This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Systems/Circuits Cerebrospinal fluid-contacting neurons sense pH changes and motion in the hypothalamus Elham Jalalvand1, Brita Robertson1, Hervé Tostivint2, Peter Löw1, Peter Wallén1 and Sten Grillner1 1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden 2Evolution des Régulations Endocriniennes, UMR 7221 CNRS, and Muséum National d'Histoire Naturelle, Paris, France. DOI: 10.1523/JNEUROSCI.3359-17.2018 Received: 24 November 2017 Revised: 4 July 2018 Accepted: 15 July 2018 Published: 23 July 2018 Author contributions: E.J., B.R., P.W., and S.G. designed research; E.J., H.T., and P.L. performed research; E.J., B.R., P.L., and P.W. analyzed data; E.J., B.R., P.W., and S.G. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by the Vetenskapsrådet VR-M-K2013-62X-03026 and VR-M-2015-02816, VR- NT-621-2013-4613, the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no 604102 (HBP), EU/Horizon 2020 under grant agreement no 720270 (HBP SGA1), StratNeuro Karolinska Institutet, the Karolinska Institutet's Research Funds and the Centre National de la Recherche Scientifique and the Muséum National d'Histoire Naturelle. We thank Dr. Charlotta Borgius for assistance with the in situ hybridization, and Dr. Liang Wang for conducting experiments on dopamine CSF-c neurons in the spinal cord, which did not respond to deviations in pH. Correspondence to: Prof. Sten Grillner, The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden., E-mail: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3359-17.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Cerebrospinal fluid-contacting neurons sense pH changes and motion in the 2 hypothalamus 3 Abbreviated title: Hypothalamic CSF-c neurons sense pH and motion 4 Elham Jalalvand1, Brita Robertson1, Hervé Tostivint2, Peter Löw1, Peter Wallén1 and Sten 5 Grillner1* 6 1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, 7 SE-171 77 Stockholm, Sweden and 2Evolution des Régulations Endocriniennes, UMR 7221 8 CNRS, and Muséum National d'Histoire Naturelle, Paris, France. 9 *Correspondence to: Prof. Sten Grillner, The Nobel Institute for Neurophysiology, 10 Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 11 E-mail: [email protected] 12 Number of pages: 33 13 Number of figures: 6 14 Number of words in Abstract: 237 15 Number of words in Introduction: 595 16 Number of words in Discussion: 1766 17 We declare no conflict of interest. 18 Authors contributions: S.G., P.W., B.R. and E.J. designed research; E.J., P.L. and H.T. 19 performed experiments; E.J., P.W. P.L. and B.R. analyzed data; E.J., B.R., P.W. and S.G. 20 wrote the paper. 21 22 Acknowledgements: This work was supported by the Vetenskapsrådet VR-M-K2013-62X- 23 03026 and VR-M-2015-02816, VR-NT-621-2013-4613, the European Union Seventh 24 Framework Programme (FP7/2007-2013) under grant agreement no 604102 (HBP), 25 EU/Horizon 2020 under grant agreement no 720270 (HBP SGA1), StratNeuro Karolinska 26 Institutet, the Karolinska Institutet’s Research Funds and the Centre National de la Recherche 27 Scientifique and the Muséum National d’Histoire Naturelle. We thank Dr. Charlotta Borgius 28 for assistance with the in situ hybridization, and Dr. Liang Wang for conducting experiments 29 on dopamine CSF-c neurons in the spinal cord, which did not respond to deviations in pH. 30 31 ABSTRACT 32 Cerebrospinal fluid-contacting (CSF-c) cells are present in the walls of the brain ventricles 33 and the central canal of the spinal cord and found throughout the vertebrate phylum. We 34 recently identified ciliated somatostatin/GABA-expressing CSF-c neurons in the lamprey 35 spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and 36 alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. 37 Here, we investigate the functional properties of the ciliated somatostatin/GABA-positive 38 CSF-c neurons in the hypothalamus, by performing whole-cell recordings in hypothalamic 39 slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c 40 neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, 41 however, with membrane potential depolarization and trains of action potentials to small 42 deviations in pH, in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the 43 acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the 44 alkaline response appears to depend on connexin hemichannels, and not PKD2L1 channels. 45 We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced 46 by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like 47 channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally and 48 laterally, but as yet the effects exerted on hypothalamic circuits are unknown. With similar 49 neurons being present in rodents, the pH- and mechano-sensing ability of hypothalamic CSF-c 50 neurons is most likely conserved throughout vertebrate phylogeny. 51 Keywords: CSF-c neurons; hypothalamus; somatostatin; pH sensor; ASIC3; mechanosensor. 52 53 2 54 Significance statement 55 CSF-contacting neurons are present in all vertebrates, located mainly in the hypothalamic area 56 and the spinal cord. Here, we report that the somatostatin/GABA-expressing CSF-c neurons 57 in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH, and do so 58 via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic 59 CSF-c neurons have extensive axonal ramifications and may decrease the level of motor 60 activity via release of somatostatin. In conclusion, hypothalamic somatostatin/GABA- 61 expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic 62 mechanism, designed to sense any deviation from physiological pH and thus constitute a 63 feedback regulatory system, intrinsic to the CNS, possibly serving a protective role from 64 damage caused by changes in pH. 65 INTRODUCTION 66 All organisms are sensitive to changes in the extracellular pH and consequently, for their 67 survival, it is necessary to maintain the pH stable within the physiological range. Variations in 68 extracellular or intracellular pH in brain tissue modulates neuronal excitability and function 69 (Ruusuvuori and Kaila, 2014). Increased CO2 levels, as in ischemia, will result in acidosis, 70 while conversely hyperventilation due to low O2 will result in alkalosis, and similarly 71 metabolic events can influence the pH in both directions (Levin and Buck, 2015). Moreover, 72 during high levels of neuronal activity, pH within the CNS itself is lowered through enhanced 73 levels of lactate that is produced by astrocytes (Magistretti and Allaman, 2015, 2018). 74 We recently showed that the ciliated somatostatin/GABA-expressing CSF-c neurons 75 located at the lateral aspect of the central canal in the lamprey spinal cord act as pH sensors. 76 The same cell detects both acidic and alkaline deviations from around pH 7.4, through the 77 acid-sensing ion channel 3 (ASIC3)-like and the polycystic kidney disease (PKD)-protein-2- 78 like 1 (PKD2L1) channel, respectively, resulting in an increased depolarization and firing of 3 79 action potentials (Jalalvand et al., 2016a, b). Similarly, CSF-c neurons in the mouse dorsal 80 vagal complex respond to alkaline pH via the PKD2L1 channel (Orts-Del’Immagine et al., 81 2012, 2016). 82 Even moderate changes in the extracellular pH, whether in the alkaline or acidic 83 direction (0.3 pH units or less) will enhance CSF-c neuronal spike firing, which in turn will 84 suppress the locomotor activity by negative feedback to the spinal cord circuits. The response 85 to any pH change in the spinal cord will thus be a reduced motor activity, which should help 86 the organism to recover its normal pH (Jalalvand et al., 2016a, b). 87 CSF-c neurons come in many different forms that express different transmitters and 88 peptides. They are present in all major groups of vertebrates, from cyclostomes to mammals, 89 and line the wall of the brain ventricles and the central canal (Vigh-Teichmann et al., 1983a; 90 Brodin et al., 1990; Vigh et al., 1977, 2004; Vigh and Vigh-Teichmann, 1998; Russo et al., 91 2008; Marichal et al., 2009; Orts-Del’Immagine et al., 2012, 2016; Jalalvand et al., 2014). 92 The largest number of CSF-c neurons in the brain is found in the third ventricle in the 93 diencephalon, mainly in the hypothalamus (Vigh and Vigh-Teichmann, 1998). Hypothalamus 94 is an evolutionarily conserved area of the brain that regulates a variety of homeostatic 95 mechanisms such as osmoregulation, food intake and thermoregulation (Boulant and Dean, 96 1986; Hofman and Swaab, 1993; Broberger, 2005; Saper at al., 2002, 2005; Ball, 2007; 97 DiMicco and Zaretsky, 2007). Hypothalamic cultured neurons (mouse) have been reported to 98 be sensitive to acidic pH changes through ASICs (Wang et al., 2007). 99 The hypothalamic CSF-c neurons have a ciliated bulb-like ending that protrudes into 100 the third ventricle (Vigh et al., 1980) and their axons branch and extend laterally, dorsally and 101 ventrally.