Cerebral blood flow is highly sensitive to changes in CO2/H+ where an increase in CO2/H+ causes vasodilation and increased blood flow. Tissue CO2/H+ also functions as the main stimulus for breathing by activating chemosensitive neurons that control respiratory output. Considering that CO2/H+-induced vasodilation would accelerate removal of CO2/H+ and potentially counteract the drive to breathe, we hypothesize that chemosensitive brain regions have adapted a means 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 tone during high CO2/H+ and disruption of this mechanism decreases the CO2ventilatory response. Our discovery that CO2/H+-dependent regulation of vascular tone in the RTN is the opposite to the rest of the cerebral vascular tree is novel and fundamentally important for understanding how regulation of vascular tone is tailored to support neural function and behavior, in this case the drive to breathe.https://doi.org/10.7554/eLife.25232.001
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.https://doi.org/10.7554/eLife.25232.002
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 P2Y2/4 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.
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 P2Y2/4 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 P2Y2/4 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.
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 P2Y2/4 can vary depending on which cells express the receptor (Burnstock and Ralevic, 2014), it remains possible that differential expression of P2Y2/4 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.
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.
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.
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 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 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 DiaEMG 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 P2Y2/4 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 P2Y2/4 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.
All procedures were performed in accordance with National Institutes of Health and University of Connecticut Animal Care and Use Guidelines. A total of 93 adult Sprague Dawley rats (60–100 days of age) were used for in vitro experiments. Animals were decapitated under isoflurane anesthesia, and transverse brainstem slices (200 μm) were prepared using a vibratome in ice-cold substituted artificial 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 to imaging slices were incubated for 1 hr with 10 µg/ml TRITC-lectin conjugate (Sigma-Aldrich, St. Louis, MO) or 6 µg/ml DyLight 594 Isolectin B4 conjugate (Vector Labortories) to label endothelial cells as previously described (Mishra et al., 2014). Slicing solutions were equilibrated with a high oxygen carbogen gas (95% O2-5% CO2) (Mulkey et al., 2001).
An 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 min−1) with aCSF bubbled with 5% CO2, 21% O2 (balance N2; pHo ~7.35; 37⁰ C) (Gordon et al., 2008). Hypercapnic solution was made by equilibrating aCSF with 15% CO2, 21% O2 (balance N2; pHo ~6.90; 37⁰ C). Arterioles were identified as previously described (Mishra et al., 2014; Filosa et al., 2004) by the following criteria: clear evidence of vasomotion under IR-DIC, bulky fluorescent labeling and a thick layer of smooth muscle surrounding the vessel lumen. Vessels that appeared collapsed and unhealthy were excluded, as were those with little fluorescence staining and thin walls, indicative of a lack of smooth muscle (Mishra et al., 2014). All arterioles selected for analysis had a resting luminal diameter of between 8–45 µm; RTN vessels were located within 200 µm of the ventral surface and below the caudal 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 water objective, a Clara CCD Andor camera and NIS Elements software. To induce a partially constricted state 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 vasoconstriction (Filosa et al., 2004; Girouard et al., 2010). In the continued presence of U-46619, we then characterized the effects of hypercapnia, ATP (100 µM; Sigma-Aldrich, St. Louis, MO), α,β-methyleneATP (100 µM), UTPγS (0.5 µM), and adenosine (1 µM), or the mGluR agonist t-ACPD ((±)−1-aminocyclopentane-trans-1,3-dicarboxylic acid; 50 µM) alone or in the presence of P2-recepetor blocker PPADS (5 µM; Tocris Bioscience, Minneapolis, MN), the P1 receptor antagonist 8-Phenyltheophylline (8-PT; 10 µM; Sigma) or the Ecto-NTPDase antagonist sodium metatungstate (POM 1; 100 µM; Tocris). In a subset of experiments we also tested CO2, ATP and PPADS in the presence of TTX (0.5 µM; Alomone Laboratories). As previously described (Girouard et al., 2010), at the end of each experiment we assessed arteriole viability by inducing a constriction with a high K+ solution (60 mM) and then maximal dilation with a Ca2+ free solution containing EGTA (5 mM), papaverine (200 μM, a phosphodiesterase inhibitor) and diltiazem (50 µM, to block L-type Ca channels). Vessels from both regions of interest (RTN and cortex) show similar responses to these conditions, and vessels that did not respond were excluded from analysis.
Rat brain slices were prepared from three rats and labelled with DyLight 594 Isolectin B4 conjugate as 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 sections were treated to unmask epitopes with 0.2 mg/ml pepsin (Sigma-Aldrich, St. Louis, MO) in 0.2 M HCl for 10 mins at 37°C (Corteen et al., 2011) followed by three washes in PBS for P2Y4 labelling only. A blocking stage was then performed by incubating tissue in 10% normal horse serum in PBS with 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 1 hr at room temperature (RT). Sections were 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) (Alomone Labs, Alomone Labs, Jerusalem Israel), 1:200 chicken anti-glial fibrillary acidic protein (RRID: AB_177521) (Chemicon) and 1:500 mouse anti-α-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 secondary 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 Immunoresearch Laboratories). Sections were washed in PBS again before being mounted with Vectasheild (VectorLabs). Images were acquired using a Nikon A1R confocal microscope (Nikon Instruments), with minimal background staining observed in the control reactions where primary antibodies were omitted or P2 receptor antibodies were pre-absorbed with control antigen prior to exposure to tissues. For confocal photomicrographs, two-dimensional flattened images of the projected z-stacks are presented.
Animal use was in accordance with guidelines approved by the University of São Paulo Animal Care and Use Committee. A total of 21 adult male Wistar rats (60–90 days of age; 270–310 g) were used for in vivo experiments. General anesthesia was induced with 5% halothane in 100% O2. A tracheostomy 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 Commodities) for measurement of arterial pressure (AP). The femoral vein was cannulated for administration of fluids and drugs. Rats were placed supine onto a stereotaxic apparatus (Type 1760; Harvard Apparatus) on a heating pad and core body temperature was maintained at a minimum of 36.5°C via a thermocouple. The trachea was cannulated. Respiratory flow was monitored via a flow head connected to a transducer (GM Instruments) and CO2 via a capnograph (CWE, Inc,) connected to the tracheal tube. Paired EMG wire electrodes (AM-System) were inserted into the diaphragm muscle to record respiratory-related activity. After the anterior neck muscles were removed, a basio-occipital craniotomy exposed the ventral medullary surface, and the dura was resected. After bilateral vagotomy, the exposed tissue around the neck and the mylohyoid muscle was covered with mineral oil to prevent drying. Baseline parameters were allowed to stabilize for 30 min prior to recording.
Mean 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 processed off-line with version 6 of Spike 2 software (Cambridge Electronic Design). Integrated diaphragm activity (∫DiaEMG) was collected after rectifying and smoothing (τ = 0.03) the original signal, which was acquired with a 300–3000 Hz bandpass filter. Noise was subtracted from the recordings prior to performing any calculations of evoked changes in DiaEMG. A direct physiological comparison of the absolute level of DiaEMG activity across nerves is not possible because of non-physiological factors (e.g., muscle electrode contact, size of muscle bundle) and the ambiguity in interpreting how a given increase in voltage in one EMG relates to an increase in voltage in another EMG. Thus, muscle 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 the magnitude of increases or decreases across muscle from those physiological baselines.
Each in vivo experiment began by testing responses to hypercapnia by adding pure CO2 to the breathing air supplied by artificial ventilation. In each rat, the addition of CO2 was monitored to reach a maximum end-expiratory CO2 between 9.5% and 10%, which corresponds with an estimated arterial 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 then replaced by 100% O2.
To determine whether local regulation of vascular tone in the region of the RTN contributes to the CO2/H+-dependent drive to breathe, we made bilateral injections of saline, phenylephrine (Phe, 1 µM), U46619 (1 µM) or sodium nitroprusside (SNP, 1 µM) while monitoring DiaEMG amplitude and frequency. These drugs were diluted to 1 µM with sterile saline (pH 7.4) and applied using single-barrel glass pipettes (tip diameter of 25 µm) connected to a pressure injector (Picospritzer III, Parker Hannifin Corp, Cleveland, OH). For each injection, we delivered a volume of 100 nl over a period of 5s. Injections in the VMS region were placed 1.9 mm lateral from the basilar artery, 0.9 mm rostral from the most rostral hypoglossal nerve rootlet, and at the VMS. The second injection was made 1–2 min later at the same level on the contralateral side. In separate series of experiments saline, ATP (1 mM), UTPγS (1 mM) or PPADS (10 µM) 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 window was prepared using standard protocols previously described (Kim et al., 2015). Briefly, a dental drill (Midwest Stylus Mini 540S, Dentsply International) was used to thin a circumference of a 4–5 mm-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 neck muscles were removed, a basio-occipital craniotomy exposed the ventral medullary surface, and the dura was resected. Pial vessels in the VMS had an average and were located 1.9 mm lateral from the basilar artery and 0.9 mm rostral to the most rostral portion of the hypoglossal nerve rootlet. Both thinned bone were lifted with forceps. The surface of the cortex or the VMS were cleaned with buffer containing (in mmol/L) the following: 135 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.3., and a chamber (home-made 1.1-cm-diameter plastic ring was glued with dental acrylic cement attached 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 microscope was used for vessel imaging (x40 magnification).
Vessel diameter was determined offline using ImageJ. For in vitro experiments, stack registration and selection of linear regions of interest (ROIs) (three regions per arteriole) was carried out. Linear ROI’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 to determine the peak-peak distance as a measure of vessel diameter for each frame. In most cases, we also confirmed vessel diameter by manually measuring at least one point per frame. In vivo data was also analyzed using three linear ROI’s drawn perpendicular to the vessel in each image and the Diameter plug-in function in ImageJ was used to calculate changes in diameter (Kim et al., 2015; Fischer et al., 2010).
All in vitro images were calibrated and pixel distances converted to diameter (µM) and these values were used for analysis of CO2/H+ or agonist-induced changes in vessel diameter from baseline by RM-one-way ANOVA and Fishers LSD test or paired t-test. Hash marks were used to identity differences from baseline (vasoconstriction or vasodilation). Mean percent changes in vessel diameter was used to compare between agonist responses or CO2/H+ responses under control conditions vs during purinergic receptor blockade or in the presence of an ectonucleotidase inhibitor by one-way ANOVA and Fishers LSD test or t-test. Asterisks were used to identify differences in % change in vessel diameter. For in vivo experiments, respiratory muscle activity was calculated as the mean amplitude of the integrated DiaEMG over 20 respiratory cycles. To obtain control values, the 20 cycles preceding each experimental manipulation for all parameters were averaged. Under hypercapnic conditions, measurements from the 20 cycles preceding stimulus cessation were averaged. Respiratory frequency (fR) was (1/(inspiratory time + expiratory time). Differences in the ventilatory response to CO2 were determined using either paired t-test or one-way analysis of variance (ANOVA) followed by the Bonferroni multiple-comparisons as appropriate. Power analysis was used to determine sample size, all data sets were tested for normality using Shapiro-Wilk test. All data values are expressed as means ± SEM and specific statistical test and relevant p values are reported in the text and figure legends.
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Jan-Marino RamirezReviewing Editor; Seattle Children's Research Institute and University of Washington, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Purinergic signaling provides specialized control of vascular tone to support the drive to breathe" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor) and three reviewers, one of whom, Jan-Marino Ramirez (Reviewer #1), is a member of our Board of Reviewing Editors.
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
The reviewers saw the importance of your significance, but felt that the pharmacology was not sufficiently convincing to support your conclusions. They suggest testing your hypothesis with additional drugs that are more specific.
The study by Hawkins and collaborators demonstrates that the effect of CO2/H+ on the vascular tone in the RTN, the presumed site for central chemoreception, is significantly different from the vascular control in the cortex.
This is interesting for two main reasons:
A) It is the first demonstration that CO2 does not cause a uniform vasodilation throughout the nervous system, but that it differentially controls an area that senses CO2.
B) The study is also interesting as it links chemosensation to concurrent changes in vascular tone. This is an interesting and novel concept that will trigger further studies aimed at better defining this interaction.
In this study, the role of purinergic modulation of vascular tone is primarily explored by pharmacological manipulations, as is the in vivo demonstration. The use of phenylephrine for example will affect vascular tone, but this manipulation might also have other indirect effects. Given the novelty of this observation, I am not too concerned that most mechanistic insights are based on pharmacological manipulations. However, being primarily a pharmacological study the authors cannot definitely demonstrate e.g. that it is the asctrocytic ATP which antagonizes the CO2/H+ vasodilation.
This is a potentially interesting paper that makes the case that ATP signaling mediates a specialized form of signaling in the brain areas that control breathing. Overall, I found this finding to be a useful contribution, but was not convinced that it was sufficiently novel for a general audience. In some ways, it seems appropriate for a specialized audience of researchers working on breathing.
My major concern for the paper is the use of 100 μm suramin and PPADS to implicate purinergic signaling. At these concentrations, neither drug is specific and their ability to block many other receptors is well described in published work. Moreover, much better blockers are available that target distinct ATP receptors. My suggestion is to repeat the key experiments with more selective antagonists and/or to repeat the experiments with suramin and PPADS at 3-10 uM, at which doses they are considered more selective for ATP receptors over receptors for other neurotransmitters. Without a more complete evaluation of ATP's involvement, the central message of this paper appears to be on weak foundations.
Finally, some of the traces in Figure 1 showing changes in blood vessel diameter over time were not particularly clear or convincing (e.g. Figure 1D1, A2, E2). Perhaps bolster these experiments with another in vitro approach to be sure the metrics used are reliable.
This is manuscript begins to address the intriguing idea that vascular tone in the chemosensitive area, the retrotrapezoid nucleus (RTN) responds differently to increasing CO2/H+, and thus, likely contributing to the chemosensitivity response. The authors used both in vitro and in vivo preparations to examine changes in arteriole diameter on the ventrolateral medullary surface during increased CO2/H+ and after blocking purinergic receptors with suramin or PPADS. Cumulatively, the authors suggest that their results demonstrate that the arterioles near the RTN respond to ATP released from astrocytes to prevent vasodilation in response to increasing CO2/H+. This is a very interesting and important idea; however, two major issues fail to be addressed in this manuscript. Firstly, the authors also fail to address why different purinergic receptor antagonists are used in the in vitro (suramin) and in vivo (PPADS) experiments. Secondly, the authors fail to acknowledge that suramin, and to a lesser extent PPADS, are associated with non-specific effects, particularly at glutamate receptors in the concentrations used here (for example, see Gu et al., 1998, Neuroscience Letters; Motin and Bennett, 1995, Br. J. Pharmacol.; Nakazawa et al., 1995, Naunyn Schmiedebergs Arch. Pharmacol; Lambrecht, 2000, Naunyn Schmiedebergs Arch. Pharmacol). These two issues at a minimum need to be addressed in the Discussion. In addition, the authors need to address how these non-specific actions could alter their conclusions. Lastly, the authors state clearly in the Introduction that they hypothesize "CO2/H+-evoked astrocyte ATP release will antagonize CO2/H+-vasodilation in the RTN…"; however, this is not actually the hypothesis that the authors test since they do not directly test the involvement of astrocytes. While this is a logical conclusion based on previous work and in conjunction with the studies tested here, the hypothesis stated needs to accurately describe the experiments herein. In other words, the hypothesis tested is that ATP released during increased CO2/H+ does not cause vasodilation in the RTN. Despite these issues, the data presented is very interesting and has the potential to foster additional studies to examine the interaction between astrocytes and the vasculature in this area, similar to work in the cortex.
[Editors’ note: a second version of this study was rejected after peer review, but the authors submitted for reconsideration. The decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Purinergic signaling provides specialized control of vascular tone to support the drive to breathe" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom, Jan-Marino Ramirez (Reviewer #1), is a member of our Board of Reviewing Editors.
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
The reviewers continue to believe that the study is potentially of general interest, and that your finding is significant. But, the reviewers felt that the authors continue to overstate their findings as detailed in the reviews.
Most importantly, the reviewers remain unconvinced that the study demonstrates the purinergic modulation. The study still reports experiments in which the drugs are used at concentrations that are non-specific. Moreover, it is not always clear what concentrations were used. One reviewer suggests additional experiments with P2X and P2Y receptor agonists and antagonists that would more specifically test the proposed mechanism. As also suggested, the addition of immunohistochemistry would clearly strengthen the study by demonstrating that there is indeed the expression of the relevant receptors. Inhibitors of ectoATPases could be tried to explore endogenous ATP involvement, and apyrase could be used to degrade ATP. Although, one reviewer suggested the use of knock-out mice, there was not consensus that this is really necessary for the main conclusion.
This revised manuscript has greatly improved and the authors provide various convincing arguments why this study is interesting for a general readership. I agree with the authors that the local control of vascular tone has general implication and is not only relevant for those interested in respiration. Moreover, the authors have been very responsive and added various new experiments that strengthen their conclusions.
The authors used for example an additional vasoconstrictor that has been frequently used, thus addressing a previous concern, and strengthening the conclusion that the RTN is involved in regulating vascular tone. The use of PPADS was suggested by the reviewers and the authors added new experiments to strengthen their pharmacological characterization of purinergic signaling, which is consistent with the findings obtained with the exogenous application of ATP.
Another strength is the combination of in vitro and in vivo approaches. The authors added new figures and re-arranged figures to enhance the clarity of the manuscript. Thus, the authors have addressed all my previous concerns.
The manuscript by Hawkins et al. is vastly improved by the revisions. The authors addressed the concerns brought forth by the reviewers. And this reviewer remains interested in the results; however, a few lingering issues remain that decrease the clarity of the manuscript. Mainly, some of the results of the current experiments continue to be overstated (see below). As discussed in the previous review, while these conclusions are plausible given previously published results, the results from the present experiments do not directly lead to such conclusions. Wording adjustments are necessary to make clear what is actually shown in the current paper. Secondly, inconsistencies between the data reported in the text of the results and the figures (specifically the bar graphs) is concerning (see below for details). These discrepancies make it challenging to accurately evaluate the manuscript. Overall, this manuscript remains of interest to this reviewer with a few more revisions.
1) Concluding sentence overstatements
"Together, these results suggest that astrocytes and purinergic signaling have fundamentally different roles in the regulation of vascular tone in the RTN and cortex, with the RTN being specialized to support chemoreceptor function." The reviewer would agree with the conclusion about purinergic signaling, but these data do not directly address involvement of astrocytes. The authors measure arterioles, not astrocyte activity.
"These results provide clear evidence that increasing or decreasing vascular tone in the RTN will influence the response to CO2. Therefore, we conclude that regulation of vascular tone in the RTN contributes to the chemoreflex." The conclusion for these results is more appropriately worded in the response to reviewers. Since the authors did not verify vascular tone in the experiments related to this conclusion, this conclusion is overstated for what is shown. Despite the addition of the U46619 data, the data does not indicate these drugs ONLY altered blood vessels. Based on the data presented, they cannot rule out diffusion of the drugs and altering astrocytes or neurons in VLM.
2) Data inconsistencies
Figure 1 and Results. Overall, it is unclear which statistical tests were run and which data were involved in which tests. The Methods indicate both t-tests and ANOVAs were run, but it is not clear when each was run or how data were grouped to determine significance.
3) It is unclear what "control" is for Figure 1. Is control the response to CO2 for all data in Figure 1? In specific, is "con" referring to the CO2 response or the t-ACPD in Figure 1E1? The same question for Figure 1F2. If in both cases, "con" refers to the CO2 response and if that is included when running the statistics, why does this bar not appear in the summary data?
4) Figure 1F1. – Data in text indicate constriction of 3.6 +/- 1.3%, but average bar appears at -5% for t-ACPD. Additionally, figure legend indicates p<0.05, but p-value reported in the text is p=0.05.
5) Figure 1G1. – What is the ATP data compared to? A 0% baseline, CO2 response or t-ACPD? Additionally, the p-value in the text is denoted as p=0.0018, but is labeled as P<0.001 in the figure legend.
6) Figure 2 and Results. Given how the results are presented in the text (as changes from saline), it is unclear what numbers were used to run the statistics (%ampl or the δ numbers)? If run on the δ numbers, a justification is needed.
7) Based on the% ampl numbers presented in Figure 2C-E, the data are convincing, but the necessity of presenting the numbers differently in the results remains unclear.
8) Why is there only one p-value reported for Figure 2C?
9) Response to reviewers indicated additional data had been added to Figure 2B-D, but the U46619 data is not shown in 2B.
11) The corresponding frequency and amplitude% data are not reported in the text of the results.
The authors have performed some additional experiments to tackle the issue of non-specific effects of suramin and PPADS. However, the original concerns remain – the evidence to implicate a purinergic mechanism is based on non-specific pharmacology. I agree the data are suggestive of a purinergic mechanism, but as shown the data do not provide strong evidence to prove this. This weakness also challenges the impact of the observations and the study as a whole for eLife.
1) The authors have included the use of 10 μm PPADS in Figure 1 and 3. However, in Figure 1 they still report data with 100 μm suramin. This is perplexing as at this concentration the drug is non-specific.
2) In Figure 3 new data are added with 10 μm PPADS (Figure 3A), but the data in Figure 3D-F are still based on 100 μm PPADS, which is a non-specific concentration. In the last paragraph of the Results and Discussion, they state low concentrations of PPADS are specific, which suggests that they know 100 μm PPADS is non-specific. Why report data with its use at this concentration?
Overall, the manuscript has improved, but the study still relies on high concentrations of drugs that are well known to be non-specific.
Additional experiments with P2X and P2Y receptor agonists and antagonists need to be tried to really prove the mechanism that is proposed. Immunohistochemistry needs to be shown to demonstrate expression of the relevant receptors. Inhibitors of ectoATPases can be tried to explore endogenous ATP involvement. Apyrase could be used to degrade ATP. Ideally, key experiments could be tried in a knock-out mouse of the relevant receptor.
In short, the central finding to implicate ATP is not convincing and more work is needed. The current level of proof (suramin, ATP and PPADS) is where the field was 20-30 years ago. Better tools, drugs and mouse models are available. They should be tried.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for submitting your article "Purinergic regulation of vascular tone in the retrotrapezoid nucleus is specialized to support the drive to breathe" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom, Jan-Marino Ramirez (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Jerome Dempsey (Reviewer #3).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
We congratulate you to your discovery of a differential control of the vascular system within the RTN, the region critical for the sensing of CO2. It is well established that in response to increased CO2/H+ the brain, and body in general, responds with vasodilation, a physiological response necessary to increase blood flow and to facilitate oxygenation and removal of CO2/H+. Your study convincingly demonstrates that the arterioles in the RTN constrict in response to CO2/H+, which is mediated by purinergic receptors. The RTN is the presumed site for chemoreception and it seems advantageous that the RTN has a differential vasomotor response compared to other sites in the body, a response that seems to be adapted to allow optimal chemoreception. Whether the RTN is the only central nervous system site to differentially control the vasomotor response cannot be deduced from these experiments. But, the study supports the notion that the vascular response is differentially regulated to adapt the blood supply to the specific function of a given CNS region.
We have a few discussion points listed under "Essential revisions" that we would like you to address to further improve the impact of your study.
1) We would like you to discuss other studies and observations that are potentially consistent with a local control of vascular reactivity within the brain. We believe that the following studies could be relevant for the concept that you propose in the present study.
A) In healthy humans indomethacin administration reduces the CBF and "cerebrovascular" response to hyper-/hypocapnia in the middle cer artery and many other brain and brain stem regions (see Xie et al. 2006, 2009 ref and references) with corresponding increases in eupneic ventilation and the ventilatory response slope to hyperoxic hypercapnia and hypocapnia.
B) Similar increases in the CO2 ventilatory response occurred with carotid occlusion in the goat (Parisi, 1992, Chapman, 1979).
C) Clinically, in post-transplantation anaemia and associated congestive heart failure, cerebrovascular responses to CO2 have steep ventilatory responses to CO2 contributing to breathing instability and apneas.
Could this (C) and the other findings (A, B) mean that under certain conditions that influence cerebral vascular reactivity "globally", there are marked influences on chemoreceptor responsiveness and ventilatory control stability, because they influence the PCO2 locally at the level of the chemoreceptors? Would these observations be compatible with a qualitatively different vascular responsiveness to CO2/H+ at the level of the central chemoreceptors as your data supports? A discussion like this could enhance the clinical impact of your interesting finding.
2) We would like you to discuss/clarify the following consideration: Your contention that the unique RTN vascular responsiveness to CO2/H+ "enhances chemo function","…contributes to the drive to breathe", "…supports neural function and behavior" seems to imply that you believe this specialized function to be beneficial to the organism. However the function of both the ventilatory response and (at least most of) the cerebral vascular response to hyper/hypocapnia is to minimize the disturbance in CO2/H+.….so would not having the vasodilation/vasoconstriction responses in the RTN mean that the burden is now completely on the (more expensive?) ventilatory response to regulate local H+?
3) It would help to include n values in the figures and/or figure legends.
4) The use of "con" and "control" is still confusing. The reviewer thanks the authors for clarifying "con" in the response to reviewers. It would help the readers of the manuscript to benefit from such clarification. This reviewer recommends labeling it as baseline, instead of "con" or "control". Particularly, since the CO2 group becomes the control group in comparisons.
5) Figure 1B. The figure legend describes that 1B shows the response in PPADs, but only "con" and CO2" are shown.
6) Results/Discussion, "under control conditions", "baseline" would be more informative.
8) Excellent work from Brian MacVicar's group has gone a long way to helping understand the role of astrocytes in cerebral blood flow, yet reference to this group is only made in the Methods section. Additional acknowledgement to their body of work is needed, including their most recent paper (see below).
9) Results/Discussion, first paragraph: N=34 – is this n=34 animals or 34 vessels? Please clarify.
10) Results/Discussion, second paragraph: Did the authors also do immuno for P2X receptors? Are they expressed there, but not involved? If the authors are eliminating their involvement, it would be useful to know whether or not they are expressed there.
11) Results/Discussion, fifth paragraph: MRS2179 data not shown in figures. Please indicate in the text.
12) Also, a reference to a recent study from MacVicar's group needs to indicate that more is known about dilation in the hippocampal-neocortical/barrel cortex (Howarth et al. 2017 J Neurosci).
15) Figure 2: Parts C and D are not addressed in the figure legend.
16) Figure 5: Saline should be open/white bars, not black bars.
17) Methods – your use of "peak" di EMG to quantify "resp muscle activity" is partly dependent on breath timing changes alone. Wouldn't a more appropriate metric be either mean amplitude (total area over TI) or rate of rise of di EMG?
18) You chose to test the "response to hypercapnia" in vivo by raising FetCO2 to 10% or almost twice the air br value in the rodent and showing that the vessel diam changed 3-4%. Given the highly sensitive response of CBF to CO2 in the cer vasc (3-4% per mmHg δ PCO2) did you try more physiologic perturbations to show the sensitivity of your prep? Also, given the absence of protein buffers in the cerebral ECF your pH in the RTN is probably in the 6-6.5 range with your level of hypercapnia.https://doi.org/10.7554/eLife.25232.008
- Daniel K Mulkey
- Mark T Nelson
- Mark T Nelson
- Mark T Nelson
- Daniel K Mulkey
- Ana C Takakura
- Ana C Takakura
- Virginia E Hawkins
- Mark T Nelson
- Mark T Nelson
- Mark T Nelson
- Thiago S Moreira
- Thiago S Moreira
- Milene R Malheiros-Lima
- Thiago S Moreira
- Ana C Takakura
- Thiago S Moreira
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank David Attwell and Anusha Mishra (University College London), Catherine Hall (University of 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), and public funding from the São Paulo Research Foundation (FAPESP) Grants 2014/22406-1 (ACT), 2016/22069–0 (TSM), 2015/23376–1 (TSM) and FAPESP fellowship 2014/07698-6 (MRML); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); grant: 471263/2013–3 (ACT) and 471283/2012–6 (TSM). CNPq fellowship 301651/2013–2 (ACT) and 301904/2015–4 (TSM).
Animal experimentation: All in vitro procedures were performed in accordance with National Institutes 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 of São Paulo Animal Care and Use Committee (protocol # 112/2015).
- Jan-Marino Ramirez, Reviewing Editor, Seattle Children's Research Institute and University of Washington, United States
© 2017, Hawkins et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.