Steffensen AB , Oernbo EK , Stoica A , Gerkau NJ , Barbuskaite D , Tritsaris K , Rose CR , MacAulay N .

	Fig. 1. Cotransporter-mediated active water transport against an osmotic gradient. a Image of calcein fluorescence in mouse choroid plexus (CP, top) and the converted image (bottom) with indication of the region of interest (red box). Scale bar = 60 µm. b Volume changes of CPs challenged with a 100 mOsm gradient of mannitol (M), indicated by the extended bars (n = 5 CPs from five mice). c CPs challenged with a 100 mOsm gradient of first mannitol (M), then NaCl, and finally mannitol, indicated by the extended bars (n = 6 CPs from six mice). d CPs challenged with a 100 mOsm gradient of first mannitol (M), then KCl, and finally mannitol, indicated by the extended bars (n = 6 CPs from six mice). e Summary of the second volume change induced by identical osmotic gradients. f CPs challenged with 100 mOsm KCl alone (control, black symbols, data from d) or in the presence of 1 mM furosemide (purple symbols, n = 6 CPs from six mice). Inset magnifies the area of interest where furosemide blocks the KCl-induced swelling. g CPs challenged with 100 mOsm alone (control, black symbols, data from d) or in the presence of 10 µM bumetanide (blue symbols, n = 6 CPs from six mice). Inset magnifies the area of interest where bumetanide blocks the KCl-induced swelling. h CPs challenged with 100 mOsm KCl (control, black symbols, data from d) and in the absence of Na+ in the test solution (green symbols, n = 4 CPs from four mice). Inset magnifies the area of interest where lack of Na+ blocks the KCl-induced swelling. i Summary of the second volume change induced by KCl alone or with drug application/Na+ omission. Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test. In panel e, the asterisks refer to a comparison to the mannitol-mediated shrinkage and in panel i, the asterisks refer to a comparison to the KCl-mediated swelling. **P < 0.01, ***P < 0.001, NS not significant
	Fig. 2. NKCC1 and KCC1 are expressed in mouse choroid plexus. a mRNA expression levels of NKCC1 and the four KCC isoforms in mouse choroid plexuses. The five target genes were normalized to two reference genes, GAPDH and H2AFZT, and presented as relative expression; NKCC1 = 60.0 ± 3.9, KCC1 = 21.2 ± 1.8, KCC2 = 0.2 ± 0.1, KCC3 = 1.2 ± 0.1, and KCC4 = 3.2 ± 0.2 (n = 6 mice), error bars represent standard error of the mean. b Representative western blots to verify antibody specificity. NKCC1 and KCC1-4 were expressed in Xenopus laevis oocytes and purified membranes from these and uninjected oocytes were exposed to SDS-PAGE followed by western blot (n = 3 experiments). c−f Western blots of lysates from mouse choroid plexus demonstrated expression of NKCC1 (c) and KCC1 (d), while KCC2 (e) and KCC4 (f) were not detected. Beta-tubulin was employed as loading control (n = 3 experiments)
	Fig. 3. NKCC1 is located at the luminal membrane facing the ventricles. a Immunostaining of mouse choroid plexus illustrated expression of NKCC1 in the membrane facing the lumen (green) with E-cadherin (red) as a basolateral marker and nuclei in blue. Scale bar = 10 µm. Previously unpublished image generously provided by Dr. Jeppe Praetorius. b Schematic illustration of the luminal surface biotinylation of mouse choroid plexus in situ. c Representative western blots showing expression of NKCC1, KCC1, and E-cadherin in total tissue fraction (Ft) and purified biotinylated (luminal) membrane protein fraction (Fl). Below is depicted the quantification of protein abundance represented as luminal membrane/total protein fraction (n = 4 mice). NKCC1 was expressed significantly higher in the purified luminal membrane fraction while KCC1 and E-cadherin were predominantly in the total fraction. Error bars represent standard error of the mean and statistical significance was determined with one-sample t test and comparison to equal distribution; Fl/Ft = 1 and the asterisks refer to a value significantly different from 1. **P < 0.01, ***P ≤ 0.001
	Fig. 4. NKCC1 is poised for outward transport in the luminal membrane. a Image of SBFI fluorescence of the choroid plexus taken with wide-field fluorescence microscope. Scale bar = 40 µm. b Representative sodium signals of choroid plexus during 10 min baseline, 10 min application of the NKCC1 inhibitor bumetanide (10 μM), and 20 min washout. Gray lines represent the SBFI ratio obtained from 40 single cells during one experiment, black line represents an average of these cells. c Average bumetanide-induced increase in SBFI ratio of choroid plexus cells, indicative of increased intracellular sodium concentration (n = 10 CPs from ten mice). d Representative images of calcein fluorescence of the choroid plexus either after 10 min in aCSF (top panel) or after 10 min of bumetanide treatment (bottom panel, time point marked as * in b). Scale bar = 40 µm. e Loss of 86Rb+ from choroid plexus as a function of time in control settings (black, n = 7 CPs from seven mice) or with treatment of either bumetanide (20 μM, blue, n = 5 CPs from five mice) or furosemide (1 mM, violet, n = 5 CPs from five mice). Y-axis is the natural logarithm of amount of 86Rb+ left in choroid plexus at time t (At) divided by the amount at time 0 (A0). f Efflux rate constants for 86Rb+ in control (0.041 ± 0.003 s−1, n = 7 CPs from seven mice), in the presence of bumetanide (0.008 ± 0.001 s−1, n = 5 CPs from five mice), or furosemide (0.005 ± 0.001 s−1, n = 5 CPs from five mice). Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test and the asterisks above the bars indicate comparison to the control while the comparison between the two test solutions is indicated with a line above the relevant bars. ***P < 0.0001, NS not significant (P = 0.526)
	Fig. 5. NKCC1 acts as a significant contributor to in vivo CSF production. a Schematic drawing of the infusion of aCSF containing dextran (dark red cannula) into the right lateral ventricle (RV) and collection of the diluted dextran (light red cannula) at cisterna magna (CM) of a ventilated mouse. b Representative time course of the fluorescence ratio of dextran (outflow/inflow) during a control ventriculo-cisternal perfusion. After stable baseline (60 min), the dextran/DMSO aCSF-solution was changed within 5–10 min. Inset depicts the average CSF production rate (n = 18 mice). c Summarized data from ventriculo-cisternal perfusion illustrating CSF production (percentage of baseline) as a function of time. Data normalized to the average of the two last samples before solution change. Control perfusion with the DMSO-vehicle is shown in black (n = 6 mice), treatment with bumetanide in blue (n = 6 mice), and furosemide (n = 6 mice) in purple. Inset illustrates the summarized CSF production rates after 60 min exposure to vehicle (black), bumetanide (blue), or furosemide (purple) normalized to own control. d Mid-sagittal section of a mouse brain after unilateral injection of Evans Blue revealed staining mainly in the injected, right lateral ventricle (top panel) compared to the contralateral ventricle (bottom image). Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test with asterisks above the bars indicating comparison to control perfusion and comparison between test solutions indicated by lines above the respective bars. *P < 0.05, ***P < 0.001
	Fig. 6. Live imaging determines significant NKCC1 contribution to in vivo CSF production. a Illustration of pseudo-color fluorescence superimposed on a white light image from a white mouse after ventricular injection of 10 μM IRDye 800CW carboxylate dye. The dye content is quantified in the red box, placed in line with lambda. The intensity scale is arbitrary units and applies to all images. b Intensity of images were quantified and normalized to the first image (0 min) for control (vehicle, black), bumetanide (blue), or furosemide (purple) and plotted as a function of time. Inset illustrates summarized data at the 10-min time point; the fluorescence intensity of control mice reached 1.95 ± 0.12 (n = 8 mice), furosemide-treated 1.65 ± 0.05 (n = 6 mice), and bumetanide 1.63 ± 0.07 (n = 8 mice). c Unilateral injection of Evans Blue revealed staining mainly in the injected, right lateral ventricle (red oval) compared to the contralateral ventricle. d Representative images at 0, 5, and 10 min for control condition (top panels), bumetanide treatment (middle panels), and furosemide treatment (bottom panels). Error bars represent standard error of the mean and statistical significance was evaluated with two-way ANOVA (RM) followed by Tukey’s multiple comparisons test. Asterisks above the bars indicate comparison to control while comparisons between test solutions are indicated with a line above the respective bars. **P < 0.01, NS: not significant (P = 0.98)
	Fig. 7. Schematic drawing of transporters in a choroid plexus epithelial cell. a The drawing depicts the selective expression of NKCC1 at the luminal membrane and indicates its unique outward transport direction, due to the high intracellular concentration of Na+ and Cl− in this tissue (ion concentrations given in mM). The NKCC1-mediated cotransport of water is indicated with a dashed arrow. The Na+/K+-ATPase and AQP114 are indicated on the luminal membrane of the choroid plexus epithelium (CPE), while KCC1 is localized to the basolateral membrane facing the vascular compartment (this study). b The drawing includes the many other coupled transporters localized to the choroidal epithelial membranes1. Note that NCBE may also be referred to as NBCn2

Abbott, Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. 2004, Pubmed

Abbott, Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. 2004, Pubmed
Ames, Effects of Pco2 acetazolamide and ouabain on volume and composition of choroid-plexus fluid. 1965, Pubmed
Boettger, Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4. 2002, Pubmed
Boettger, Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. 2003, Pubmed
Bratosin, Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. 2005, Pubmed
Bu, Mechanisms of hydrocephalus after intraventricular haemorrhage in adults. 2016, Pubmed
Carey, Effect of systemic arterial hypotension on the rate of cerebrospinal fluid formation in dogs. 1974, Pubmed
Charan, How to calculate sample size in animal studies? 2013, Pubmed
Choe, Water permeation through the sodium-dependent galactose cotransporter vSGLT. 2010, Pubmed
Christensen, Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice. 2013, Pubmed
Cong, Ion transporters in brain tumors. 2015, Pubmed
Cserr, Physiology of the choroid plexus. 1971, Pubmed
Curl, Transport of water and electrolytes between brain and ventricular fluid in the rabbit. 1968, Pubmed
Damkier, Cerebrospinal fluid secretion by the choroid plexus. 2013, Pubmed
Damkier, Nhe1 is a luminal Na+/H+ exchanger in mouse choroid plexus and is targeted to the basolateral membrane in Ncbe/Nbcn2-null mice. 2009, Pubmed
Davson, The effects of some inhibitors and accelerators of sodium transport on the turnover of 22Na in the cerebrospinal fluid and the brain. 1970, Pubmed
Delpire, Human and murine phenotypes associated with defects in cation-chloride cotransport. 2002, Pubmed
Ducros, Headache arising from idiopathic changes in CSF pressure. 2015, Pubmed
HEISEY, Bulk flow and diffusion in the cerebrospinal fluid system of the goat. 1962, Pubmed
Haas, The Na-K-Cl cotransporter of secretory epithelia. 2000, Pubmed
Hallaert, Endoscopic coagulation of choroid plexus hyperplasia. 2012, Pubmed
Hamann, Cotransport of water by the Na+-K+-2Cl(-) cotransporter NKCC1 in mammalian epithelial cells. 2010, Pubmed
Hladky, Fluid and ion transfer across the blood-brain and blood-cerebrospinal fluid barriers; a comparative account of mechanisms and roles. 2016, Pubmed
Hübner, Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. 2001, Pubmed
Iliff, A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. 2012, Pubmed
Javaheri, Bumetanide decreases canine cerebrospinal fluid production. In vivo evidence for NaCl cotransport in the central nervous system. 1993, Pubmed
Johanson, Active transport of sodium and potassium by the choroid plexus of the rat. 1974, Pubmed
KISTER, Carbonic anhydrase inhibition. VI. The effect of acetazolamide on cerebrospinal fluid flow. 1956, Pubmed
Kanaka, The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system. 2001, Pubmed
Karadsheh, Localization of the KCC4 potassium-chloride cotransporter in the nervous system. 2004, Pubmed
Karimy, Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus. 2017, Pubmed
Keep, Potassium cotransport at the rat choroid plexus. 1994, Pubmed
Kimelberg, Astrocytic swelling in traumatic-hypoxic brain injury. Beneficial effects of an inhibitor of anion exchange transport and glutamate uptake in glial cells. 1989, Pubmed
Kurihara, Characterization of a phosphorylation event resulting in upregulation of the salivary Na(+)-K(+)-2Cl(-) cotransporter. 1999, Pubmed
Larsen, Contributions of the Na⁺/K⁺-ATPase, NKCC1, and Kir4.1 to hippocampal K⁺ clearance and volume responses. 2014, Pubmed , Xenbase
Li, Patterns of cation-chloride cotransporter expression during embryonic rodent CNS development. 2002, Pubmed
Lun, Spatially heterogeneous choroid plexus transcriptomes encode positional identity and contribute to regional CSF production. 2015, Pubmed
Lykke, The search for NKCC1-selective drugs for the treatment of epilepsy: Structure-function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A. 2016, Pubmed , Xenbase
MacAulay, Water transport between CNS compartments: contributions of aquaporins and cotransporters. 2010, Pubmed
Macri, Preferential vasoconstrictor properties of acetazolamide on the arteries of the choroid plexus. 1966, Pubmed
Markkanen, Distribution of neuronal KCC2a and KCC2b isoforms in mouse CNS. 2014, Pubmed
Meier, Properties of the new fluorescent Na+ indicator CoroNa Green: comparison with SBFI and confocal Na+ imaging. 2006, Pubmed
Milhorat, Cerebrospinal fluid production by the choroid plexus and brain. 1971, Pubmed
Murphy, Na(+)-H+ exchange in choroid plexus and CSF in acute metabolic acidosis or alkalosis. 1990, Pubmed
Oresković, The formation of cerebrospinal fluid: nearly a hundred years of interpretations and misinterpretations. 2010, Pubmed
Oshio, Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1. 2005, Pubmed
Payne, Cation-chloride co-transporters in neuronal communication, development and trauma. 2003, Pubmed
Pearson, Localization of the K(+)-Cl(-) cotransporter, KCC3, in the central and peripheral nervous systems: expression in the choroid plexus, large neurons and white matter tracts. 2001, Pubmed
Philp, Mouse MCT3 gene is expressed preferentially in retinal pigment and choroid plexus epithelia. 2001, Pubmed
Plotkin, Expression of the Na(+)-K(+)-2Cl- cotransporter BSC2 in the nervous system. 1997, Pubmed
Pollay, Secretion of cerebrospinal fluid by the ventricular ependyma of the rabbit. 1967, Pubmed
Praetorius, Transport across the choroid plexus epithelium. 2017, Pubmed
Praetorius, Distribution of sodium transporters and aquaporin-1 in the human choroid plexus. 2006, Pubmed
Rennels, Evidence for a 'paravascular' fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space. 1985, Pubmed
Rust, Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice. 2007, Pubmed
Spector, A balanced view of choroid plexus structure and function: Focus on adult humans. 2015, Pubmed
TSCHIRGI, Inhibition of cerebrospinal fluid formation by a carbonic anhydrase inhibitor, 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide (diamox). 1954, Pubmed
Untiet, Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation. 2017, Pubmed
Vogh, The effect of furosemide and bumetanide on cerebrospinal fluid formation. 1981, Pubmed
Vogh, The effect of carbonic anhydrase inhibitors and other drugs on sodium entry to cerebrospinal fluid. 1981, Pubmed
WELCH, SECRETION OF CEREBROSPINAL FLUID BY CHOROID PLEXUS OF THE RABBIT. 1963, Pubmed
Welch, Volume flow across choroidal ependyma of the rabbit. 1966, Pubmed
Zeuthen, Cotransport of water by Na⁺-K⁺-2Cl⁻ cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2. 2012, Pubmed , Xenbase
Zeuthen, Cotransport of K+, Cl- and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus. 1994, Pubmed
Ziebell, Flow-regulated versus differential pressure-regulated shunt valves for adult patients with normal pressure hydrocephalus. 2013, Pubmed
de Los Heros, The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters. 2014, Pubmed