 (C, F) Quantification of single cells Gpr4 and Task2 mRNA fluorescence staining intensity along the rostro-caudal extension of RTN in Nmb+/PHOX2B+ (red filled dot) and Nmb+/PHOX2B- (red empty dot) neurons in PHOX2B-shRNA rats (C, p=0.022); (F, p=0.029). Data are shown as average cell fluorescence value at different rostro-caudal levels of the RTN. Mean corrected total cell fluorescence (CTCF) value ± SEM combined (naive, n=4; NT-shRNA, n=10; PHOX2B-shRNA, n=6) (see Materials and methods for detail). One-way ANOVA repeated measures.
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Figure 6—source data 1
Gpr4 and Task2 mRNA expression within the RTN area in naive, and PHOX2B-shRNA injected rats 4 weeks post viral shRNA injection.
- https://cdn.elifesciences.org/articles/94653/elife-94653-fig6-data1-v1.xlsx
Additional files
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Supplementary file 1
Details on the main statistical analyses reported in the indicated figures.
- https://cdn.elifesciences.org/articles/94653/elife-94653-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94653/elife-94653-mdarchecklist1-v1.docx
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Knockdown of PHOX2B in the retrotrapezoid nucleus reduces the central CO2 chemoreflex in rats
eLife 13:RP94653.
https://doi.org/10.7554/eLife.94653.3
