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Background: Optimal management of anesthesia-induced respiratory depression requires identification of the neural pathways that are most effective in maintaining breathing during anesthesia. Lesion studies point to the brainstem retrotrapezoid nucleus. We therefore examined the respiratory effects of common anesthetic/analgesic agents in mice with selective genetic loss of retrotrapezoid nucleus neurons (Phox2b mice, hereafter designated "mutants")., Methods: All mice received intraperitoneal ketamine doses ranging from 100 mg/kg at postnatal day (P) 8 to 250 mg/kg at P60 to P62. Anesthesia effects in P8 and P14 to P16 mice were then analyzed by administering propofol (100 and 150 mg/kg at P8 and P14 to P16, respectively) and fentanyl at an anesthetic dose (1 mg/kg at P8 and P14 to P16)., Results: Most mutant mice died of respiratory arrest within 13 min of ketamine injection at P8 (12 of 13, 92% vs. 0 of 8, 0% wild type; Fisher exact test, P < 0.001) and P14 to P16 (32 of 42, 76% vs. 0 of 59, 0% wild type; P < 0.001). Cardiac activity continued after terminal apnea, and mortality was prevented by mechanical ventilation, supporting respiratory arrest as the cause of death in the mutants. Ketamine-induced mortality in mutants compared to wild types was confirmed at P29 to P31 (24 of 36, 67% vs. 9 of 45, 20%; P < 0.001) and P60 to P62 (8 of 19, 42% vs. 0 of 12, 0%; P = 0.011). Anesthesia-induced mortality in mutants compared to wild types was also observed with propofol at P8 (7 of 7, 100% vs. 0 of 17,7/7, 100% vs. 0/17, 0%; P < 0.001) and P14 to P16 (8 of 10, 80% vs. 0 of 10, 0%; P < 0.001) and with fentanyl at P8 (15 of 16, 94% vs. 0 of 13, 0%; P < 0.001) and P14 to P16 (5 of 7, 71% vs. 0 of 11, 0%; P = 0.002)., Conclusions: Ketamine, propofol, and fentanyl caused death by respiratory arrest in most mice with selective loss of retrotrapezoid nucleus neurons, in doses that were safe in their wild type littermates. The retrotrapezoid nucleus is critical to sustain breathing during deep anesthesia and may prove to be a pharmacologic target for this purpose.

Phox2b is an essential transcription factor for the development of the autonomic nervous system. Mice carrying one invalidated Phox2b allele (Phox2b(+/-)) show mild autonomic disorders including sleep apneas, and impairments in chemosensitivity and thermoregulation that recover within 10days of postnatal age. Because Phox2b is not expressed above the pons nor in the cerebellum, this mutation is not expected to affect brain development and cognitive functioning directly. However, the transient physiological disorders in Phox2b(+/-) mice might impair neurodevelopment. To examine this possibility, we conducted a behavioral test battery of emotional, motor, and cognitive functioning in adult Phox2b(+/-) mice and their wildtype littermates (Phox2b(+/+)). Adult Phox2b(+/-) mice showed altered exploratory behavior in the open field and in the elevated plus maze, both indicative of anxiety. Phox2b(+/-) mice did not show cognitive or motor impairments. These results suggest that also mild autonomic control deficits may disturb long-term emotional development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O(2), CO(2), and pH homeostasis. In mammals, it is generated by a neuronal network in the lower brainstem, the respiratory rhythm generator (Feldman et al., 2003). A century-old tenet in respiratory physiology posits that the respiratory chemoreflex, the stimulation of breathing by an increase in partial pressure of CO(2) in the blood, is indispensable for rhythmic breathing. Here we have revisited this postulate with the help of mouse genetics. We have engineered a conditional mouse mutant in which the toxic PHOX2B(27Ala) mutation that causes congenital central hypoventilation syndrome in man is targeted to the retrotrapezoid nucleus, a site essential for central chemosensitivity. The mutants lack a retrotrapezoid nucleus and their breathing is not stimulated by elevated CO(2) at least up to postnatal day 9 and they barely respond as juveniles, but nevertheless survive, breathe normally beyond the first days after birth, and maintain blood PCO(2) within the normal range. Input from peripheral chemoreceptors that sense PO(2) in the blood appears to compensate for the missing CO(2) response since silencing them by high O(2) abolishes rhythmic breathing. CO(2) chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO(2) is dispensable for life-sustaining breathing and maintaining CO(2) homeostasis in the blood.

Congenital central hypoventilation syndrome (CCHS) is a rare disease with variable severity, generally present from birth and chiefly characterized by impaired chemosensitivity to hypercapnia. The main cause of CCHS is a mutation in the PHOX2B gene, which encodes a transcription factor involved in the development of autonomic medullary reflex pathways. Temperature regulation is abnormal in many patients with CCHS. Here, we examined whether ambient temperature influenced CO(2) sensitivity in a mouse model of CCHS. A weak response to CO(2) at thermoneutrality (32 degrees C) was noted previously in 2-day-old mice with an invalidated Phox2b allele (Phox2b+/-), compared with wild-type littermates. We exposed Phox2b+/- pups to 8% CO(2) at three ambient temperatures (TAs): 29 degrees C, 32 degrees C, and 35 degrees C. We measured breathing variables and heart rate (HR) noninvasively using a novel whole body flow plethysmograph equipped with contact electrodes. Body temperature and baseline breathing increased similarly with TA in mutant and wild-type pups. The hypercapnic ventilatory response increased linearly with TA in both groups, while remaining smaller in mutant than in wild-type pups at all TAs. The differences between the absolute increases in ventilation in mutant and wild-type pups become more pronounced as temperature increased above 29 degrees C. The ventilatory abnormalities in mutant pups were not associated with significant impairments of heart rate control. In both mutant and wild-type pups, baseline HR increased with TA. In conclusion, TA strongly influenced the hypercapnic ventilatory response in Phox2b+/- mutant mice. These findings suggest that abnormal temperature regulation may contribute to the severity of respiratory impairments in CCHS patients.

Heterozygous mutations of the transcription factor PHOX2B have been found in most patients with central congenital hypoventilation syndrome, a rare disease characterized by sleep-related hypoventilation and impaired chemosensitivity to sustained hypercapnia and sustained hypoxia. PHOX2B is a master regulator of autonomic reflex pathways, including peripheral chemosensitive pathways. In the present study, we used hyperoxic tests to assess the strength of the peripheral chemoreceptor tonic drive in Phox2b+/-newborn mice. We exposed 69 wild-type and 67 mutant mice to two hyperoxic tests (12-min air followed by 3-min 100% O2) 2 days after birth. Breathing variables were measured noninvasively using whole body flow plethysmography. The initial minute ventilation decrease was larger in mutant pups than in wild-type pups: -37% (SD 13) and -25% (SD 18), respectively, P<0.0001. Furthermore, minute ventilation remained depressed throughout O2 exposure in mutants, possibly because of their previously reported impaired CO2 chemosensitivity, whereas it returned rapidly to the normoxic level in wild-type pups. Hyperoxia considerably increased total apnea duration in mutant compared with wild-type pups (P=0.0001). A complementary experiment established that body temperature was not influenced by hyperoxia in either genotype group and, therefore, did not account for genotype-related differences in the hyperoxic ventilatory response. Thus partial loss of Phox2b function by heterozygosity did not diminish the tonic drive from peripheral chemoreceptors.