Key pointsr Neurons of the retrotrapezoid nucleus (RTN) and medullary serotonin (5-HT) neurons are both candidates for central CO 2 /pH chemoreceptors, but it is not known how interactions between them influence their responses to pH.r We found that RTN neurons in brain slices were stimulated by exogenous 5-HT and by heteroexchange release of endogenous 5-HT, and these responses were blocked by antagonists of 5-HT 7 receptors.r The pH response of RTN neurons in brain slices was markedly reduced by the same antagonists of 5-HT 7 receptors.r Similar results were obtained in dissociated, primary cell cultures prepared from the ventral medulla, where it was also found that the pH response of RTN neurons was blocked by preventing 5-HT synthesis and enhanced by blocking 5-HT reuptake. Exogenous 5-HT did not enable latent intrinsic RTN chemosensitivity.r RTN neurons may play more of a role as relays from other central and peripheral chemoreceptors than as CO 2 sensors.Abstract Phox2b-expressing neurons in the retrotrapezoid nucleus (RTN) and serotonin (5-HT) neurons in the medullary raphe have both been proposed to be central respiratory chemoreceptors. How interactions between these two sets of neurons influence their responses to acidosis is not known. Here we recorded from mouse Phox2b+ RTN neurons in brain slices, and found that their Yuanming Wu, MD, received his MD from Hubei Medical College Xianning Branch, China, and his MS from Tongji Medical University, China. He joined the laboratory of George B Richerson, MD, PhD, in the neurology department at Yale University from 1997 to 2010. He then moved to the University of Iowa in 2010, where he has risen to the rank of Research Scientist. He is highly experienced and skilled at patch clamp recordings from neurons in cell culture and brain slices, and has a deep interest in synaptic transmission, chemoreception, respiratory control and epilepsy. Katherine Proch received her BS in Neuroscience and German in 2008 from Allegheny College, Meadville, PA, where her senior thesis introduced her to CO 2 chemosensation. She continued research in that field as a member of the Medical Scientist Training Program at the University of Iowa, and defended her thesis, Effects of serotonergic input on Phox2b neuron chemosensitivity in the RTN, in March 2018. She is expected to receive her medical degree and PhD in May 2019. * These authors contributed equally. Y. Wu and others J Physiol 597.10response to moderate hypercapnic acidosis (pH 7.4 to ß7.2) was markedly reduced by antagonists of 5-HT 7 receptors. RTN neurons were stimulated in response to heteroexchange release of 5-HT, indicating that RTN neurons are sensitive to endogenous 5-HT. This electrophysiological behaviour was replicated in primary, dissociated cell cultures containing 5-HT and RTN neurons grown together. In addition, pharmacological inhibition of 5-HT synthesis in culture reduced RTN neuron chemosensitivity, and blocking 5-HT reuptake enhanced chemosensitivity. The effect of 5-HT on RTN neuron chemosensitivity was ...

Biological functions take place within tightly controlled parameters, including pH, which is managed in part through the ventilatory chemoreflex. This reflex is mediated by central respiratory chemoreceptors (CRCs) specialized to detect blood pH/CO 2 . Two neuronal populations are thought to mediate this response: the serotonergic (5-HT) neurons of the medullary raphé, and the Phox2b expressing neurons of the retrotrapezoid nucleus (RTN). These groups are both responsive to CO 2 stimuli in vivo and in vitro. There are also apparent one-way connections from the raphé to the RTN, which is sensitive to 5-HT. Due to its complex innervation, study of RTN neurons while isolated from other cells, especially 5-HT neurons, has been limited. Here, we developed a culture model that simplifies this circuit, limiting cell types to those found in the rostral ventral medulla. This protocol yielded healthy RTN and 5-HT neurons in vitro, as well as other cell types from that area. Upon study with patch-clamp electrophysiology, cultured RTN neurons responded to CO 2 and 5-HT in similar ways to what is reported for different RTN neuron preparations. Using this model, RTN neuron chemosensitivity was significantly decreased during application of 5-HT 7 antagonists (SB258719, SB269970) and a 5-HT 2A antagonist (MDL 11,939). The effect of 5-HT 7 antagonists was recapitulated in slice recordings. Therefore, signaling at 5-HT 7 and 5-HT 2A receptors is necessary for RTN neuron chemosensitivity. Exogenous 5-HT application also increased RTN neuron firing rate without potentiating the response to CO 2 , most likely indicating that the necessary 5-HT stimulation must come from neurons that can alter their activity during acidosis. We conclude that RTN neuron chemosensitivity is largely driven by chemosensitive 5-HT neurons, and should be considered an integrative or relay center, rather than an independently chemosensitive one.iii Public AbstractBreathing is central to the survival of humans and many other animals because it brings oxygen into the bloodstream and expels waste carbon dioxide from the body.Breathing can also regulate the acidity (pH) of the blood, and is tightly controlled to keep all of these factors in balance. This control involves neurons in the brain that are specialized to detect pH and carbon dioxide levels in the blood; these are called chemoreceptors. Malfunction of these chemoreceptors has been related to sudden infant death syndrome (SIDS) and sudden unexpected death in epilepsy (SUDEP).It is not fully understood how these chemoreceptors work or how much they communicate with one another, however there are two prime candidates for this function: serotonin neurons and retrotrapezoid nucleus (RTN) neurons. It is known that serotonin neurons send signals to RTN neurons, but whether that affects their ability to detect carbon dioxide remains unknown.To answer this question, we simplified this system by growing these serotonin and RTN neurons together in cell cultureessentially in a petri dish. We were then able to stud...