Acute perturbation of Pet1-neuron activity in neonatal mice impairs cardiorespiratory homeostatic recovery

  1. Ryan T Dosumu-Johnson
  2. Andrea E Cocoran
  3. YoonJeung Chang
  4. Eugene Nattie
  5. Susan M Dymecki  Is a corresponding author
  1. Harvard Medical School, United States
  2. The Geisel School of Medicine at Dartmouth, United States
7 figures, 3 tables and 1 additional file

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…

https://doi.org/10.7554/eLife.37857.003
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’’’

https://doi.org/10.7554/eLife.37857.004
Figure 2 with 3 supplements
Acute perturbation of Pet1 neurons alters baseline cardiorespiratory values.

(A–B’) A and B represent minute ventilation (V˙E, ml min−1 •kg−1) and heart rate (HR, beats per minute, bpm), respectively, in sibling controls harboring the unrecombined RC-FDi allele …

https://doi.org/10.7554/eLife.37857.006
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 …

https://doi.org/10.7554/eLife.37857.007
Figure 2—figure supplement 2
Changes in baseline heart rate in relation to ventilation.

(A–A’) Linear regression of pre-CNO minute ventilation (V˙E•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 ΔV˙E

https://doi.org/10.7554/eLife.37857.008
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 (V˙E/V˙O2) and maximal oxygen consumption (V˙O2, ml • min−1 • g−1), respectively, in sibling controls harboring the unrecombined RC-FDi allele …

https://doi.org/10.7554/eLife.37857.009
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 …

https://doi.org/10.7554/eLife.37857.010
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 …

https://doi.org/10.7554/eLife.37857.011
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 …

https://doi.org/10.7554/eLife.37857.012
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 …

https://doi.org/10.7554/eLife.37857.013
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-…

https://doi.org/10.7554/eLife.37857.014
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; …

https://doi.org/10.7554/eLife.37857.015
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; …

https://doi.org/10.7554/eLife.37857.016
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 …

https://doi.org/10.7554/eLife.37857.018

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.

https://doi.org/10.7554/eLife.37857.005
RC-FDi(Control-Di)n=15Pet1-Flpe;RC-FDi(Pet1-Di)n=22
Mouse baseline characteristicsMeanSDMeanSDt-test
p value
Weight (BW) (g)5.5031.1155.041.2750.2616
Breathing frequency (ƒ) (breaths • min−1)241.626.87246.330.930.6406
Tidal volume (VT)
(ml • g−1)
4.511.0344.7571.7840.6322
Minute ventilation (V˙E)
(ml • min−1 • g−1)
1070169.91139331.90.4618
Heart rate (HR) (bpm)634.640.88612.842.010.1271
Oxygen consumption
(V˙O2) (ml • min−1 • g−1)
77.4319.1971.2514.390.2705
Ventilatory equivalents
(V˙E/V˙O2)
14.332.98916.133.7410.1281
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.

https://doi.org/10.7554/eLife.37857.017
Baseline characteristicOdds ratioConfidence intervalp-value*
Heart rate1.0170.9941.0400.15
Ventilation1.0010.9991.0040.38
Maximum Oxygen Consumption0.9650.9071.0260.25
Ventilatory Equivalents1.3991.0391.8830.027
  1. *logistic regression, controlling for genotype

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Pet1-FlpePMID: 18344997Dr. Susan Dymecki (Department of Genetics, Harvard Medical School)
Genetic reagent (M. musculus)RC-FDi
Gt(ROSA)26Sortm(CAG-FSF-CHRM4*(Di))Dym
PMID: 21798952derivative of MGI:5790683
Gt(ROSA)26Sortm9(CAG-mCherry,-CHRM4*)Dym
Dr. Susan Dymecki (Department of Genetics, Harvard Medical School)
AntibodyRabbit polyclonal anti-Tph2Novus BiologicalCat. #: NB100-74555
RRID:AB_1049988
IHC (1:1000)
AntibodyRat monoclonal anti-HAChromotekCat. # 7C9
RRID: AB_2631399
IHC (1:200)
AntibodyGoat polyclonal anti-5-HTAbcamCat. # ab66047
RRID:AB_1142794
IHC (1:1000)
AntibodyDonkey anti-rabbit IgG-Alexa Fluor 488ThermoFisher ScientificCat. # A-21206
RRID:AB_2535792
IHC (1:500)
AntibodyDonkey anti-rat IgG-Alexa Fluor 594ThermoFisher ScientificCat. # A-21209
RRID:AB_2535795
IHC (1:500)
AntibodyDonkey anti-goat igG-Alexa Fluor 647ThermoFisher ScientificCat. # A-21447
RRID:AB_2535864
IHC (1:500)
Recombinant DNA reagentFlpe forward primer5’-GCATCTGGGAGATCACTGAG-3’PCR genotyping
Recombinant DNA reagentFlpe reverse primer5’-CCCATTCCATGCGGGGTATCG-3’PCR genotyping
Recombinant DNA reagentFDi forward primer5’-CGAATTCGGAAACATAACTTCG-3’PCR genotyping
Recombinant DNA reagentFDi reverse primer5’-GGCAATGAAGACTTTCCACCG-3’PCR genotyping
Chemical compound, drugclozapine-N-oxide (CNO)SigmaCat. # C0832fresh stock solution 1 mg/ml in saline
Chemical compound, drugDAPI (4', 6-diamidino-2-phenylindole)ThermoFisher ScientificCat. # D1306nuclear counter staining, final concentraion at 5 µg/mL

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