Active, neuronal-activity-dependent trans-membrane water cycling detected by NMR
Ruiliang Bai1, Charles S Springer2, Dietmar Plenz3, and Peter J Basser4

1Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy For Advanced Studies, Zhejiang University, Hangzhou, China, 2Advanced Imaging Research Center, Oregon Health & Science University, Portland, OR, United States, 3Section on Critical Brain Dynamics, LSN, NIMH, National Institutes of Health, Bethesda, MD, United States, 4Section on Quantitative Imaging and Tissue Sciences, DIBGI, NICHD, National Institutes of Health, Bethesda, MD, United States


Transmembrane water cycling has long been thought an entirely passive, diffusion-dominated process. Recent studies suggest that an energetically active water cycling (AWC) mechanism, driven by Na+-K+-ATPase (NKA) pump activity, also occurs in different cell types, including neurons and astrocytes. Here we hypothesize that monitoring AWC could provide a new, more direct physical indicator of neuronal activity, which involves much ion cycling and enhanced NKA activity. By investigating neuronal populations in vitro under resting conditions with spontanotanous activity, and perturbed with tetrodotoxin (TTX), we found TTX simultanously reduces neuronal spiking activity and AWC (by >63%) suggesting AWC directly reflects neuronal activity.


Water homeostasis and transport play important roles in brain function, e.g., ion homeostasis, neuronal excitability, cell volume regulation. However, specific mechanisms of water transport across cell membranes in neuronal tissue have not been completely elaborated. In addition to passive water cycling by water diffusing across cell plasma membranes, energetically active water cycling (AWC) also occurs in different cell types1,2, including neurons and astrocytes3,4. The AWC is largely driven by cell membrane Na+-K+-ATPase pump (NKA) activity, where water is co-transported with ion cycling across membrane using secondary active cotransporters to exit and enter the cell (Figure 1)2,5,6.

Neuronal activity is a highly metabolically demanding process that involves ion transport and recycling: NKA plays an essential role restoring ion gradients7. Recently, glutamate transporter activity was found to be strongly coupled to, and regulated by, NKA activity8. Here we hypothesize that AWC is postively correlated with neuronal activity, and monitoring AWC could provide a new, more direct physical indicator or measure of neuronal activity in vivo. To test this hypothesis, we investigated neuronal populations (organotypic cortical cultures (OCC) from newborn rat somatosensory cortex) under resting conditions in which spontanotanous activity is observed, and then treated with tetrodotoxin (TTX, a voltage-gated sodium channel (VGSC) blocker), to depress neuronal activity. We simultaneously measure the AWC and neuronal actitivity changes.


According to our hypothesis, the unidirectional rate constant for steady-state cellular water efflux, kio, has two components:

kio = kio(a) + kio(p) (1)

where a and p represent active and passive kio contributions, respectively. For passive exchange, kio(p) = <A/V>•PW(p), where <A/V> is the mean cell surface area/volume ratio, and PW(p) is the diffusion-driven passive membrane water permeability coefficient1,2. The active rate constant, kio(a), depends on the cellular metabolic rate of NKA, cMRNKA [e.g., pmol(ATP)/s/cell]. Thus, we rewrite kio as:

kio = {x/([H2Oi]•<V>)}cMRNKA + <A/V>•P (p) (2)

where [H2Oi] is the intracellular water concentration and x is the water stoichiometric multiplier (x = 500 to 1000 per co-transporter (I, IV in Fig. 1). In order to detect cMRNKA changes, kio(p) changes should be simultaneously estimated.

The kinetics of water exchange in OCC was measured using simultaneous, real-time fluorescence optical imaging and NMR. NMR signals were acquired during perfusion with a paramagnetic agent, Gadoteridol, which enables the determination of kio, and the mole fraction of intracellular water (pi), related to the average cell volume (V). Changes in intracellular calcium concentration, [Cai2+] were used as a proxy for neuronal activity and were monitored by fluorescence. Detailed experimental methods have been reported4.


With normal artificial cerebrospinal fluid (ACSF) perfusion, the OCC exhibited spontaneous neuronal activity, demonstrated by the intracellular calcium fluorescence signal (Figure 2A), which exhibited brief (0.5 – 3 s) periods of neuronal excitation separated by ~10 s of very low activity9. This baseline spontaneous activity is largely reduced by adding 0.2 µM TTX (Fig. 2A). Interestingly, kio is also reduced, by 63%, from 2.05 (± 1.67) s-1 to 0.75 (± 1.13) s-1 (p = 0.03, Fig. 2B). The mean cell volume also was also reduced during TTX perfusion, with pi dropping from 0.070 (± 0.018) to 0.057 (± 0.015) (p = 8 x 10-4).

Assuming PW(p) and cell shape do not change, the kio(p) change from baseline to TTX can be estimated from Eq. 2:

{kio(p,TTX)/kio(p, baseline)} = {Vbaseline/VTTX}1/3 = 1/0.95 = 1.05 (3)

In this case, kio(p) increased by 5%, and, thus, kio(a) independently decreased by more than 63%.

Discussions and Conclusions

In this study, we found an AWC component in OCC, which is related to neuronal activity. With TTX perturbations, kio decreased by >63% while cell diameter (d) decreased by 5%. In a previous study with ouabain perturbation, kio decreased by 45% while d increased by a maximum of 23% (Figure 3)4. The two experiments demonstrate that d has no correlation with kio (Eq. 2), i.e., the active pathway must exist in neuronal tissue.

TTX is frequently used to modulate neuronal spiking activity by blocking VGSC, a Na+ influx channel. Here we show that TTX simultaneously reduces neuronal spiking activity and AWC (by at least 63%). A direct consequence of TTX binding would be to reduce Na+ influx, and thus decrease NKA activity, which pumps out Na+ to maintain the ion’s gradient, and to decrease the NKA activity-driven AWC component. It is probable that VGSC is one of the major III transporters shown in Fig. 1. Since its major activity is generally thought to occur during the action potential, our results suggest that kio, and specifically kio(a), directly reflects neuronal activity: cMRNKA increases during the spikes.


RB and PJB were supported by the Intramural Research Program (IRP) of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. RB was also supported by the 985 Program at Zhejiang University. We thank our colleague Craig Steward for helping prepare organotypic cultures. CSS acknowledges Drs. Craig Jahr, Thomas Barbara, Christopher Kroenke, Deborah Burstein, Robert Balaban, and Suzanne Scarlata for stimulating discussions, and the NIH [Grants UO1‑CA154602 and R44-CA180425] and the Paul G. Allen Family Foundation [Grant 12079] for his support. DP was supported by the Intramural Research Program (IRP) of the National Institute of Mental Health (ZIAMH002797), NIH.


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Figure 1. Illustration of the passive (p) and active (a) water cycling pathways in biological tissue. kio and koi are the steady-state cellular water efflux and influx rate constants, respectively. II and III represent transporters K+ uses to re‑exit and Na+ uses to re-enter the cell, respectively. I and IV represent water co-transporters that H2O uses to exit and enter the cell, respectively. po and pi, the respective mole fractions of extra- and intracellular water, PH, the MRI contrast agent ProHance. The NKA substrate ATPi and inhibitor ouabain, and Na+ channel blocker TTX are also indicated, in red.

Figure 2. Na+ channel blocker TTX. (A) Representative calcium fluorescence signal of spontaneous neuronal activity in normal ACSF (left), reduced neuronal activity in response to TTX perfusion, and recovery of neuronal activity in the washout with normal ACSF. (B) The results of the mean neuronal activity ([100ΔF/F0], left), the cellular water efflux rate constant kio (middle), and the intracellular water mole fraction pi (right) in two phases: Baseline (N = 13) and TTX perfusion (N = 13).

Figure 3. Comparing percentage water exchange rate constant (kio, blue bar) changes with simultaneous mean cell diameter (V1/3, yellow bar) changes in response to perfusion with ouabain (NKA inhibitor, details in ref4) or TTX (VGSC blocker, current study).

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)