Breathing is our first act upon birth and the last action we complete before death. The first to last breath taken, is in fact, how we define someone’s life. Since it was first reported that the blood concentration of CO2istightly controlled, and provides the dominant drive to breathe, the search for the cells that regulate it began. It took almost 60 years for the identification of the first central chemosensitive areas, regions within the brain that respond to specific chemical stimuli (such as CO2or its proxy H+), found at the ventrolateral surface of the medulla (VLM). Since then the debate over which cells in these areas are responsible for detectingCO2and signalling its fluctuations to the respiratory oscillators, has been extensive and heated. Chemosensitive cells are thought to have cell bodies located in, or close to, the VLM with dendrites in close apposition to blood vessels to better detect changes in blood gases. Several candidates fulfil this criteria, including the retrotrapezoid nucleus (RTN) and medullary raphe

Huckstepp, Robert T. R.

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Your input is a breath of fresh air! A chemosensory microcircuit of medullary raphe and RTN neurons

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     warwick.ac.uk/lib-publications      Manuscript version: Author’s Accepted Manuscript The version presented in WRAP is the author’s accepted manuscript and may differ from the published version or Version of Record.  Persistent WRAP URL: http://wrap.warwick.ac.uk/115923                              How to cite: Please refer to published version for the most recent bibliographic citation information.  If a published version is known of, the repository item page linked to above, will contain details on accessing it.  Copyright and reuse: The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions.   Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.  Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way.  Publisher’s statement: Please refer to the repository item page, publisher’s statement section, for further information.  For more information, please contact the WRAP Team at: wrap@warwick.ac.uk.   This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/JP277972. This article is protected by copyright. All rights reserved. DOI: 10.1113/JP277972   Title Page   Your input is a breath of fresh air! A chemosensory microcircuit of medullary raphe and RTN neurons Huckstepp, Robert T.R.  School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK. Tel: +44 24 765 75097; E-Mail: R.Huckstepp@warwick.ac.uk  Total number of Pages: 4 (including title page)   Figures: 0   Total number of words: 1005 excluding title page and references     Breathing is our first act upon birth and the last action we complete before death. The first to last breath taken, is in fact, how we define someone’s life. Since it was first reported that the blood concentration of CO2 is tightly controlled, and provides the dominant drive to breathe, the search for the cells that regulate it began. It took almost 60 years for the identification of the first central chemosensitive areas, regions within the brain that respond to specific chemical stimuli (such as CO2 or its proxy H+), found at the ventrolateral surface of the medulla (VLM). Since then the debate over which cells in these areas are responsible for detecting CO2 and signalling its fluctuations to the respiratory oscillators, has been extensive and heated. Chemosensitive cells are thought to have cell bodies located in, or close to, the VLM with dendrites in close apposition to blood vessels to better detect changes in blood gases. Several candidates fulfil this criteria, including the retrotrapezoid nucleus (RTN) and medullary raphe. The RTN gained traction as a central chemoreceptor with the discovery that it expresses the homeobox gene, Phox2b, linking it to congenital central hypoventilation syndrome (CCHS) (Stornetta et al., 2006); a respiratory disorder, characterised by a complete lack of chemosensitivity and an absence of the drive to breathe during sleep. Whilst CCHS can be replicated by targeted loss of   This article is protected by copyright. All rights reserved. Phox2b neurons in the RTN, respiratory responses to CO2 partially recover by 3 months of age (Ramanantsoa et al., 2011). Therefore the importance of RTN neurons to the hypercapnic ventilatory response (HVCR) diminishes with age, with other nuclei assuming this role. Contrary to the RTN, the medullary raphe become more responsive to hypercapnia with postnatal development (Cerpa et al., 2017). Thus age must be considered when choosing a model for testing the chemosensitivity of these 2 nuclei (Huckstepp & Dale, 2011). In this issue of the Journal of Physiology, Wu et al., demonstrate the chemosensitive response of juvenile to young adult RTN neurons is largely dependent on serotonergic input from the medullary raphe (Wu et al., 2019). Furthermore, they were able to recapitulate the in vivo connectivity of the raphe and RTN (Mulkey et al., 2007), in a co-culture of neurons from these regions upon a bed of glia. This provides a novel reduced model for studying this important microcircuit without confounding afferents from other regions; following antagonism of serotonergic signalling the residual response to hypercapnic acidosis of RTN neurons in culture was considerably lower than that of RTN neurons in the more intact circuitry of brain slices (Wu et al., 2019). Given the response of serotonergic neurons to hypercapnic acidosis and their influence on RTN neurons (Wu et al., 2019), it is not surprising that loss of serotonergic neurons leads to a 50% reduction in the HCVR of adult mice (Hodges et al., 2008). Intriguingly, the HCVR of these mice can be rescued by intracerebroventricular administration of 5-HT (Hodges et al., 2008). Investigating this further, Wu et al. show blockade of the serotonin transporter increased the response of RTN neurons to hypercapnic acidosis, whilst depletion of serotonin by the tryptophan hydroxylase inhibitor, PCPA, blunted it. Interestingly, exogenous 5-HT added to the milieu of PCPA treated neurons did not restore the hypercapnic response of RTN neurons when hyperpolarising current was added to restore membrane potential to normal resting levels. Therefore, it appears that 5-HT could increase the HVCR by 2 mechanisms; 1) signalling the increased activity of the raphe in response to H+/CO2, and 2) inducing a depolarising current that brings the membrane potential of neurons in other respiratory nuclei (e.g. RTN) into a more excitable range, allowing typically subthreshold currents to induce neuronal firing.  Following antagonism of both 5-HT2 and 5-HT7 receptors at room temperature, and 5-HT7 at a more physiologically relevant temperature, there was a residual response to hypercapnic acidosis in RTN neurons in culture of 20% and 17% respectively (Wu et al., 2019). As these antagonists were not able to fully prevent the response of RTN neurons to exogenously applied 5-HT, it may be that the remaining response to hypercapnic acidosis comes from other serotonergic pathways that do not involve these 5-HT receptor sub-types. However, depletion of serotonin by PCPA reduced the hypercapnic response of RTN neurons by a similar amount (18%) (Wu et al., 2019), suggesting this is not the case.  So where might this residual chemosensitivity come from? Firstly, the medullary raphe are still able to excite RTN neurons via thyrotropin-releasing hormone and/or substance-P (Mulkey et al., 2007), and both pathways remain intact throughout the experiments performed by Wu et al. Another potential interpretation is stimulation of RTN neurons by gliotransmitters (Huckstepp & Dale, 2011), as the RTN responds to glial-derived signalling molecules and glia are present in Wu et al.'s cultures. Alternatively, it may be due to an intrinsic chemosensitivity of RTN neurons. The acid-sensing channel, GPR4, was recently identified as a pH-sensor in the RTN (Kumar et al., 2015). However, this   This article is protected by copyright. All rights reserved. channel fails the test of a chemosensory transducer because its presence is not sufficient to convey chemosensitivity (Huckstepp & Dale, 2011), as removal of GPR4 in the raphe did not affect the CO2-response of these neurons (Kumar et al., 2015). Moreover, expression of GPR4 in the RTN is relatively low and it’s pH-sensitive range is not conducive to driving the respiratory response to CO2 (Hosford et al., 2018), posing serious doubts over the role of this channel as a physiologically relevant CO2/H+ transducer. Thus, for intrinsic chemosensitivity of RTN neurons to be a convincing possibility, more conclusive evidence for GPR4 bestowing acid sensitivity to these neurons, or a new candidate chemosensory transducer, must be found. In summary, the multiple roles of the medullary raphe in the respiratory response to hypercapnia of juvenile and young adult mice has been further illuminated by this comprehensive and compelling study by Wu et al. The burning question now is - what underlies the hypercapnic response of RTN neurons that persists in the absence of 5-HT7 and 5-HT2a signalling, and depletion of serotonin?  References Cerpa VJ, Wu Y, Bravo E, Teran FA, Flynn RS & Richerson GB. (2017). Medullary 5-HT neurons: Switch from tonic respiratory drive to chemoreception during postnatal development. Neuroscience 344, 1-14.  Hodges MR, Tattersall GJ, Harris MB, McEvoy SD, Richerson DN, Deneris ES, Johnson RL, Chen ZF & Richerson GB. (2008). Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons. J Neurosci 28, 2495-2505.  Hosford PS, Mosienko V, Kishi K, Jurisic G, Seuwen K, Kinzel B, Ludwig MG, Wells JA, Christie IN, Koolen L, Abdala AP, Liu BH, Gourine AV, Teschemacher AG & Kasparov S. (2018). CNS distribution, signalling properties and central effects of G-protein coupled receptor 4. Neuropharmacology 138, 381-392.  Huckstepp RTR & Dale N. (2011). Redefining the components of central CO2 chemosensitivity – towards a better understanding of mechanism. J Physiol 589, 5561-5579.  Kumar NN, Velic A, Soliz J, Shi Y, Li K, Wang S, Weaver JL, Sen J, Abbott SBG, Lazarenko RM, Ludwig M-G, Perez-Reyes E, Mohebbi N, Bettoni C, Gassmann M, Suply T, Seuwen K, Guyenet PG, Wagner CA & Bayliss DA. (2015). Regulation of breathing by CO₂ requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons. Science 348, 1255-1260.  Mulkey DK, Rosin DL, West G, Takakura AC, Moreira TS, Bayliss DA & Guyenet PG. (2007). Serotonergic neurons activate chemosensitive retrotrapezoid nucleus neurons by a pH-independent mechanism. J Neurosci 27, 14128-14138.  Ramanantsoa N, Hirsch MR, Thoby-Brisson M, Dubreuil V, Bouvier J, Ruffault PL, Matrot B, Fortin G, Brunet JF, Gallego J & Goridis C. (2011). Breathing without CO2 chemosensitivity in conditional phox2b mutants. J Neurosci 31, 12880-12888.   This article is protected by copyright. All rights reserved.  Stornetta RL, Moreira TS, Takakura AC, Kang BJ, Chang DA, West GH, Brunet JF, Mulkey DK, Bayliss DA & Guyenet PG. (2006). Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J Neurosci 26, 10305-10314.  Wu Y, Proch K, Teran F, Lechtenberg R, Kothari H & Richerson G. (2019). Chemosensitivity of Phox2b-expressing retrotrapezoid neurons is mediated in part by input from 5-HT neurons. J Physiol.   

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Your input is a breath of fresh air! A chemosensory microcircuit of medullary raphe and RTN neurons

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