(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…
(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’’’…
(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 …
(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 …
(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 …
(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 …
(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 …
(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 …
(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 …
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 …
(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-…
(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; …
(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; …
(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 …
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 |
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 |
*logistic regression, controlling for genotype
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 |