Abstract
CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical 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 hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.
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 in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.
Introduction
All 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 extracellular or intracellular pH in brain tissue modulates neuronal excitability and function (Ruusuvuori and Kaila, 2014). Increased CO2 levels, as in ischemia, will result in acidosis, whereas hyperventilation due to low O2 will result in alkalosis; similarly, metabolic events can influence the pH in both directions (Levin and Buck, 2015). Moreover, during high levels of neuronal activity, pH within the CNS itself is lowered through enhanced levels of lactate 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 central canal in the lamprey spinal cord act as pH sensors. The same cell detects both acidic and alkaline deviations from ∼pH 7.4 through the acid-sensing ion channel 3 (ASIC3)-like and the polycystic kidney disease (PKD)-protein-2-like 1 (PKD2L1) channel, respectively, resulting in an increased depolarization and firing of action potentials (Jalalvand et al., 2016a,b). Similarly, CSF-c neurons in the mouse dorsal vagal complex respond to alkaline pH via the PKD2L1 channel (Orts-Del'Immagine et al., 2014, 2016).
Even moderate changes in the extracellular pH, whether in the alkaline or acidic direction (0.3 pH units or less), will enhance CSF-c neuronal spike firing, which in turn will suppress the locomotor activity by negative feedback to the spinal cord circuits. The response to any pH change in the spinal cord will thus be a reduced motor activity, which should help the organism to recover its normal pH (Jalalvand et al., 2016a,b).
CSF-c neurons come in many different forms that express different transmitters and peptides. They are present in all major groups of vertebrates, from cyclostomes to mammals, and line the 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 et al., 2009; Orts-Del'Immagine et al., 2012, 2014, 2016; Jalalvand et al., 2014). The largest number of CSF-c neurons in the brain is found in the third ventricle in the diencephalon, mainly in the hypothalamus (Vígh and Vígh-Teichmann, 1998). Hypothalamus is an evolutionarily conserved area of the brain that regulates a 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). Hypothalamic cultured neurons (mouse) have been reported to be sensitive to acidic pH changes through ASICs (Wang et al., 2007).
The hypothalamic CSF-c neurons have a ciliated bulb-like ending that protrudes into the third ventricle (Vígh et al., 1980) and their axons branch and extend laterally, dorsally, and ventrally. Our goal here is to investigate the functional role of the somatostatin-/GABA-expressing subpopulation of CSF-c neurons in the lamprey hypothalamus and to determine whether they respond to the composition of the CSF. Patch-clamp recordings were performed while the cells were subjected to moderate alterations in the extracellular pH or to mechanical stimulation by applied fluid movement. In contrast to non-somatostatin-expressing CSF-c neurons in the third ventricle, the somatostatin-/GABA-expressing hypothalamic CSF-c neurons responded to both acidic and alkaline pH and to mechanical fluid motion. The acidic and mechanical responses are mediated through ASIC3-like channels, whereas the alkaline response appears to be mediated through the connexin hemichannel. These results also show that the hypothalamic somatostatin/GABA CSF-c neurons play a role as pH sensors as well as mechanosensors.
Materials and Methods
Animals.
Experiments were performed on a total of 44 adult river lampreys (Lampetra fluviatilis) of both sexes that were collected from the Ljusnan River, Hälsingland, Sweden. The experimental procedures were approved by the local ethical committee (Stockholm's Norra Djurförsöksetiska Nämnd) and were in accordance with the Policy on the Use of Animals in Neuroscience Research (Society for Neuroscience). During the investigation, every effort was made to minimize animal suffering and to reduce the number of animals used.
Electrophysiology.
Animals (n = 32) were deeply anesthetized through immersion in 0.01 m PBS containing tricane methanesulfonate (MS-222; 100 mg L−1; Sigma-Aldrich). Following decapitation, the exposed brain was removed and placed in ice-cold artificial CSF (aCSF; extracellular solution) of the following composition (in mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 25 NaHCO3, and 10 glucose, pH 7.4. The aCSF was oxygenated continuously with 95% O2 and 5% CO2 and osmolarity was udjusted to 290 mOsm L−1 with glucose. Transverse slices (300 μm) 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 cooled aCSF. Responses to deviations in extracellular pH were recorded by adjusting 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 the fluid in the chamber, it has the advantage of revealing the exact pH applied. Previous investigators have ejected solutions from a micropipette (Marichal et al., 2009; Orts-Del'Immagine et al., 2016), which has the advantage of short latencies, but the exact pH levels cannot be determined. For mechanical fluid motion stimulation, glass pipettes (2–4 MΩ) were filled with aCSF. Fluid pulse stimuli were given by applying 5–20 psi pressure pulses of 10–80 ms duration by a PicoSpritzer II unit (Parker Hannifin). Patch electrodes (8–12 MΩ) were prepared from borosilicate glass microcapillaries (Hilgenberg) using a two-stage puller (PP-830; Narishige) and filled with an intracellular solution of the following 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 the solution 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 configuration and in current-clamp or voltage-clamp mode using a Multiclamp 700B amplifier (Molecular Devices). The gigaseal resistance during recording was >10 GΩ, at least five times higher than the input resistance of the cells. Access to the cell in voltage-clamp mode was repeatedly confirmed during the course of the experiment. Cells were visualized with differential interference contrast/infrared optics. The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2, an extract of the sea anemone, Anthopleura elegantissima toxin-2, (1 μm; catalog #500527; Calbiochem), the connexin hemichannel blocker lanthanum chloride (100 μm; catalog #298182; Sigma-Aldrich), the GABAA receptor antagonist gabazine (20 μm; catalog #1262; Tocris Bioscience), the NMDA receptor antagonist AP5 (50 μm; catalog #0105; Tocris Bioscience), the AMPA receptor antagonist CNQX (40 μm; catalog #1045; Tocris Bioscience), and tetrodotoxin (TTX, 1.5 μm; catalog #T8024; Sigma-Aldrich).
Immunohistochemistry.
The brains (n = 8) were fixed by immersion in 4% formalin and 14% saturated picric acid solution in 0.1 m phosphate buffer (PB; pH 7.4; 1% glutaraldehyde was added for GABA immunohistochemistry) for 12–24 h at 4°C and subsequently cryoprotected in 20% sucrose in PB for 3–12 h. Then, 20 μm transverse sections were cut on a cryostat (catalog #HM 560; Microm), collected on gelatin-coated slides, and kept at −20°C until processing. For detection of somatostatin (n = 2), coexpression of somatostatin/GABA (n = 2), coexpression of somatostatin/α-tubulin (n = 2), and somatostatin/connexin 35/36 (n = 2), hypothalamic sections were incubated overnight with a rat monoclonal anti-somatostatin antibody (1:200; MAB354; RRID:AB_2255365; Millipore), a mouse monoclonal anti-GABA antibody (0.1 μg/ml; MAB3A12; RRID:AB_2314450; a generous gift from Prof. Peter Streit, Zürich, Switzerland), a mouse monoclonal anti-acetylated α-tubulin antibody (1:500; 6-11B-1; RRID:AB_477585; Sigma-Aldrich) or a mouse monoclonal anti-connexin 35/36 antibody (1:200; MAB3045; RRID:AB_94632; Millipore). The sections were subsequently rinsed thoroughly in 0.01 m PBS. To visualize coexpression of somatostatin and GABA, the sections were incubated with a mixture of Cy3-conjugated donkey anti-rat IgG (1:500; catalog #712-165-150; RRID:AB_2340667; Jackson ImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200; catalog #715-545-150; RRID:AB_2340846; Jackson ImmunoResearch) for 2 h. For coexpression of somatostatin and α-tubulin or connexin 35/36, the sections were incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG (1:200; catalog #712-545-153; RRID:AB_2340684; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-mouse IgG (1:500; catalog #715-165-150; RRID:AB_2315777; Jackson ImmunoResearch). The sections were then rinsed in 0.01 m PBS and mounted with glycerol containing 2.5% diazabicyclooctane (DABCO) (catalog #D27802; Sigma-Aldrich).
To examine the morphology of hypothalamic CSF-c neurons, cells were intracellularly labeled with 0.3% Neurobiotin (catalog #SP-1120; Vector Laboratories) during the whole-cell recording. The hypothalamic slices 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 incubated with Alexa Fluor 488-conjugated streptavidin (1:1000; catalog #016-640-084; Jackson ImmunoResearch) for 3 h. The sections were then rinsed in 0.01 m PBS and mounted with glycerol containing 2.5% DABCO. Alternatively, after fixation and rinsing in PBS, slices were incubated in Vectastain (catalog #PK6100; Vector Laboratories) followed by diaminobenzidine (DAB) (ImmPACT, catalog #SK-4100; Vector Laboratories) for 5 min and then rinsed and dehydrated in alcohol before mounting in Entellan (Merck).
To investigate whether the intracellularly Neurobiotin-labeled CSF-c cells express somatostatin, the slices were incubated overnight with a rat monoclonal anti-somatostatin antibody (1:200; MAB354; RRID:AB_2255365; Millipore) and then rinsed thoroughly in 0.01 m PBS and incubated 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 secondary antibodies 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 as described above and the brain and rostral spinal cord (the spinal cord was used 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-μm-thick cryostat sections were made and stored in −80°C until processed. Single-stranded digoxigenin-labeled sense and antisense pkd2l1 riboprobes were generated by in vitro transcription of the previously cloned pkd2l1 cDNA using the Digoxigenin RNA Labeling kit (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 μg/ml, salmon sperm DNA, 250 μg/ml yeast RNA) at 60°C. Sections incubated with the heat-denaturated digoxigenin-labeled riboprobe were hybridized overnight at 60°C. Following 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 at room temperature. After blocking in 0.5% blocking reagent (PerkinElmer), the sections were incubated overnight in anti-DIG antibody coupled to HRP (1:2000; catalog #RRID:AB_514497; Roche Diagnostics) at 4°C. The probe was then visualized by TSA Cy3 Plus Evaluation Kit (catalog #NEL763E001; PerkinElmer). The specificity of the hybridization procedure was verified by incubating sections with the sense riboprobe (data not shown). The sections were rinsed thoroughly in 0.01 m PBS and then incubated with a rat monoclonal anti-somatostatin antibody (1:200; MAB354; Millipore) overnight at 4°C, rinsed in PBS, and incubated 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 primary and secondary antibodies were diluted in 1% BSA and 0.3% Triton X-100 in 0.1 m PB.
Western blot.
Lamprey brains (n = 2) were solubilized by sonication in 1% Na-SDS, 0.32 m sucrose, 20 mm HEPES, pH 7.6, and a mixture of protease inhibitors. Samples were then subjected to SDS-PAGE on Bio-Rad gradient gels (4–15%) and subsequently transferred to PVDF membrane according to the manufacturer's instructions. The membrane was incubated with mouse anti-connexin 35/36 (1:1000; MAB3045; RRID:AB_94632; Millipore) overnight at 4°C and then with goat anti-mouse IgG-HRP (1:5000; P0447; DAKO) for 2 h at room temperature. It was developed with SuperSignal West Dura (Thermo Scientific).
Morphological analysis.
Photomicrographs were taken with an Olympus 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 scanning microscope (LSM 510 NLO; Zeiss) and processed using Zeiss LSM software. Illustrations were prepared using Adobe Illustrator and Photoshop CS6. Images were adjusted only for brightness and contrast.
Experimental design and statistical analysis.
Hypothalamic CSF-c neurons were recorded in whole-cell configuration and in current-clamp or voltage-clamp mode. Resting membrane potentials were determined in current-clamp mode. Bridge balance and pipette capacitance compensation were adjusted and signals were digitized and recorded using Clampex software and analyzed in Clampfit (pCLAMP 10; Molecular Devices). Membrane and firing properties were analyzed by injection of short (20 ms) and long (500 or 1000 ms) depolarizing and hyperpolarizing current pulses. Responses to deviations in extracellular pH were recorded as changes in membrane potential and firing frequency (current clamp) and in the frequency of inward current events (voltage-clamp). Effects were analyzed by comparing the responses in the same neuron at the control condition at physiological pH (7.4) and at acidic or alkaline pH, 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 presented as means ± SD or SEM and statistical comparisons were made using Student's paired two-tailed t test. ***p < 0.001, **p < 0.01 significant difference compared with control; n.s.: no significant difference (for details, see figure legends).
Results
Somatostatin-/GABA-expressing CSF-c neurons in the hypothalamus
An area in the rostral hypothalamus contains numerous somatostatin-positive CSF-c neurons (Fig. 1A,B). These neurons have a short, thick apical process with a bulb-like ending (Fig. 1C) from which an α-tubulin-immunoreactive cilium protrudes into the CSF (Fig. 1D, arrow). To further identify the phenotype of the hypothalamic CSF-c neurons, we investigated whether these cells coexpress somatostatin and GABA, similar to spinal CSF-c neurons. Most of the somatostatin-positive neurons within the hypothalamus were CSF-c cells (Fig. 1B,C) that coexpressed GABA (Fig. 1E, arrows). However, some CSF-c neurons in this region were only immunoreactive to GABA (Fig. 1E, arrowhead). In the lamprey, the periventricular area of hypothalamus is the main source of somatostatin. Intracellular Neurobiotin labeling of recorded hypothalamic CSF-c neurons showed that their axonal branches extend laterally, dorsally, and ventrally (Fig. 1F). Somatostatin-positive fibers and terminals were found throughout the brain, including a dense innervation of motor-related areas such as the deep layer of the optic tectum and the reticulospinal nuclei (Fig. 1G,H). Conversely, sensory areas throughout the brain were devoid of somatostatin innervation, as shown in the retinorecipient superficial layer of the optic tectum and the brainstem alar plate (Fig. 1G,H).
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 of transverse sections through the hypothalamic area. B, Photomicrograph of somatostatin-immunopositive CSF-c neurons in the hypothalamus (arrows). Scale bar, 200 μm. C, High-magnification photomicrograph 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 μm. D, Confocal image of a somatostatin-expressing hypothalamic CSF-c neuron (green) with an α-tubulin-immunoreactive cilium (magenta, arrow). Scale bar, 5 μm. E, Somatostatin (magenta) and GABA (green) are colocalized in hypothalamic CSF-c neurons (arrows). Some of the CSF-c neurons only expressed GABA (arrowhead). Scale bar, 20 μm. F, Illustration of a reconstructed intracellularly labeled hypothalamic CSF-c neuron with a bulb-like ending protruding into the ventricle and axonal branches extending dorsally, laterally, and ventrally. G, Somatostatin-immunoreactive fibers/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 μm. H, Rich somatostatin labeling in the ventral brainstem at the level of MRRN and the trigeminal nucleus (nV). Scale bar, 200 μm. 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.
Presence of gap junctions in somatostatin-expressing CSF-c neurons
Intracellular injection of Neurobiotin into a single somatostatin-expressing CSF-c neuron in hypothalamus sometimes resulted in staining of one or two additional CSF-c neurons in close vicinity to the injected cell, suggesting dye coupling via gap junctions (n = 4; Fig. 2A). To investigate this possibility further, we applied the neuronal connexin-35/36 antibody, a gap junction marker (O'Brien et al., 1998; Srinivas et al., 1999). Somatostatin-positive CSF-c neurons coexpressed the connexin protein (Fig. 2B–D), suggesting that gap junctions are present between at least some CSF-c neurons in the hypothalamus. Figure 2E is a Western blot of the anti-connexin 35/36 antibody (immunogen recombinant perch connexin 35), showing that this antibody recognizes a band at around 35 kDa and is thus specific for the lamprey brain and a band 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 ortholog for lamprey connexin (PMZ_00001234-RA), with more than 80% identity to both mouse and perch connexin 35/36 (results not shown).
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 DAB staining. Scale bar, 20 μm. B, Somatostatin-expressing hypothalamic CSF-c neurons. Scale bar, 20 μm. C, Connexin 35/36 immunoreactivity in the same hypothalamic area as in B. Scale bar, 20 μm. D, Merged image showing connexin 35/36-immunoreactivity (arrows) coexpressed with somatostatin in hypothalamic CSF-c neurons. Scale bar, 20 μm. E, Western blot of a lamprey brain extract detected with a monoclonal mouse anti-connexin 35/36 antibody showing a distinct band around 35 kDa.
Neuronal properties of hypothalamic CSF-c neurons
To examine the membrane properties of somatostatin-expressing CSF-c neurons in the hypothalamus, we performed whole-cell recordings in voltage- or current-clamp mode (n = 45 cells). The hypothalamic CSF-c neurons had a mean resting membrane potential of −65 ± 4 mV, a mean input resistance of 1.9 ± 0.4 GΩ (n = 15), and a mean spike threshold of −45 ± 1.8 mV (n = 15). In contrast to spinal CSF-c neurons, hypothalamic CSF-c neurons did not fire action potentials spontaneously under control conditions. However, spiking was readily 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 by an afterhyperpolarization (Fig. 3A, left trace). A train of action potentials was evoked with longer current injections (20 pA, 500 ms; Fig. 3A, right trace). Hypothalamic CSF-c neurons generally showed spontaneous GABAergic and glutamatergic postsynaptic potentials, the latter mediated by both the AMPA and NMDA receptors (Fig. 3B). The voltage responses recorded during depolarizing current steps in current-clamp mode showed reliable spiking with spike frequency adaptation (Fig. 3C). The I–V curve revealed a linear current–voltage relationship (Fig. 3D).
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 current injection (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- and glutamate-mediated postsynaptic potentials (top trace) that were successively blocked by gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm; bottom traces), respectively. C, Voltage responses to 12 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.
Hypothalamic CSF-c neurons expressing somatostatin respond to acidic and alkaline pH
To determine whether hypothalamic somatostatin-expressing CSF-c neurons respond to changes of the extracellular pH, acidic (pH 6.5) (Fig. 4A, red trace) or alkaline (pH 8.0) (Fig. 4A, blue trace) solutions were bath-applied to hypothalamic slices during whole-cell recording. To exclude the possibility of indirect effects through synaptic inputs, blockers of GABA (gabazine) and glutamate (AP5, CNQX) receptors were applied throughout these experiments. In 30 of 45 tested CSF-c neurons, a decrease as well as an increase of pH in the same cell depolarized the membrane potential by 10–12 mV, eliciting a sustained discharge of action potentials (Fig. 4A). After application of TTX, the membrane potential depolarization induced by acidic or alkaline pH remained (n = 12; Fig. 4B).
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 the membrane 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 all subsequent recordings, gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) were applied to exclude any indirect, synaptically mediated effects. B, In the presence of TTX (1.5 μm), both acidic and alkaline 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 with Neurobiotin (green, arrow), showing that the cell expressed somatostatin (magenta). Scale bars, 20 μm. 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 inward current 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 recorded at acidic and alkaline pH. TTX (1.5 μm) was present in B, E–G.
These results thus suggest that the response to both acidic and alkaline extracellular pH is due to a direct effect on the CSF-c neuron. The recorded cells were intracellularly labeled with Neurobiotin and costained for somatostatin, verifying that the pH-sensing CSF-c cells express somatostatin (Fig. 4C). None of the recorded CSF-c neurons that were nonresponsive to acidic or alkaline extracellular pH expressed somatostatin (n = 15). Figure 4D illustrates the mean membrane potential changes of CSF-c neurons recorded in whole-cell mode, showing that individual CSF-c neurons in the hypothalamus respond with membrane potential 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 a U-shaped response curve (p < 0.001, paired t test; n = 15 cells).
The response to acidic and alkaline pH was also analyzed in voltage-clamp mode (Fig. 4E–G). Only a few low-amplitude current events were seen at pH 7.4 (AP5, CNQX, gabazine, and TTX present; Fig. 4E). Upon decreasing or increasing the extracellular pH to 6.5 or 8.0, respectively, the frequency of inward current events increased markedly (p < 0.001, paired t test; n = 7 cells; Fig. 4E,F). We also recorded unitary inward current events, presumably corresponding to single-channel openings, at acidic and alkaline pH (Fig. 4G).
At the transition from the brainstem to the spinal cord, where the central canal starts, there is a small region in which CSF-c neurons coexpress somatostatin and dopamine. These cells also responded to acidic and alkaline pH deviations (n = 3; not illustrated). However, CSF-c neurons at both the obex level and in the spinal cord that only express dopamine did not respond to pH changes (n = 4). It thus appears that the common factor is an expression of somatostatin in CSF-c neurons responding to deviations of the extracellular pH.
Specific blockade of ASIC3 eliminates the response of hypothalamic CSF-c neurons to acidic pH
To investigate whether the sensitivity of hypothalamic CSF-c neurons to decreases in pH depends on the ASIC3 subtype, as is the 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 to acidic or alkaline resulted in firing of action potentials, as well as a net depolarization of the membrane potential (Fig. 5A). Following application of APETx2, both aspects of the response were eliminated at acidic pH, whereas the alkaline response was not affected (n = 5; Fig. 5A). Changing the pH of the extracellular solution to 6.5 or 8.0 in voltage-clamp mode (gabazine, CNQX, AP5, and TTX present) caused inward current deflections that were abolished by APETx2 at acidic pH, whereas the current events induced by alkaline pH remained (pH 6.5 vs 7.4 nonsignificant; 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 channels represent the acid sensor in somatostatin-expressing hypothalamic CSF-neurons.
Response to acidic and alkaline pH is mediated by different channels. A, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm). Application of APETx2 (1 μm) 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 at pH 7.4 (black traces) in the presence of gabazine (20 μm), AP5 (50 μm), CNQX (40 μm), and TTX (1.5 μm). Inward current deflections appeared after a decrease or increase in the extracellular pH. The current events recorded in acidic pH (6.5) were completely blocked in the presence of APETx2 (1 μm; red trace), whereas those recorded in alkaline pH (8.0; blue trace) remained. C, Mean frequency increase 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 μm. E, Spinal somatostatin-CSF-c neurons (green) coexpress PKD2L1 (arrows; magenta). Scale bar, 20 μm. cc, Central canal. F, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) 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 μm) 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 lanthanum chloride (100 and 70 μm) [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 at 8 (p = 9.43 × 10−5, t4 = 15.77)]. In the presence of lanthanum, a complete (100 μm) or partial (70 μm) blockade of the spiking response to alkaline pH was seen [means ± SEM; n = 5; Student's paired t test: ***p < 0.001 significant difference at pH 8 vs control in the presence of lanthanum at 100 μm (p = 9.4 × 10−5, t4 = 15.77; as well as at 70 μm (p = 9.3 × 10−4, t4 = 8.76)]. A tendency 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 μm (p = 0.06, t4 = 2.58). H, The recorded CSF-c neuron in F intracellularly labeled with Neurobiotin expressed somatostatin. Scale bars, 10 μm. I, Whole-cell current clamp of a hypothalamic CSF-c neuron in control condition and in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) 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 I intracellularly labeled with Neurobiotin did not express somatostatin. Scale bars, 10 μm.
Specific blockade of connexin hemichannels eliminates the response of hypothalamic CSF-c neurons to alkaline pH
To investigate which type of channel that might be sensing alkaline pH in hypothalamic CSF-c neurons, we performed in situ hybridization for PKD2L1 (no specific PKD2L1 antagonist exists). This channel was a main candidate because it is considered to mediate the alkaline response in spinal somatostatin-positive CSF-c neurons (Jalalvand et al., 2016b). However, no hypothalamic somatostatin-expressing nor any other hypothalamic CSF-c neurons expressed the PKD2L1 channel (Fig. 5D). As a positive control, we performed in situ hybridization on spinal cord sections that were mounted on the same slide as the hypothalamic sections. Strong PKD2L1 expression was observed in spinal CSF-c neurons (Fig. 5E), consistent with our previous findings (Jalalvand et al., 2016b). Therefore, PKD2L1 is not the alkaline sensor in hypothalamic somatostatin-expressing CSF-c neurons.
The connexin hemichannels, which can be present in the cell membrane even though there is no coupling to an adjacent cell, corresponds structurally to one-half of the gap junction channel and represents another candidate that has been suggested to sense alkaline pH (Schalper et al., 2010). The somatostatin-positive hypothalamic CSF-c neurons are immunoreactive to connexin 35/36 (Fig. 2), so we tested whether the connexin hemichannel blocker lanthanum (La3+) could block the alkaline response. Lanthanum blocks connexin hemichannels but have no effect on the gap junction channels themselves at the concentrations used (100 and 70 μm; Contreras et al., 2002; Spray et al., 2006). In a CSF-c neuron that responded to both acidic and alkaline pH, application of LaCl3 (100 μm) completely blocked responses to alkaline pH, including the net depolarization of the membrane potential, as well as the firing of action potentials (control: pH 6.5 and pH 8 vs pH 7.4, significant difference p < 0.001; lanthanum, 100 μm: pH 8 vs control, significant difference p < 0.001, paired t test; n = 5; Fig. 5F,G). LaCl3 at 100 μm, however, also tended to reduce the acidic response (nonsignificant, p = 0.06, paired t test; n = 5). At 70 μm, there was no effect on the acidic response, but a clear and significant reduction of the alkaline response (p < 0.001, paired t test; n = 5; Fig. 5G). The recorded CSF-c neurons were intracellularly labeled with Neurobiotin and costained for somatostatin, verifying that the hypothalamic CSF-c neurons responding to the changes of extracellular pH express somatostatin (Fig. 5H).
Some hypothalamic CSF-c neurons did not respond to either alkaline or acidic pH (Fig. 5I) and none of these non-pH-sensitive CSF-c neurons expressed somatostatin (Fig. 5J). These somatostatin-negative CSF-c neurons exhibited spontaneous postsynaptic potentials (Fig. 5I, top trace), as well as action potential firing upon current injection.
Together, these results suggest that the alkaline response in hypothalamic, somatostatin-expressing CSF-c neurons is mediated via connexin hemichannels.
Hypothalamic CSF-c neurons sense fluid movements via ASIC3-like channels
To investigate whether hypothalamic CSF-c neurons are mechanosensitive to fluid movements, brief pressure pulses were applied to an aCSF-filled micropipette placed close to the bulb-like protrusion of the CSF-c neuron with its cilium (Fig. 1E) while performing patch recordings (Fig. 6A). To verify that the responses were not indirect or synaptically evoked, GABAergic and glutamatergic synaptic transmission was blocked by bath application of gabazine, AP5, and CNQX. Graded receptor potential responses were reliably elicited by fluid pulses (duration 40–100 ms) in all CSF-c neurons tested (n = 8; Fig. 6B,C). The receptor potential amplitude increased with increasing pulse magnitudes (Fig. 6B, 10–20 psi) and action potentials were evoked when the membrane potential was held at −55 mV (Fig. 6B, top trace).
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 third ventricle. An aCSF-filled pressure pipette was placed close to a bulb-like ending of a recorded hypothalamic CSF-c neuron. Scale bar, 20 μm. B, A short fluid-pulse (80 ms) elicited receptor potential responses and action potentials while holding the membrane potential at −65 mV and −55 mV, respectively, in the presence of gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm). C, Receptor potential elicited by fluid pulse stimulation (20 psi, 80 ms) was blocked by application of the ASIC3 blocker APETx2 (1 μm). 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 (with GABA and glutamate receptor antagonists present).
Because ASIC3-like channels mediate the mechanosensitivity underlying the fluid pulse response in spinal CSF-c neurons (Jalalvand et al., 2016a), we investigated whether this channel may also act as a mechanosensor in hypothalamic CSF-c neurons that express somatostatin. The ASIC3 blocker APETx2 was bath applied 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 of APETx2 (Fig. 6C,D). Therefore, the mechanosensitivity of the hypothalamic CSF-c neurons also appears to be mediated by ASIC3-like channels. The same CSF-c neuron responded to decreased (pH 6.5) and increased (pH 8) extracellular pH (Fig. 6E).
Discussion
An accurate control of the acid–base balance is one of the main homeostatic tasks of an organism. With regard to the brain, it is important to sense the pH of the CSF because it integrates the overall impact of pH changes within the brain. To have pH sensors in the wall of the third ventricle at the hypothalamic level would seem to be an optimal location given the role of hypothalamus in a variety of other homeostatic mechanisms. A high level of neuronal activity can by itself lead to an extracellular acid shift through the glucose–lactate shuttle in the astrocytes to furnish neurons with lactate as a source of energy Chesler and Kaila, 1992; Magistretti and Allaman, 2015, 2018). Moreover, increased CO2 levels (Voipio and Kaila, 1993), as in ischemia, will lead to acidosis, whereas hyperventilation due to low O2 levels will result in alkalosis. Similarly, metabolic events can influence the pH in both directions (Levin and Buck, 2015). Somatostatin/GABA CSF-c cells lining an area within the third ventricle, as shown here, sense both acidic and alkaline deviations from the control level. This also applies to the same type of CSF-c neurons located 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 the spinal cord appears to be exclusive to the somatostatin CSF-c subpopulation of neurons because somatostatin-negative neurons do not respond to pH changes. In the spinal cord, acidic and alkaline deviations of the extracellular pH lead to an activation of the CSF-c neurons and a release of somatostatin in the spinal cord, which in turn exerts a depressing effect on the network generating locomotor activity (Jalalvand et al., 2016a,b). The response to any pH change in the spinal cord will thus be a reduced motor activity, which should help the organism to recover its normal pH.
Do the hypothalamic somatostatin-positive CSF-c neurons have a similar role as their spinal counterparts? There is a very rich somatostatin innervation in the motor areas of the brain extending from the forebrain to the brainstem, whereas sensory areas have little or no innervation (see also below). Although not addressed here, this raises the possibility that the hypothalamic somatostatin-/GABA-expressing CSF-c-neurons could act at the supraspinal level to reduce motor activity and thereby help to restore the pH level.
pH sensitivity of the CSF-c neurons in relation to physiological and pathophysiological conditions
The U-shaped curves of pH sensitivity of both hypothalamic and spinal CSF-c neurons show a significant depolarization at pH 7.1 or 7.7 from a neutral value of 7.4, but a much larger response occurs outside this area at pH 6.5 and 8.3 (cf. Fig. 4D). Whereas pH values between 7.1 and 7.7 can be regarded as being within the physiological range, values outside of this range would imply pathophysiology. The somatostatin CFS-c neurons will thus respond steeply if the pH shifts to pathophysiological levels, but will also detect smaller changes within the physiological range. In lamprey, the pH of the blood can change with as much as 0.5 units during exercise (Tufts et al., 1992) and the pH values in the interstitial fluid is somewhat lower than in the blood ∼pH 7.3 to 7.4 (Chesler, 1986). During epilepsy or ischemia, pH values of 6.5 have been reported in mammalian brain tissue (Rehncrona, 1985; Siesjö et al., 1985). In the blood of patients with severe acidosis, pH just <7 and for alkalosis >7.65 have been reported (Thorén, 1960). Also, under physiological conditions, high levels of neuronal activity can lead to deviations in the acidic direction due to the glucose–lactate shuttle in the astrocytes, which provides energy to neurons through lactate (Chesler and Kaila, 1992; Magistretti and Allaman, 2015).
The fact that the somatostatin CSF-c neurons respond to both acidic and alkaline deviations with increased activity can be regarded as an elegant evolutionary solution to respond to and master a life-threatening condition. The response in both cases can result in reduction of the motor activity. In the case of metabolic demands during ischemia or hyperactivity, this will lead to a restoration of the pH. Similarly, during conditions with low pO2, when hyperventilation will cause a lower pCO2 and thereby an alkaline pH, a reduction of the motor activity will also help to restore the pH. This applies to spinal cord somatostatin CSF-c neurons and may also apply to the hypothalamic somatostatin CSF-c cell subpopulation.
Response to alkaline pH is mediated by different molecular mechanisms in spinal and hypothalamic CSF-c neurons
PKD2L1 is the alkaline sensor in brainstem and spinal CSF-c neurons (Jalalvand et al., 2016a,b; Orts-Del'Immagine et al., 2016) and was therefore considered a main candidate for the hypothalamic CSF-c neurons. Unexpectedly, we could not detect any expression of PKD2L1 in hypothalamic CSF-c neurons (Fig. 5D), suggesting that another unidentified channel is responsible for the alkaline response in hypothalamus. This is consistent with studies in the mouse, in which PKD2L1 channels are not expressed at a level just rostral to obex (Orts-Del′Immagine et al., 2014). A very small group of mouse hypothalamic cells in cell culture were shown to be PKD2L1-positive, but were not identified as CSF-c cells (Huang et al., 2006).
Different extracellular alkaline pH sensors have been described in the vertebrate brain, including connexin hemichannels, insulin receptor-related receptors, and two-pore-domain K+ (K2P) channels (for review, see Murayama and Maruyama, 2015). Because the connexin 35/36 protein is expressed in hypothalamic CSF-c neurons (Fig. 2B–D), in contrast to the cells around the central canal, we considered connexin hemichannels a possibility. The connexin hemichannels are sensitive to pH changes (Francis et al., 1999; Contreras et al., 2002; Sáez et al., 2005; Spray et al., 2006; Yu et al., 2007) and extracellular alkalization increases intracellular Ca2+ levels in cells expressing connexin hemichannels (Schalper et al., 2010).
The somatostatin CSF-c neurons in hypothalamus express connexin 35/36 and the alkaline response could be totally blocked with lanthanum, suggesting that connexin hemichannels are sensors for alkaline pH. We used two different concentrations of lanthanum, 70 and 100 μm. At 70 μm, lanthanum did not affect the acidic response but very significantly reduced the alkaline response, whereas at 100 μm, a complete blockade of the alkaline response was observed, together with a nonsignificant reduction of the acidic response. At very high concentrations (≥100 μm), lanthanum has an inhibitory effect on many channels including kainate and AMPA receptors (at concentrations of 1–30 mm; Reichling and MacDermott, 1991; Hong et al., 2004), as well as TRP channels (at concentrations >1 mm; Zhao et al., 2015) and thus at such high levels may also affect ASIC3 in hypothalamic CSF-c neurons.
Another difference between lamprey hypothalamic and spinal CSF-c neurons is that the former have a lower mean resting potential (−65 mV vs −52 mV for spinal CSF-c neurons) at pH 7.4 and therefore are rarely spontaneously active at this pH. Presumably as a consequence of this difference, hypothalamic CSF-c neurons showed a more prominent membrane potential depolarization 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 to fluid motion
The acidic response in hypothalamic CSF-c neurons, as in spinal CSF-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, ASIC3 has been reported to be expressed throughout the hypothalamus, including the suprachiasmatic nucleus (Wang et al., 2007; Chen et al., 2009; Meng et al., 2009). Lamprey hypothalamic CSF-c neurons are also mechanosensitive and activated by fluid movements such as motion of the CSF. This response is also mediated by ASIC3-like channels, as in the spinal cord (Jalalvand et al., 2016b). In CSF-c neurons of the most caudal part of the mouse brainstem, the acidic response was shown to be mediated through the general class of ASICs (Orts-Del'Immagine et al., 2016). It would therefore seem that vertebrate CSF-c neurons responsive to acidic pH have developed this capacity through the expression of ASICs, in contrast to the alkaline sensors, which can take different forms.
Role of somatostatin CSF-c neurons in the brain and spinal cord
In the lamprey brain, the main source of somatostatin is the hypothalamic CSF-c neurons, although small populations exist in the ventral thalamus and the isthmic region and of course in the cells around the central canal (Wright, 1986; Buchanan et al., 1987; Christenson et al., 1991; Yáñez et al., 1992 our unpublished observations). Periventricular somatostatin-expressing neurons are also highly concentrated along the third ventricle in other vertebrates, 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), and fish (Vígh-Teichmann et al., 1983b; Sas and Maler, 1991). The lamprey pallium and striatum have a rich somatostatin innervation originating from CSF-c neurons in hypothalamus (Suryanarayana et al., 2017), but no intrinsic somatostatin interneurons, in contrast to the mammalian cortex and striatum (Liguz-Lecznar et al., 2016).
The hypothalamic somatostatin CSF-c neurons innervate most parts of the brain, however, with a higher density of fibers and/or terminals in motor areas, including the output layer of the optic tectum and the reticulospinal nuclei, but not in sensory areas. Interestingly, somatostatin-expressing CSF-c neurons at the obex level, which coexpress dopamine, also respond to pH changes, whereas dopaminergic CSF-c neurons that do not express somatostatin are unresponsive. This further strengthens the notion of a functional link between somatostatin expression in CSF-c neurons and pH sensitivity. In the lamprey spinal cord, the suppression of motor activity induced by activation of the somatostatin-/GABA-expressing CSF-c cells following a change of extracellular pH is mediated by a release of somatostatin (Jalalvand et al., 2016a,b). The hypothalamic CSF-c neurons may well have a similar role.
Conclusion
Together, somatostatin-expressing CSF-c neurons in the hypothalamus, like in the spinal cord, may serve as pH sensors as well as mechanosensors. They will sense any pH deviation and motion in the CSF of the third ventricle and respond by membrane potential depolarization and action potential firing. The acidic response and mechanosensitivity are mediated by ASIC3-like channels, whereas the alkaline response appears to be mediated through connexin hemichannels, in contrast to their spinal counterparts, in which PKD2L1 is responsible. Activation of hypothalamic CSF-c neurons will cause a release of somatostatin in the different target areas, many of which are motor related. As in the spinal cord, this may contribute to restoring the deviation in pH and thus to homeostasis.
Footnotes
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 Grant 604102 to H.B.P.), EU/Horizon 2020 Grant 720270 to H.B.P. specific grant agreement 1), StratNeuro Karolinska Institutet, the Karolinska Institutet's Research Funds, 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 that 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 of Neuroscience, Karolinska Institutet, Solnavägen 9, SE-171 77 Stockholm, Sweden. sten.grillner{at}ki.se
