Systems/Circuits

Cerebrospinal Fluid-Contacting Neurons Sense pH Changesand Motion in the Hypothalamus

X Elham Jalalvand,1 X Brita Robertson,1 X Herve Tostivint,2 X Peter Low,1 Peter Wallen,1 and X Sten Grillner1

1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden and 2Evolution desRegulations Endocriniennes, Unite Mixte de Recherche 7221 Centre National de la Recherche Scientifique, and Museum National d’Histoire Naturelle,Paris, France

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughoutthe vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that actas pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like andPKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neuronsin the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked actionpotentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, withmembrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Likein spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline responseappears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond tomechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels.The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamiccircuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-cneurons is most likely conserved throughout vertebrate phylogeny.

Key words: ASIC3; CSF-c neurons; hypothalamus; mechanosensor; pH sensor; somatostatin

IntroductionAll organisms are sensitive to changes in the extracellular pH;therefore, for their survival, it is necessary to maintain the pH

stable within the physiological range. Variations in extracellularor intracellular pH in brain tissue modulates neuronal excitabil-ity and function (Ruusuvuori and Kaila, 2014). Increased CO2

levels, as in ischemia, will result in acidosis, whereas hyperventi-lation due to low O2 will result in alkalosis; similarly, metabolicevents can influence the pH in both directions (Levin and Buck,2015). Moreover, during high levels of neuronal activity, pHwithin the CNS itself is lowered through enhanced levels of lac-

Received Nov. 24, 2017; revised July 4, 2018; accepted July 15, 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.This work was supported by the Vetenskapsrådet (Grants VR-M-K2013-62X-03026, VR-M-2015-02816,

and VR-NT-621-2013-4613), the European Union Seventh Framework Programme (FP7/2007-2013 Grant604102 to H.B.P.), EU/Horizon 2020 Grant 720270 to H.B.P. specific grant agreement 1), StratNeuro Karolin-ska Institutet, the Karolinska Institutet’s Research Funds, the Centre National de la Recherche Scientifique,and the Museum National d’Histoire Naturelle. We thank Dr. Charlotta Borgius for assistance with the in situhybridization and Dr. Liang Wang for conducting experiments on dopamine CSF-c neurons in the spinal cordthat did not respond to deviations in pH.

The authors declare no competing financial interests.

Correspondence should be addressed to Sten Grillner, The Nobel Institute for Neurophysiology, Department ofNeuroscience, Karolinska Institutet, Solnavägen 9, SE-171 77 Stockholm, Sweden. E-mail: sten.grillner@ki.se.

DOI:10.1523/JNEUROSCI.3359-17.2018Copyright © 2018 the authors 0270-6474/18/387713-12$15.00/0

Significance Statement

CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here,we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations inthe extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-cneurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclu-sion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeo-static mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory systemintrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.

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tate that is produced by astrocytes (Magistretti and Allaman,2015, 2018).

We recently showed that the ciliated somatostatin-/GABA-expressing CSF-c neurons located at the lateral aspect of the centralcanal in the lamprey spinal cord act as pH sensors. The same celldetects both acidic and alkaline deviations from �pH 7.4 throughthe acid-sensing ion channel 3 (ASIC3)-like and the polycystic kid-ney disease (PKD)-protein-2-like 1 (PKD2L1) channel, respectively,resulting in an increased depolarization and firing of action poten-tials (Jalalvand et al., 2016a,b). Similarly, CSF-c neurons in themouse dorsal vagal complex respond to alkaline pH via the PKD2L1channel (Orts-Del’Immagine et al., 2014, 2016).

Even moderate changes in the extracellular pH, whether in thealkaline or acidic direction (0.3 pH units or less), will enhanceCSF-c neuronal spike firing, which in turn will suppress the lo-comotor activity by negative feedback to the spinal cord circuits.The response to any pH change in the spinal cord will thus be areduced motor activity, which should help the organism to re-cover its normal pH (Jalalvand et al., 2016a,b).

CSF-c neurons come in many different forms that expressdifferent transmitters and peptides. They are present in all majorgroups of vertebrates, from cyclostomes to mammals, and linethe wall of the brain ventricles and the central canal (Vígh et al.,1977, 2004; Vígh-Teichmann et al., 1983a; Brodin et al., 1990;Vígh and Vígh-Teichmann, 1998; Russo et al., 2008; Marichal etal., 2009; Orts-Del’Immagine et al., 2012, 2014, 2016; Jalalvand etal., 2014). The largest number of CSF-c neurons in the brain isfound in the third ventricle in the diencephalon, mainly in thehypothalamus (Vígh and Vígh-Teichmann, 1998). Hypothala-mus is an evolutionarily conserved area of the brain that regulatesa variety of homeostatic mechanisms such as osmoregulation,food intake, and thermoregulation (Boulant and Dean, 1986;Hofman and Swaab, 1993; Saper at al., 2002, 2005; Broberger,2005; Ball, 2007; Dimicco and Zaretsky, 2007). Hypothalamiccultured neurons (mouse) have been reported to be sensitive toacidic pH changes through ASICs (Wang et al., 2007).

The hypothalamic CSF-c neurons have a ciliated bulb-like end-ing that protrudes into the third ventricle (Vígh et al., 1980) and theiraxons branch and extend laterally, dorsally, and ventrally. Our goalhere is to investigate the functional role of the somatostatin-/GABA-expressing subpopulation of CSF-c neurons in the lamprey hypo-thalamus and to determine whether they respond to thecomposition of the CSF. Patch-clamp recordings were performedwhile the cells were subjected to moderate alterations in the ex-tracellular pH or to mechanical stimulation by applied fluidmovement. In contrast to non-somatostatin-expressing CSF-cneurons in the third ventricle, the somatostatin-/GABA-expressing hypothalamic CSF-c neurons responded to bothacidic and alkaline pH and to mechanical fluid motion. Theacidic and mechanical responses are mediated through ASIC3-like channels, whereas the alkaline response appears to be medi-ated through the connexin hemichannel. These results also showthat the hypothalamic somatostatin/GABA CSF-c neurons play arole as pH sensors as well as mechanosensors.

Materials and MethodsAnimals. Experiments were performed on a total of 44 adult river lam-preys (Lampetra fluviatilis) of both sexes that were collected from theLjusnan River, Halsingland, Sweden. The experimental procedures wereapproved by the local ethical committee (Stockholm’s Norra Djurforsok-setiska Namnd) and were in accordance with the Policy on the Use ofAnimals in Neuroscience Research (Society for Neuroscience). Duringthe investigation, every effort was made to minimize animal sufferingand to reduce the number of animals used.

Electrophysiology. Animals (n � 32) were deeply anesthetized throughimmersion in 0.01 M PBS containing tricane methanesulfonate (MS-222;100 mg L �1; Sigma-Aldrich). Following decapitation, the exposed brainwas removed and placed in ice-cold artificial CSF (aCSF; extracellularsolution) of the following composition (in mM): 125 NaCl, 2.5 KCl, 1MgCl2, 1.25 NaH2PO4, 2 CaCl2, 25 NaHCO3, and 10 glucose, pH 7.4. TheaCSF was oxygenated continuously with 95% O2 and 5% CO2 and os-molarity was udjusted to 290 mOsm L �1 with glucose. Transverse slices(300 �m) of the hypothalamic area were cut using a vibrating microtome(HM 650V; Microm International), and mounted in a cooled (5– 8°C)recording chamber. The chamber was continuously perfused with cooledaCSF. Responses to deviations in extracellular pH were recorded by ad-justing the pH of the perfusate to various pH values (6.5, 6.8, 7.1, 7.4, 7.7,8.0, and 8.3) with HCl or NaOH. Although it takes 2 min to exchange thefluid in the chamber, it has the advantage of revealing the exact pHapplied. Previous investigators have ejected solutions from a micropi-pette (Marichal et al., 2009; Orts-Del’Immagine et al., 2016), which hasthe advantage of short latencies, but the exact pH levels cannot be deter-mined. For mechanical fluid motion stimulation, glass pipettes (2– 4M�) were filled with aCSF. Fluid pulse stimuli were given by applying5–20 psi pressure pulses of 10 – 80 ms duration by a PicoSpritzer II unit(Parker Hannifin). Patch electrodes (8 –12 M�) were prepared fromborosilicate glass microcapillaries (Hilgenberg) using a two-stage puller(PP-830; Narishige) and filled with an intracellular solution of the fol-lowing composition (in mM): 130 K-gluconate, 5 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, and 10 phosphocreatine sodium salt. The pH of thesolution was adjusted to 7.4 with KOH and osmolarity to 270 mOsm L �1

with water.CSF-c cells in the hypothalamic area were recorded in whole-cell con-

figuration and in current-clamp or voltage-clamp mode using a Multi-clamp 700B amplifier (Molecular Devices). The gigaseal resistanceduring recording was �10 G�, at least five times higher than the inputresistance of the cells. Access to the cell in voltage-clamp mode wasrepeatedly confirmed during the course of the experiment. Cells werevisualized with differential interference contrast/infrared optics. The fol-lowing drugs were added to the extracellular solution and applied by bathperfusion: the specific ASCI3 blocker APETx2, an extract of the seaanemone, Anthopleura elegantissima toxin-2, (1 �M; catalog #500527;Calbiochem), the connexin hemichannel blocker lanthanum chloride(100 �M; catalog #298182; Sigma-Aldrich), the GABAA receptor antago-nist gabazine (20 �M; catalog #1262; Tocris Bioscience), the NMDA re-ceptor antagonist AP5 (50 �M; catalog #0105; Tocris Bioscience), theAMPA receptor antagonist CNQX (40 �M; catalog #1045; Tocris Biosci-ence), and tetrodotoxin (TTX, 1.5 �M; catalog #T8024; Sigma-Aldrich).

Immunohistochemistry. The brains (n � 8) were fixed by immersion in4% formalin and 14% saturated picric acid solution in 0.1 M phosphatebuffer (PB; pH 7.4; 1% glutaraldehyde was added for GABA immuno-histochemistry) for 12–24 h at 4°C and subsequently cryoprotected in20% sucrose in PB for 3–12 h. Then, 20 �m transverse sections were cuton a cryostat (catalog #HM 560; Microm), collected on gelatin-coatedslides, and kept at �20°C until processing. For detection of somatostatin(n � 2), coexpression of somatostatin/GABA (n � 2), coexpression ofsomatostatin/�-tubulin (n � 2), and somatostatin/connexin 35/36 (n �2), hypothalamic sections were incubated overnight with a rat monoclo-nal anti-somatostatin antibody (1:200; MAB354; RRID:AB_2255365;Millipore), a mouse monoclonal anti-GABA antibody (0.1 �g/ml;MAB3A12; RRID:AB_2314450; a generous gift from Prof. Peter Streit,Zurich, Switzerland), a mouse monoclonal anti-acetylated �-tubulin an-tibody (1:500; 6-11B-1; RRID:AB_477585; Sigma-Aldrich) or a mousemonoclonal anti-connexin 35/36 antibody (1:200; MAB3045; RRID:AB_94632; Millipore). The sections were subsequently rinsed thoroughlyin 0.01 M PBS. To visualize coexpression of somatostatin and GABA, thesections were incubated with a mixture of Cy3-conjugated donkey anti-rat IgG (1:500; catalog #712-165-150; RRID:AB_2340667; JacksonImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200; catalog #715-545-150; RRID:AB_2340846; Jack-son ImmunoResearch) for 2 h. For coexpression of somatostatin and�-tubulin or connexin 35/36, the sections were incubated with AlexaFluor 488-conjugated donkey anti-rat IgG (1:200; catalog #712-545-153;

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RRID:AB_2340684; Jackson ImmunoResearch) and Cy3-conjugateddonkey anti-mouse IgG (1:500; catalog #715-165-150; RRID:AB_2315777;Jackson ImmunoResearch). The sections were then rinsed in 0.01 M PBS andmounted with glycerol containing 2.5% diazabicyclooctane (DABCO) (cat-alog #D27802; Sigma-Aldrich).

To examine the morphology of hypothalamic CSF-c neurons, cellswere intracellularly labeled with 0.3% Neurobiotin (catalog #SP-1120;Vector Laboratories) during the whole-cell recording. The hypothalamicslices were fixed overnight in 4% formalin and 14% picric acid in 0.1 M

PB. They were subsequently rinsed thoroughly in 0.01 M PBS and incu-bated with Alexa Fluor 488-conjugated streptavidin (1:1000; catalog#016-640-084; Jackson ImmunoResearch) for 3 h. The sections werethen rinsed in 0.01 M PBS and mounted with glycerol containing 2.5%DABCO. Alternatively, after fixation and rinsing in PBS, slices were in-cubated in Vectastain (catalog #PK6100; Vector Laboratories) followedby diaminobenzidine (DAB) (ImmPACT, catalog #SK-4100; Vector Lab-oratories) for 5 min and then rinsed and dehydrated in alcohol beforemounting in Entellan (Merck).

To investigate whether the intracellularly Neurobiotin-labeled CSF-ccells express somatostatin, the slices were incubated overnight with a ratmonoclonal anti-somatostatin antibody (1:200; MAB354; RRID:AB_2255365; Millipore) and then rinsed thoroughly in 0.01 M PBS andincubated with a mixture of Alexa Fluor 488-conjugated streptavidin(1:1000; catalog #016-640-084; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-rat IgG (1:500; catalog #712-165-150; RRID:AB_2340667; Jackson ImmunoResearch). All primary and secondaryantibodies were diluted in 1% bovine serum albumin (BSA) with 0.3%Triton X-100 in 0.1 M PB.

In situ hybridization. Animals (n � 2) were deeply anesthetized asdescribed above and the brain and rostral spinal cord (the spinal cord wasused as a positive control) were removed, fixed in 4% formalin in 0.1 M

PB overnight at 4°C and then cryoprotected in 20% sucrose in 0.1 M PB.Then, 10- and 20-�m-thick cryostat sections were made and stored in�80°C until processed. Single-stranded digoxigenin-labeled sense andantisense pkd2l1 riboprobes were generated by in vitro transcription ofthe previously cloned pkd2l1 cDNA using the Digoxigenin RNA Labelingkit (Jalalvand et al., 2016b; catalog #11 277 073 910; Roche Diagnostics).Briefly, sections were incubated for 1 h in prehybridization mix (50%formamide, 5� SSC, 1% Denhardt’s, 50 �g/ml, salmon sperm DNA, 250�g/ml yeast RNA) at 60°C. Sections incubated with the heat-denaturateddigoxigenin-labeled riboprobe were hybridized overnight at 60°C. Fol-lowing the hybridization, the sections were rinsed twice in 1�SSC,washed twice in 1�SSC (30 min each) at 60°C and twice in 0.2�SSC atroom temperature. After blocking in 0.5% blocking reagent (PerkinEl-mer), the sections were incubated overnight in anti-DIG antibody cou-pled to HRP (1:2000; catalog #RRID:AB_514497; Roche Diagnostics) at4°C. The probe was then visualized by TSA Cy3 Plus Evaluation Kit(catalog #NEL763E001; PerkinElmer). The specificity of the hybridiza-tion procedure was verified by incubating sections with the sense ribo-probe (data not shown). The sections were rinsed thoroughly in 0.01 M

PBS and then incubated with a rat monoclonal anti-somatostatin anti-body (1:200; MAB354; Millipore) overnight at 4°C, rinsed in PBS, andincubated with Alexa Fluor 488-conjugated donkey anti-rat IgG (1:200;catalog #712-545-153; RRID:AB_2340684; Jackson ImmunoResearch)for 2 h and mounted with glycerol containing 2.5% DABCO. All primaryand secondary antibodies were diluted in 1% BSA and 0.3% Triton X-100in 0.1 M PB.

Western blot. Lamprey brains (n � 2) were solubilized by sonication in1% Na-SDS, 0.32 M sucrose, 20 mM HEPES, pH 7.6, and a mixture ofprotease inhibitors. Samples were then subjected to SDS-PAGE on Bio-Rad gradient gels (4 –15%) and subsequently transferred to PVDF mem-brane according to the manufacturer’s instructions. The membrane wasincubated with mouse anti-connexin 35/36 (1:1000; MAB3045; RRID:AB_94632; Millipore) overnight at 4°C and then with goat anti-mouseIgG-HRP (1:5000; P0447; DAKO) for 2 h at room temperature. It wasdeveloped with SuperSignal West Dura (Thermo Scientific).

Morphological analysis. Photomicrographs were taken with an Olym-pus XM10 digital camera mounted on an Olympus BX51 fluorescence/bright-field microscope (Olympus ). Intracellularly labeled cells were

drawn using a camera lucida tube attached to a bright field microscope(Leitz). Confocal images were obtained using a Zeiss confocal laser scan-ning microscope (LSM 510 NLO; Zeiss) and processed using Zeiss LSMsoftware. Illustrations were prepared using Adobe Illustrator and Photo-shop CS6. Images were adjusted only for brightness and contrast.

Experimental design and statistical analysis. Hypothalamic CSF-c neu-rons were recorded in whole-cell configuration and in current-clamp orvoltage-clamp mode. Resting membrane potentials were determined incurrent-clamp mode. Bridge balance and pipette capacitance compensa-tion were adjusted and signals were digitized and recorded using Clam-pex software and analyzed in Clampfit (pCLAMP 10; MolecularDevices). Membrane and firing properties were analyzed by injection ofshort (20 ms) and long (500 or 1000 ms) depolarizing and hyperpolariz-ing current pulses. Responses to deviations in extracellular pH were re-corded as changes in membrane potential and firing frequency (currentclamp) and in the frequency of inward current events (voltage-clamp).Effects were analyzed by comparing the responses in the same neuron atthe control condition at physiological pH (7.4) and at acidic or alkalinepH, respectively, or in the absence or presence of an ion channel blocker.In all cases, paired comparisons were made between two conditions (e.g.,control vs acidic pH or absence vs presence of blocker). Data are pre-sented as means � SD or SEM and statistical comparisons were madeusing Student’s paired two-tailed t test. ***p 0.001, **p 0.01 signif-icant difference compared with control; n.s.: no significant difference(for details, see figure legends).

ResultsSomatostatin-/GABA-expressing CSF-c neurons inthe hypothalamusAn area in the rostral hypothalamus contains numeroussomatostatin-positive CSF-c neurons (Fig. 1A,B). These neuronshave a short, thick apical process with a bulb-like ending (Fig. 1C)from which an �-tubulin-immunoreactive cilium protrudes intothe CSF (Fig. 1D, arrow). To further identify the phenotype of thehypothalamic CSF-c neurons, we investigated whether these cellscoexpress somatostatin and GABA, similar to spinal CSF-c neu-rons. Most of the somatostatin-positive neurons within the hy-pothalamus were CSF-c cells (Fig. 1B,C) that coexpressed GABA(Fig. 1E, arrows). However, some CSF-c neurons in this regionwere only immunoreactive to GABA (Fig. 1E, arrowhead). In thelamprey, the periventricular area of hypothalamus is the mainsource of somatostatin. Intracellular Neurobiotin labeling of re-corded hypothalamic CSF-c neurons showed that their axonalbranches extend laterally, dorsally, and ventrally (Fig. 1F).Somatostatin-positive fibers and terminals were found through-out the brain, including a dense innervation of motor-relatedareas such as the deep layer of the optic tectum and the reticu-lospinal nuclei (Fig. 1G,H). Conversely, sensory areas through-out the brain were devoid of somatostatin innervation, as shownin the retinorecipient superficial layer of the optic tectum and thebrainstem alar plate (Fig. 1G,H).

Presence of gap junctions in somatostatin-expressingCSF-c neuronsIntracellular injection of Neurobiotin into a single somatostatin-expressing CSF-c neuron in hypothalamus sometimes resulted instaining of one or two additional CSF-c neurons in close vicinityto the injected cell, suggesting dye coupling via gap junctions(n � 4; Fig. 2A). To investigate this possibility further, we appliedthe neuronal connexin-35/36 antibody, a gap junction marker(O’Brien et al., 1998; Srinivas et al., 1999). Somatostatin-positiveCSF-c neurons coexpressed the connexin protein (Fig. 2B–D),suggesting that gap junctions are present between at least someCSF-c neurons in the hypothalamus. Figure 2E is a Western blotof the anti-connexin 35/36 antibody (immunogen recombinant

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perch connexin 35), showing that this antibody recognizes a bandat around 35 kDa and is thus specific for the lamprey brain and aband a75 kDa, probably representing a dimer (O’Brien et al.,2004). Searching the genome build for Petromyzon_marinus-7.0

(petMar2) using mouse connexin, we also found a potential or-tholog for lamprey connexin (PMZ_00001234-RA), with morethan 80% identity to both mouse and perch connexin 35/36 (re-sults not shown).

Figure 1. Ciliated CSF-c neurons in the hypothalamus express GABA and somatostatin. A, Location of somatostatin-expressing CSF-c neurons (red dots) indicated in schematic drawings oftransverse sections through the hypothalamic area. B, Photomicrograph of somatostatin-immunopositive CSF-c neurons in the hypothalamus (arrows). Scale bar, 200 �m. C, High-magnificationphotomicrograph of the boxed area in C. CSF-c neurons expressing somatostatin have short, thick apical processes with a bulb-like ending protruding into the ventricle (arrows). Scale bar, 20 �m.D, Confocal image of a somatostatin-expressing hypothalamic CSF-c neuron (green) with an �-tubulin-immunoreactive cilium (magenta, arrow). Scale bar, 5 �m. E, Somatostatin (magenta) andGABA (green) are colocalized in hypothalamic CSF-c neurons (arrows). Some of the CSF-c neurons only expressed GABA (arrowhead). Scale bar, 20 �m. F, Illustration of a reconstructed intracellularlylabeled hypothalamic CSF-c neuron with a bulb-like ending protruding into the ventricle and axonal branches extending dorsally, laterally, and ventrally. G, Somatostatin-immunoreactivefibers/terminals at the level of the optic tectum. Note the dense innervation of the deep layer (DL) and absence of labeling in the retinorecipient superficial layer (SL). Scale bar, 400 �m. H, Richsomatostatin labeling in the ventral brainstem at the level of MRRN and the trigeminal nucleus (nV). Scale bar, 200 �m. Th, Thalamus; DL, deep layer; cpo, postoptic commissure; Hb, habenula; Hyp,hypothalamus; LPal, lateral pallium; MPal, medial pallium; MRRN, middle rhombencephalic reticular nucleus; ncpo, nucleus of the postoptic commissure; nh, neurohypophysis; ot, optic tract; SL,superficial layer.

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Neuronal properties of hypothalamic CSF-c neuronsTo examine the membrane properties of somatostatin-expressingCSF-c neurons in the hypothalamus, we performed whole-cellrecordings in voltage- or current-clamp mode (n � 45 cells).The hypothalamic CSF-c neurons had a mean resting mem-brane potential of �65 � 4 mV, a mean input resistance of1.9 � 0.4 G� (n � 15), and a mean spike threshold of �45 �1.8 mV (n � 15). In contrast to spinal CSF-c neurons, hypo-thalamic CSF-c neurons did not fire action potentials sponta-neously under control conditions. However, spiking wasreadily evoked in response to depolarizing current injection.For a current pulse of short duration (20 pA, 20 ms), the

neuron responded with a single action potential followed byan afterhyperpolarization (Fig. 3A, left trace). A train of actionpotentials was evoked with longer current injections (20 pA,500 ms; Fig. 3A, right trace). Hypothalamic CSF-c neuronsgenerally showed spontaneous GABAergic and glutamatergicpostsynaptic potentials, the latter mediated by both the AMPAand NMDA receptors (Fig. 3B). The voltage responses re-corded during depolarizing current steps in current-clampmode showed reliable spiking with spike frequency adaptation(Fig. 3C). The I–V curve revealed a linear current–voltagerelationship (Fig. 3D).

Figure 2. Hypothalamic CSF-c neurons express connexin. A, Intracellular injection of Neurobiotin in a hypothalamic CSF-c neuron (patched cell) resulted in two filled neurons revealed by DABstaining. Scale bar, 20 �m. B, Somatostatin-expressing hypothalamic CSF-c neurons. Scale bar, 20 �m. C, Connexin 35/36 immunoreactivity in the same hypothalamic area as in B. Scale bar, 20 �m.D, Merged image showing connexin 35/36-immunoreactivity (arrows) coexpressed with somatostatin in hypothalamic CSF-c neurons. Scale bar, 20 �m. E, Western blot of a lamprey brain extractdetected with a monoclonal mouse anti-connexin 35/36 antibody showing a distinct band around 35 kDa.

Figure 3. Electrophysiological properties of hypothalamic CSF-c neurons. A, Whole-cell current-clamp recording of a hypothalamic CSF-c neuron showing its firing pattern. A brief currentinjection (20 pA, 20 ms; left) elicited a single action potential, whereas longer current injections (20 pA, 500 ms; right) generated repetitive firing. B, CSF-c neuron showing spontaneous GABA- andglutamate-mediated postsynaptic potentials (top trace) that were successively blocked by gabazine (20 �M), CNQX (40 �M), and AP5 (50 �M; bottom traces), respectively. C, Voltage responses to12 consecutive hyperpolarizing and depolarizing current injections. The red and green traces illustrate a single action potential and spike frequency adaptation evoked by depolarizing steps,respectively. D, I–V curve showing a linear current–voltage relationship.

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Hypothalamic CSF-c neurons expressing somatostatinrespond to acidic and alkaline pHTo determine whether hypothalamic somatostatin-expressingCSF-c neurons respond to changes of the extracellular pH, acidic(pH 6.5) (Fig. 4A, red trace) or alkaline (pH 8.0) (Fig. 4A, bluetrace) solutions were bath-applied to hypothalamic slices during

whole-cell recording. To exclude the possibility of indirect effectsthrough synaptic inputs, blockers of GABA (gabazine) and glu-tamate (AP5, CNQX) receptors were applied throughout theseexperiments. In 30 of 45 tested CSF-c neurons, a decrease as wellas an increase of pH in the same cell depolarized the membranepotential by 10 –12 mV, eliciting a sustained discharge of action

Figure 4. Hypothalamic CSF-c neurons are activated by both acidic and alkaline pH. A, Change to acidic (pH 6.5) as well as to alkaline (pH 8.0) extracellular pH (gray area) depolarized themembrane potential (10 –12 mV) and triggered action potentials. Upon return to pH 7.4, firing ceased and the membrane potential repolarized back to the control value (�63 mV). In this and allsubsequent recordings, gabazine (20 �M), AP5 (50 �M), and CNQX (40 �M) were applied to exclude any indirect, synaptically mediated effects. B, In the presence of TTX (1.5 �M), both acidic andalkaline pH resulted in depolarization of the membrane potential that recovered after return to pH 7.4. C, Photomicrographs of the CSF-c neuron recorded in A after intracellular labeling withNeurobiotin (green, arrow), showing that the cell expressed somatostatin (magenta). Scale bars, 20 �m. D, Mean membrane potential changes in hypothalamic CSF-c neurons for each pH condition[mean � SD; n � 15; Student’s paired t test: ***p 0.001 significant difference vs pH 7.4 at 6.5 ( p � 1.43 � 10 �19, t14 � 22.7), at 6.8 ( p � 5.56 � 10 �18, t14 � 19.76), at 7.1 ( p � 1.97 �10 �14, t14 � 14.35), at 7.7 ( p � 2.20 � 10 �13, t14 � 13.0), at 8.0 ( p � 2.24 � 10 �20, t14 � 24.33), and at 8.3 ( p � 1.38 � 10 �21, t14 � 26.99)]. E, In voltage-clamp mode, frequent inwardcurrent deflections appeared at pH 6.5 and 8.0 with a maximal amplitude of �10 pA. F, Mean frequency increase of events (5–15 pA) in response to acidic and alkaline pH [mean � SEM; n � 7;Student’s paired t test: ***p 0.001 significant difference vs pH 7.4 at pH 6.5 ( p � 2.34 � 10 �7; t6 � 25.59) and at 8 ( p � 4.48 � 10 �8; t6 � 33.79)]. G, Unitary current deflections recordedat acidic and alkaline pH. TTX (1.5 �M) was present in B, E–G.

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potentials (Fig. 4A). After application of TTX, the membranepotential depolarization induced by acidic or alkaline pH re-mained (n � 12; Fig. 4B).

These results thus suggest that the response to both acidic andalkaline extracellular pH is due to a direct effect on the CSF-cneuron. The recorded cells were intracellularly labeled with Neu-robiotin and costained for somatostatin, verifying that the pH-sensing CSF-c cells express somatostatin (Fig. 4C). None of therecorded CSF-c neurons that were nonresponsive to acidic oralkaline extracellular pH expressed somatostatin (n � 15). Figure4D illustrates the mean membrane potential changes of CSF-cneurons recorded in whole-cell mode, showing that individualCSF-c neurons in the hypothalamus respond with membranepotential depolarization (and firing) to step changes in pH (6.5,6.8, 7.1, 7.7, 8.0, and 8.3) from the normal value of 7.4, yielding aU-shaped response curve (p 0.001, paired t test; n � 15 cells).

The response to acidic and alkaline pH was also analyzed involtage-clamp mode (Fig. 4E–G). Only a few low-amplitude cur-rent events were seen at pH 7.4 (AP5, CNQX, gabazine, and TTXpresent; Fig. 4E). Upon decreasing or increasing the extracellularpH to 6.5 or 8.0, respectively, the frequency of inward currentevents increased markedly (p 0.001, paired t test; n � 7 cells;Fig. 4E,F). We also recorded unitary inward current events, pre-sumably corresponding to single-channel openings, at acidic andalkaline pH (Fig. 4G).

At the transition from the brainstem to the spinal cord, wherethe central canal starts, there is a small region in which CSF-cneurons coexpress somatostatin and dopamine. These cells alsoresponded to acidic and alkaline pH deviations (n � 3; not illus-trated). However, CSF-c neurons at both the obex level and in thespinal cord that only express dopamine did not respond to pHchanges (n � 4). It thus appears that the common factor is anexpression of somatostatin in CSF-c neurons responding to de-viations of the extracellular pH.

Specific blockade of ASIC3 eliminates the response ofhypothalamic CSF-c neurons to acidic pHTo investigate whether the sensitivity of hypothalamic CSF-cneurons to decreases in pH depends on the ASIC3 subtype, as isthe case with their spinal counterparts (Jalalvand et al., 2016a),the specific ASIC3 blocker APETx2 was applied. In current-clamp mode, changing the pH of the extracellular solution toacidic or alkaline resulted in firing of action potentials, as well asa net depolarization of the membrane potential (Fig. 5A). Follow-ing application of APETx2, both aspects of the response wereeliminated at acidic pH, whereas the alkaline response was notaffected (n � 5; Fig. 5A). Changing the pH of the extracellularsolution to 6.5 or 8.0 in voltage-clamp mode (gabazine, CNQX,AP5, and TTX present) caused inward current deflections thatwere abolished by APETx2 at acidic pH, whereas the currentevents induced by alkaline pH remained (pH 6.5 vs 7.4 nonsig-nificant; pH 8 vs 7.4 p 0.01, paired t test; n � 3 cells; Fig. 5B,C).Therefore, as in the spinal CSF-c neurons, ASIC3-like channelsrepresent the acid sensor in somatostatin-expressing hypotha-lamic CSF-neurons.

Specific blockade of connexin hemichannels eliminates theresponse of hypothalamic CSF-c neurons to alkaline pHTo investigate which type of channel that might be sensing alka-line pH in hypothalamic CSF-c neurons, we performed in situhybridization for PKD2L1 (no specific PKD2L1 antagonist ex-ists). This channel was a main candidate because it is consideredto mediate the alkaline response in spinal somatostatin-positive

CSF-c neurons (Jalalvand et al., 2016b). However, no hypotha-lamic somatostatin-expressing nor any other hypothalamicCSF-c neurons expressed the PKD2L1 channel (Fig. 5D). As apositive control, we performed in situ hybridization on spinalcord sections that were mounted on the same slide as the hypo-thalamic sections. Strong PKD2L1 expression was observed inspinal CSF-c neurons (Fig. 5E), consistent with our previousfindings (Jalalvand et al., 2016b). Therefore, PKD2L1 is not thealkaline sensor in hypothalamic somatostatin-expressing CSF-cneurons.

The connexin hemichannels, which can be present in the cellmembrane even though there is no coupling to an adjacent cell,corresponds structurally to one-half of the gap junction channeland represents another candidate that has been suggested to sensealkaline pH (Schalper et al., 2010). The somatostatin-positivehypothalamic CSF-c neurons are immunoreactive to connexin35/36 (Fig. 2), so we tested whether the connexin hemichannelblocker lanthanum (La 3) could block the alkaline response.Lanthanum blocks connexin hemichannels but have no effect onthe gap junction channels themselves at the concentrations used(100 and 70 �M; Contreras et al., 2002; Spray et al., 2006). In aCSF-c neuron that responded to both acidic and alkaline pH,application of LaCl3 (100 �M) completely blocked responses toalkaline pH, including the net depolarization of the membranepotential, as well as the firing of action potentials (control: pH 6.5and pH 8 vs pH 7.4, significant difference p 0.001; lanthanum,100 �M: pH 8 vs control, significant difference p 0.001, pairedt test; n � 5; Fig. 5F,G). LaCl3 at 100 �M, however, also tended toreduce the acidic response (nonsignificant, p � 0.06, paired t test;n � 5). At 70 �M, there was no effect on the acidic response, buta clear and significant reduction of the alkaline response (p 0.001, paired t test; n � 5; Fig. 5G). The recorded CSF-c neuronswere intracellularly labeled with Neurobiotin and costained forsomatostatin, verifying that the hypothalamic CSF-c neurons re-sponding to the changes of extracellular pH express somatostatin(Fig. 5H).

Some hypothalamic CSF-c neurons did not respond to eitheralkaline or acidic pH (Fig. 5I) and none of these non-pH-sensitive CSF-c neurons expressed somatostatin (Fig. 5J). Thesesomatostatin-negative CSF-c neurons exhibited spontaneouspostsynaptic potentials (Fig. 5I, top trace), as well as action po-tential firing upon current injection.

Together, these results suggest that the alkaline response inhypothalamic, somatostatin-expressing CSF-c neurons is medi-ated via connexin hemichannels.

Hypothalamic CSF-c neurons sense fluid movements viaASIC3-like channelsTo investigate whether hypothalamic CSF-c neurons are mecha-nosensitive to fluid movements, brief pressure pulses were ap-plied to an aCSF-filled micropipette placed close to the bulb-likeprotrusion of the CSF-c neuron with its cilium (Fig. 1E) whileperforming patch recordings (Fig. 6A). To verify that the re-sponses were not indirect or synaptically evoked, GABAergic andglutamatergic synaptic transmission was blocked by bath appli-cation of gabazine, AP5, and CNQX. Graded receptor potentialresponses were reliably elicited by fluid pulses (duration 40 –100ms) in all CSF-c neurons tested (n � 8; Fig. 6B,C). The receptorpotential amplitude increased with increasing pulse magnitudes(Fig. 6B, 10 –20 psi) and action potentials were evoked when themembrane potential was held at �55 mV (Fig. 6B, top trace).

Because ASIC3-like channels mediate the mechanosensitivityunderlying the fluid pulse response in spinal CSF-c neurons

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Figure 5. Response to acidic and alkaline pH is mediated by different channels. A, Whole-cell current clamp in the presence of gabazine (20 �M), AP5 (50 �M), and CNQX (40 �M). Application ofAPETx2 (1 �M) abolished the response to acidic pH but not to alkaline pH in the same cell. B, Recordings from a hypothalamic CSF-c neuron in voltage-clamp mode. No current events were seen atpH 7.4 (black traces) in the presence of gabazine (20 �M), AP5 (50 �M), CNQX (40 �M), and TTX (1.5 �M). Inward current deflections appeared after a decrease or increase in the extracellular pH. Thecurrent events recorded in acidic pH (6.5) were completely blocked in the presence of APETx2 (1 �M; red trace), whereas those recorded in alkaline pH (8.0; blue trace) remained. C, Mean frequencyincrease of events (5–15 pA) in response to acidic and alkaline pH in the presence of APETx2 [mean � SEM; n � 3; Student’s paired t test: no significant difference vs pH 7.4 at 6.5 ( p � 1, t2 � 0),but a significant difference vs pH 7.4 at 8 (**p 0.01, p � 0.007, t2 � 11.55)]. D, Hypothalamic somatostatin-immunopositive CSF-c neurons (green) do not express the PKD2L1 channel(magenta). Scale bar, 20 �m. E, Spinal somatostatin-CSF-c neurons (green) coexpress PKD2L1 (arrows; magenta). Scale bar, 20 �m. cc, Central canal. F, Whole-cell current clamp in the presenceof gabazine (20 �M), AP5 (50 �M), and CNQX (40 �M) showing the response to both acidic (pH 6.5; red trace) and alkaline (pH 8.0; blue trace) pH. The connexin hemichannel blocker lanthanum (100�M) abolished the alkaline response but not the acidic response. G, Mean frequency increase of action potential firing in response to acidic and alkaline pH before and after application of lanthanumchloride (100 and 70 �M) [before application (control): means � SEM; n � 5; Student’s paired t test: ***p 0.001 significant difference vs pH 7.4 at 6.5 ( p � 1.31 � 10 �4, t4 � 14.51) and at8 ( p � 9.43 � 10 �5, t4 � 15.77)]. In the presence of lanthanum, a complete (100 �M) or partial (70 �M) blockade of the spiking response to alkaline pH was seen [means � SEM; n � 5; Student’spaired t test: ***p 0.001 significant difference at pH 8 vs control in the presence of lanthanum at 100 �M ( p � 9.4 � 10 �5, t4 � 15.77; as well as at 70 �M ( p � 9.3 � 10 �4, t4 � 8.76)]. Atendency for a small, nonsignificant (n.s.) frequency reduction was also observed at acidic pH (6.5) versus control in the presence of lanthanum at 100 �M ( p�0.06, t4 �2.58). H, The recorded CSF-cneuron in F intracellularly labeled with Neurobiotin expressed somatostatin. Scale bars, 10 �m. I, Whole-cell current clamp of a hypothalamic CSF-c neuron in control condition and in the presenceof gabazine (20 �M), AP5 (50 �M), and CNQX (40 �M) showing that this cell did not respond to either acidic (pH 6.5; red trace) or alkaline (pH 8.0; blue trace) pH. J, The recorded CSF-c neuron in Iintracellularly labeled with Neurobiotin did not express somatostatin. Scale bars, 10 �m.

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(Jalalvand et al., 2016a), we investigated whether this channelmay also act as a mechanosensor in hypothalamic CSF-c neuronsthat express somatostatin. The ASIC3 blocker APETx2 was bathapplied while recording the mechanical response to fluid pulses.In all cells tested (n � 4), all of which were somatostatin-positive(data not shown), the response was eliminated in the presence ofAPETx2 (Fig. 6C,D). Therefore, the mechanosensitivity of thehypothalamic CSF-c neurons also appears to be mediated byASIC3-like channels. The same CSF-c neuron responded to de-creased (pH 6.5) and increased (pH 8) extracellular pH (Fig. 6E).

DiscussionAn accurate control of the acid– base balance is one of the mainhomeostatic tasks of an organism. With regard to the brain, it isimportant to sense the pH of the CSF because it integrates theoverall impact of pH changes within the brain. To have pH sen-sors in the wall of the third ventricle at the hypothalamic levelwould seem to be an optimal location given the role of hypothal-amus in a variety of other homeostatic mechanisms. A high levelof neuronal activity can by itself lead to an extracellular acid shiftthrough the glucose–lactate shuttle in the astrocytes to furnishneurons with lactate as a source of energy Chesler and Kaila,1992; Magistretti and Allaman, 2015, 2018). Moreover, increasedCO2 levels (Voipio and Kaila, 1993), as in ischemia, will lead toacidosis, whereas hyperventilation due to low O2 levels will resultin alkalosis. Similarly, metabolic events can influence the pH inboth directions (Levin and Buck, 2015). Somatostatin/GABACSF-c cells lining an area within the third ventricle, as shownhere, sense both acidic and alkaline deviations from the controllevel. This also applies to the same type of CSF-c neuronslocated around the central canal (Jalalvand et al., 2016a,b) and

mammalian CSF-c neurons at the level of obex (Orts-Del’Immagine et al., 2016).

The pH-sensing capacity in both the hypothalamus and thespinal cord appears to be exclusive to the somatostatin CSF-csubpopulation of neurons because somatostatin-negative neu-rons do not respond to pH changes. In the spinal cord, acidic andalkaline deviations of the extracellular pH lead to an activation ofthe CSF-c neurons and a release of somatostatin in the spinalcord, which in turn exerts a depressing effect on the networkgenerating locomotor activity (Jalalvand et al., 2016a,b). The re-sponse to any pH change in the spinal cord will thus be a reducedmotor activity, which should help the organism to recover itsnormal pH.

Do the hypothalamic somatostatin-positive CSF-c neuronshave a similar role as their spinal counterparts? There is a veryrich somatostatin innervation in the motor areas of the brainextending from the forebrain to the brainstem, whereas sensoryareas have little or no innervation (see also below). Although notaddressed here, this raises the possibility that the hypothalamicsomatostatin-/GABA-expressing CSF-c-neurons could act at thesupraspinal level to reduce motor activity and thereby help torestore the pH level.

pH sensitivity of the CSF-c neurons in relation tophysiological and pathophysiological conditionsThe U-shaped curves of pH sensitivity of both hypothalamic andspinal CSF-c neurons show a significant depolarization at pH 7.1or 7.7 from a neutral value of 7.4, but a much larger responseoccurs outside this area at pH 6.5 and 8.3 (cf. Fig. 4D). WhereaspH values between 7.1 and 7.7 can be regarded as being within thephysiological range, values outside of this range would imply

Figure 6. The same hypothalamic CSF-c neuron is sensitive to both fluid movement and pH changes. A, In vitro preparation of the hypothalamus with CSF-c neurons protruding into the thirdventricle. An aCSF-filled pressure pipette was placed close to a bulb-like ending of a recorded hypothalamic CSF-c neuron. Scale bar, 20 �m. B, A short fluid-pulse (80 ms) elicited receptor potentialresponses and action potentials while holding the membrane potential at �65 mV and �55 mV, respectively, in the presence of gabazine (20 �M), CNQX (40 �M), and AP5 (50 �M). C, Receptorpotential elicited by fluid pulse stimulation (20 psi, 80 ms) was blocked by application of the ASIC3 blocker APETx2 (1 �M). D, Complete blockade of responses after application of APETx2 (n � 4).E, In the same hypothalamic CSF-c neuron as in C, exposure to acidic (pH 6.5) as well as alkaline (pH 8.0) pH depolarized the membrane potential (10 –12 mV) and triggered action potentials (withGABA and glutamate receptor antagonists present).

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pathophysiology. The somatostatin CFS-c neurons will thus re-spond steeply if the pH shifts to pathophysiological levels, but willalso detect smaller changes within the physiological range. Inlamprey, the pH of the blood can change with as much as 0.5 unitsduring exercise (Tufts et al., 1992) and the pH values in the in-terstitial fluid is somewhat lower than in the blood �pH 7.3 to 7.4(Chesler, 1986). During epilepsy or ischemia, pH values of 6.5have been reported in mammalian brain tissue (Rehncrona,1985; Siesjo et al., 1985). In the blood of patients with severeacidosis, pH just 7 and for alkalosis �7.65 have been reported(Thoren, 1960). Also, under physiological conditions, high levelsof neuronal activity can lead to deviations in the acidic directiondue to the glucose–lactate shuttle in the astrocytes, which pro-vides energy to neurons through lactate (Chesler and Kaila, 1992;Magistretti and Allaman, 2015).

The fact that the somatostatin CSF-c neurons respond to bothacidic and alkaline deviations with increased activity can be re-garded as an elegant evolutionary solution to respond to andmaster a life-threatening condition. The response in both casescan result in reduction of the motor activity. In the case of met-abolic demands during ischemia or hyperactivity, this will lead toa restoration of the pH. Similarly, during conditions with lowpO2, when hyperventilation will cause a lower pCO2 and therebyan alkaline pH, a reduction of the motor activity will also help torestore the pH. This applies to spinal cord somatostatin CSF-cneurons and may also apply to the hypothalamic somatostatinCSF-c cell subpopulation.

Response to alkaline pH is mediated by different molecularmechanisms in spinal and hypothalamic CSF-c neuronsPKD2L1 is the alkaline sensor in brainstem and spinal CSF-cneurons (Jalalvand et al., 2016a,b; Orts-Del’Immagine et al.,2016) and was therefore considered a main candidate for thehypothalamic CSF-c neurons. Unexpectedly, we could not detectany expression of PKD2L1 in hypothalamic CSF-c neurons (Fig.5D), suggesting that another unidentified channel is responsiblefor the alkaline response in hypothalamus. This is consistent withstudies in the mouse, in which PKD2L1 channels are not ex-pressed at a level just rostral to obex (Orts-Del�Immagine et al.,2014). A very small group of mouse hypothalamic cells in cellculture were shown to be PKD2L1-positive, but were not identi-fied as CSF-c cells (Huang et al., 2006).

Different extracellular alkaline pH sensors have been de-scribed in the vertebrate brain, including connexin hemichan-nels, insulin receptor-related receptors, and two-pore-domainK (K2P) channels (for review, see Murayama and Maruyama,2015). Because the connexin 35/36 protein is expressed in hypo-thalamic CSF-c neurons (Fig. 2B–D), in contrast to the cellsaround the central canal, we considered connexin hemichannelsa possibility. The connexin hemichannels are sensitive to pHchanges (Francis et al., 1999; Contreras et al., 2002; Saez et al.,2005; Spray et al., 2006; Yu et al., 2007) and extracellular alkaliza-tion increases intracellular Ca 2 levels in cells expressing con-nexin hemichannels (Schalper et al., 2010).

The somatostatin CSF-c neurons in hypothalamus expressconnexin 35/36 and the alkaline response could be totally blockedwith lanthanum, suggesting that connexin hemichannels are sen-sors for alkaline pH. We used two different concentrations oflanthanum, 70 and 100 �M. At 70 �M, lanthanum did not affectthe acidic response but very significantly reduced the alkalineresponse, whereas at 100 �M, a complete blockade of the alkalineresponse was observed, together with a nonsignificant reductionof the acidic response. At very high concentrations (�100 �M),

lanthanum has an inhibitory effect on many channels includingkainate and AMPA receptors (at concentrations of 1–30 mM;Reichling and MacDermott, 1991; Hong et al., 2004), as well asTRP channels (at concentrations �1 mM; Zhao et al., 2015) andthus at such high levels may also affect ASIC3 in hypothalamicCSF-c neurons.

Another difference between lamprey hypothalamic and spinalCSF-c neurons is that the former have a lower mean resting po-tential (�65 mV vs �52 mV for spinal CSF-c neurons) at pH 7.4and therefore are rarely spontaneously active at this pH. Presum-ably as a consequence of this difference, hypothalamic CSF-cneurons showed a more prominent membrane potential depo-larization in response to a change in pH than spinal CSF-c cells(Jalalvand et al., 2016a).

ASIC3-like channels mediate the response to acidic pH and tofluid motionThe acidic response in hypothalamic CSF-c neurons, as in spinalCSF-c neurons (Jalalvand et al., 2016a), is mediated by ASIC3-like channels because APETx2, a specific antagonist of ASIC3(Diochot et al., 2004), blocks acidic sensing. In rodents, ASIC3has been reported to be expressed throughout the hypothalamus,including the suprachiasmatic nucleus (Wang et al., 2007; Chenet al., 2009; Meng et al., 2009). Lamprey hypothalamic CSF-cneurons are also mechanosensitive and activated by fluid move-ments such as motion of the CSF. This response is also mediatedby ASIC3-like channels, as in the spinal cord (Jalalvand et al.,2016b). In CSF-c neurons of the most caudal part of the mousebrainstem, the acidic response was shown to be mediatedthrough the general class of ASICs (Orts-Del’Immagine et al.,2016). It would therefore seem that vertebrate CSF-c neuronsresponsive to acidic pH have developed this capacity through theexpression of ASICs, in contrast to the alkaline sensors, which cantake different forms.

Role of somatostatin CSF-c neurons in the brain andspinal cordIn the lamprey brain, the main source of somatostatin is thehypothalamic CSF-c neurons, although small populations existin the ventral thalamus and the isthmic region and of course inthe cells around the central canal (Wright, 1986; Buchanan et al.,1987; Christenson et al., 1991; Yanez et al., 1992 our unpublishedobservations). Periventricular somatostatin-expressing neuronsare also highly concentrated along the third ventricle in othervertebrates, including rat (Kronheim et al., 1976; Finley et al.,1981; Johansson et al., 1984), reptiles (Fasolo and Gaudino, 1982;Wang et al., 2016), amphibians (Fasolo and Gaudino, 1981), andfish (Vígh-Teichmann et al., 1983b; Sas and Maler, 1991). Thelamprey pallium and striatum have a rich somatostatin innerva-tion originating from CSF-c neurons in hypothalamus (Sury-anarayana et al., 2017), but no intrinsic somatostatin interneurons,in contrast to the mammalian cortex and striatum (Liguz-Lecznar etal., 2016).

The hypothalamic somatostatin CSF-c neurons innervatemost parts of the brain, however, with a higher density of fibersand/or terminals in motor areas, including the output layer of theoptic tectum and the reticulospinal nuclei, but not in sensoryareas. Interestingly, somatostatin-expressing CSF-c neurons atthe obex level, which coexpress dopamine, also respond to pHchanges, whereas dopaminergic CSF-c neurons that do not ex-press somatostatin are unresponsive. This further strengthens thenotion of a functional link between somatostatin expression inCSF-c neurons and pH sensitivity. In the lamprey spinal cord, the

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suppression of motor activity induced by activation of thesomatostatin-/GABA-expressing CSF-c cells following a changeof extracellular pH is mediated by a release of somatostatin (Ja-lalvand et al., 2016a,b). The hypothalamic CSF-c neurons maywell have a similar role.

ConclusionTogether, somatostatin-expressing CSF-c neurons in the hypo-thalamus, like in the spinal cord, may serve as pH sensors as wellas mechanosensors. They will sense any pH deviation and motionin the CSF of the third ventricle and respond by membrane po-tential depolarization and action potential firing. The acidic re-sponse and mechanosensitivity are mediated by ASIC3-likechannels, whereas the alkaline response appears to be mediatedthrough connexin hemichannels, in contrast to their spinal coun-terparts, in which PKD2L1 is responsible. Activation of hypotha-lamic CSF-c neurons will cause a release of somatostatin in thedifferent target areas, many of which are motor related. As in thespinal cord, this may contribute to restoring the deviation in pHand thus to homeostasis.

ReferencesBall SG (2007) Vasopressin and disorders of water balance: the physiology

and pathophysiology of vasopressin. Ann Clin Biochem 44:417– 431.CrossRef Medline

Boulant JA, Dean JB (1986) Temperature receptors in the central nervoussystem. Annu Rev Physiol 48:639 – 654. CrossRef Medline

Broberger C (2005) Brain regulation of food intake and appetite: moleculesand networks. J Intern Med 258:301–327. CrossRef Medline

Brodin L, Hokfelt T, Grillner S, Panula P (1990) Distribution of histaminergicneurons in the brain of the lamprey lampetra fluviatilis as revealed byhistamine-immunohistochemistry. J Comp Neurol 292:435–442. CrossRefMedline

Buchanan JT, Brodin L, Hokfelt T, Van Dongen PA, Grillner S (1987) Sur-vey of neuropeptide-like immunoreactivity in the lamprey spinal cord.Brain Res 408:299 –302. CrossRef Medline

Chen CH, Hsu YT, Chen CC, Huang RC (2009) Acid-sensing ion channelsin neurones of the rat suprachiasmatic nucleus. J Physiol 587:1727–1737.CrossRef Medline

Chesler M (1986) Regulation of intracellular pH in reticulospinal neuronesof the lamprey, Petromyzon marinus. J Physiol 381:241–261. CrossRefMedline

Chesler M, Kaila K (1992) Modulation of pH by neuronal activity. TrendsNeurosci 15:396 – 402. CrossRef Medline

Christenson J, Alford S, Grillner S, Hokfelt T (1991) Co-localized GABAand somatostatin use different ionic mechanisms to hyperpolarize targetneurons in the lamprey spinal cord. Neurosci Lett 134:93–97. CrossRefMedline

Contreras JE, Sanchez HA, Eugenin EA, Speidel D, Theis M, Willecke K,Bukauskas FF, Bennett MV, Saez JC (2002) Metabolic inhibition in-duces opening of unapposed connexin 43 gap junction hemichannels andreduces gap junctional communication in cortical astrocytes in culture.Proc Natl Acad Sci U S A 99:495–500. CrossRef Medline

Dimicco JA, Zaretsky DV (2007) The dorsomedial hypothalamus: a newplayer in thermoregulation. Am J Physiol Regul Integr Comp Physiol292:R47–R63. CrossRef Medline

Diochot S, Baron A, Rash LD, Deval E, Escoubas P, Scarzello S, Salinas M,Lazdunski M (2004) A new sea anemone peptide, APETx2, inhibitsASIC3, a major acid-sensitive channel in sensory neurons. EMBO J 23:1516 –1525. CrossRef Medline

Fasolo A, Gaudino G (1981) Somatostatin immunoreactive neurons andfibers in the hypothalamus of the newt. Gen Comp Endocrinol 44:256 –263. CrossRef Medline

Fasolo A, Gaudino G (1982) Immunohistochemical localization ofsomatostatin-like immunoreactivity in the hypothalamus of the liz-ard, Lacerta sicula. Gen Comp Endocrinol 48:205–212. CrossRefMedline

Finley JC, Maderdrut JL, Roger LJ, Petrusz P (1981) The immunocytochem-ical localization of somatostatin-containing neurons in the rat centralnervous system. Neuroscience 6:2173–2192. CrossRef Medline

Francis D, Stergiopoulos K, Ek-Vitorín JF, Cao FL, Taffet SM, Delmar M(1999) Connexin diversity and gap junction regulation by pHi. DevGenet 24:123–136. CrossRef Medline

Hofman MA, Swaab DF (1993) Diurnal and seasonal rhythms of neuronalactivity in the suprachiasmatic nucleus of humans. J Biol Rhythms 8:283–295. CrossRef Medline

Hong Z, Wang DS, Lu SY, Li JS (2004) Suppression of kainate-evokedAMPA receptor mediated responses by lanthanum in rat sacral dorsalcommissural neurons. Neurosignals 13:258 –264. CrossRef Medline

Huang AL, Chen X, Hoon MA, Chandrashekar J, Guo W, Trankner D, RybaNJ, Zuker CS (2006) The cells and logic for mammalian sour taste de-tection. Nature 442:934 –938. CrossRef Medline

Jalalvand E, Robertson B, Wallen P, Hill RH, Grillner S (2014) Laterallyprojecting cerebrospinal fluid-contacting cells in the lamprey spinal cordare of two distinct types. J Comp Neurol 522:1753–1768. CrossRefMedline

Jalalvand E, Robertson B, Wallen P, Grillner S (2016a) Ciliated neuronslining the central canal sense both fluid movement and pH throughASIC3. Nat Commun 7:10002. CrossRef Medline

Jalalvand E, Robertson B, Tostivint H, Wallen P, Grillner S (2016b) Thespinal cord has an intrinsic system for the control of pH. Curr Biol 26:1346 –1351. CrossRef Medline

Johansson O, Hokfelt T, Elde RP (1984) Immunohistochemical distribu-tion of somatostatin-like immunoreactivity in the central nervous systemof the adult rat. Neuroscience 13:265–339. CrossRef Medline

Kronheim S, Berelowitz M, Pimstone BL (1976) A radioimmunoassay forgrowth hormone release-inhibiting hormone: method and quantitativetissue distribution. Clin Endocrinol (Oxf) 5:619 – 630. CrossRef Medline

Levin LR, Buck J (2015) Physiological roles of acid-base sensors. Annu RevPhysiol 77:347–362. CrossRef Medline

Liguz-Lecznar M, Urban-Ciecko J, Kossut M (2016) Somatostatin andsomatostatin-containing neurons in shaping neuronal activity and plas-ticity. Front Neural Circuits 10:48. CrossRef Medline

Magistretti PJ, Allaman I (2015) A cellular perspective on brain energy me-tabolism and functional imaging. Neuron 86:883–901. CrossRef Medline

Magistretti PJ, Allaman I (2018) Lactate in the brain: from metabolic end-product to signaling molecule. Nat Rev Neurosci 19:235–249. CrossRefMedline

Marichal N, García G, Radmilovich M, Trujillo-Cenoz O, Russo RE (2009)Enigmatic central canal contacting cells: immature neurons in “standbymode”? J Neurosci 29:10010 –10024. CrossRef Medline

Meng QY, Wang W, Chen XN, Xu TL, Zhou JN (2009) Distribution ofacid-sensing ion channel 3 in the rat hypothalamus. Neuroscience 159:1126 –1134. CrossRef Medline

Murayama T, Maruyama IN (2015) Alkaline pH sensor molecules. J Neu-rosci Res 93:1623–1630. CrossRef Medline

O’Brien J, Nguyen HB, Mills SL (2004) Cone photoreceptors in bass retinause two connexins to mediate electrical coupling. J Neurosci 24:5632–5642. CrossRef Medline

O’Brien J, Bruzzone R, White TW, Al-Ubaidi MR, Ripps H (1998) Cloningand expression of two related connexins from the perch retina define adistinct subgroup of the connexin family. J Neurosci 18:7625–7637.CrossRef Medline

Orts-Del’Immagine A, Wanaverbecq N, Tardivel C, Tillement V, DallaportaM, Trouslard J (2012) Properties of subependymal cerebrospinal fluidcontacting neurones in the dorsal vagal complex of the mouse brainstem.J Physiol 590:3719 –3741. CrossRef Medline

Orts-Del’Immagine A, Kastner A, Tillement V, Tardivel C, Trouslard J, Wa-naverbecq N (2014) Morphology, distribution and phenotype of poly-cystin kidney disease 2-like 1-positive cerebrospinal fluid contactingneurons in the brainstem of adult mice. PLoS One 9:e87748. CrossRefMedline

Orts-Del’Immagine A, Seddik R, Tell F, Airault C, Er-Raoui G, Najimi M,Trouslard J, Wanaverbecq N (2016) A single polycystic kidney disease2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem. Neuropharmacology 101:549 –565. CrossRef Medline

Rehncrona S (1985) Brain acidosis. Ann Emerg Med 14:770 –776. CrossRefMedline

Reichling DB, MacDermott AB (1991) Lanthanum actions on excitatoryamino acid-gated currents and voltage-gated calcium currents in rat dor-sal horn neurons. J Physiol 441:199 –218. CrossRef Medline

Jalalvand et al. • Hypothalamic CSF-c Neurons Sense pH and Motion J. Neurosci., August 29, 2018 • 38(35):7713–7724 • 7723

Russo RE, Reali C, Radmilovich M, Fernandez A, Trujillo-Cenoz O (2008)Connexin 43 delimits functional domains of neurogenic precursors in thespinal cord. J Neurosci 28:3298 –3309. CrossRef Medline

Ruusuvuori E, Kaila K (2014) Carbonic anhydrases and brain pH in thecontrol of neuronal excitability. Subcell Biochem 75:271–290. CrossRefMedline

Saez JC, Retamal MA, Basilio D, Bukauskas FF, Bennett MV (2005) Connexin-based gap junction hemichannels: gating mechanisms. Biochim Biophys Acta1711:215–224. CrossRef Medline

Saper CB, Chou TC, Elmquist JK (2002) The need to feed: homeostatic andhedonic control of eating. Neuron 36:199 –211. CrossRef Medline

Saper CB, Scammell TE, Lu J (2005) Hypothalamic regulation of sleep andcircadian rhythms. Nature 437:1257–1263. CrossRef Medline

Sas E, Maler L (1991) Somatostatin-like immunoreactivity in the brain of anelectric fish (Apteronotus leptorhynchus) identified with monoclonal anti-bodies. J Chem Neuroanat 4:155–186. CrossRef Medline

Schalper KA, Sanchez HA, Lee SC, Altenberg GA, Nathanson MH, Saez JC(2010) Connexin 43 hemichannels mediate the Ca 2 influx induced byextracellular alkalinization. Am J Physiol Cell Physiol 299:C1504 –C1515.CrossRef Medline

Siesjo BK, von Hanwehr R, Nergelius G, Nevander G, Ingvar M (1985)Extra- and intracellular pH in the brain during seizures and in the recov-ery period following the arrest of seizure activity. J Cereb Blood FlowMetab 5:47–57. CrossRef Medline

Spray DC, Ye ZC, Ransom BR (2006) Functional connexin “hemichannels”:a critical appraisal. Glia 54:758 –773. CrossRef Medline

Srinivas M, Rozental R, Kojima T, Dermietzel R, Mehler M, Condorelli DF,Kessler JA, Spray DC (1999) Functional properties of channels formedby the neuronal gap junction protein connexin36. J Neurosci 19:9848 –9855. CrossRef Medline

Suryanarayana SM, Robertson B, Wallen P, Grillner S (2017) The lampreypallium provides a blueprint of the mammalian layered cortex. Curr Biol27:3264 –3277.e5. Medline

Thoren L (1960) Vatskebalans. In: Boktryckeri Aktiebolag. Uppsala, Swe-den: Almqvist and Wiksells.

Tufts BL, Bagatto B, Cameron B (1992) In vivo analysis of gas transport inarterial and venous blood of the sea lamprey Petromyzon marinus. J ExpBiol 169:105–119.

Vígh B, Vígh-Teichmann I (1998) Actual problems of the cerebrospinalfluid-contacting neurons. Microsc Res Tech 41:57– 83. CrossRef Medline

Vígh B, Vígh-Teichmann I, Aros B (1977) Special dendritic and axonal end-ings formed by the cerebrospinal fluid contacting neurons of the spinalcord. Cell Tissue Res 183:541–552. Medline

Vígh B, Vígh-Teichnann I, Sikora K, Jennes L (1980) Scanning electron mi-croscopy of the cerebrospinal fluid-contacting neurons of the central ca-nal in various vertebrate. Verh Anat Ges 74:707–714.

Vígh B, Manzano e Silva MJ, Frank CL, Vincze C, Czirok SJ, Szabo A, LukatsA, Szel A (2004) The system of cerebrospinal fluid-contacting neurons.its supposed role in the nonsynaptic signal transmission of the brain.Histol Histopathol 19:607– 628. CrossRef Medline

Vígh-Teichmann I, Vígh B (1983a) The system of cerebrospinal fluid-contacting neurons. Arch Histol Jpn 46:427– 468. CrossRef Medline

Vígh-Teichmann I, Vígh B, Korf HW, Oksche A (1983b) CSF-contactingand other somatostatin-immunoreactive neurons in the brains ofAnguilla anguilla, Phoxinus phoxinus, and Salmo gairdneri (Teleostei).Cell Tissue Res 233:319 –334. Medline

Voipio J, Kaila K (1993) Interstitial PCO2 and pH in rat hippocampal slicesmeasured by means of a novel fast CO2/H()-sensitive microelectrodebased on a PVC-gelled membrane. Pflugers Arch 423:193–201. CrossRefMedline

Wang H, Zhang R, Zhang S, Zhou Y, Wu X (2016) Immunohistochemicallocalization of somatostatin in the brain of Chinese alligator Alligatorsinensis. Anat Rec (Hoboken) 300:507–519. CrossRef Medline

Wang W, Yu Y, Xu TL (2007) Modulation of acid-sensing ion channels byCu 2 in cultured hypothalamic neurons of the rat. Neuroscience 145:631– 641. CrossRef Medline

Wright GM (1986) Immunocytochemical demonstration of growth hor-mone, prolactin and somatostatin-like immunoreactivities in the brain oflarval, young adult and upstream migrant adult sea lamprey, Petromyzonmarinus. Cell Tissue Res 246:23–31. Medline

Yanez J, Rodríguez-Moldes I, Anadon R (1992) Distribution of somatostatin-immunoreactivity in the brain of the larval lamprey (Petromyzon marinus).J Chem Neuroanat 5:511–520. CrossRef Medline

Yu J, Bippes CA, Hand GM, Muller DJ, Sosinsky GE (2007) Aminosulfonatemodulated pH-induced conformational changes in connexin26 hemi-channels. J Biol Chem 282:8895– 8904. CrossRef Medline

Zhao PY, Gan G, Peng S, Wang SB, Chen B, Adelman RA, Rizzolo LJ (2015)TRP channels localize to subdomains of the apical plasma membrane inhuman fetal retinal pigment epithelium. Invest Ophthalmol Vis Sci 56:1916 –1923. CrossRef Medline

7724 • J. Neurosci., August 29, 2018 • 38(35):7713–7724 Jalalvand et al. • Hypothalamic CSF-c Neurons Sense pH and Motion