Hawkins, VE and Takakura, AC and Trinh, A and Malheiros-Lima, MR andCleary, CM and Wenker, IC and Dubreuil, T and Rodriguez, EM and Nelson,MT and Moreira, TS and Mulkey, DK (2017)Purinergic regulation of vascu-lar tone in the retrotrapezoid nucleus is specialized to support the drive tobreathe. eLife, 6. e25232-e25232.Downloaded from: http://e-space.mmu.ac.uk/623823/Version: Published VersionPublisher: eLife Sciences PublicationsDOI: https://doi.org/10.7554/eLife.25232Usage rights: Creative Commons: Attribution 4.0Please cite the published versionhttps://e-space.mmu.ac.uk*For correspondence: tmoreira@icb.usp.br (TSM); daniel.mulkey@uconn.edu (DKM)†These authors contributedequally to this workCompeting interests: Theauthors declare that nocompeting interests exist.Funding: See page 13Received: 18 January 2017Accepted: 06 April 2017Published: 07 April 2017Reviewing editor: Jan-MarinoRamirez, Seattle Children’sResearch Institute and Universityof Washington, United StatesCopyright Hawkins et al. Thisarticle is distributed under theterms of the Creative CommonsAttribution License, whichpermits unrestricted use andredistribution provided that theoriginal author and source arecredited.Purinergic regulation of vascular tone inthe retrotrapezoid nucleus is specializedto support the drive to breatheVirginia E Hawkins1, Ana C Takakura2, Ashley Trinh1, Milene R Malheiros-Lima3,Colin M Cleary1, Ian C Wenker1, Todd Dubreuil1, Elliot M Rodriguez1,Mark T Nelson4,5, Thiago S Moreira3*†, Daniel K Mulkey1*†1Department of Physiology and Neurobiology, University of Connecticut, Storrs,United States; 2Department of Pharmacology, Institute of Biomedical Sciences,University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3Department of Physiology andBiophysics, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo,Brazil; 4Department of Pharmacology, College of Medicine, University of Vermont,Burlington, United States; 5Institute of Cardiovascular Sciences, University ofManchester, Manchester, United KingdomAbstract Cerebral blood flow is highly sensitive to changes in CO2/H+ where an increase inCO2/H+ causes vasodilation and increased blood flow. Tissue CO2/H+ also functions as the mainstimulus for breathing by activating chemosensitive neurons that control respiratory output.Considering that CO2/H+-induced vasodilation would accelerate removal of CO2/H+ and potentiallycounteract the drive to breathe, we hypothesize that chemosensitive brain regions have adapted ameans of preventing vascular CO2/H+-reactivity. Here, we show in rat that purinergic signaling,possibly through P2Y2/4 receptors, in the retrotrapezoid nucleus (RTN) maintains arteriole toneduring high CO2/H+ and disruption of this mechanism decreases the CO2ventilatory response. Ourdiscovery that CO2/H+-dependent regulation of vascular tone in the RTN is the opposite to the restof the cerebral vascular tree is novel and fundamentally important for understanding howregulation of vascular tone is tailored to support neural function and behavior, in this case the driveto breathe.DOI: 10.7554/eLife.25232.001IntroductionCerebral blood flow is highly sensitive to changes in CO2/H+. An increase in CO2/H+ causes vasodila-tion and increased blood flow, which in turn facilitates removal of excess CO2/H+. This response,known as vascular CO2 reactivity, serves to match blood flow with tissue metabolic needs(Ainslie and Duffin, 2009). Maintaining tight control of brain CO2/H+ levels is critical, as there isonly a narrow range that is conducive to normal neural function. For example, a modest alkalosis ofjust 0.2 pH units can trigger seizure activity; conversely, a similar degree of acidification can inhibitcortical activity (Schuchmann et al., 2006). Tissue CO2/H+ levels are also regulated by respiratoryactivity. This is accomplished by specialized subsets of neurons known as respiratory chemoreceptorsthat are activated by an increase in CO2/H+ (Guyenet and Bayliss, 2015). This information is thenrelayed to respiratory centers to enhance breathing, and consequently facilitate removal of arterialCO2 in the exhaled breath.The retrotrapezoid nucleus (RTN) is a region critical for respiratory chemoreception(Guyenet and Bayliss, 2015). This region contains a subset of neurons that are intrinsically sensitiveto changes in CO2/H+ (Mulkey et al., 2004: Wang et al., 2013) and relay responses to furtherHawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 1 of 16RESEARCH ARTICLErespiratory control regions, such as the ventral respiratory complex to control breathing rate, inspira-tory amplitude, active expiration and airway patency (Guyenet and Bayliss, 2015; Silva et al.,2016). Disrupting mechanisms by which RTN neurons sense CO2/H+ abolishes ventilatory responsesto CO2 and results in severe apnea (Kumar et al., 2015). Interestingly, RTN astrocytes also supportchemoreception by providing a CO2/H+-dependent purinergic drive that enhances activity of che-mosensitive neurons (Gourine et al., 2010; Wenker et al., 2012). This function of RTN astrocytes isunique to the RTN since astrocytes elsewhere do not respond similarly to changes in pH(Gourine et al., 2010; Sobrinho et al., 2014).For more than a century, vascular CO2 reactivity has been assumed to be a common feature ofthe entire cerebrovascular tree (Ainslie and Duffin, 2009; Roy and Sherrington, 1890). However, ifCO2/H+-induced vasodilation were to occur in chemosensitive regions it would accelerate removalof tissue CO2/H+ and effectively counter-regulate activity of respiratory chemoreceptors (Xie et al.,2006). Therefore, we propose that regulation of vascular tone is specialized to support local neuralnetwork function, and specifically that a chemoreceptor region like the RTN has evolved a means ofmaintaining vascular tone during exposure to high CO2/H+ in a manner that supports the drive tobreathe. Consistent with this, early studies showed that fast breath by breath changes in arterioleCO2 correspond with changes in pH measured at the ventral medullary surface (Millhorn et al.,1984), suggesting tissue pH in this region is not highly buffered, possibly because blood vessels inthis region do not dilate in response to CO2/H+. Furthermore, considering that CO2/H+-evoked ATPrelease appears to be unique feature of RTN chemoreception (Gourine et al., 2010) and since ATPcan mediate vasoconstriction in other brain regions (Kur and Newman, 2014; Peppiatt et al.,2006), we hypothesize that CO2/H+-evoked ATP release will antagonize CO2/H+-vasodilation in theRTN, and thus prevent CO2/H+ washout, further enhancing chemoreceptor function.Consistent with this hypothesis, we find that arterioles in the RTN and cortex are differentially mod-ulated by purinergic signaling during exposure to high CO2/H+. Specifically, we show in vitro and invivo that exposure to CO2/H+ caused vasoconstriction of RTN arterioles but vasodilation of corticaleLife digest We breathe to help us take oxygen into the body and remove carbon dioxide. Ourcells use the oxygen to break down food to release energy, and as they do so they produce carbondioxide as a waste product. Cells release this carbon dioxide back into the bloodstream so that itcan be transported to the lungs to be breathed out. Carbon dioxide also makes the blood moreacidic; if the blood becomes too acidic, tissues and organs may not work properly.The brain uses roughly 25% of the oxygen consumed by the body and is particularly sensitive tothe levels of gases and acidity in the blood. It has been known for more than a century thatincreased carbon dioxide causes blood vessels in the brain to widen, allowing the excess carbondioxide to be carried away quickly. More recent work has shown that increased carbon dioxide alsoactivates neurons called respiratory chemoreceptors. These in turn activate the brain centers thatdrive breathing, causing us to breathe more rapidly to help us remove surplus carbon dioxide.But this scenario contains a paradox. If high levels of carbon dioxide cause widening of the bloodvessels in the brain regions that contain respiratory chemoreceptors, this should, in theory, wash outthat important stimulus, reducing the drive to breathe. So how does the brain prevent this unhelpfulresponse? By studying the brains of adult rats, Hawkins et al. show that different rules apply to thebrain centers that control breathing compared to other areas of the brain. In one such region, if theblood becomes too acidic, support cells called astrocytes release chemical signals called purines.This counteracts the tendency of high carbon dioxide levels to widen blood vessels in this region,and instead causes these vessels to become narrower.This mechanism ensures that local levels of carbon dioxide in respiratory brain centers remain intune with the demands of local networks, thereby maintaining the drive to breathe. The nextchallenges are to identify the molecular mechanisms that control the diameter of blood vessels inbrain regions containing respiratory chemoreceptors, and to find out whether drugs that modulatethese mechanisms have the potential to treat some respiratory conditions.DOI: 10.7554/eLife.25232.002Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 2 of 16Research article Neurosciencearterioles. The CO2/H+-response of RTN arterioles was blocked by bath application of a P2 receptorblocker (pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid; PPADS) and mimicked by a P2Y2/4receptor agonist (UTPgS) but not a P2X receptor agonist (a,b-mATP), suggesting mechanism(s) under-lying this response in the RTN involve purinergic signaling and downstream activation of P2Y2 and/orP2Y4 receptors. To support the possibility that RTN vascular control contributes to respiratory behav-ior, we show that disruption of purinergic regulation of vascular tone or application of a vasodilator(SNP) to the RTN region decreased the ventilatory response to CO2, whereas application of vasocon-strictors (phenylephrine or U46619) potentiated the central chemoreflex. These results suggest for thefirst time that regulation of vascular tone in a respiratory chemoreceptor region is specialized to sup-port the drive to breathe.Results and discussionWe initially tested our hypothesis using the brain slice preparation optimized for detecting increasesor decreases in vascular tone (see Mateials and methods). For these experiments, we targeted arte-rioles based on previously described criteria (Mishra et al., 2014; Filosa et al., 2004). Vessel diame-ter was monitored continuously during exposure to 15% CO2 (pH = 6.9) under baseline conditionsand during purinergic receptor blockade with PPADS. Consistent with our hypothesis, we foundCO2/H+ differentially regulates arteriole diameter in the RTN depending on the function of puriner-gic receptors. For example, exposure to CO2/H+ under control conditions resulted in a vasoconstric-tion of 4.6 ± 0.6% (p<0.0001, N = 34 vessels) (Figure 1C) (estimated by Poiseuille’s law todecrease blood flow by ~20%). Further, we found that CO2/H+-induced constriction of RTN arterio-les was retained in the presence of tetrodotoxin (TTX; 0.5 mM) to block neuronal action potentials(6.1 ± 1.6%, p=0.0103, N = 6 vessels), thus suggesting glutamatergic CO2/H+-activated neurons(Mulkey et al., 2004) are not requisite determinants of this response. Conversely, exposure to CO2/H+ did not cause constriction of RTN arterioles during P2 receptor blockade with 5 mM PPADS (0.1± 0.9%, p=0.4876, N = 8 vessels) (Figure 1A–C). We also tested effects of exogenous ATP to con-firm that it functions as vasoconstrictor of RTN arterioles. Indeed, we found that exposure to ATP(100 mM) resulted in a 5.8 ± 1.5% constriction (p=0.0018, N = 7 vessels) (Figure 1D–E). Theseresults show that purinergic signaling contributes to CO2/H+-dependent control of RTN arterioles.Purinergic receptors are expressed by a wide variety of cell types including neurons, astrocytes,smooth muscle and endothelial cells; in the context of vascular control, the P2 receptors most com-monly implicated in vasoconstriction are several members of the P2X family of ionotropic receptorsand metabotropic P2Y2, P2Y4 and P2Y6 (Burnstock and Ralevic, 2014). Since the concentration ofPPADS used above to block purinergic modulation of RTN arterioles has highest affinities for P2Xand P2Y2 and P2Y4 (Ralevic and Burnstock, 1998), to identify candidate P2 receptors that helpmaintain RTN arteriole tone during exposure to CO2/H+, we tested effects of a selective P2Y2 andP2Y4 receptor agonist (UTPgS) (Lazarowski et al., 1996) and an agonist with high affinity for P2Xreceptors (a,b-mATP) (Burnstock and Kennedy, 1985). We found that bath application of UTPgS(0.5 mM) mimicked effects of CO2/H+ by decreasing diameter of RTN arterioles (3.4 ± 0.6%,p=0.0003, N = 8 vessels), whereas exposure to a,b-mATP (100 mM) minimally affected arteriole tone(p=0.2113, N = 9 vessels) (Figure 1D–E). These results suggest that mechanism(s) underlying puri-nergic-dependent control of RTN arterioles during high CO2/H+ involve activation of Gq-coupledP2Y2/4 receptors. To further support this possibility, we performed immunohistochemistry usingcommercially available P2Y2 and P2Y4 specific antibodies in conjunction with cell-type specificmarkers for endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), vascular smooth muscle cells(a-smooth muscle actin; a-SMA), and astrocytes (anti-glial fibrillary acidic protein; GFAP). Wedetected P2Y2 and P2Y4 immunoreactivity in close proximity to all three cell types associated withRTN arterioles. For example, P2Y2 and P2Y4 labeling appeared as numerous intensely stained punctanear endothelial cells and smooth muscle cells and as smaller more diffuse puncta near astrocytes(Figure 1F–G). The expression of these receptors together with our functional evidence suggestP2Y2/4 receptors contribute to purinergic-dependent vasoconstriction in the RTN during exposure toCO2/H+.Considering that ATP and UTP breakdown products are known to affect vascular tone in otherbrain regions (Burnstock and Ralevic, 2014), we also pharmacologically manipulated P1 receptorsand ectonucleotidase activity before or during exposure to CO2/H+. We found that application ofHawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 3 of 16Research article NeuroscienceCbaselineCO2ATP5 min1 μmAdoCO25 min1 μm PPADSCO2 Δdiameter (%)UTPγSADB########-20010 ***PPADS8-PTPOM1E Δ diameter (%)-20010#######CO2CO2*******ATPUTPγSα,β mATPAdoP2Y2P2Y4IB4 αSMA GFAPIB4 αSMA GFAPFi Fii Fiii FviGi Gii Giii GviRTNendothelial cells smooth muscle astrocytes mergeFigure 1. CO2/H+-induced vasoconstriction of RTN arterioles is mediated by a purinergic dependent mechanism involving P2Y2/4 receptors. (A) trace ofan RTN arteriole diameter show that increasing CO2 in the perfusion media from 5% to 15% (balance air, in TTX) caused vasoconstriction underbaseline conditions but not in PPADS (5 mM). (B) example vessel image under baseline conditions and corresponding fluorescent intensity profile plotsalso show that exposure to high CO2 decreased vessel diameter. Profile plot scale bars: 2000 a.u., 10 mm. (C) summarized results of RTN arterioleresponses to CO2/H+ under baseline conditions (N = 34 vessels) and when P2-receptors were blocked (5 mM PPADS; N = 8 vessels), P1-receptors wereblocked (10 mM 8-PT; N = 7 vessels), or ectonucleotidase activity was inhibited (100 mM POM1; N = 5 vessels). (D) example diameter traces show RTNarterioles constrict in response to bath application of ATP (100 mM) or the selective P2Y2/4 receptor agonist UTPgS (0.5 mM) but dilate when P1receptors are activated by adenosine (Ado; 1 mM). (E) summary data plotted as % diameter change in response to ATP (N = 7 vessels), UTP (N = 8vessels), a,b-mATP (100 mM, preferential P2X agonist; N = 9 vessels) or adenosine (N = 9 vessels). (F–G), immunoreactivity for P2Y2 (F) and P2Y4 (G)receptors was detected as brightly label puncta near endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), arteriole smooth muscle (a-smoothmuscle actin; aSMA), and astrocytes (glial fibrillary acidic protein; GFAP) associated with arterioles in the RTN (N = 3 animals). Arrows identify receptorlabeling close to endothelial or smooth muscle cells and arrowhead identifies receptor labeling of astrocyte processes. Scale bar 10 mM. Hash marksdesignate a difference in mm from baseline as determined by RM-one-way ANOVA and Fishers LSD test or paired t-test and asterisks identifydifferences in CO2/H+-induced % change under baseline conditions vs in the presence of PPADS (C) or ATP vs specific agonist-induced % change (E)(one-way ANOVA and Fishers LSD test); one symbol = p<0.05, two symbols = p<0.01, three symbols = p<0.001, four symbols = p<0.0001.DOI: 10.7554/eLife.25232.003Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 4 of 16Research article Neuroscienceadenosine (1 mM) under control conditions caused vasodilation of RTN arterioles (2.6 ± 0.6%;p=0.0027, N = 9 vessels) (Figure 1D–E); however, blockade of adenosine receptors with 8-phenyl-theophylline (8-PT; 10 mM) had negligible effects on CO2/H+-induced vasoconstriction (3.2 ± 0.4%,p=0.0002, N = 7 vessels) (Figure 1C). Likewise, incubation in sodium metatungstate (POM 1; 100mM) to inhibit ectonucleotidase activity also minimally affected the CO2/H+-vascular response ofRTN arterioles (3.1 ± 0.6%, p=0.0195, N = 5 vessels) (Figure 1C). These results suggest that nucle-oside metabolites are not essential for CO2/H+-dependent regulation of vascular tone in the RTN.Previous evidence (Gourine et al., 2010) suggests CO2/H+-evoked ATP release from RTN astro-cytes is mediated by intracellular Ca2+. Therefore, in the absence of high CO2, pharmacological acti-vation of RTN astrocytes should trigger arteriole constriction by a purinergic-dependent mechanism.We test this by bath application of t-ACPD (50 mM), an mGluR agonist used to elicit Ca2+ elevationsin cortical astrocytes (Filosa et al., 2004; Zonta et al., 2003). Exposure to t-ACPD caused vasocon-striction of RTN arterioles under baseline conditions (3.5 ± 0.5%, p=0.0007, N = 7) but not inPPADS (0.6 ± 0.4%, p=0.2163, N = 7 vessels) (Figure 2A–B,E). These results are consistent withour hypothesis that purinergic signaling, possibly from CO2/H+-sensitive RTN astrocytes(Gourine et al., 2010), serves to maintain tone of arteriole in the RTN during hypercapnia.In marked contrast to the RTN, we found that cortical arterioles dilated in response to astrocyteactivation by t-ACPD. For example, bath application of t-ACPD (50 mM) dilated cortical arterioles by5 min1 μmt-ACPDt-ACPD##5 min1 μmt-ACPDbaselinet-ACPD-15010###t-ACPD Δ diameter (%)RTN cortexRTNCortexACBD Et-ACPD t-ACPDPPADS**t-ACPDPPADSbaselineFigure 2. t-ACPD-mediated astrocyte activation has opposite effects on arteriole diameter in the RTN and cortex.(A) diameter trace of an RTN arteriole show the response of an RTN arteriole to t-ACPD (50 mM) under baselineconditions and during P2-receptor blockade with PPADS (5 mM). (B) example RTN vessel image under baselineconditions and corresponding fluorescent intensity profile plots also show that exposure to tACPD decreasedvessel diameter. (C) diameter trace of an cortical arteriole and corresponding vessel image with example profileplots (D) show that exposure to tACPD (50 mM) increase cortical arteriole diameter. Profile plot scale bars: 2000 a.u., 10 mm. (E) summary from the RTN (N = 7 vessels) and cortex (N = 5 vessels) data show that t-ACPD causedvasoconstriction of RTN arterioles under control conditions but not in the presence of PPADS, suggestingpurinergic signaling most likely from astrocytes mediate constriction of arterioles in the RTN. Conversely, in thecortex t-ACPD caused vasodilation. ##, difference in mm from baseline (paired t-test, p<0.01). ###, difference in mmfrom baseline (RM-one-way ANOVA and Fishers LSD test, p<0.001). **, difference in t-ACPD-induced % changeunder baseline conditions vs in PPADS (paired t-test, p<0.01).DOI: 10.7554/eLife.25232.004Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 5 of 16Research article Neuroscience3.2 ± 0.6% (p=0.0030, N = 5 vessels) (Figure 2C–E). This response is consistent with previous corticalstudies (Filosa et al., 2004; Zonta et al., 2003), and suggests that astrocytes in the RTN and cortexhave fundamentally different roles in regulation of vascular tone. Also consistent with previous work(Ainslie and Duffin, 2009), we confirmed that cortical arterioles dilate in response to CO2/H+(5.7 ±1.1%, p=0.0057, N = 11 vessels) (Figure 3A–C). Interestingly, we also found that the CO2/H+-vascu-lar response of cortical arterioles was reduced to 0.5 ± 0.5% in PPADS (p=0.004, N = 6 vessels)(Figure 3A–C), suggesting involvement of endogenous ATP in cortical arteriole CO2/H+-dilation. Asin the RTN, we also found that endothelial cells, smooth muscle and astrocytes associated with corti-cal arterioles were immunoreactive for P2Y2 and P2Y4 (Figure 3D–E), suggesting the differentialroles of purinergic signaling in these regions is not due to the presence or absence of P2Y2 andP2Y4. However, since the vascular responses to activation of P2Y2/4 can vary depending on whichcells express the receptor (Burnstock and Ralevic, 2014), it remains possible that differentialexpression of P2Y2/4 by endothelial and smooth muscle may mediate vasodilation in the cortex andconstriction in the RTN, respectively. It is also possible that other purinergic receptors contribute toregulation of arteriole tone in these regions. For example, endothelial P2Y1 receptors are known tomediate vasodilation in the cortex (Burnstock and Ralevic, 2014). However, we found that in vivoP2Y2P2Y4endothelial cells smooth muscle astrocytes mergeIB4 αSMA GFAPIB4 αSMA GFAPDi Dii Diii DviEi Eii Eiii Evi1 μmA CCO2BCO2 Δ diameter (%)**##CO2PPADS5 minCO2CO2 PPADSCortex-5051520baselineFigure 3. Cortical arterioles dilate in response to CO2/H+. (A) diameter trace of a cortical arteriole with an example vessel image and fluorescenceprofile plots (B) show that exposure to CO2/H+ caused vasodilation under baseline conditions and this response was blunted by PPADS (5 mM). Profileplot scale bars: 2000 a.u., 10 mm. (C) summary data show CO2/H+-induced vasodilation of cortical arterioles under bassline conditions (N = 11 vessels)but not in PPADS (N = 6 vessels). (D–E), immunoreactivity for P2Y2 (D) and P2Y4 (E) receptors was detected as brightly label puncta near endothelialcells (DyLight 594 Isolectin B4 conjugate; IB4), arteriole smooth muscle (a-smooth muscle actin; aSMA), and astrocytes (glial fibrillary acidic protein;GFAP) associated with cortical arterioles (N = 3 animals). Arrows identify receptor labeling close to endothelial or smooth muscle cells and arrowheadsidentifies receptor labeling of astrocyte processes. Scale bar 10 mM. ##, difference in mm from baseline (paired t test, p<0.01). **, difference in CO2/H+-induced % change under control conditions vs in PPADS (paired t-test, p<0.01).DOI: 10.7554/eLife.25232.005Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 6 of 16Research article Neuroscienceapplication of a selective P2Y1 receptor blocker (MRS2179, 100 mM) had no measurable effect onthe CO2/H+ response of pial vessels in the RTN (3.7 ± 0.8%, vs. saline plus CO2: 4.3 ± 0.7%;p=0.068; N = 5 vessels) or cortex (4.8 ± 0.5%, vs. saline plus CO2: 4.7 ± 0.6%; p=0.24; N = 5 vessels)(data not shown). Alternatively, arachidonic acid metabolites are also potent regulators of vasculartone (MacVicar and Newman, 2015) and recent evidence showed that CO2/H+-mediated vasodila-tion in the cortex and hippocampus involved activation of cyclooxygenase-1 and prostaglandin E2release by astrocytes (Howarth et al., 2017). Considering that purinergic signaling can elicit Ca2+responses in astrocytes to facilitate prostaglandin E2 synthesis (Xu et al., 2003), it remains possiblethat purinergic signaling contributes to cortical CO2/H+ dilation by influencing synthesis and releaseof prostaglandin E2 by astrocytes. However, currently the cellular and molecular basis of purinergicdilation in the cortex remains unknown.To determine whether regulation of vascular tone in the RTN impacts respiratory behavior, wepharmacologically manipulated RTN vessels in anesthetized rats while simultaneously measuring sys-temic blood pressure and diaphragm EMG activity (as a measure of respiratory activity) during expo-sure to high CO2. We found that localized application of the vasoconstrictors phenylephrine (Phe; 1mM) or U46619 (1 mM) to the ventral medullary surface (VMS) enhanced the ventilatory response toCO2 by increasing diaphragm electromyogram (EMG) amplitude 15 ± 2% and 18 ± 1.8%, respec-tively (Figure 4A–C) (p=0.02; N = 6 animals) but with no change in frequency (Figure 4D) (p>0.05;N = 6 animals). Also consistent with the possibility that increased blood flow will facilitate removal oftissue CO2/H+, and thus decrease the stimulus to chemosensitive neurons, we found that VMS appli-cation of the vasodilator sodium nitroprusside (SNP; 1 mM) decreased ventilatory response to CO2by decreasing diaphragm amplitude by 24 ± 2.6% (Figure 4A–C) (p=0.02; N = 6 animals) but withDiaEMG freq (%)10060MAP (mm Hg)12090ABEtCO2 (%)1020000AP (mm Hg)10DiaEMG(a.u.)10DiaEMG(a.u.)EPheU46619salineSNPPheU46619salineSNPDCDiaEMG ampl (%)10050 PheU46619salineSNP***300 sSNPU46619Phesaline SNPsalinePhe U466192 ssalinesalineFigure 4. Local constriction and dilation of RTN vessels reciprocally modulates the ventilatory response to CO2in vivo. (A) end expiratory CO2 (EtCO2),arterial pressure (AP) and diaphragm EMG (DiaEMG) traces show that application of vasoconstrictors (phenylephrine, Phe, 1 mM or U46619, 1 mM) or avasodilator (sodium nitroprusside, SNP, 1 mM) to the RTN increased and decreased the ventilatory response to 7–8% CO2, respectively. (B) diaphragmEMG (DiaEMG) traces expanded in time show that application of Phe, U46619 or SNP, to the VMS in the region of the RTN increased and decreased theDiaEMG amplitude response to 7–8% CO2. (C–E) summary data show effects of saline, SNP, Phe and U46619 applications to the VMS near the RTN onDiaEMG amplitude (N = 6 animals per group) (C), DiaEMG frequency (D) and mean arterial pressure (MAP) (E).*, difference in CO2/H+-induced % changeunder control conditions (saline) vs during vasodilation or vasoconstriction (RM-ANOVA followed by Bonferroni multiple-comparison test, p<0.05).DOI: 10.7554/eLife.25232.006Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 7 of 16Research article Neuroscienceno change in frequency (Figure 4D) (p>0.05; N = 6 animals). These treatments had negligible effecton systemic mean arterial pressure (MAP) (Phe: 110 ± 2; SNP: 110 ± 2; U4619: 108 ± 3 vs. saline:109 ± 2 mmHg; p>0.05) (Figure 4E). These results are consistent with the possibility that regulationof vascular tone in the RTN can influence respiratory output. However, we cannot exclude potentialdirect effects of these drugs on activity of neurons or astrocytes in the region. For example, phenyl-ephrine can directly stimulate activity of chemosensitive RTN neurons (Kuo et al., 2016). Therefore,effects of phenylephrine on chemoreception likely involves both vasoconstriction and direct neuralactivation. It remains to be determined whether U46619 or SNP also affect activity of neurons orastrocytes in the RTN.To determine whether purinergic signaling regulates CO2/H+-mediated constriction in vivo, wefirst measured the diameter of pial vessels on the VMS in the region of the RTN or on the surface ofthe cortex during exposure to high CO2 under control conditions and when P2 receptor are blockedwith PPADS (10 mM). Consistent with our in vitro data, we found that increasing end-expiratory CO2to 9.5–10%, which corresponds with an arterial pH of 7.2 pH units (Guyenet et al., 2005), con-stricted VMS vessels by 4.5 ± 0.5% (p=0.014, N = 5 animals) (Figure 5A–B). However, when P2-receptors are blocked with PPADS (10 mM), increasing inspired CO2 resulted in a vasodilation of 4.3± 0.4% (Figure 5A–B) (p=0.036; N = 5 animals). This suggests that in the RTN, purinergic-mediatedvasoconstriction is working against a background CO2/H+ dilation, possibly mediated by a cyclooxy-genenase/prostroglandine E2-dependent mechanism as described elsewhere in the brain(Howarth et al., 2017). Therefore, disruption of CO2/H+ dilation would be expected to enhancepurinergic-dependent vasoconstriction of RTN arterioles, and thus increase baseline breathing andthe ventilatory response to CO2. Consistent with this, administration of a cyclooxygenase inhibitor(indomethacin) has been shown to increase baseline breathing and the ventilatory response to CO2in humans (Xie et al., 2006). However, it is also possible that decreasing cerebral blood flow glob-ally by indomethacin treatment or cerebral ischemia (Chapman et al., 1979) will cause widespreadacidification leading to enhanced activation of multiple chemoreceptor regions including those out-side the RTN (Nattie and Li, 2012), thus further increasing chemoreceptor drive. It should also benoted that global disruption of cerebrovascular CO2/H+ reactivity as associated with certain patho-logical states including heart failure and stroke (Yonas et al., 1993; Howard et al., 2001) or by sys-temic administration of indomethacin can increase chemoreceptor gain to the extent that breathingbecomes unstable (Fan et al., 2010; Xie et al., 2009). These results underscore the need to under-stand how CO2/H+ regulates vascular tone at other levels of the respiratory circuit.Also consistent with our slice data, we found that exogenous application of ATP (1 mM) or UTPgS(1 mM) constricted VMS vessels by 5.1 ± 0.6% and 5.0 ± 0.5%, respectively (p=0.001; N = 5 ani-mals) (Figure 5A). Conversely, cortical pial vessels dilated in response to an increase in inspired CO2under control conditions (5 ± 0.5%) and after application of 10 mM PPADS (3.2 ± 0.3%) (Figure 5C)(p=0.03; N = 5 animals); however, the CO2/H+-induced vasodilation of cortical vessels was bluntedin the presence of PPADS (p=0.03; N = 5 animals), suggesting endogenous purinergic signaling mayfacilitate dilation of cortical vessels in response to CO2/H+. However, we failed to mimic thisresponse by exogenous application of purinergic agonists; exposure to ATP (1 mM) or UTPgS (1mM) constricted cortical vessels by 4.9 ± 0.7% and 4.1 ± 0.7%, respectively (p=0.024; N = 5 ani-mals). These divergent results are not entirely unexpected since we detected P2Y2 and P2Y4 immu-noreactivity on endothelial cells and smooth muscle of cortical arterioles (Figure 3D–E), andexogenous application of P2 agonists may activate P2 receptors not necessarily targeted by endoge-nous purinergic signaling. Future work is required to identify the source of purinergic drive andeffector P2 receptors contributing to vascular CO2/H+-reactivity in the cortex.In addition, as previously described (Gourine et al., 2005), we found that application of PPADS(10 mM) to the RTN blunted the ventilatory response to CO2 both in terms of DiaEMG frequency(82 ± 3, vs. saline: 97 ± 4%) (p=0.042, N = 5) and amplitude (83 ± 6, vs. saline: 100 ± 5%) (p=0.035,N = 5) (Figure 5D–E). Considering that RTN manipulations of vascular tone preferentially affectrespiratory amplitude (Figure 4A–D), whereas application of PPADS to the same region, which likelydisrupts both direct excitatory effects of ATP on RTN chemoreceptors (Wenker et al., 2012) andindirect effects of ATP on vascular tone (Figure 5A), blunts respiratory frequency and amplitude,suggests that purinergic signaling in the RTN might regulate discrete aspects of respiratory output.In the cortex, application of PPADS had no measurable effect on CO2-induced changes in DiaEMGfrequency (p=0.33, N = 5 animals) and amplitude (p=0.42, N = 5) (Figure 5F). Together withHawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 8 of 16Research article Neuroscienceprevious evidence, these findings suggest that purinergic signaling contributes to RTN chemorecep-tion by directly activating RTN neurons (Gourine et al., 2010) and indirectly by opposing CO2/H+-vasodilation.It should be acknowledged that our study is limited to the use of pharmacological tools thatpotentially have off-target effects. We have minimized this concern by (i) using low concentrations ofPPADS that are reported to be specific for P2 receptor’s (Lorier et al., 2004); (ii) mimicking CO2/H+-induced vasoconstriction in vitro and in vivo by exogenous application of ATP and a specificP2Y2/4 receptor agonist (UTPgS), but not by a non-specific P2X receptor agonist (a,b-mATP); (iii) con-firming that candidate P2Y2 and P2Y4 receptors are expressed in the RTN at the astrocyte-arterioleBcontrolPPADS 10% CO25% CO2basilar arteryDEtCO2 (%)1020000AP (mm Hg)10DiaEMG(a.u.)PPADScontrol50 sA0Δ vessel diameter (%)10 CortexCCO2 + PPADS (10 μM) CO2 + saline0Δ vessel diameter (%)10-10RTNATP*ECO2 ΔDiaEMG freq (%) saline PPADS (10 μM)CO2 ΔDiaEMG amp (%) F 1200CO2 ΔDiaEMG freq (%) CO2 ΔDiaEMG amp (%) 120cortexRTN120000120***UTPγSATPUTPγS-1010 ##### ###10 *Figure 5. Purinergic signaling opposes CO2/H+-dilation of VMS pial vessels in vivo and contributes to the ventilatory response to CO2. (A) summarydata plotted as % change in RTN pial vessel diameter in response to an increase in end expiratory CO2 after VMS application of saline (100 nL; N = 5animals) or PPADS (10 mM, 100 nL; N = 5 animals). Also shown are the vascular responses to VMS application of ATP (1 mM, 100 nL; N = 5) and UTPgS(1 mM, 100 nL; N = 5 animals). (B) Photomicrographs (40X) show pial vessel distribution on the VMS, arrow; representative vessel analyzed. (C) Summarydata shows the response of cortical pial vessels to CO2 after local application of saline or PPADS (10 mM, 100 nL; N = 5 animals). Also shown arevascular responses to exogenous application of ATP (1 mM - 100 nL; N = 5 animals) or UTPgS (1 mM, 100 nL; N = 5). (D) End expiratory CO2 (EtCO2),arterial pressure (AP) and diaphragm EMG (DiaEMG) traces show that bilateral VMS application of PPADS (10 mM, 100 nL) attenuated the ventilatoryresponse to CO2. (E) summary data show CO2-induced changes in DiaEMG frequency and amplitude after bilateral VMS application of saline and PPADS(10 mM; N = 5 animals). (F) summary data show that PPADS (10 mM) application to the cortex surface had no measurable effect on CO2-inducedchanges in DiaEMG frequency and amplitude (N = 5 animals). Hash marks designate a difference in mm from baseline (RM-ANOVA followed byBonferroni multiple-comparison test, #, p<0.05). Asterisks identify a difference in CO2/H+–induced % change under control conditions (saline) vs in thepresence of PPADS (RM-ANOVA followed by Bonferroni multiple-comparison test, *, p<0.05; **, p<0.01) (panels A and C) or paired t-test (panel E, *,p<0.05).DOI: 10.7554/eLife.25232.007Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 9 of 16Research article Neuroscienceinterface; and (iv) for in vitro experiments by confirming that CO2/H+-induced vasoconstriction wasretained when neuronal action potentials were blocked with TTX. Therefore, our results suggest thatpurinergic signaling possibly through P2Y2/4 receptors in the RTN provides specialized control ofvascular tone by preventing CO2/H+-induced dilation. Our results also suggest that regulation ofvascular tone in the RTN contributes functionally to the ventilatory response to CO2. This is the firstevidence to suggest that regulation of vascular tone in a chemoreceptor region contributes to thedrive to breathe. This discovery may be of fundamental importance to understanding how regulationof vascular tone impacts neural network function and ultimately behavior.Materials and methodsBrainstem slice preparationAll procedures were performed in accordance with National Institutes of Health and University ofConnecticut Animal Care and Use Guidelines. A total of 93 adult Sprague Dawley rats (60–100 daysof age) were used for in vitro experiments. Animals were decapitated under isoflurane anesthesia,and transverse brainstem slices (200 mm) were prepared using a vibratome in ice-cold substitutedartificial cerebrospinal fluid (aCSF) solution containing (in mm): 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2,1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, with 0.4 mM L-ascorbic acid added (all Sigma-Aldrich).Slices were incubated for ~30 min at 37˚C and subsequently at room temperature in aCSF. Prior toimaging slices were incubated for 1 hr with 10 mg/ml TRITC-lectin conjugate (Sigma-Aldrich, St. Louis, MO) or 6 mg/ml DyLight 594 Isolectin B4 conjugate (Vector Labortories) to labelendothelial cells as previously described (Mishra et al., 2014). Slicing solutions were equilibratedwith a high oxygen carbogen gas (95% O2-5% CO2) (Mulkey et al., 2001).Imaging of arterioles in brainstem slicesAn individual slice containing the RTN was transferred to a recording chamber mounted on a fixed-stage microscope (Zeiss Axioskop FS) and perfused continuously (~2 ml min1) with aCSF bubbledwith 5% CO2, 21% O2 (balance N2; pHo ~7.35; 370 C) (Gordon et al., 2008). Hypercapnic solutionwas made by equilibrating aCSF with 15% CO2, 21% O2 (balance N2; pHo ~6.90; 370 C). Arterioleswere identified as previously described (Mishra et al., 2014; Filosa et al., 2004) by the following cri-teria: clear evidence of vasomotion under IR-DIC, bulky fluorescent labeling and a thick layer ofsmooth muscle surrounding the vessel lumen. Vessels that appeared collapsed and unhealthy wereexcluded, as were those with little fluorescence staining and thin walls, indicative of a lack of smoothmuscle (Mishra et al., 2014). All arterioles selected for analysis had a resting luminal diameter ofbetween 8–45 mm; RTN vessels were located within 200 mm of the ventral surface and below the cau-dal end of the facial motor nucleus and cortical vessels were located in layers 1–3.For an experiment, fluorescent images were acquired at a rate of 1 frame/20 s using a x40 waterobjective, a Clara CCD Andor camera and NIS Elements software. To induce a partially constrictedstate we bath applied a thromboxane A2 receptor agonist (U46619; 125 nM; Sigma-Aldrich, St.Louis, MO). At this concentration, U46619 has been shown to decrease vessel diameter by 20–30%under similar experimental conditions, thus allowing assessment of both vasodilation and vasocon-striction (Filosa et al., 2004; Girouard et al., 2010). In the continued presence of U-46619, we thencharacterized the effects of hypercapnia, ATP (100 mM; Sigma-Aldrich, St. Louis, MO), a,b-methyle-neATP (100 mM), UTPgS (0.5 mM), and adenosine (1 mM), or the mGluR agonist t-ACPD ((±)1-amino-cyclopentane-trans-1,3-dicarboxylic acid; 50 mM) alone or in the presence of P2-recepetor blockerPPADS (5 mM; Tocris Bioscience, Minneapolis, MN), the P1 receptor antagonist 8-Phenyltheophylline(8-PT; 10 mM; Sigma) or the Ecto-NTPDase antagonist sodium metatungstate (POM 1; 100 mM; Toc-ris). In a subset of experiments we also tested CO2, ATP and PPADS in the presence of TTX (0.5 mM;Alomone Laboratories). As previously described (Girouard et al., 2010), at the end of each experi-ment we assessed arteriole viability by inducing a constriction with a high K+ solution (60 mM) andthen maximal dilation with a Ca2+ free solution containing EGTA (5 mM), papaverine (200 mM, aphosphodiesterase inhibitor) and diltiazem (50 mM, to block L-type Ca channels). Vessels from bothregions of interest (RTN and cortex) show similar responses to these conditions, and vessels that didnot respond were excluded from analysis.Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 10 of 16Research article NeuroscienceImmunohistochemistryRat brain slices were prepared from three rats and labelled with DyLight 594 Isolectin B4 conjugateas above followed by immersion fixation overnight in 1% paraformaldehyde in pH 7.4 PBS at 4˚C.Excess fixative was removed by three washes in PBS, and prior to antibody incubations, tissue sec-tions were treated to unmask epitopes with 0.2 mg/ml pepsin (Sigma-Aldrich, St. Louis, MO) in 0.2M HCl for 10 mins at 37˚C (Corteen et al., 2011) followed by three washes in PBS for P2Y4 labellingonly. A blocking stage was then performed by incubating tissue in 10% normal horse serum in PBSwith 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 1 hr at room temperature (RT). Sectionswere then incubated overnight at RT with primary antibodies diluted in blocking solution as follows:1:200 rabbit anti-P2Y2 (RRID: AB_2040078) or P2Y4 receptor (RRID: AB_2040080) (AlomoneLabs, Alomone Labs, Jerusalem Israel), 1:200 chicken anti-glial fibrillary acidic protein (RRID: AB_177521) (Chemicon) and 1:500 mouse anti-a-smooth muscle actin (RRID: AB_262054) (Sigma-Aldrich,St. Louis, MO). After washes in PBS, tissues were incubated for 1 hr at RT with the appropriate sec-ondary antibodies raised in donkey, conjugated with 488DyLight 1:800 (RRID: AB_2492289),405DyLight 1:200 (RRID: AB_2340373) or Cy5 1:500 (RRID: AB_2340820) (Jackson ImmunoresearchLaboratories). Sections were washed in PBS again before being mounted with Vectasheild (Vector-Labs). Images were acquired using a Nikon A1R confocal microscope (Nikon Instruments), with mini-mal background staining observed in the control reactions where primary antibodies were omittedor P2 receptor antibodies were pre-absorbed with control antigen prior to exposure to tissues. Forconfocal photomicrographs, two-dimensional flattened images of the projected z-stacks arepresented.In vivo preparationAnimal use was in accordance with guidelines approved by the University of Sa˜o Paulo Animal Careand Use Committee. A total of 21 adult male Wistar rats (60–90 days of age; 270–310 g) were usedfor in vivo experiments. General anesthesia was induced with 5% halothane in 100% O2. A tracheos-tomy was made and the halothane concentration was reduced to 1.4–1.5% until the end of surgery.The femoral artery was cannulated (polyethylene tubing, 0.6 mm o.d., 0.3 mm i.d., Scientific Com-modities) for measurement of arterial pressure (AP). The femoral vein was cannulated for administra-tion of fluids and drugs. Rats were placed supine onto a stereotaxic apparatus (Type 1760; HarvardApparatus) on a heating pad and core body temperature was maintained at a minimum of 36.5˚C viaa thermocouple. The trachea was cannulated. Respiratory flow was monitored via a flow head con-nected to a transducer (GM Instruments) and CO2 via a capnograph (CWE, Inc,) connected to thetracheal tube. Paired EMG wire electrodes (AM-System) were inserted into the diaphragm muscle torecord respiratory-related activity. After the anterior neck muscles were removed, a basio-occipitalcraniotomy exposed the ventral medullary surface, and the dura was resected. After bilateral vagot-omy, the exposed tissue around the neck and the mylohyoid muscle was covered with mineral oil toprevent drying. Baseline parameters were allowed to stabilize for 30 min prior to recording.In vivo recordings of physiological variablesMean arterial pressure (MAP), diaphragm muscle activity (DiaEMG) and end-expiratory CO2 (etCO2)were digitized with a micro1401 (Cambridge Electronic Design), stored on a computer, and proc-essed off-line with version 6 of Spike 2 software (Cambridge Electronic Design). Integrated dia-phragm activity (ÐDiaEMG) was collected after rectifying and smoothing (t = 0.03) the original signal,which was acquired with a 300–3000 Hz bandpass filter. Noise was subtracted from the recordingsprior to performing any calculations of evoked changes in DiaEMG. A direct physiological comparisonof the absolute level of DiaEMG activity across nerves is not possible because of non-physiologicalfactors (e.g., muscle electrode contact, size of muscle bundle) and the ambiguity in interpreting howa given increase in voltage in one EMG relates to an increase in voltage in another EMG. Thus, mus-cle activity was defined according to its baseline physiological state, just prior to their activation.The baseline activity was normalized to 100%, and the percent change was used to compare themagnitude of increases or decreases across muscle from those physiological baselines.Hawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 11 of 16Research article NeuroscienceIn vivo experimental protocolEach in vivo experiment began by testing responses to hypercapnia by adding pure CO2 to thebreathing air supplied by artificial ventilation. In each rat, the addition of CO2 was monitored toreach a maximum end-expiratory CO2 between 9.5% and 10%, which corresponds with an estimatedarterial pH of 7.2 based on the following equation: pHa = 7.955–0.7215 log10 (ETCO2)(Guyenet et al., 2005). This maximum end-expiratory CO2 was maintained for 5 min and thenreplaced by 100% O2.To determine whether local regulation of vascular tone in the region of the RTN contributes tothe CO2/H+-dependent drive to breathe, we made bilateral injections of saline, phenylephrine (Phe,1 mM), U46619 (1 mM) or sodium nitroprusside (SNP, 1 mM) while monitoring DiaEMG amplitude andfrequency. These drugs were diluted to 1 mM with sterile saline (pH 7.4) and applied using single-barrel glass pipettes (tip diameter of 25 mm) connected to a pressure injector (Picospritzer III, ParkerHannifin Corp, Cleveland, OH). For each injection, we delivered a volume of 100 nl over a period of5s. Injections in the VMS region were placed 1.9 mm lateral from the basilar artery, 0.9 mm rostralfrom the most rostral hypoglossal nerve rootlet, and at the VMS. The second injection was made 1–2min later at the same level on the contralateral side. In separate series of experiments saline, ATP (1mM), UTPgS (1 mM) or PPADS (10 mM) were applied similarly to the VMS to test the effect of P2-blockade on vascular CO2/H+ reactivity and the ventilatory response to CO2. A cranial optical win-dow was prepared using standard protocols previously described (Kim et al., 2015). Briefly, a dentaldrill (Midwest Stylus Mini 540S, Dentsply International) was used to thin a circumference of a 4–5mm-diameter circular region of the skull over somatosensory cortex (stereotaxic coordinates: AP:1.8 mm from bregma and ML: 2.8 mm lateral to the midline). For the VMS, the anterior neckmuscles were removed, a basio-occipital craniotomy exposed the ventral medullary surface, and thedura was resected. Pial vessels in the VMS had an average and were located 1.9 mm lateral from thebasilar artery and 0.9 mm rostral to the most rostral portion of the hypoglossal nerve rootlet. Boththinned bone were lifted with forceps. The surface of the cortex or the VMS were cleaned withbuffer containing (in mmol/L) the following: 135 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH7.3., and a chamber (home-made 1.1-cm-diameter plastic ring was glued with dental acrylic cementattached to a baseplate). The chamber was sealed with a circular glass coverslip (#1943–00005,Bellco). The baseplate was affixed to the Digital Camera (Sony, DCR-DVD3-5) and a light microscopewas used for vessel imaging (x40 magnification).Image analysisVessel diameter was determined offline using ImageJ. For in vitro experiments, stack registrationand selection of linear regions of interest (ROIs) (three regions per arteriole) was carried out. LinearROI’s were used to create a fluorescent intensity profile plot as described previously (Mishra et al.,2014) and a macro (available at https://github.com/omsai/blood-vessel-diameter [Nanda, 2017];copy archived at https://github.com/elifesciences-publications/blood-vessel-diameter).was used todetermine the peak-peak distance as a measure of vessel diameter for each frame. In most cases, wealso confirmed vessel diameter by manually measuring at least one point per frame. In vivo data wasalso analyzed using three linear ROI’s drawn perpendicular to the vessel in each image and theDiameter plug-in function in ImageJ was used to calculate changes in diameter (Kim et al., 2015;Fischer et al., 2010).Data analysis and statisticsAll in vitro images were calibrated and pixel distances converted to diameter (mM) and these valueswere used for analysis of CO2/H+ or agonist-induced changes in vessel diameter from baseline byRM-one-way ANOVA and Fishers LSD test or paired t-test. Hash marks were used to identity differ-ences from baseline (vasoconstriction or vasodilation). Mean percent changes in vessel diameter wasused to compare between agonist responses or CO2/H+ responses under control conditions vs dur-ing purinergic receptor blockade or in the presence of an ectonucleotidase inhibitor by one-wayANOVA and Fishers LSD test or t-test. Asterisks were used to identify differences in % change in ves-sel diameter. For in vivo experiments, respiratory muscle activity was calculated as the mean ampli-tude of the integrated DiaEMG over 20 respiratory cycles. To obtain control values, the 20 cyclespreceding each experimental manipulation for all parameters were averaged. Under hypercapnicHawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 12 of 16Research article Neuroscienceconditions, measurements from the 20 cycles preceding stimulus cessation were averaged. Respira-tory frequency (fR) was (1/(inspiratory time + expiratory time). Differences in the ventilatory responseto CO2 were determined using either paired t-test or one-way analysis of variance (ANOVA) fol-lowed by the Bonferroni multiple-comparisons as appropriate. Power analysis was used to determinesample size, all data sets were tested for normality using Shapiro-Wilk test. All data values areexpressed as means ± SEM and specific statistical test and relevant p values are reported in the textand figure legends.AcknowledgementsWe thank David Attwell and Anusha Mishra (University College London), Catherine Hall (Universityof Sussex) and Pariksheet Nanda (University of Connecticut) for assistance with vessel image analysis.This work was supported by funds from the National Institutes of Health Grants HL104101 (DKM),HL126381 (VEH), and P01-HL-095488, R01-HL-121706, R37-DK-053832 and R01-HL-131181 (MTN).Additional support comes from the Totman Medical Research Trust (MTN), Fondation Leducq(MTN), EC Horizon 2020 (MTN), Connecticut Department of Public Health Grant 150263 (DKM), andpublic funding from the Sa˜o Paulo Research Foundation (FAPESP) Grants 2014/22406-1 (ACT),2016/22069–0 (TSM), 2015/23376–1 (TSM) and FAPESP fellowship 2014/07698-6 (MRML); ConselhoNacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq); grant: 471263/2013–3 (ACT) and471283/2012–6 (TSM). CNPq fellowship 301651/2013–2 (ACT) and 301904/2015–4 (TSM).Additional informationFundingFunder Grant reference number AuthorNational Institutes of Health HL104101 Daniel K MulkeyTotman Medical ResearchTrustMark T NelsonFondation Leducq Mark T NelsonEuropean Commission Horizon 2020 Mark T NelsonConnecticut department ofpublic health150263 Daniel K MulkeyFundac¸a˜o de Amparo a` Pes-quisa do Estado de Sa˜o Paulo2014/22406-1 Ana C TakakuraConselho Nacional de Desen-volvimento Cientı´fico e Tecno-lo´gico471263/2013-3 Ana C TakakuraNational Institutes of Health HL126381 Virginia E HawkinsNational Institutes of Health HL095488 Mark T NelsonNational Institutes of Health DK053832 Mark T NelsonNational Institutes of Health HL131181 Mark T NelsonFundac¸a˜o de Amparo a` Pes-quisa do Estado de Sa˜o Paulo2016/22069-0 Thiago S MoreiraFundac¸a˜o de Amparo a` Pes-quisa do Estado de Sa˜o Paulo2015/23376-1 Thiago S MoreiraFundac¸a˜o de Amparo a` Pes-quisa do Estado de Sa˜o Paulo2014/07698-6 Milene R Malheiros-LimaConselho Nacional de Desen-volvimento Cientı´fico e Tecno-lo´gico471283/2012-6 Thiago S MoreiraConselho Nacional de Desen-volvimento Cientı´fico e Tecno-lo´gico30165½013-2 Ana C TakakuraHawkins et al. eLife 2017;6:e25232. DOI: 10.7554/eLife.25232 13 of 16Research article NeuroscienceConselho Nacional de Desen-volvimento Cientı´fico e Tecno-lo´gico301904/2015-4 Thiago S MoreiraThe funders had no role in study design, data collection and interpretation, or the decision tosubmit the work for publication.Author contributionsVEH, Data curation, Formal analysis, Supervision, Project administration, Writing—review and edit-ing; ACT, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Proj-ect administration, Writing—review and editing; AT, TD, Data curation, Formal analysis,Methodology, Writing—review and editing; MRM-L, CMC, EMR, Data curation, Formal analysis,Investigation, Writing—review and editing; ICW, Conceptualization, Data curation, Formal analysis,Investigation, Writing—review and editing; MTN, Conceptualization, Supervision, Funding acquisi-tion, Investigation, Methodology, Writing—review and editing; TSM, Conceptualization, Data cura-tion, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration,Writing—review and editing; DKM, Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editingAuthor ORCIDsVirginia E Hawkins, http://orcid.org/0000-0001-9505-6776Daniel K Mulkey, http://orcid.org/0000-0002-7040-3927EthicsAnimal experimentation: All in vitro procedures were performed in accordance with National Insti-tutes of Health and University of Connecticut Animal Care and Use Guidelines (protocol # A16-034).All in vivo procedures were performed in accordance with guidelines approved by the University ofSa˜o Paulo Animal Care and Use Committee (protocol # 112/2015).ReferencesAinslie PN, Duffin J. 2009. 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CO(2) in the spotlight

Abstract

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CO(2) in the spotlight

Abstract

Extracted data

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