["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend Central respiratory chemoreceptors (CRCs) are critical for maintaining normal systemic levels of pH and PCO2. A CRC must respond to pH via intrinsic mechanisms. Here we quantified intrinsic chemosensitivity of medullary 5‐HT neurones and retrotrapezoid nucleus (RTN) neurones using acute dissociation. Top – RTN neurones were acutely dissociated and recorded with patch‐clamp recordings. Most RTN neurones (84%), including those that expressed Neuromedin B (Nmb), did not respond to respiratory acidosis. Middle – In contrast, 48% of 5‐HT neurones were stimulated by acidosis. Bottom – RTN neurones were co‐cultured with 5‐HT neurones that expressed channelrhodopsin. When recordings were made from RTN neurones, a subset was stimulated by respiratory acidosis. These same neurones were stimulated by light, indicating that 5‐HT neurones had formed synapses on them. These results indicate that a subset of medullary 5‐HT neurones are important CRCs. RTN neurones may integrate and relay pH information from 5‐HT neurones but contribute less to direct pH sensation.\n\n\n\n\n\n\n\n\n\nAbstract\nCentral respiratory chemoreceptors (CRCs) are critically important for maintaining normal systemic levels of pH and PCO2. Their specific identity has been controversial. It is widely acknowledged that CRCs must respond to small changes in pH via cell‐autonomous (intrinsic) mechanisms. However most studies designed to identify CRCs have only blocked a limited subset of synaptic mechanisms. Here we used patch‐clamp recordings to compare chemosensitivity of two candidates for CRCs: (1) Phox2b‐expressing neurones of the retrotrapezoid nucleus (RTN) and (2) serotonin (5‐HT)‐producing neurones of the medullary raphe nuclei. We used acute dissociation to ensure responses were intrinsic. In response to a change in CO2 from 5% to 9% (pH 7.4 to ≈7.2) 48% of medullary 5‐HT neurones (n = 118) increased their firing rate by more than 20% with a mean increase of 139%, whereas 16% of RTN neurones (n = 93) increased their firing rate by more than 20% with a mean increase of 46%. RTN neurones that expressed Neuromedin B (Nmb) (a proposed biomarker of RTN CRCs) were not more likely to have a larger pH response. After 3 days in culture many RTN neurones began to receive excitatory synaptic drive from 5‐HT neurones and also responded to acidosis. These results support the conclusion that a subset of medullary 5‐HT neurones are CRCs. Many RTN neurones may play a role in integration and relay of pH information from 5‐HT neurones and other chemoreceptor sites but contribute less to direct pH sensation.\n\n\n\n\n\n\n\n\n\nKey points\n\nCentral respiratory chemoreceptors in the brainstem detect changes in CO2 and pH via intrinsic mechanisms and induce changes in breathing to maintain pH homoeostasis. The identity of these cells is controversial.\nWe measured the pH response of two prominent candidates for these chemoreceptors after they were acutely isolated from all other cells to ensure that their responses were intrinsic.\nA small percentage of neurones from the retrotrapezoid nucleus had a small response to acidosis. A much larger percentage of serotonin neurones were stimulated by acidosis with a large increase in firing rate.\nOver the first few days in cultured neuronal preparations serotonin neurones formed synapses on retrotrapezoid neurones and stimulated them in response to acidosis.\nThese results reveal that a large subset of serotonin neurones have properties consistent with central chemoreceptors, whereas a small number of retrotrapezoid neurones have a small response, and may be more important as relays of chemoreceptor information rather than as direct sensors of pH themselves.\n\n\n"]