Vascular control of the CO2/H+ dependent drive to breathe

Respiratory chemoreceptors regulate breathing in response to changes in tissue CO2/H+. Blood flow is a fundamental determinant of tissue CO2/H+, yet little is known regarding how regulation of vascular tone in chemoreceptor regions contributes to respiratory behavior. Previously, we showed in rat that CO2/H+-vasoconstriction in the retrotrapezoid nucleus (RTN) supports chemoreception by a purinergic-dependent mechanism (Hawkins et al. 2017). Here, we show in mice that CO2/H+ dilates arterioles in other chemoreceptor regions, thus demonstrating CO2/H+ vascular reactivity in the RTN is unique. We also identify P2Y2 receptors in RTN smooth muscle cells as the substrate responsible for this response. Specifically, pharmacological blockade or genetic deletion of P2Y2 from smooth muscle cells blunted the ventilatory response to CO2, and re-expression of P2Y2 receptors only in RTN smooth muscle cells fully rescued the CO2/H+ chemoreflex. These results identify P2Y2 receptors in RTN smooth muscle cells as requisite determinants of respiratory chemoreception. Significance Statement Disruption of vascular control as occurs in cardiovascular disease leads to compromised chemoreceptor function and unstable breathing. Despite this, virtually nothing is known regarding how regulation of vascular tone in chemoreceptor regions contributes to respiratory behavior. Here, we identify P2Y2 receptors in RTN vascular smooth muscle cells as a novel vascular element of respiratory chemoreception. Identification of this mechanism may facilitate development of treatments for breathing problems including those associated with cardiovascular disease.


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To determine whether P2Y 2 receptors in the RTN regulate CO 2 /H + vascular reactivity in vivo, we 172 measured the diameter of pial vessels on the ventral medullary surface (VMS) in the region of 173 the RTN during exposure to high CO 2 under control conditions (saline) and when P2Y 2 receptor 174 are blocked with AR-C118925. Consistent with our slice data, we found under saline control 175 conditions that increasing end-expiratory CO 2 to 9-10%, which corresponds with an arterial pH 176 of 7.2 pH units (16), constricted VMS vessels by -7.8 ± 0.7 % (p=0.01, N = 7 animals) ( Fig. 2A). 177 However, when P2Y 2 -receptors are blocked by application of AR-C118925 (10 µM) to the 178 VMS, increasing inspired CO 2 resulted in a vasodilation of 5.2 ± 0.9% ( Fig. 2A) (p=0.05; N = 7 animals). Thus, in the absence of functional P2Y 2 receptors, RTN vessels respond to CO 2 /H + in 180 a manner similar to other brain regions. Also consistent with our slice data, we found that 181 activation of P2Y 2 receptors by application of PSB1114 (100 µM) to the VMS constricted 182 vessels in the region of the RTN by -6.3 ± 0.9 % (p=0.05, N = 7 animals) (Fig. 2B).

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To correlate P2Y 2 -dependent vasoconstriction in the RTN region with respiratory behavior, we 185 simultaneously measured systemic blood pressure and external intercostal electromyogram 186 (Int EMG ) activity (as a measure of respiratory activity) in urethane-anesthetized mice during 187 exposure to low and high CO 2 following VMS application of saline or AR-C118925. We found 188 that VMS application of AR-C118925 (1 mM) minimally affected the CO 2 /H + apneic threshold 189 (3.3 ± 0.4% vs. saline: 3.1 ± 0.5% (p > 0.05; two-way RM ANOVA; N = 7). However at high 190 levels of CO 2 (9-10% etCO 2 ) AR-C118925 decreased intercostal EMG amplitude by 23 ± 5%   Table 2). This chemoreceptor deficit involves a diminished capacity to increase 242 both respiratory frequency (Fig. 4D) and tidal volume (Fig. 4E) during exposure to CO 2 (Table   243 2). Similar results were obtained in P2Y 2 cKO mice generated using a different smooth muscle 244 cre line (smMHC cre/eGFP ) (Supplemental Fig. 3) immunoreactive cells in the RTN are also GFP + (Fig. 4B, Supplemental Fig. 6). Some 263 background eGFP labeling was observed, but not associated with DAPI labeling (Supplemental 264 Fig. 6aii). We also confirmed RTN smooth muscle cells from P2Y 2 rescue mice show increased 265 P2Y 2 transcript levels compared to P2Y 2 cKO (p=0.0073) but not to the same level as cells from 266 control mice (p = 0.0219) (Fig. 4A). Importantly, re-expression of P2Y 2 receptors in RTN 267 smooth muscle cells resulted in a full rescue of the minute ventilatory response to CO 2 ( Fig. 4C each region of interest and pharmacological manipulation of candidate mechanisms independent of potential confounding effects of blood pressure on myogenic tone. However, because blood 295 vessels in brain slice have limited myogenic tone these experiments were performed in the 296 presence of U-46619 to pre-constrict vessels ~30% (2). We also used a large stimulus (15%

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CO 2 ) to characterize CO 2 /H + vascular reactivity in vitro. For these reasons, we also confirmed in 298 vivo in the absence of U-46691 that more physiological levels of CO 2 (9-10%) constricted 299 arterioles in the RTN by a P2Y 2 -dependent mechanism. Second, we and others (41)  In contrast to the RTN, we also found that CO 2 /H + (Fig. 1C) and astrocyte activation by bath 369 application of t-ACPD ( Fig. 1F) (Fig. 3). However, further work is needed to understand whether and how differential CO 2 /H + vascular reactivity in these chemoreceptor regions contributes to breathing problems in disease 387 states.

Taqman probe P2ry2
ThermoFisher Mm02619978_s1 Sequence-based reagent Taqman probe P2ry4 ThermoFisher Mm00445136_s1 Sequence-based reagent Taqman probe P2ry6 ThermoFisher Mm02620937_s1 Sequence-based reagent Taqman probe P2ry12 ThermoFisher Mm01950543_s1 Sequence-based reagent Taqman probe P2ry13 ThermoFisher Mm01950543_s1 Sequence-based reagent of the cNTS arterioles were small (average diameter was 9 µm) and ROb vasculature did not  2017]) was used to determine peak to peak distance using fluorescence intensity profile plots for 586 all slices of the data file.  System) were inserted into the external intercostal muscle to record respiratory-related activity.

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After the anterior neck muscles were removed, a basio-occipital craniotomy exposed the ventral 666 medullary surface, and the dura was resected. After bilateral vagotomy, the exposed tissue 667 around the neck and the mylohyoid muscle was covered with mineral oil to prevent drying.

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Baseline parameters were allowed to stabilize for 30 min prior to recording. A cranial optical window was prepared using standard protocols. For the VMS, the anterior neck 707 muscles were removed, a basio-occipital craniotomy exposed the ventral medullary surface, and 708 the dura was resected. Pial vessels in the VMS were located 1.9 mm lateral from the basilar Three linear ROIs were drawn over the pial vasculature we recognize as supplying blood flow to 732 the RTN, similar to the in vitro methods described above. Bright field imaging was analyzed 733 using a macro in ImageJ. Briefly, the macro measured the drop and rise of the local maxima and 734 minima, correlating to vessel boundaries. These boundaries were then used to make a linear 735 measurement between the two points which was vessel diameter.