Breathing stability during sleep and during hypercapnia is dependent on the retrotrapezoid nucleus in adult mice

Background: Eupneic breathing during sleep is determined by the chemical drive arising from central and peripheral chemoreceptors. The retrotrapezoid nucleus (RTN) contains chemoreceptor neurons that are proposed to be critical for ventilatory responses to CO 2 . RTN chemoreceptor neurons can be identified by expression of transcripts for Neuromedin B ( Nmb + ). We hypothesized that the ablation of RTN Nmb + neurons cause alveolar hypoventilation at rest and breathing instability during sleep. Normally, in intact subjects, increasing respiratory drive with hypercapnia produces a robust and more stable breathing pattern compared to resting condition. We furhter hypothesized that in the absence of RTN, hypercapnia will not increase breathing stability and the ventilatory response to CO 2 will be impaired. Methods: Bilateral microinjections of a designer cell-ablation virus, AAV5-Flex-Casp3-TEVP, were placed in the RTN in heterozygous Cre-positive (RTN lesion) and Cre-negative (controls) Nmb-Cre mice and EEG/neck EMG electrodes were implanted. One month later, mice were tested in an unrestrained plethysmography chamber for breathing and sleep/wake recordings in normoxic conditions. Mice were exposed to hyperoxic hypercapnia (F I CO 2 up to 0.09) to assess the central respiratory CO 2 chemoreflex after RTN lesion. Arterial blood gases were also collected in unrestrained unanesthetized conditions. Values are presented as mean ±SD. Results: Ablation of the RTN was complete (<1% of total RTN Nmb + neurons remaining in RTN lesion group) and selective based on counts of neighboring catecholaminergic and serotonergic neurons. Arterial PCO 2 was increased in RTN-lesion mice (n=6) compared to controls (n=7) (49 ± 5 vs. 39 ± 3 mmHg, p<0.001) and arterial PO 2 was reduced (75 ± 11 vs. 89 ± 7 mmHg, p=0.019). Minute-ventilation was reduced in RTN-lesion mice (n=13) compared to control (n=13) (1.7 ± 0.4 vs. 2.4 ± 0.3 mL/min/g, p<0.0001) due to a reduction in tidal volume that was most evident during NREM sleep. Breathing variability was increased in RTN-lesion mice (n=9) compared to controls (n=10) during NREM (22 ± 5 vs. 10 ± 1% of inter-breath variability, p<0.001) and REM sleep (37 ± 8 vs. 25 ±7 % of inter-breath variability, p<0.01) but not during wakefulness (28 ± 9 vs. 21 ±10 % of inter-breath variability, p=0.32). The ventilatory response to CO 2 was attenuated in RTN lesion mice compared to controls (3.2 ±1 vs. 10.4 ±2 mL/min/g at FICO2 0.09, p<0.0001).Finally, during hypercapnia, breathing was more variable in RTN-lesion mice (23 ±10 vs. 3.1 ±0.4 % of inter-breath variability, p<0.0001) compared to control. Summary and conclusion: In sum, RTN neurons contribute to resting alveolar ventilation and eupneic breathing, especially during sleep. The hypercapnic ventilatory response is severely blunted and hypercapnia does not increase breathing stability in the absence of RTN. We conclude that breathing stability during sleep and during hypercapnia is dependent on the RTN. This work is funded by National Institutes of Health, R01HL148004 to Stephen BG Abbott and R01HL108609 to Douglas A Bayliss. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.