Acute perturbation of Pet1-neuron activity in neonatal mice impairs cardiorespiratory homeostatic recovery
- Harvard Medical School, United States
- The Geisel School of Medicine at Dartmouth, United States
Figures
Figure 1 with 1 supplement
Genetic mouse model and postneonatal autoresuscitation assay.
(A–C”’) HA-tagged Di receptor expression targeted to Pet1-raphe serotonergic neurons in double-transgenic Pet1-Flpe; RC-FDi (referred to as Pet1-Di) pups at P8, as shown previously in adult mice (Bru…
Figure 1—figure supplement 1
Additional examples of HA-Di expression in medullary brain tissue of Pet1-Di pups from independent litters.
(A–D”’) HA-tagged Di receptor expression targeted to Pet1-raphe serotonergic neurons in double-transgenic Pet1-Flpe; RC-FDi (referred to as Pet1-Di) pups at P8, as shown in Figure 1. A-A’’ and C-C’’’…
Figure 2 with 3 supplements
Acute perturbation of Pet1 neurons alters baseline cardiorespiratory values.
(A–B’) A and B represent minute ventilation (, ml min−1 •kg−1) and heart rate (HR, beats per minute, bpm), respectively, in sibling controls harboring the unrecombined RC-FDi allele …
Figure 2—figure supplement 1
Variation in heart rate and respiratory rate.
(A–A’) Plotted coefficient of variation for respiratory rate determined across the 30 s stable trace analyzed in Figure 2 (and described in the Methods section) for pre- versus CNO exposure …
Figure 2—figure supplement 2
Changes in baseline heart rate in relation to ventilation.
(A–A’) Linear regression of pre-CNO minute ventilation (•min−1•kg−1) plotted against change in heart rate (HR, bpm) for control-Di pups (A) and Pet1-Di pups (A’). (B–B’) Linear regression of Δ …
Figure 2—figure supplement 3
Analyses of ventilatory equivalents for oxygen and oxygen consumption before and during CNO exposure.
(A–B’) A and B represent ventilatory equivalents for oxygen () and maximal oxygen consumption (, ml • min−1 • g−1), respectively, in sibling controls harboring the unrecombined RC-FDi allele …
Figure 3
Acutely disrupting Pet1-neuron activity impairs pup ability to recover from multiple apneas.
(A) Percent mortality after repeated asphyxia-induced apneas plotted as a function of genotype (one-sided Fisher’s Exact Test with Lancaster’s Mid P correction p=0.041). (B) Example breathing and …
Figure 4
Acutely disrupting Pet1-neuron activity primarily affects respiratory components during apnea recovery
(A) Compressed schematic of autoresuscitation assay timeline indicating asphyxia-apnea bouts and data extraction windows; open windows a’-d’ reflect bout-specific baseline measurements, filled …
Figure 5 with 1 supplement
Pet1-Di-CNO pups show a decoupling of heart rate and breathing rate recovery times.
(A–A”) Scatter plot of recovery τf (seconds, s) versus τHR (s) that includes all possible apnea recoveries of both groups, with data points from Pet1-Di-CNO pups in red, and control-Di-CNO pups in …
Figure 5—figure supplement 1
Example traces showing differential breathing and HR recovery responses to asphyxia-induced apnea in a Pet1-Di-CNO pup (A) as compared to control (B).
In each panel (A, B), the top trace reflects breathing response expressed as the raw tracing with millivolts (mV) reflecting breath size; bottom trace reflects HR in beats per min (bpm). Gray-shaded …
Figure 6 with 2 supplements
Pet1-Di-CNO pups demonstrate a disordered gasp response to the initial asphyxic bout and induced apnea.
(A) Example respiratory traces immediately following the first asphyxia-induced apnea: top trace, from a Pet1-Di-CNO pup that goes on to die during a subsequent bout; bottom trace, from a control-Di-…
Figure 6—figure supplement 1
Variability for the baseline room air interbreath interval in Control-Di and Pet1-Di pups prior to and during CNO exposure.
(A–C) Control-Di pups; (A’–C’) Pet1-Di pups; gray dots reflect pups that survived all four asphyxia-induced apnea challenges; red dots, pups that died during the assay; black dots reflect the mean; …
Figure 6—figure supplement 2
Variability for the baseline room air interbeat interval in Control-Di and Pet1-Di pups prior to and during CNO exposure.
(A–C) Control-Di pups; (A’–C’) Pet1-Di pups; gray dots reflect pups that survived all four asphyxia-induced apnea challenges; red dots, pups that died during the assay; black dots reflect the mean; …
Figure 7
Proposed Pet1-neuron involvement in the postneonatal autoresuscitation response to apneas.
(A) In control pups (unrecombined RC-FDi allele harboring, non-Di expressors exposed to CNO), an apnea (with associated bradycardia) would lead to blood/tissue oxygen tension (PO2) reduction and …
Tables
Table 1
Baseline cardiorespiratory values prior to CNO-induced disruption of Pet1-neurons.
Data (mean ±standard deviation) for each time point were obtained prior to CNO-induced silencing (Figure 1G open window a). Student’s t-test was used to assess differences between genotypes.
| RC-FDi(Control-Di)n=15 | Pet1-Flpe;RC-FDi(Pet1-Di)n=22 | ||||
|---|---|---|---|---|---|
| Mouse baseline characteristics | Mean | SD | Mean | SD | t-test p value |
| Weight (BW) (g) | 5.503 | 1.115 | 5.04 | 1.275 | 0.2616 |
| Breathing frequency (ƒ) (breaths • min−1) | 241.6 | 26.87 | 246.3 | 30.93 | 0.6406 |
| Tidal volume (VT) (ml • g−1) | 4.51 | 1.034 | 4.757 | 1.784 | 0.6322 |
| Minute ventilation () (ml • min−1 • g−1) | 1070 | 169.9 | 1139 | 331.9 | 0.4618 |
| Heart rate (HR) (bpm) | 634.6 | 40.88 | 612.8 | 42.01 | 0.1271 |
| Oxygen consumption () (ml • min−1 • g−1) | 77.43 | 19.19 | 71.25 | 14.39 | 0.2705 |
| Ventilatory equivalents () | 14.33 | 2.989 | 16.13 | 3.741 | 0.1281 |
Table 2
Mortality by homeostatic characteristics prior to neuron perturbation.
Logistic regression results of different homeostatic characteristics and mortality outcomes of Pet1-Di-CNO pups.
| Baseline characteristic | Odds ratio | Confidence interval | p-value* | |
|---|---|---|---|---|
| Heart rate | 1.017 | 0.994 | 1.040 | 0.15 |
| Ventilation | 1.001 | 0.999 | 1.004 | 0.38 |
| Maximum Oxygen Consumption | 0.965 | 0.907 | 1.026 | 0.25 |
| Ventilatory Equivalents | 1.399 | 1.039 | 1.883 | 0.027 |
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*logistic regression, controlling for genotype
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Pet1-Flpe | PMID: 18344997 | Dr. Susan Dymecki (Department of Genetics, Harvard Medical School) | |
| Genetic reagent (M. musculus) | RC-FDi Gt(ROSA)26Sortm(CAG-FSF-CHRM4*(Di))Dym | PMID: 21798952 | derivative of MGI:5790683 Gt(ROSA)26Sortm9(CAG-mCherry,-CHRM4*)Dym | Dr. Susan Dymecki (Department of Genetics, Harvard Medical School) |
| Antibody | Rabbit polyclonal anti-Tph2 | Novus Biological | Cat. #: NB100-74555 RRID:AB_1049988 | IHC (1:1000) |
| Antibody | Rat monoclonal anti-HA | Chromotek | Cat. # 7C9 RRID: AB_2631399 | IHC (1:200) |
| Antibody | Goat polyclonal anti-5-HT | Abcam | Cat. # ab66047 RRID:AB_1142794 | IHC (1:1000) |
| Antibody | Donkey anti-rabbit IgG-Alexa Fluor 488 | ThermoFisher Scientific | Cat. # A-21206 RRID:AB_2535792 | IHC (1:500) |
| Antibody | Donkey anti-rat IgG-Alexa Fluor 594 | ThermoFisher Scientific | Cat. # A-21209 RRID:AB_2535795 | IHC (1:500) |
| Antibody | Donkey anti-goat igG-Alexa Fluor 647 | ThermoFisher Scientific | Cat. # A-21447 RRID:AB_2535864 | IHC (1:500) |
| Recombinant DNA reagent | Flpe forward primer | 5’-GCATCTGGGAGATCACTGAG-3’ | PCR genotyping | |
| Recombinant DNA reagent | Flpe reverse primer | 5’-CCCATTCCATGCGGGGTATCG-3’ | PCR genotyping | |
| Recombinant DNA reagent | FDi forward primer | 5’-CGAATTCGGAAACATAACTTCG-3’ | PCR genotyping | |
| Recombinant DNA reagent | FDi reverse primer | 5’-GGCAATGAAGACTTTCCACCG-3’ | PCR genotyping | |
| Chemical compound, drug | clozapine-N-oxide (CNO) | Sigma | Cat. # C0832 | fresh stock solution 1 mg/ml in saline |
| Chemical compound, drug | DAPI (4', 6-diamidino-2-phenylindole) | ThermoFisher Scientific | Cat. # D1306 | nuclear counter staining, final concentraion at 5 µg/mL |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.37857.019
