Breathing is an essential homeostatic function and its disruption leads to disability, brain damage, and death. Serotonin (5-hydroxytryptamine; 5-HT) neurons in the brainstem play an important role in control of breathing. Medullary 5-HT neurons are stimulated by increased CO₂ and subsequently stimulate respiratory nuclei to increase ventilation and maintain normal blood gas levels. Anesthetic-induced breathing dysfunction is a serious concern in healthcare settings. In research settings, experiments are often performed under anesthesia, and therefore it is important to understand how these drugs affect animal physiology. Unfortunately, little is known about how anesthetics modulate 5-HT neurons, breathing, and CO₂ chemoreception in mice, as many of the previous studies have been performed in different species. Characterizing how anesthetics commonly used in both research and clinical settings affect 5-HT neurons, breathing and CO₂ chemoreception is valuable to the broader field of neuroscience since these drugs are so ubiquitously used in research. Breathing dysfunction and defects in the serotonergic system have been implicated in disorders, such as sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS), which means better characterizing the role of 5-HT neurons in breathing has translational impact as well.
Here I examine whether halogenated inhalational anesthetics, which potentiate TWIK-related acid-sensitive K⁺ (TASK) currents and GABAA receptors, could mask an effect of CO₂ on 5-HT neurons. During in vivo plethysmography in mice, a therapeutic level of isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) in all mouse strains tested. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized 5-HT neurons, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro, and markedly decreases the HCVR in vivo.
Next, I demonstrate that ketamine-xylazine or urethane anesthesia also significantly reduced the HCVR in mice at both therapeutic and sub-therapeutic doses. However, mice treated with a sub-therapeutic dose of anesthesia decreased their O₂ consumption in parallel, and thus matched their ventilation to metabolic demands. Mice that were anesthetized with the therapeutic dose did not sufficiently match their breathing and metabolic demands, and thus anesthesia induced hypoventilation. Recordings from 5-HT neurons in culture indicated that neither ketamine nor urethane affected 5-HT neuron chemosensitivity. These data demonstrate that anesthetics with different molecular targets similarly reduce the HCVR in mice, but not all of their effects are mediated via 5-HT neurons. Moreover, both ketamine-xylazine and urethane anesthesia altered baseline breathing in different ways, suggesting they targeted different parts of the respiratory network.
Finally I show that isoflurane anesthesia in neonatal mice caused depression of resting ventilation, which was different from isoflurane-anesthetized adults. This effect was more pronounced in wildtype mice compared to littermates with genetic deletion of 5-HT neurons. Isoflurane-induced breathing depression decreased and mice fully recovered following washout of isoflurane at P8. I observed that genetic deletion of 5-HT neurons in mice with a congenic C57Bl/6 background led to a more severe phenotype than previously described in mixed genetic background strains. These mice had decreased survival, severe growth retardation, and reduced baseline ventilation. These results indicate that 5-HT neurons have a different role during the neonatal period and that some mouse strains are more sensitive to genetic deletion of 5-HT neurons; thus, background genetics play an important role in phenotype presentation.
In summary, different classes of anesthetics each strongly depress chemoreception. Isoflurane seems to affect breathing, in part, by hyperpolarizing 5-HT neurons and masking their chemosensitivity, whereas ketamine and urethane have less effect on 5-HT neurons. However, both ketamine-xylazine and urethane anesthesia alter baseline breathing. Isoflurane anesthesia decreases baseline ventilation in neonates, but this effect is absent in adults, which suggests that the effects of isoflurane on breathing changes as mice age. These data are important for the field of respiratory physiology because they highlight the sensitivity of breathing to the effects of anesthetics. These results are valuable to the broader field of neuroscience, because anesthetics are widely used during in vivo research. Additionally, some transgenic mouse strains are more sensitive to 5-HT neuron deletion depending on their genetic background. In the future it will be critical to characterize the molecular mechanisms that underlie these phenomena.