Fig 1: P2Y1R-dependent signaling is necessary for spontaneous and hypoxia-induced sigh generation in the preBötC.a Integrated preBötC activity depicting time course for P2Y1R manipulation. MRS2365 significantly increased the number of sighs from baseline, while P2Y1R antagonist MRS2279 abolished all sighs (n = 5, one-way ANOVA). P2XR antagonist (TNP-ATP) did not significantly affect sigh frequency (n = 8, one-way ANOVA); no significant effect on eupneic frequency was observed with any drug additions. Analysis of sigh and eupnea burst number is reported as number of bursts/20 min period (one-way ANOVA). b P2Y1R antagonist MRS2279 eliminated the βAR agonist induced increase in sighs (n = 8, one-way ANOVA). c Representative traces from transverse preBötC slices during the initial bout of severe hypoxia (SH, 6 mins), followed by a recovery period and second hypoxic challenge with MRS2279 (n = 9). The lower panel depicts control with two consecutive bouts of severe hypoxic challenge. Gaps in recordings were made to align control and experimental traces. Inset preBötC integrated traces have time scales of 10 s. MRS2279, in addition to severe hypoxia, significantly impaired the hypoxic response by eliminating sighs (left) and reducing the augmenting phase of the eupneic rhythm (right, both two-tailed paired t-tests). Data are presented as mean values ± SEM.
Fig 2: Chr2 activation of preBӧtC astrocytes drives sighing in vitro and in vivo.a PreBötC slice depicting electrode and laser placement and representative traces comparing spontaneous (light green, gray) and photo-evoked (dark green, dark gray) sighs and eupnea, with the expanded view above. The turquoise bar represents 200 ms light pulse. b Evoked sighs withheld the same characteristics as spontaneous sighs with a smaller duration, given the larger number of evoked monophasic sighs (paired t-test, n = 12). c Optogenetically evoked sighs are blocked by Cadmium (4 µM) and MRS2279 (20 µM). Representative traces depicting differences between baseline evoked sighs and ‘sigh attempts’ and eupnea in drug conditions. d Cadmium, but not MRS2279, significantly reduced the probability of evoking a sigh. Sigh characteristics were absent from evoked bursts in cadmium and MRS2279, including a reduction in burst amplitude, burst duration, and post-burst interval (paired t-test, n = 12). Graphs show that ‘sigh attempts’ did not significantly differ from eupnea in most characteristics. Missing symbols in Cadmium or MRS2279 represent that no sigh attempts were evoked. e Schematic depicting in vivo anesthetized preparation and f corresponding (shortened) evoked sweeps as recorded from the hypoglossal (XII) nerve. g Evoked sighs in vivo were best distinguished by diaphragm amplitude and generally retained sigh characteristics that were significantly different from evoked eupnea, apart from burst duration and post-burst interval (n = 11). h Hypothesis of sigh rhythm generation. Changes in PO2 levels increase intracellular Ca2+ in astrocytes, resulting in ATP release, which is converted to ADP. ADP binds to purinergic P2Y1Rs leading to a Ca2+ increase in astrocytes and neurons through g-protein signaling cascades. P2Y1R-driven sighs in vivo can be overridden by neuromodulation from upstream pathways (e.g., Norepinephrine, neuromedin B, gastrin-releasing peptide) to provide further sigh modulation depending on metabolic state. Data are presented as mean values ± SEM.
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