This is the first reported data of CSF sodium chronobiology in the human nervous system showing that [Na+]csf has a 12 h rhythm that peaks around 08.00 and 18.10 h and troughs around 03.20 and 09.50 h. [Na+]csf also has a 100 min rhythm. The invasiveness of the procedure in humans has limited the sample size. However, we present evidence that the fluctuations we observed are biological because firstly, reproducibility was good and much less than for the 24-h sample variations. The measures of osmolarity at each time point were made on single assays, but the reproducibility of this assay was extremely good. Secondly, the cation calibration standards were used frequently to detect and correct for drift and thirdly, sodium rhythms were not significantly correlated with total protein or osmolarity. We interpret the lack of correlation with protein to exclude a mechanical inflammation that would have led to a rise of total protein. We interpret the lack of correlation with osmolarity to exclude any major evaporation or dehydration of samples.
Data from 144 samples per subject over 24 h allows rhythms at 20 min or greater to be detected. The principal rhythm with a 12-h period is visible in Figures 4 &5. We have less confidence in the 24-h period, since this involved the entire time of our sampling. This would require further testing in a longer duration study. We did not confidently identify the other significant period at 1.65 h (100 min) in the raw data (we were not looking for it), but it is significant in all the spectral analyses (Figure 6 and Additional Files 1 and 2). The primary source of sodium is dietary, but since the two meal times of these participants did not coincide with the principal 12- or 1.6-h cycles, their diet is not likely to be responsible for these rhythms.
In spite of these reassurances, this first report of CSF sodium chronobiology could reflect many other variables, as yet untested. Both a longer duration study, with more individuals, and with other species would be informative. Animal model systems, such as sheep or non-human primates, would be conducive to a more expansive study.
Although the fluctuations in K+ were minimal, the positive instead of negative correlation of [K+] with [Na+] was surprising, since NKAT activity at the blood-brain barrier should lead to an inverse relationship. We suggest that the positive correlation of Na+ with K+ could be a reflection of differences between their methods of regulation. We have proposed that capillary endothelial cell NKAT activity is the primary source for the overall higher [Na+] and lower [K+] in brain interstitial fluid compared to plasma. However, brain tissue is more intolerant of changes in [K+] than [Na+] and will stabilize [K+] levels rapidly; for example, increased neuronal firing from elevated [Na+]e may increase [K+]e, and active glial mechanisms effect local [K+] homeostasis to minimize these changes. To explain our current data, we propose that the cation levels derived from the brain interstitial fluid/capillary NKAT environment are modified by the time they reach the lumbar CSF. Thus the [Na+] changes are still evident while the brain tissue has stabilized the [K+]. This is reflected in our more dispersed [Na+] than [K+] data. Clearly, more experiments are required to investigate this dynamic cation biochemistry.
Sodium chronobiology has been studied in blood and urine from young, healthy volunteers and was shown to decrease from midnight to early morning [20–22]. Elderly healthy volunteers also had a similar circadian rhythm for blood sodium, though the amplitude of change was reduced compared to younger persons . More frequent sampling in rats demonstrated increasing blood plasma sodium during sleep, with a decrease prior to waking . These results suggest that brain/CSF sodium rhythms are differentially regulated from the systemic circulation. Furthermore, none of these blood plasma sodium references have 10 minute sampling similar to our study, precluding identification of shorter rhythms in these studies, such as the 1.65 h (100 min) -period that we identified in CSF.
The individual PSD analyses reveal individual variability (Additional File 1). We expect different people will have different rhythms and that these will fluctuate in individuals as sleep and other behaviors vary. We have confined our study to look for physiological rhythms that are reasonably common, because of the small number of participants. Hence our emphasis on the averaged PSDs that reveal overall common rhythms. A nocturnal rhythm was not surprising, since many analytes are known to change between sleep and wake states. The afternoon rise was perhaps less expected, since this has not been reported from previous studies of other CSF analytes. The shorter 1.65-h period was least expected, but tissue rhythms of this time (100 min) are well known, and correspond both with the timing of known rhythms of biological events such as cognition  and sleep periods ) and, at a molecular level, with NKAT activity .
The regulation responsible for these CSF sodium rhythms is of interest. Sensory circumventricular organs are involved in brain homeostasis . Subfornical organ Nax channels sense sodium, and osmolarity is assessed by transient receptor potential vanilloid type 1 channels in neuronal cells and volume-regulated anion channels in glial cells, both in the supraoptic and paraventricular nuclei . Sodium Potassium ATPase (NKAT) is the principal regulator of [Na+], consuming nearly one half of the energy of the brain , pumping 3 Na+ out of and 2 K+ into cells. NKAT rhythms have been reported in the mammalian brain , and NKAT is clearly involved in [Na+]csf regulation [31–33]. Our data does not reveal inverse changes of the Na+/K+ that would be consistent with NKAT rhythms being the effector of our reported sodium rhythms and have provided an explanation for this discrepancy above. In addition, we cannot assess rhythms < 20 min in duration, many other regulatory factors in brain tissue, or the effects of CSF circulation to the lumbar site of collection.
The change of CSF [Na+] that we report (10 mM from the data in Figure 5, and over 40 mM in Figure 1) will reflect changes in brain sodium. Radioactive Na+ distribution studies reveal that although [Na+]csf is modified at different points along the neuraxis [15, 34], it is reasonable to assume it reflects [Na+]e, since equilibration of [Na+]e with lumbar CSF occurs rapidly [13–15]. We expect the changes in [Na+]e may be greater in specific brain regions, since the samples obtained here will have been diluted with normal CSF before reaching the lumbar site of collection.
The magnitude of change of CSF [Na+] (mean shift of 10 mM) is sufficient to have physiological effect on neuronal excitability. Moreover, this data raises the possibility that efforts to reduce the natural sodium variation or to administer drugs to coincide with sodium chronobiology may reduce migraine in susceptible people. Hodgkin and Katz  demonstrated that the action potential rises at a rate roughly proportional to the rise of [Na+]e. When a neuron is at rest, the Na+ influx through voltage-gated Na+ channels is low, as these channels are usually closed or inactivated. However, the channel gate is displaced when [Na+]e increases . Higher [Na+]e speeded recovery from the inactivation state, enabling an earlier action potential and leading to hyperexcitability . Higher [Na+]csf caused a sympathetic hyperactivity response (increasing blood pressure and heart rate) through increasing ouabain-like substances and activating the brain renin-angiotensin- aldosterone system [37, 38].