We breathe to help us take oxygen into the body and remove carbon dioxide. Our cells use the oxygen to break down food to release energy, and as they do so they produce carbon dioxide as a waste product. Cells release this carbon dioxide back into the bloodstream so that it can be transported to the lungs to be breathed out. Carbon dioxide also makes the blood more acidic; 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 to the levels of gases and acidity in the blood. It has been known for more than a century that increased carbon dioxide causes blood vessels in the brain to widen, allowing the excess carbon dioxide to be carried away quickly. More recent work has shown that increased carbon dioxide also activates neurons called respiratory chemoreceptors. These in turn activate the brain centers that drive 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 blood vessels in the brain regions that contain respiratory chemoreceptors, this should, in theory, wash out that important stimulus, reducing the drive to breathe. So how does the brain prevent this unhelpful response? By studying the brains of adult rats, Hawkins et al. show that different rules apply to the brain centers that control breathing compared to other areas of the brain. In one such region, if the blood 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 in tune with the demands of local networks, thereby maintaining the drive to breathe. The next challenges are to identify the molecular mechanisms that control the diameter of blood vessels in brain regions containing respiratory chemoreceptors, and to find out whether drugs that modulate these mechanisms have the potential to treat some respiratory conditions.

Cerebral blood flow is highly sensitive to changes in CO₂/H⁺. An increase in CO₂/H⁺ causes vasodilation 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 is only a narrow range that is conducive to normal neural function. For example, a modest alkalosis of just 0.2 pH units can trigger seizure activity; conversely, a similar degree of acidification can inhibit cortical activity (Schuchmann et al., 2006). Tissue CO₂/H⁺ levels are also regulated by respiratory activity. This is accomplished by specialized subsets of neurons known as respiratory chemoreceptors that are activated by an increase in CO₂/H⁺ (Guyenet and Bayliss, 2015). This information is then relayed to respiratory centers to enhance breathing, and consequently facilitate removal of arterial CO₂ 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 sensitive to changes in CO₂/H⁺ (Mulkey et al., 2004: Wang et al., 2013) and relay responses to further respiratory control regions, such as the ventral respiratory complex to control breathing rate, inspiratory 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 responses to CO₂ and results in severe apnea (Kumar et al., 2015). Interestingly, RTN astrocytes also support chemoreception by providing a CO₂/H⁺-dependent purinergic drive that enhances activity of chemosensitive neurons (Gourine et al., 2010; Wenker et al., 2012). This function of RTN astrocytes is unique 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 of the entire cerebrovascular tree (Ainslie and Duffin, 2009; Roy and Sherrington, 1890). However, if CO₂/H⁺-induced vasodilation were to occur in chemosensitive regions it would accelerate removal of 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 neural network function, and specifically that a chemoreceptor region like the RTN has evolved a means of maintaining vascular tone during exposure to high CO₂/H⁺ in a manner that supports the drive to breathe. Consistent with this, early studies showed that fast breath by breath changes in arteriole CO₂ 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 in this region do not dilate in response to CO₂/H⁺. Furthermore, considering that CO₂/H⁺-evoked ATP release appears to be unique feature of RTN chemoreception (Gourine et al., 2010) and since ATP can 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 the RTN, 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 modulated by purinergic signaling during exposure to high CO₂/H⁺. Specifically, we show in vitro and in vivo that exposure to CO₂/H⁺ caused vasoconstriction of RTN arterioles but vasodilation of cortical arterioles. The CO₂/H⁺-response of RTN arterioles was blocked by bath application of a P2 receptor blocker (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid; PPADS) and mimicked by a P2Y_2/4 receptor agonist (UTPγS) but not a P2X receptor agonist (α,β-mATP), suggesting mechanism(s) underlying this response in the RTN involve purinergic signaling and downstream activation of P2Y₂ and/or P2Y₄ receptors. To support the possibility that RTN vascular control contributes to respiratory behavior, 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 vasoconstrictors (phenylephrine or U46619) potentiated the central chemoreflex. These results suggest for the first time that regulation of vascular tone in a respiratory chemoreceptor region is specialized to support the drive to breathe.

We initially tested our hypothesis using the brain slice preparation optimized for detecting increases or decreases in vascular tone (see Mateials and methods). For these experiments, we targeted arterioles based on previously described criteria (Mishra et al., 2014; Filosa et al., 2004). Vessel diameter was monitored continuously during exposure to 15% CO₂ (pH = 6.9) under baseline conditions and during purinergic receptor blockade with PPADS. Consistent with our hypothesis, we found CO₂/H⁺ differentially regulates arteriole diameter in the RTN depending on the function of purinergic receptors. For example, exposure to CO₂/H⁺ under control conditions resulted in a vasoconstriction of −4.6 ± 0.6% (p<0.0001, N = 34 vessels) (Figure 1C) (estimated by Poiseuille’s law to decrease blood flow by ~20%). Further, we found that CO₂/H⁺-induced constriction of RTN arterioles was retained in the presence of tetrodotoxin (TTX; 0.5 µM) 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 µM PPADS (−0.1 ± 0.9%, p=0.4876, N = 8 vessels) (Figure 1A–C). We also tested effects of exogenous ATP to confirm that it functions as vasoconstrictor of RTN arterioles. Indeed, we found that exposure to ATP (100 µM) resulted in a −5.8 ± 1.5% constriction (p=0.0018, N = 7 vessels) (Figure 1D–E). These results show that purinergic signaling contributes to CO₂/H⁺-dependent control of RTN arterioles.

Figure 1

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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 commonly implicated in vasoconstriction are several members of the P2X family of ionotropic receptors and metabotropic P2Y₂, P2Y₄ and P2Y₆ (Burnstock and Ralevic, 2014). Since the concentration of PPADS used above to block purinergic modulation of RTN arterioles has highest affinities for P2X and P2Y₂ and P2Y₄ (Ralevic and Burnstock, 1998), to identify candidate P2 receptors that help maintain RTN arteriole tone during exposure to CO₂/H⁺, we tested effects of a selective P2Y₂ and P2Y₄ receptor agonist (UTPγS) (Lazarowski et al., 1996) and an agonist with high affinity for P2X receptors (α,β-mATP) (Burnstock and Kennedy, 1985). We found that bath application of UTPγS (0.5 µM) mimicked effects of CO₂/H⁺ by decreasing diameter of RTN arterioles (−3.4 ± 0.6%, p=0.0003, N = 8 vessels), whereas exposure to α,β-mATP (100 µM) minimally affected arteriole tone (p=0.2113, N = 9 vessels) (Figure 1D–E). These results suggest that mechanism(s) underlying purinergic-dependent control of RTN arterioles during high CO₂/H⁺ involve activation of Gq-coupled P2Y_2/4 receptors. To further support this possibility, we performed immunohistochemistry using commercially available P2Y₂ and P2Y₄ specific antibodies in conjunction with cell-type specific markers for endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), vascular smooth muscle cells (α-smooth muscle actin; α-SMA), and astrocytes (anti-glial fibrillary acidic protein; GFAP). We detected P2Y₂ and P2Y₄ immunoreactivity in close proximity to all three cell types associated with RTN arterioles. For example, P2Y₂ and P2Y₄ labeling appeared as numerous intensely stained puncta near 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 suggest P2Y_2/4 receptors contribute to purinergic-dependent vasoconstriction in the RTN during exposure to CO₂/H⁺.

Considering that ATP and UTP breakdown products are known to affect vascular tone in other brain regions (Burnstock and Ralevic, 2014), we also pharmacologically manipulated P1 receptors and ectonucleotidase activity before or during exposure to CO₂/H⁺. We found that application of adenosine (1 µM) 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-phenyltheophylline (8-PT; 10 µM) 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; 100 µM) to inhibit ectonucleotidase activity also minimally affected the CO₂/H⁺-vascular response of RTN arterioles (−3.1 ± 0.6%, p=0.0195, N = 5 vessels) (Figure 1C). These results suggest that nucleoside 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 astrocytes is mediated by intracellular Ca²⁺. Therefore, in the absence of high CO₂, pharmacological activation of RTN astrocytes should trigger arteriole constriction by a purinergic-dependent mechanism. We test this by bath application of t-ACPD (50 µM), an mGluR agonist used to elicit Ca²⁺ elevations in cortical astrocytes (Filosa et al., 2004; Zonta et al., 2003). Exposure to t-ACPD caused vasoconstriction of RTN arterioles under baseline conditions (−3.5 ± 0.5%, p=0.0007, N = 7) but not in PPADS (−0.6 ± 0.4%, p=0.2163, N = 7 vessels) (Figure 2A–B,E). These results are consistent with our 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.

Figure 2

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In marked contrast to the RTN, we found that cortical arterioles dilated in response to astrocyte activation by t-ACPD. For example, bath application of t-ACPD (50 µM) dilated cortical arterioles by 3.2 ± 0.6% (p=0.0030, N = 5 vessels) (Figure 2C–E). This response is consistent with previous cortical studies (Filosa et al., 2004; Zonta et al., 2003), and suggests that astrocytes in the RTN and cortex have 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⁺-vascular 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. As in the RTN, we also found that endothelial cells, smooth muscle and astrocytes associated with cortical arterioles were immunoreactive for P2Y₂ and P2Y₄ (Figure 3D–E), suggesting the differential roles of purinergic signaling in these regions is not due to the presence or absence of P2Y₂ and P2Y₄. However, since the vascular responses to activation of P2Y_2/4 can vary depending on which cells express the receptor (Burnstock and Ralevic, 2014), it remains possible that differential expression of P2Y_2/4 by endothelial and smooth muscle may mediate vasodilation in the cortex and constriction in the RTN, respectively. It is also possible that other purinergic receptors contribute to regulation of arteriole tone in these regions. For example, endothelial P2Y₁ receptors are known to mediate vasodilation in the cortex (Burnstock and Ralevic, 2014). However, we found that in vivo application of a selective P2Y₁ receptor blocker (MRS2179, 100 µM) had no measurable effect on the 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 vascular tone (MacVicar and Newman, 2015) and recent evidence showed that CO₂/H⁺-mediated vasodilation in the cortex and hippocampus involved activation of cyclooxygenase-1 and prostaglandin E2 release 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 possible that purinergic signaling contributes to cortical CO₂/H⁺ dilation by influencing synthesis and release of prostaglandin E2 by astrocytes. However, currently the cellular and molecular basis of purinergic dilation in the cortex remains unknown.

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To determine whether regulation of vascular tone in the RTN impacts respiratory behavior, we pharmacologically manipulated RTN vessels in anesthetized rats while simultaneously measuring systemic blood pressure and diaphragm EMG activity (as a measure of respiratory activity) during exposure to high CO₂. We found that localized application of the vasoconstrictors phenylephrine (Phe; 1 µM) or U46619 (1 µM) to the ventral medullary surface (VMS) enhanced the ventilatory response to CO₂ by increasing diaphragm electromyogram (EMG) amplitude 15 ± 2% and 18 ± 1.8%, respectively (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 of tissue CO₂/H⁺, and thus decrease the stimulus to chemosensitive neurons, we found that VMS application of the vasodilator sodium nitroprusside (SNP; 1 µM) decreased ventilatory response to CO₂ by decreasing diaphragm amplitude by 24 ± 2.6% (Figure 4A–C) (p=0.02; N = 6 animals) but with no change in frequency (Figure 4D) (p>0.05; N = 6 animals). These treatments had negligible effect on 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 regulation of vascular tone in the RTN can influence respiratory output. However, we cannot exclude potential direct effects of these drugs on activity of neurons or astrocytes in the region. For example, phenylephrine can directly stimulate activity of chemosensitive RTN neurons (Kuo et al., 2016). Therefore, effects of phenylephrine on chemoreception likely involves both vasoconstriction and direct neural activation. It remains to be determined whether U46619 or SNP also affect activity of neurons or astrocytes in the RTN.

Figure 4

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To determine whether purinergic signaling regulates CO₂/H⁺-mediated constriction in vivo, we first measured the diameter of pial vessels on the VMS in the region of the RTN or on the surface of the cortex during exposure to high CO₂ under control conditions and when P2 receptor are blocked with PPADS (10 µM). Consistent with our in vitro data, we found that increasing end-expiratory CO₂ to 9.5–10%, which corresponds with an arterial pH of 7.2 pH units (Guyenet et al., 2005), constricted 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 µM), 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-mediated vasoconstriction is working against a background CO₂/H⁺ dilation, possibly mediated by a cyclooxygenenase/prostroglandine E2-dependent mechanism as described elsewhere in the brain (Howarth et al., 2017). Therefore, disruption of CO₂/H⁺ dilation would be expected to enhance purinergic-dependent vasoconstriction of RTN arterioles, and thus increase baseline breathing and the 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 CO₂ in humans (Xie et al., 2006). However, it is also possible that decreasing cerebral blood flow globally by indomethacin treatment or cerebral ischemia (Chapman et al., 1979) will cause widespread acidification leading to enhanced activation of multiple chemoreceptor regions including those outside the RTN (Nattie and Li, 2012), thus further increasing chemoreceptor drive. It should also be noted that global disruption of cerebrovascular CO₂/H⁺ reactivity as associated with certain pathological states including heart failure and stroke (Yonas et al., 1993; Howard et al., 2001) or by systemic administration of indomethacin can increase chemoreceptor gain to the extent that breathing becomes unstable (Fan et al., 2010; Xie et al., 2009). These results underscore the need to understand how CO₂/H⁺ regulates vascular tone at other levels of the respiratory circuit.

Figure 5

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Also consistent with our slice data, we found that exogenous application of ATP (1 mM) or UTPγS (1 mM) constricted VMS vessels by −5.1 ± 0.6% and −5.0 ± 0.5%, respectively (p=0.001; N = 5 animals) (Figure 5A). Conversely, cortical pial vessels dilated in response to an increase in inspired CO₂ under control conditions (5 ± 0.5%) and after application of 10 µM PPADS (3.2 ± 0.3%) (Figure 5C) (p=0.03; N = 5 animals); however, the CO₂/H⁺-induced vasodilation of cortical vessels was blunted in the presence of PPADS (p=0.03; N = 5 animals), suggesting endogenous purinergic signaling may facilitate dilation of cortical vessels in response to CO₂/H⁺. However, we failed to mimic this response by exogenous application of purinergic agonists; exposure to ATP (1 mM) or UTPγS (1 mM) constricted cortical vessels by −4.9 ± 0.7% and −4.1 ± 0.7%, respectively (p=0.024; N = 5 animals). These divergent results are not entirely unexpected since we detected P2Y₂ and P2Y₄ immunoreactivity on endothelial cells and smooth muscle of cortical arterioles (Figure 3D–E), and exogenous application of P2 agonists may activate P2 receptors not necessarily targeted by endogenous purinergic signaling. Future work is required to identify the source of purinergic drive and effector 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 µM) to the RTN blunted the ventilatory response to CO₂ both in terms of Dia_EMG 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 affect respiratory amplitude (Figure 4A–D), whereas application of PPADS to the same region, which likely disrupts both direct excitatory effects of ATP on RTN chemoreceptors (Wenker et al., 2012) and indirect 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 Dia_EMG frequency (p=0.33, N = 5 animals) and amplitude (p=0.42, N = 5) (Figure 5F). Together with previous evidence, these findings suggest that purinergic signaling contributes to RTN chemoreception 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 that potentially have off-target effects. We have minimized this concern by (i) using low concentrations of PPADS 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 specific P2Y_2/4 receptor agonist (UTPγS), but not by a non-specific P2X receptor agonist (α,β-mATP); (iii) confirming that candidate P2Y₂ and P2Y₄ receptors are expressed in the RTN at the astrocyte-arteriole interface; and (iv) for in vitro experiments by confirming that CO₂/H⁺-induced vasoconstriction was retained when neuronal action potentials were blocked with TTX. Therefore, our results suggest that purinergic signaling possibly through P2Y_2/4 receptors in the RTN provides specialized control of vascular tone by preventing CO₂/H⁺-induced dilation. Our results also suggest that regulation of vascular tone in the RTN contributes functionally to the ventilatory response to CO₂. This is the first evidence to suggest that regulation of vascular tone in a chemoreceptor region contributes to the drive to breathe. This discovery may be of fundamental importance to understanding how regulation of vascular tone impacts neural network function and ultimately behavior.

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Author details

1. Virginia E Hawkins

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   VEH, Data curation, Formal analysis, Supervision, Project administration, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9505-6776

2. Ana C Takakura

   Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

   Contribution

   ACT, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Ashley Trinh

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   AT, Data curation, Formal analysis, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

4. Milene R Malheiros-Lima

   Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

   Contribution

   MRM-L, Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Colin M Cleary

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   CMC, Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

6. Ian C Wenker

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   ICW, Conceptualization, Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

7. Todd Dubreuil

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   TD, Data curation, Formal analysis, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Elliot M Rodriguez

   Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

   Contribution

   EMR, Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Mark T Nelson

   1. Department of Pharmacology, College of Medicine, University of Vermont, Burlington, United States
   2. Institute of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   MTN, Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

10. Thiago S Moreira

    Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Contribution

    TSM, Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration, Writing—review and editing

    Contributed equally with

    Daniel K Mulkey

    For correspondence

    tmoreira@icb.usp.br

    Competing interests

    The authors declare that no competing interests exist.

11. Daniel K Mulkey

    Department of Physiology and Neurobiology, University of Connecticut, Storrs, United States

    Contribution

    DKM, Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    Contributed equally with

    Thiago S Moreira

    For correspondence

    daniel.mulkey@uconn.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-7040-3927

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