Carotid body function in tyrosine hydroxylase conditional Olfr78 knockout mice

FUNCTION, 2024, 5(3): zqae010 https://doi.org/10.1093/function/zqae010 Advance Access Publication Date: 22 February 2024 Original Research RESEARCH ARTICLE Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice Olalla Colinas1,2,3, Peter Mombaerts4,Jos´ eL´ opez-Barneo1,2,3,*, Patricia Ortega-S´ aenz1,2,3,* 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Roc´ ıo/CSIC/Universidad de Sevilla, Sevile 41013, Spain, 2Departamento de Fisiolog´ ıa M´ edica y Biof´ ısica, Facultad de Medicina, Universidad de Sevilla, Seville 41009, Spain, 3Centro de Investigaci´ on Biom´ edica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Seville 41013, Spain and 4Max Planck Research Unit for Neurogenetics, Frankfurt 60438, Germany ∗Address correspondence to P.O-S. (e-mail: gorteg[email protected]); J.L-B. (e-mail: [email protected]) Abstract The Olfr78 gene encodes a G-protein-coupled olfactory receptor that is expressed in several ectopic sites. Olfr78 is one of the most abundant mRNA species in carotid body (CB) glomus cells. These cells are the prototypical oxygen (O2)sensitive arterial chemoreceptors, which, in response to lowered O2tension (hypoxia), activate the respiratory centers to induce hyperventilation. It has been proposed that Olfr78 is a lactate receptor and that glomus cell activation by the increase in blood lactate mediates the hypoxic ventilatory response (HVR). However, this proposal has been challenged by several groups showing that Olfr78 is not a physiologically relevant lactate receptor and that the O2-based regulation of breathing is not affected in constitutive Olfr78 knockout mice. In another study, constitutive Olfr78 knockout mice were reported to have altered systemic and CB responses to mild hypoxia. To further characterize the functional role of Olfr78 in CB glomus cells, we here generated a conditional Olfr78 knockout mouse strain and then restricted the knockout to glomus cells and other catecholaminergic cells by crossing with a tyrosine hydroxylase-specific Cre driver strain (TH-Olfr78 KO mice). We find that TH-Olfr78 KO mice have a normal HVR. Interestingly, glomus cells of TH-Olfr78 KO mice exhibit molecular and electrophysiological alterations as well as a reduced dopamine content in secretory vesicles and neurosecretory activity. These functional characteristics resemble those of CB neuroblasts in wild-type mice. We suggest that, although Olfr78 is not essential for CB O2sensing, activation of Olfr78-dependent pathways is required for maturation of glomus cells. Submitted: 9 November 2023; Revised: 9 February 2024; Accepted: 20 February 2024 C The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 2FUNCTION, 2024, Vol. 5, No. 3 carotid body neuroblast mature glomus cell Olfr78 expression Olfr78 knockout TH expression quantal dopamine content quan sensitivity to low O2/glucose Na+ channels voltage-dependent Key words: carotid body; glomus cells; chemoreception; oxygen sensing; olfactory receptor; Olfr78 knockout; hypoxic ventilatory response; neuroblasts; responsiveness to hypoxia Introduction Olfactory receptor 78 (Olfr78) is a G-protein-coupled odorant receptor that is atypically expressed outside the olfactory epithelium. Olfr78 is expressed at high levels in the carotid body (CB),1–3 the prototypical acute oxygen (O2) sensor, and main organ responsible for the hypoxic ventilatory response (HVR).4,5 One group reported that Olfr78 acts as a lactate receptor in mice and that the HVR in constitutive Olfr78 knockout mice (Olfr78 −/−mice) is abolished and the CB responses to hypoxia blunted.1Based on their findings, this group proposed a model whereby an increase in lactatemia during hypoxia and the subsequent endocrine activation of Olfr78-expressing CB glomus cells are responsible for the O2regulation of breathing.1This model has been challenged by several groups showing that lactate is a poor agonist for Olfr78,2,6that Olfr78 is not a physiologically relevant lactate receptor,7,8and that the HVR is not significantly altered in several strains of Olfr78 −/−mice.7It is well established that O2sensing is an intrinsic property of CB glomus (or type I) cells9–11 and that these polymodal chemoreceptors are activated by low O2tension and other stimuli, including lactate, via mechanisms that are unrelated to Olfr78.4,12 Nonetheless, one study reported that Olfr78 −/−mice have impaired HVR and glomus cell responses to mild hypoxia8and a reduction in sympathetic activation and systemic hypertension induced by chronic intermittent hypoxia.13 Olfr78 is one of the most abundant mRNA species expressed in CB glomus cells, but its functional significance remains poorly understood. As is the case for other genes relevant for CB glomus cell function,14 Olfr78 mRNA levels also depend on the constitutively high expression of hypoxia-inducible factor 2 alpha (HIF2α).15 Therefore, it is conceivable that, although not essential for O2sensing, Olfr78 has a role in glomus cell homeostasis. Importantly, all previous reports were about mice with a constitutive (or global) knockout of the Olfr78 gene16 and therefore the phenotypes observed could result from pleiotropic effects on the various CB cell types and/or on various organs.17 In addition, the properties of Olfr78-deficient CB glomus cells have not been analyzed in detail. Here, we report the generation and characterization of a C57BL/6-inbred conditional Olfr78 knockout mouse strain, which we crossed with a gene-targeted TH-IRES-Cre driver strain in order to restrict the knockout of the gene to CB glomus cells and other catecholaminergic cell types. Offspring of this cross are henceforward referred to as TH-Olfr78 KO mice. We did not observe significant changes in the HVR of TH-Olfr78 KO mice. However, they exhibited a subtle but clearly reproducible glomus cell phenotype, with changes in mRNA expression, electrophysiological features, intracellular Ca2+dynamics, and secretory activity that suggest alterations in the trophic maintenance of these cells. We speculate that Olfr78 contributes to maintaining a high basal activity of the adenyl cyclase-cyclic AMP pathway, which may be required for phenotypic specification of mature glomus cells. Materials and Methods Ethical Approval In Frankfurt, mouse experiments were carried out in accordance with guidelines of the German Animal Welfare Act, the Directive 2010/63/EU of the European Parliament and of the Council, and the institutional ethical and animal welfare guidelines of the Max Planck Research Unit for Neurogenetics. Approval came from the Regierungspra¨sidium Darmstadt (Germany), and the Veterina¨ramt of the City of Frankfurt (Germany). In Seville, the Institutional Committee for Animal Care and Use at the University of Seville (21/04/2020/053 and 27/01/2017/018) approved all procedures used in this study. Handling of the animals was conducted in accordance with the European Community Council directive of September 22, 2010 (Directive 2010/63/EU) and the implementations of June 5, 2019 (Regulation 2019/2010) for the Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 Colinas et al. 3 care and use of laboratory animals. To minimize the number of animals used in the experiments, mice subjected to in vivo analyses were subsequently euthanized and used for the in vitro studies. As all measurements of physiological constants done on mice were painless, analgesia was not used. For in vitro experiments, mice were killed by intraperitoneal injection with a lethal dose of anaesthetic (sodium thiopental, 120-200 mg/kg) Mouse Models Generation of Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice The coding region of exon 4 of the Olfr78 gene was flanked by loxP sites by gene targeting in the C57BL/6-derived embryonic stem cell line Bruce4. After obtaining germline transmission, the neo-selectable marker flanked by FRT sites was removed from the targeted mutation by crossing with Oz flp, a C57BL/6- inbred strain that carries a knockin of the site-specific recombinase flp in the ROSA26 locus driven by the UbiC promoter; the Oz flp allele was crossed out subsequently. The strain carrying the floxed Olfr78 allele without the Oz flip allele is publicly available from The Jackson Laboratory as stock #32801 with official strain name B6.Cg-Or51e2<tm3.1Mom>. Mice carrying the conditional Olfr78 knockout allele in the heterozygous state (Olfr78 f/+) or homozygous state (Olfr78 f/f) were crossed with TH-IRES- Cre mice.18 In this Cre driver strain, the gene-targeted Th-IRES- Cre knockin mutation provides Cre expression without disturbing expression of the tyrosine hydroxylase (Th) gene and affords efficient in vivo Cre-mediated recombination in catecholaminergic cells.19,20 Generation of Conditional HIF2αKnockout Mice The Epas1 (encoding HIF2α) conditional knock-out mice (EPAS1 KO: Epas1 f/−, Cre) were generated by crossing mice carrying an Epas1 floxed allele (The Jackson Laboratory, stock #008407)21 with mice carrying a tamoxifen-inducible Cre recombinase transgene.22 To induce recombination 2-mo-old mice were fed with tamoxifen-containing diet (TAM400/CreER; tamoxifen citrate, 400 mg/kg; Envigo) for a month. Thereafter, mice were kept on a normal diet for another 1.5 to 2 mo before experiments. Animal Care and Exposure of Mice to Chronic Hypoxia Mice were housed at 22◦C±1◦C in a 12-h light/12-h dark cycle with ad libitum access to food (Teklad global 14% protein, Envigo) and water. Both male and female mice were used. In TH-Olfr78 KO mice maintained under chronic hypoxia (10% O2atmosphere for 14 d), the experiments were performed using a hermetic chamber with control of O2,CO 2, humidity, and temperature (Coy Laboratory Products). For the experiments, mice (2-3 moold) were maintained in standard rodent cages placed into the hypoxia chamber, with ad libitum access to pellet food and water, and within a 12-h light/12-h dark cycle. Plethysmography Plethysmography was used to study respiratory function in conscious unrestricted mice as previously described.23 Briefly, mice were placed in plethysmographic chambers (EMKA Technologies) and perfused at a constant flow rate (1 L/min) with air (21% O2, normoxia), and various gas mixtures: 10% O2, 12% O2 (hypoxia, maintained for 5 min once O2percentage reached 10% or 12%), or 5% CO2(hypercapnia, maintained during 1 min when CO2percentage reached 5%). The hermetic chambers were provided with O2and CO2sensors to monitor the gas composition in parallel with changes in respiratory frequency recorded by a pressure sensor during the experiment. Data acquisition was performed using Iox2 (RRID:SCR 022973; EMKA Technologies). To calculate changes in respiratory frequency, the basal, hypoxic, and hypercapnic respiratory frequencies were estimated in each animal. Basal respiratory frequency was calculated by averaging the values acquired during the 160 s previous to exposure to hypoxia. Peak respiratory frequency (indicated by “p”inthe figures) during hypoxia was calculated by averaging the values obtained during 110 s at the peak of the hypoxic response. The total average respiratory frequency (indicated by “a” in the figures) during exposure to hypoxia (10% or 12% O2) was estimated by the mean of values measured during the last 400 s of exposure to stimuli. Respiratory frequency during exposure to hypercapnia was estimated by averaging the values obtained during 90 s after reaching 4%-5% CO2in the chamber before returning to normoxia. Hematocrit Hematocrit was measured in blood samples collected from the carotid artery using a hematocrit tube located near the incision site (where the bifurcation was dissected) to allow blood to fill the tube by capillary action. Following blood collection, tubes were sealed with bone wax, placed in a microhematocrit centrifuge, and spun for 5 min at 3 g . Hematocrit was calculated manually by measuring the length of the column of packed red cells and total blood length and expressed as the percentage of erythrocytes relative to total blood volume (100%). Immunohistochemistry and Estimation of CB Volume For immunohistochemical studies, mice were first perfused with phosphate buffered saline (PBS) and then with 4% paraformaldehyde in PBS before tissue dissection. Carotid bifurcations were fixed with 4% paraformaldehyde in PBS for 2 h, cryoprotected overnight with 30% sucrose in PBS, and embedded in O.C.T. (Cat# 4583, Tissue-Tek). Tissue sections of 10 μmwerecut with a cryostat (Leica, Wetzlar) and incubated overnight at 4◦C with primary antibodies: TH (1:100 dilution, Cat#AB1542, Sigma-Aldrich) and Ki67 (1:100 dilution, Cat#RM-9106, Epredia). This was followed by incubation with fluorescent secondary antibodies: Alexa Fluor 488 or Alexa Fluor 568 (1:500 or 1:1000 dilution, Cat#A11008 and Cat#A11004, Invitrogen, RRID: AB 143165 and RRID: AB 2534072). Nuclei were labeled with 4,6- diamidino-2-phenylindole (DAPI). Immunofluorescence images were obtained using a Nikon A1R+confocal microscope. To estimate CB volume, serial sections of CBs were immunostained and the area within each CB section was calculated using Image Jsoftware(RRID:SCR 003070). Carotid body volume was calculated considering CB area in each section, section thickness, and total number of sections per CB. Carotid Body and Superior Cervical Ganglion Resection and Molecular Analyses Mice were sacrificed by intraperitoneal administration of a lethal dose of sodium thiopental (120-150 mg/kg). Carotid bifurcations were removed and placed in cold PBS. Carotid bodies and superior cervical ganglion (SCG) were dissected. For real-time quantitative polymerase chain reaction (PCR) analysis, total RNA was isolated from CB and SCG using RNeasy Micro kit (Qiagen). For Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 4FUNCTION, 2024, Vol. 5, No. 3 SCG B carotid body C D mRNA relative to WT mRNA relative to WT 0.0004 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0006 A TH-Olfr78 KO mouse model Olfr78 null Th-IRES-Cre Olfr78 flox coding sequence LoxP LoxP X EF mRNA relative to WT < 0.0001 0.0228 mRNA relative to WT 0.0649 < 0.0001 0.0002 WT TH-Olfr78 KO WT TH-Olfr78 KO WT Epas1 KO Olfr78 Epas1 Cox8b Higd1C Rgs5 Cox4i2 Pcx Th Hif1 Olfr78Epas1 LoxP 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)LevelsofOlfr78 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 (<0.05 or near this value) calculated with unpaired two-tailed t-test (D) and with Mann-Whitney test (E) are indicated. Mean ±SEM values in panel D:Olfr78 (WT: 1 ±0.14; KO; 0.0004 ±0.00005); Epas1 (WT: 1 ±0.22; KO: 0.96 ±0.05), Cox8b (WT: 1 ±0.21; KO: 1.1 ±0.21), Higd1C (WT: 1 ±0.18; KO: 2 ±0.33), and Rgs5 (WT: 1 ±0.21; KO: 1 ±0.20); n=6 replicates/group. Mean ±SEM values in panel Eare Cox4i2 (WT: 1 ±0.26; KO: 0.57 ±0.11), Pcx (WT: 1 ±0.31; KO: 0.78 ±0.48), and Th (WT: 1 ±0.30; KO: 0.45 ±0.07); n=6 replicates/group. (F) Levels of mRNA of Epas1,Hif1α, and Olfr78 in CB samples of conditional Epas1 KO mice relative to WT mice. Data are expressed as mean ±SEM with all data values superimposed. Values are Epas1 (WT: 1 ±0.14; KO: 0.03 ±0.005), Hif1α(WT: 1 ±0.24; KO: 1.05 ±0.015), and Olfr78 (WT: 1 ±0.18 and KO, 0.21 ±0.06); n=10-12 replicates/group. P-values (<0.05) calculated by unpaired two-tailed t-test are indicated. the analysis of CB gene expression profile in TH-Olfr78 KO and wild-type (WT) mice, each CB replicate was pooled from 3 mice to obtain enough amount of mRNA. Complementary RNA (cRNA) was then amplified from CB total RNA using the GeneChip WT PLUS Reagent Kit (Affymetrix). Total RNA (500 ng; or amplified cRNA in the case of CB) was reverse-transcribed into complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Cat#205311, Qiagen). Real-time quantitative PCR was performed on a 7500 Fast Real Time PCR System (Applied Biosystem). PCR was performed in duplicate in a total volume of 20 μL containing 1 μL of cDNA solution and 1 μLofTaqManprobeof the specific genes (Thermo Fisher Scientific). Gapdh mRNA was measured in each sample to normalize the amount of total RNA (or cRNA) input to perform relative quantifications. Enzymatic Dispersion of CB Cells and Preparation of CB Slices Dispersed CB glomus cells were prepared as described previously.24 Briefly, dissected CBs were incubated for 20 min at 37◦C in enzymatic solution [PBS pH 7.4 supplemented with 50 μmCaCl 2, 0.6 mg/mL collagenase II (Cat#C6885, Sigma), 0.27 mg/mL trypsin (Cat#T8003, Sigma), and 1.25 U/mL porcine elastase (Cat#324682, Millipore)]. Then, CBs were mechanically stretched with needles and incubated for another 5 min at 37◦C. After digestion, cells were mechanically dispersed by pipetting and centrifuged for 5 min at 300 g. The cell pellet was resuspended in culture medium DMEM (0 glucose)/DMEM- F-12 (Cat#11966-025/21331-020, Gibco) medium (3:1) supplemented with 100 U/mL penicillin and 10 mg/mL streptomycin (Cat#15140-122, Gibco), 2 mm l-glutamine (Cat#25030- 024, Gibco), 10% fetal bovine serum (Cat#10270-106 Gibco), 84 U/L insulin (Actrapid, Cat#EU/1/02/230/011, Novonordisk), and plated on glass cover slips treated with poly-l-lysine (Cat#P1524, Sigma). Dispersed cells were maintained at 37◦Cina 5% CO2and 21% O2incubator and used after 24 h of incubation. To prepare CB slices, dissected CBs were placed in ice-cooled modified Tyrode solution and processed as described previously.25,26 Briefly, CB were included at 42oC in 1% low melting point agarose (prepared in PBS). Agarose block containing CBs were glued to the platform a vibratome (VT1000S, Leica) to prepare sections of 150 μm. Carotid body sections were digested in enzymatic solution (the same used for dispersed glomus cells), during 5 min, in a water bath at 37oCwithshaking.Finally, slices were washed with PBS and incubated in the same culture medium used for dispersed glomus cells to which 1.2 U/mL of erythropoietin (Cat#EU 1/07/410/028, Sandoz) were added. Slices were incubated at 37◦Cin5%CO 2for 24 h before use. Amperometry To monitor single-cell secretory activity, dopamine secretion from glomus cells in CB slices was measured by amperometry.23 Secretory events were recorded with a 10 μm carbon fiber electrode polarized to +750 mV (to favor dopamine oxidation) using Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 Colinas et al. 5 150 200 250 300 breaths/min TH-Olfr78 KO hypoxic ventilatory response hypercapnic ventilatory response 5% CO2 E 1min 5 0 % CO2 5% CO2 basal F 100 150 200 250 350 300 p: peak hypoxic response a: average hypoxic response CO 2 basal CO 2 WT TH-Olfr78 KO 100 150 200 250 300 breaths/min 10 21 5min 10% O2 % O2 A 10% O2 C 100 150 200 250 300 breaths/min basal basal 10% O 2 10% O 2 <0.0001 0.0089 0.0008 <0.0001 100 150 200 250 breaths/min WT 12% O2 B 12% O2 12 21 % O2 D 100 150 200 250 300 breaths/min ap basal basal 12% O 2 12% O 2 ap <0.0001 <0.0001 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,Aand 12% O2,B) and hypercapnia (5% CO2,E)inWTmice(10%O 2,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 % O2and % CO2over 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,Cand 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 Aand B) and during the whole stimuli (box and “a” in panel Aand 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% O2: WT, 201.3 ±11; KO, 216.0 ±6; and CO2: WT, 248.1 ±9; KO, 262.7 ±11). Statistically significant P-values calculated by one-way ANOVA followed by Tukey’s multiple comparisons test are indicated. an external voltameter connected to the EPC-7 amplifier. Amperometric currents were recorded with an EPC-7 patch-clamp amplifier (HEKA Electronics, Lambrecht/Pfaltz). The signal was filtered at 100 Hz and digitized at 250 Hz before storage on computer. Data acquisition and analysis were carried out with an ITC-16 interface (Instrutech Corporation) and PULSE/PULSEFIT software (HEKA Electronics). For the experiments, a slice was transferred to the recording chamber of an upright microscope (Axioscope, Zeiss) and continuously perfused with extracellular solution (see the “Recording Solutions” section). The secretion rate (given in fC/min or pC/min) was calculated as the amount of charge transferred to the recording electrode during a given period of time. Experiments were performed at ∼35◦C Microfluorimetric Measurements of Intracellular Ca2+ Microfluorimetric measurement of intracellular changes in Ca2+ concentration was performed in single dispersed glomus cells as previously described.11,19 Dispersed CB cells were loaded with Fura2-AM (TefLabsMW1002, Molecular Probes), 4 mm in DMEM/F-12 without serum at 37◦C for 30 min and subsequently incubated for 15 min in complete medium to remove excess Fura2-AM. To perform the experiments, a coverslip with Fura 2- AM loaded cells was placed on a recording chamber mounted on the stage of a microscope equipped with epifluorescence and photometry. This set up consists of an inverted microscope (Nikon Eclipse Ti) equipped with a 40x/0.60 NA objective, a monochromator (Polychrome V, Till Photonics), and a CCD camera, controlled by Aquacosmos software (version 2.6, Hamamatsu Photonics). Alternating excitation wavelengths of 340 and 380 nm with emission wavelength of 510 nm were used to obtain the F340/F380 ratio.11 Background fluorescence was subtracted before obtaining the F340/F380 ratio. A dichroic FF409-Di03 (Semrock) and a band-pass filter FF01-510/84 (Semrock) were used. Cytosolic Ca2+signals were digitized at a sampling interval of 500 ms. Experiments were performed at ∼35◦C. Patch Clamp Recording and Electrophysiological Analyses Macroscopic ionic currents were recorded from dispersed mouse glomus cells using the whole cell configuration of the patch clamp technique as adapted in our laboratory.24,27 Patch clamp electrodes (2-3 M) were pulled from capillary glass tubes Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 6FUNCTION, 2024, Vol. 5, No. 3 0 14 21% PO2 chronic hypoxia protocol A days WT TH-Olfr78 KO 8 0 6 4 2 CB volume (x10 8 μm 3 ) 4000 0 3000 2000 1000 number of cells TH CD TH/Ki67 Ki67 WT TH-Olfr78 KO WT TH-Olfr78 KO THTH Ki67 DAPI Ki67 B CB SCG carotid body bifurcation 50 μm 10% carotid body structure 0 20 40 60 80 Hx Nx Hx Nx 0.0357 0.0238 hematocrit after chronic hypoxia E 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 chronic hypoxia (Hx). 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 normoxia, WT, 50.7 ±0.5 (n=5); KO, 54.2 ±1.2 (n=6); chronic hypoxia, WT, 64.4 ±1.7 (n=3); KO, 67.4 ±1(n=3). P-values (<0.05) calculated with Mann-Whitney t-test are indicated. The increase in hematocrit is similar in both groups of mice. (Kimax, Kimble Products) with a horizontal pipette puller (Sutter instruments model P-1000) and fire-polished with a microforge (MF-830, Narishige). Voltage-clamp recordings were obtained with an EPC-7 patch clamp amplifier (HEKA Electronik) using standard voltage-clamp protocols designed with PULSE/PULSEFIT software (HEKA Electronik). The signal was filtered (10 kHz), subsequently digitized with an analog/digital converter (ITC-16 Instrutech Corporation), and finally sent to a computer. Data acquisition and storage were performed using the PULSE/PULSEFIT software (HEKA, Electronics) at a sampling interval of 20 μs. Experiments were performed at ∼35◦C. Recording Solutions Dispersed cells or slices used for in vitro amperometric or microfluorimetric recordings were transferred to the recording chamber and continuously perfused with a control solution containing 125 mm NaCl, 4.5 mm KCl, 23 mm NaHCO3,1mmMgCl 2, 2.5 mm CaCl2, 5 mm glucose, and 5 mm sucrose at ∼35◦C. In 40 mm K+solution, NaCl was replaced equimolarly with KCl. The “normoxic” solution was bubbled with a gas mixture of 20% O2,5%CO 2, and 75% N2(O2tension ∼145 mmHg). The “hypoxic” solution was bubbled with 5% CO2and 95% N2toreachanO 2ten- sion of ∼10 to 15 mmHg in the chamber. Osmolality of solutions was ∼300 mosm/Kg with pH 7.4. The hypercapnic solution was bubbled with a gas mixture of 20% O2, 20% CO2. The low glucose experiments were done upon exposure of the cells to solutions in which sucrose replaced glucose (125 mM NaCl, 4.5 mM KCl, 23 mM NaHCO3,1mMMgCl 2, 2.5 mM CaCl2, 0 mM glucose, and 10 mM sucrose or 0.5 mM glucose and 9.5 mM sucrose). To monitor macroscopic Ca2+,Na +,andK +currents, glomus cells were perfused with external solutions and dialyzed with internal solutions. Solutions used to record whole-cell currents through Na+and Ca2+channels (using Ba2+as a charge carrier) contained the following: external solution: 140 mm NaCl,10 mm BaCl2, 4.7 mm KCl, 10 mm Hepes, and 10 mm glucose (pH 7.4) (osmolality, 300 mOsm/kg); internal solution: 130 mm CsCl, 10 mm EGTA, 10 mm Hepes, and 4 mm adenosine triphosphate (ATP-Mg) (pH 7.2; osmolality, 285 mOsm/kg). In some experiments, a 0 Na+solution was used, in which external Na+was completely replaced with the impermeant cation N-methyl D- glucamine. The external solution used to record whole-cell K+ currents was the same control solution used for amperometric and microflourimetric analysis, and the internal solution contained 80 mm K+glutamate, 50 mm KCl, 1 mm MgCl2,10mm Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 Colinas et al. 7 500 pA 500 pA -50 -30 -10 10 30 50 100 200 300 400 500 600 Vm (mV) current density (pA/pF) TH-Olfr78 KO CD 10 ms potassium currents -70 mV 50 mV WT TH-Olfr78 KO K+ current density (pA/pF) + 40 mV E 0 200 400 600 800 1000 cell capacity (pF) input resistance (GOhm) AB 0 2 4 6 8 10 12 0 2 4 6 8 10 passive electrophysiological constants WT 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 point is the average of 11 (WT) and 10 (KO) different experiments. (E) Average maximal K+current density (in pA/pF) recorded in response to depolarizing pulses to +40 mV in isolated glomus cells of WT mice and TH-Olfr78 KO mice. Data are expressed as mean ±SEM. Values are TH+(WT, 344 ±64, n=11; KO, 487 ±67, n=10). Hepes, 4 mm ATP-Mg, and 5 mm EGTA (pH 7.2). In the high K+ solutions, KCl replaced NaCl equimolarly. In experiments where short chain lipid compounds were used, they also replaced NaCl equimolarly at the concentrations indicated. Statistical Analysis Statistical analysis was carried out using Prism Version 8.2.1. (279) for MacOS. Normality of the data sets obtained in the experiments was tested with the Shapiro-Wilk, D’Agostino & Pearson, or Kolmogorov-Smirnov test. In some cases, a log transformation was performed to normalize the distribution prior to parametric analysis. All tests, parametric and nonparametric, were two-tailed. A P<0.05 was considered statistically significant. Data with normal distribution are described as mean, and standard error of the mean with the number (n)of experiments indicated. For graphical representation of data, bar diagrams are used, with indication of the mean ±SEM, and scatter plots of the data points superimposed. For graphical representation of data that do not follow a normal distribution, box plots are used with indication of median, quartiles, and outliers. To facilitate comparisons, mean and SEM values are also given in the figure legends. Results Expression of Genes Relevant to CB Function in TH-Olfr78 Knockout Mice Mice carrying the newly generated floxed Olfr78 allele were crossed with gene-targeted knockin mice expressing Cre recombinase under the control of the tyrosine hydroxylase (Th)promoter.18,19 The resulting mice (TH-Olfr78 KO mice; Figure 1A) showed a complete absence of Olfr78 mRNA in CB glomus cells (Figure 1B) and other catecholaminergic tissues such as the SCG (Figure 1C). Next, we studied differences in the mRNA expression levels of a set of genes that are known to be relevant for CB glomus cell function.2,3,14,28,29 These included Epas1 (encoding HIF2α), 3 atypical mitochondrial complex IV (MCIV) subunit isoforms (Cox4i2,Cox8b,andHigd1C), pyruvate carboxylase (Pcx), regulator of G-protein-signaling 5 (Rgs5), and tyrosine Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 8FUNCTION, 2024, Vol. 5, No. 3 50 pA 50 pA -40 -30 -20 -10 0 10 20 30 40 -25 -20 -10 -5 Vm (mV) -40 -30 -20 -10 0 10 20 30 40 -40 -30 -10 Vm (mV) density (pA/pF) WT TH-Olfr78 KO B C E D 2 ms WT TH-Olfr78 KO -70 -60 -50 -40 -30 -20 -10 0 -60 -40 -20 0 + 10 mV + 10 mV FG 0.043 density (pA/pF) INa density (pA/pF) INa density (pA/pF) ICa Na+ and Ca2+ currents -70 mV 10 mV 0 Na+ control 40 pA INa -70 mV 10 mV A 2 ms 2 ms ICa ICa 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)AverageNa +(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)AverageNa +(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 (WT, −16.5 ±2.8, n=18; KO, −25.8 ±3.6, n=21); Ca2+current (WT, −14.6 ±2.9, n=19; KO, −13.3 ±2.2, n=21). P-values (<0.05) calculated with unpaired two-tailed t-test are indicated in panels (E) and (G). hydroxylase (Th), which is expressed in the highly dopaminergic mature glomus cells. Among these genes, Higd1C, which has been suggested to contribute to the characteristically high sensitivity of glomus cell mitochondria to hypoxia,28 was significantly upregulated in Olfr78-deficient cells (Figure 1D and E). There was also a trend for Th mRNA to be downregulated in TH-Olfr78 KO mice but the data do not reach statistical significance (Figure 1E). As is the case for other genes critically involved in adult CB function,14,15 Olfr78 mRNA expression appears to be dependent on HIF2α(Figure 1F). In Vivo Responses to Acute and Chronic Hypoxia The CB is the prototypical acute O2-sensing organ in adult mammals and main organ that is responsible for the HVR.4,5To evaluate the responsiveness of WT mice and TH-Olfr78 KO mice to hypoxia, we tested the acute effects of O2/CO2on ventilation and the changes in CB structure induced by sustained hypoxia. Mice exposed acutely to either 10% or 12% O2responded with rapid graded increases in respiration rate that have a peak (lasting for ∼1 min) at the onset of the response, followed by a progressive decline due to the inhibitory effect of hypocapnia caused by hyperventilation (Figure 2A-D). Neither the peak (“p” in the figure) nor the average (“a” in the figure) increases in breathing frequency induced by lowering environmental O2ten- sion from normal air (21% O2) to either 10% O2or 12% O2are different between WT mice and TH-Olfr78 KO mice (Figure 2A-D). Exposure to hypercapnia (5% CO2in a 21% O2atmosphere) produced a robust and sustained increase in breathing frequency, which is also similar in the 2 mouse models (Figure 2EandF). These data confirm our findings in mice with a constitutive (or Downloaded from https://academic.oup.com/function/article/5/3/zqae010/7612970 by guest on 16 September 2024 Colinas et al. 9 WT TH-Olfr78 KO A Ca2+ current deactivation (tail currents) deactivation time course BC ms τf 0.0 0.1 0.2 0.3 0 1 2 3 4 τs 0 20 40 60 80 100 0 20 40 60 80 100 % of tail current amplitude AfAs DE fast exponential fit slow fast -70 mV 10 mV double exponential fit slow exponential fit 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). global) knockout of the Olfr78 gene7and indicate that Olfr78 deficiency does not alter O2/CO2regulation of breathing within the rangeofO 2tensions that are normally used to monitor the HVR. The CB grows severalfold in size upon exposure to chronic (days/weeks) hypoxia.30 This well-known adaptive response ensures the sustained activation of the respiratory center that is required for acclimatization to hypoxic environments. Carotid body growth upon chronic hypoxia requires the activation of CB glomus cells,31 which release transmitters that rapidly induce proliferation and maturation of neighboring TH-positive CB neuroblasts (immature glomus cells) followed by activation of pluripotent CB stem cells.17,32 We found that the number of TH- positive cells, proliferating cells (Ki67 positive), and the CB volume are comparable in WT mice and TH-Olfr78 KO mice maintained in a hypoxic environment (10% O2)for2wk(Figure 3A-D). Moreover, the increase in hematocrit after exposure to chronic hypoxia, a parameter that is augmented in mice with altered CB O2sensing and no hyperventilation in response to hypoxia,20 is similar in WT mice and TH-Olfr78 KO mice (Figure 3E). Taken together, we did not detect any differences of note in the in vivo adaptive responses to sustained hypoxia between WT mice and TH-Olfr78 KO mice. Electrophysiology of Glomus Cells The electrical properties of dispersed glomus cells of WT mice and TH-Olfr78 KO mice were recorded with the whole-cell configuration of the patch clamp technique. Average passive electrical parameters (cell capacitance and input resistance) are similar in the two cell types, although there is a trend for cell capacitance (proportional to cell size) to be smaller in Olfr78- deficient cells (Figure 4A and B). All cells showed robust macroscopic voltage-dependent K+currents. The time course of the K+currents varied among glomus cells of WT and TH-Olfr78 KO mice, suggesting that different subtypes of voltage-gated K+channels with variable activation and inactivation kinetics are expressed in these cells (Figure 4C). The peak K+current amplitude (presented as current density) at membrane potentials >10 mV tends to be larger in Olfr78-deficient cells (Figure 4D and E). 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