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Department of Physiology, Chang Gung University School of Medicine, Kwei-San, Tao-Yuan, Taiwan
Submitted 20 January 2004; accepted in final form 27 May 2004
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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). Along this line of thinking, regional differences in Na+/K+-ATPase activity have been described in the heart (Wang et al. 1993) and in the auditory thalamus (Senatorov and Hu 1997) as well as its developmental regulation in skeletal muscle (see Clausen 1986). Alterations in Na+/K+-ATPase activity have also been implicated in nervous disorders such as epilepsy (Brines et al. 1995; McNamara 1994) and Alzheimer's disease (Kairane et al. 2002) and in mood control and ethanol addiction (see Bargrov et al. 2002).
The suprachiasmatic nucleus (SCN) neurons, with the majority being clock neurons, change their firing rates in a circadian manner (Green and Gillette 1982; Inouye and Kawamura 1979) and coordinate the peripheral oscillators in controlling physiological and behavioral rhythms in mammals (see Klein et al. 1991; Reppert and Weaver 2002). In addition to having higher firing activity, which would likely lead to greater dissipation of Na+ and K+ gradients, the SCN neurons also have higher intracellular calcium levels during the day (Colwell 2000; Ikeda et al. 2003). As such, the pumping activity of the Na+/K+-ATPase might also be altered accordingly to meet the daily fluctuations in the ionic environments. Here we examine this notion by studying the SCN neurons taken from the retinorecipient (ventral "core") region of the nucleus. The ventral SCN (vSCN) neurons in such a reduced preparation have been demonstrated to maintain diurnal rhythms in the spontaneous firing rate and in the calcium channel activity (Yang et al. 2003). We show that the vSCN neurons diurnally regulate the Na+/K+-ATPase, apparently by altering maximum activity of this enzyme and, perhaps, its ability to block by the cardiac glycoside strophanthidin.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of Chang Gung University School of Medicine. Sprague-Dawley rats (17–26 days old) were kept in a temperature-controlled room under a 12:12 light:dark cycle (light on at 0700–1900 h). An animal was killed by decapitation, and the brain was put in an ice-cold artificial cerebrospinal fluid (ACSF) prebubbled with 95% O2-5% CO2. The ACSF contained (in mM) 125 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 26 NaCO3, 1.2 NaH2PO4, and 10 glucose. A coronal slice (200–300 µm) containing the SCN and the optic chiasm was cut with a Vibroslice (Campden Instruments) and was then incubated at room temperature (22–26°C) in the incubation solution, which contained (in mM) 150 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, bubbled with 100% O2. The reduced vSCN preparation was obtained by using a fine needle (No. 26002-10, Fine Science Tools) to excise a small piece of tissue from the ventral region of the SCN. The excised reduced preparation, with dimensions of 
100 x 100 x tens of µm, was then transferred to a recording chamber for recording.
Electrical recordings and data analysis
The spontaneous firing was recorded at room temperature in the cell-attached mode. The perfusion bath solution contained (in mM) 150 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The patch electrode solution was the same as the bath solution. Ni2+ and higher concentrations of K+ were directly added to the bath to achieve the final concentrations. The spike counts always began only after stable recordings were made and were taken over a period of 
5 min, counted in 6-s epochs. At least 1 or 2 min of spontaneous firing rate was counted in control before the application of drugs. For recording the nighttime firing rate, the animal was killed 2 h before lights off (ZT 12). For recording the daytime firing rate, the animal was killed near ZT 4 (4 h into lights on at ZT 0). Membrane currents were recorded with the patch-clamp technique in the whole cell mode at room temperature as described previously (Huang 1995). The bath solution contained (in mM) 150 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The patch electrode contained (in mM) 20 NaCl, 1 CaCl2, 2 MgCl2, 110 K-gluconate, 11 EGTA, 10 HEPES, 3 Na-ATP, and 0.3 Na-GTP, pH 7.4. The measured liquid junction potential was –17 mV (Neher 1992) and was not corrected for in the measurements of voltages. Pipette resistance was typically 4–6 M
. The signal was low-pass filtered at 1–5 kHz (8-pole Bessel) and digitized on-line at 1–10 kHz via a 12-bit A/D digitizing board (Data Translation DT2821F-DI) with a custom-made program written in C Language. Data were analyzed and plotted also with custom-made programs written in Visual Basic 6.0.
Statistical analysis was performed with the commercial software GraphPad PRISM (GraphPad Software, San Diego, CA). The Kolmogorov-Smirnov test was used to judge the normality of distribution for the spontaneous firing rates and the strophanthidin-induced membrane currents. For the spontaneous firing rates, a total of 79 daytime and 80 nighttime cells were used to perform and were judged to pass the normality test, with the KS distance values of 0.050 (P > 0.10) and 0.14 (P > 0.10), respectively. Data were thus analyzed with repeated-measures ANOVAs, followed by Bonferroni's test for selected pairs comparison (Figs. 1–3). For the strophanthidin-induced membrane currents, a total of 23 daytime and 18 nighttime cells were used to perform and were judged to pass the normality test, with the KS distance values of 0.10 (P > 0.10) and 0.15 (P > 0.10), respectively. Data were thus analyzed using the Student's t-test (Figs. 4 and 5).
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Strophanthidin (Sigma, St. Louis, MO) was dissolved in 100% ethanol as stocked solutions (9 or 10 mM) and stored at –20°C and was then added to the bath to achieve the final concentrations during experiments. Solutions containing ethanol at concentrations 
0.33%, which was the dissolving solution for 30 µM strophanthidin, had little effect on holding currents. However, the solution containing 1% ethanol, which was the dissolving solution for 100 µM strophanthidin, induced an outward current averaging 2.09 ± 0.30 (SE) pA (n = 12), and thus the solution containing 100 µM strophanthidin was always preceded by applying 1% ethanol solution as control from which the strophanthidin-induced current was then subtracted from. Ouabain was used only in the initial experiments due to its poor reversibility (results not shown).
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RESULTS |
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Figure 1 shows the effects of 9 µM strophanthidin, an aglycone cardioactive agent formerly used as digitalis, on spontaneous firing of vSCN neurons. Two representative cells are shown to indicate the higher spontaneous firing rate during the day (Fig. 1A1) and lower at night (Fig. 1B1). For the two neurons, strophanthidin increased the daytime firing rate from 3 to 3.5 Hz (Fig. 1A2) and increased the nighttime firing rate from 0.8 to 2.2 Hz (Fig. 1B2). A summary of these experiments is shown in Fig. 1, C and D. As shown in the figure, the drug effect developed gradually and reached a steady state in 40–80 s after strophanthidin application (Fig. 1C). On average, 9 µM strophanthidin increased the daytime (ZT 6–10) firing rates from 2.86 ± 0.24 to 3.88 ± 0.03 Hz (P < 0.001, ANOVA, followed by Bonferroni's test for selected pairs comparison) and increased the nighttime (ZT 12–17) firing rates from 0.99 ± 0.19 to 2.47 ± 0.05 Hz (P < 0.001, ANOVA, followed by Bonferroni's test for selected pairs comparison; Fig. 1D). The results indicate that strophanthidin invariably increases the spontaneous firing rates of the vSCN neurons day or night. However, the extent to which the firing rate was increased by strophanthidin appeared to be different between day and night. This can be seen by taking the ratio of firing rates in the presence of strophanthidin to those in control, yielding a daytime value of 1.36 (3.88/2.86 Hz) and a nighttime value of 2.50 (2.47/0.99 Hz), respectively. Alternatively, one can also compare the day/night ratio of firing rates in control (2.86/0.99 Hz = 2.89) and in the presence of strophanthidin (3.88/2.47 Hz = 1.57). The results suggest that blocking the Na+/K+-ATPase with strophanthidin increases firing rates of vSCN neurons differently, depending on the time of day.
Strophanthidin in the presence of Ni2+ induces the vSCN neurons to fire action potentials in a diurnal rhythm
However, the nonlinear dependence of firing rates on the membrane potential and channel activity precludes us from making inference as to whether there is a day-night difference in the Na+/K+-ATPase activity. We have thus applied 1 mM Ni2+ to block spontaneous firing of these neurons, and then examined the effect of strophanthidin on excitability. Figure 2 compares the results thus obtained from two representative cells, one during the day (Fig. 2A) and the other at night (Fig. 2B). As indicated, addition of 1 mM Ni2+ blocked spontaneous firing in cells recorded in daytime (Fig. 2A, 1 and 2) and nighttime (Fig. 2B, 1 and 2), most likely due to its blockade of low-voltage-activated calcium currents and perhaps high-voltage-activated calcium currents as well (Yang et al. 2003). Further addition of strophanthidin, however, evoked the silenced cells to fire action potentials in a diurnal rhythm with higher firing rate during the day (Fig. 2A3) and lower at night (Fig. 2B3). Figure 2, C and D, summarizes the results of these experiments. As shown in Fig. 2C, 1 mM Ni2+ rapidly inhibited the spontaneous firing, and further addition of 9 µM strophanthidin again gradually stimulated these cells to fire action potentials in a diurnal manner (P < 0.001, ANOVA, followed by Bonferroni's test for selected pairs comparison). On average, the daytime (ZT 6–10) firing rates in control, in 1 mM Ni2+, and in the combined presence of 1 mM Ni2+ and 9 µM strophanthidin were 2.96 ± 0.28 Hz (n = 6), 0.01 ± 0.01 Hz (n = 6), and 2.91 ± 0.04 Hz (n = 6), respectively, and the nighttime (ZT 12–17) firing rates were 0.91 ± 0.27 Hz (n = 6), 0.01 ± 0.01 Hz (n = 6), and 1.04 ± 0.03 Hz (n = 6), respectively (Fig. 2D).
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Because 1 mM Ni2+ blocked most of the low- and high-voltage-activated calcium currents in the vSCN neurons (Yang et al. 2003), the results of strophanthidin-induced diurnal firing rhythm in 1 mM Ni2+ might be explained in terms of combined actions of membrane potential and channel activity other than the calcium currents. To test this idea, we raised the external concentrations of K+, in the presence of 1 mM Ni2+, to stimulate the silenced cells to fire action potentials and then determined if the vSCN neurons remain rhythmic under this condition. Figure 3 summarizes the results obtained from these experiments. As indicated, in the presence of 1 mM Ni2+ to block spontaneous firing, increasing external concentrations of K+ induced the vSCN neurons to fire action potentials in a dose-dependent manner day and night. However, the firing evoked by each concentration of K+ did not show day-night difference (P > 0.05, ANOVA, followed by Bonferroni's test for selected pairs comparison). The lack of diurnal firing rhythm induced by higher concentrations of external K+ is in sharp contrast to the presence of diurnal firing rhythm induced by strophanthidin. Taken together, the results suggest that the strophanthidin-induced firing rhythm is most likely due to a day-night difference in the Na+/K+-ATPase with higher activity during the day than at night.
vSCN neurons exhibit a diurnal rhythm in pump currents
Indeed under voltage-clamp conditions, pump currents blocked by strophanthidin varied in a diurnal manner (Fig. 4). For the experiment, the cell was held at –40 mV, and a 500-ms pulse from –40 to –60 mV was then applied every 7 s to measure the input resistance. On average, the daytime holding current was –1.13 ± 0.32 pA (n = 34), more negative than the nighttime average of 0.03 ± 0.42 pA (n = 25; P < 0.05, t-test); the daytime input resistance was 2.19 ± 0.15 G (n = 34), larger than the nighttime average of 1.49 ± 0.12 G (n = 25; P < 0.005, t-test). Strophanthidin blocked the electrogenic pump, thereby increasing inward holding currents (Fig. 4, A, 1 and 2, and B, 1 and 2) but did not appear to affect the input resistances. The lack of effect of strophanthidin on the input resistances of vSCN neurons is consistent with those observed in the midbrain dopamine neurons (Shen and Johnson 1998) and in the heart cells (Gadsby and Nakao 1989). The strophanthidin effect was fully reversible (Fig. 4, A3 and B3). Figure 4C summarizes the results demonstrating the daily variation of pump currents. The daytime (ZT 6–10) and nighttime (ZT 12–17) pump currents averaged –4.62 ± 0.40 pA (n = 14) and –1.51 ± 0.17 pA (n = 12; P < 0.001, t-test), respectively.
Ni2+ has no effect on pump currents
Figure 5 shows the effects of 1 mM Ni2+ on the membrane currents and pump currents. Application of 1 mM Ni2+ induced an inward current at day (Fig. 5A, 1 and 2) and at night (Fig. 5B, 1 and 2), averaging –1.55 ± 0.39 pA (n = 6) and –1.67 ± 0.88 pA (n = 6), respectively. In addition, the day- and nighttime input resistance was slightly increased from 2.03 ± 0.28 G (n = 6) to 2.32 ± 0.24 G (n = 6) and from 1.39 ± 0.19 G (n = 6) to 1.66 ± 0.27 G (n = 6), respectively. Further addition of 9 µM strophanthidin again induced inward currents (Fig. 5, A3 and B3) in a reversible manner (Fig. 5, A4 and B4). Figure 5C summarizes the results. The day- and nighttime pump currents in the presence of 1 mM Ni2+ averaged –4.42 ± 0.25 pA (n = 6) and –1.15 ± 0.33 pA (n = 6; P < 0.001, t-test), respectively, similar to those obtained in the absence of Ni2+ (Fig. 4).
Dose-dependent effects of strophanthidin on holding currents
The dose-dependent effects of strophanthidin on holding currents are shown in Fig. 6. Compared with the holding currents in control (Fig. 6, A1 and B1), strophanthidin at concentrations of 1 µM (Fig. 6, A2 and B2), 3 µM (Fig. 6, A3 and B3), 10 µM (Fig. 6, A4 and B4), and 30 µM (Fig. 6, A5 and B5) dose-dependently increased inward holding currents. The effect of 100 µM strophanthidin was determined after the application of its dissolving solution that contained 1% ethanol (see METHODS), and the data points were then included in Fig. 6D. The curves were calculated with the theoretic equation assuming one-to-one binding of strophanthidin to and blockade of Na+/K+-ATPase. The daytime (ZT 6–10) values for maximum pump current and half-block concentration of strophanthidin were –6 pA and 4 µM, respectively, and the nighttime (ZT 12–17) values were –3.5 pA and 8 µM, respectively. The results suggest that the diurnal variations in the strophanthidin effects involve changes in the maximum activity of Na+/K+-ATPase and perhaps its ability to be blocked by strophanthidin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Diurnal modulation of the Na+/K+-ATPase in vSCN neurons
Two principal observations lead us to conclude that the vSCN neurons exhibit a diurnal rhythm in the Na+/K+-ATPase. First, in the presence of 1 mM Ni2+ to block most of the calcium currents so as to silence the vSCN neurons, raising external concentrations of K+ evokes the cells to fire action potentials, but the firing rates at each concentration of K+ did not differ during the day and at night. In sharp contrast, blocking the Na+/K+-ATPase with 9 µM strophanthidin induced the silenced cells to fire action potentials in a diurnal manner with the daytime firing rate being approximately three times as fast as the nighttime firing rate (2.91 ± 0.04 vs. 1.04 ± 0.03 Hz). Second, the voltage-clamp experiments then demonstrate that the daytime pump current blocked by 9 µM strophanthidin is also approximately three times as lager as the nighttime (–4.62 ± 0.40 vs. –1.51 ± 0.17 pA) and is little affected by the presence of 1 mM Ni2+ (–4.42 ± 0.25 vs. –1.15 ± 0.33 pA).
The day-night difference in pump currents may involve changes in the maximum Na+/K+-ATPase activity and the blocking ability of strophanthidin. For the maximum Na+/K+-ATPase activity, it amounts to 6 pA of pump current during the day and drops to 3.5 pA at night. Although the mechanism is not known, it could be due to a daily rhythm in the number of functional pumps. Alternatively, it might involve changes in the substrate affinity for the enzyme and/or interactions with membrane-associated components (Therien and Blostein 2000). Incidentally, daily rhythms in glucose uptake (Schwartz and Gainer 1977; Newman et al. 1992) and in ATP contents (Yamazaki et al. 1994) have also been reported. In addition to a decrease in the maximum Na+/K+-ATPase activity, the blocking ability of strophanthidin also appears to decrease at night as indicated by the increase in the half-block concentration of strophanthidin from 4 µM in daytime to 8 µM at night. It is known that the cardioactive steroids bind specifically to the extracellular site of the Na+/K+-ATPase, and factors that can influence its apparent binding affinity include the 
-subunit and extracellular K+ (see Kaplan 2002; Scheiner-Bobis 2002). It is also possible that vSCN neurons might contain multiple forms of 
subunit with different binding affinity for ouabain and with different regulatory mechanisms as demonstrated in the cardiac cells (Gao et al. 1999; Juhaszova and Blaustein 1997).
The low half-block concentration of strophanthidin (4 or 8 µM in the presence of [Na+]i = 20 mM and [K+]o = 3.5 mM) suggests that the catalytic () subunit of the sodium pump in vSCN neurons is of the high (2 or 3) ouabain affinity isoforms (Berreri-Bertrand et al. 1990; Urayama et al. 1989). A similar, low value of strophanthidin EC50 (8.8 µM in the presence of [Na+]i = 20 mM and [K+]o = 2.5 mM) has also been reported in the midbrain dopamine neurons, which also express relatively high levels of 3-isoform (Shen and Johnson 1998). The sodium pump of these neurons generates current at a high density of 20 µA cm–2, consistent with its postulated role in regulating the firing pattern of dopamine neurons (Johnson et al. 1992). For comparison, taking the value of 10 µm (van den Pol 1980; Welsh et al. 1995) or 12 µm (Huang 1993) as the averaged diameter for the vSCN neurons and the daytime value of 6 pA (or 3.5 pA at night) as the maximum pump current, the calculated current density would be 1.3–1.9 µA cm–2 during the day (or 0.8–1.1 µA cm–2 at night). This density is 10 times lower than that in the dopamine neurons but is comparable to a value of 1.1 µA cm–2 in cardiac myocytes (De Weer et al. 1988; Glitsch et al. 1989), whereas the current density in the auditory thalamic neurons is most likely somewhere in between [estimated from their published values for pump currents in Senatorov et al. (1997) and for cell sizes in Senatorov and Hu (1997)].
Functional implications
The vSCN neurons are the retinorecipient clock neurons that receive light information from the retina via the retinohypothalamic tract. These neurons generate a circadian rhythm in spontaneous firing that appears to depend on the low-threshold calcium currents (Yang et al. 2003) in contrast to a similar role for the L-type calcium current in the dorsal "shell" SCN neurons (Pennartz et al. 2002) and for ryanodine receptor-mediated Ca2+ release from internal stores in organotypic cultures of SCN (Ikeda et al. 2003). No matter what the mechanisms mediating the firing rhythms are, these clock neurons have to face daily fluctuations in the ionic environments on two accounts. First, the intracellular calcium concentration is higher during the day as a result of calcium release from ryanodine-sensitive internal stores (Ikeda et al. 2003) and/or calcium influx via the activation of calcium channels (Colwell 2000). Second, the higher daytime firing activity will also cause a greater dissipation of ion gradients during the day.
Our findings of diurnal modulation of the maximum Na+/K+-ATPase activity fit well with the functional roles of this enzyme. On one hand, the higher daytime pumping activity of this enzyme will help to restore the Na+ and K+ gradients that are dissipated by the higher daytime firing activity of these neurons. On the other hand, indirectly via the Na+-Ca2+ exchanger, the enzyme will also help to maintain homeostasis of intracellular Ca2+, the daytime concentration of which is also higher (Colwell 2000; Ikeda et al. 2003). In addition, the Na+/K+-ATPase might also participate in regulating the resting membrane potentials and hence firing rates of vSCN neurons. For example, during the day, the maximum Na+/K+-ATPase activity generates 6 pA of outward pump current, which, in theory, would amount to 12 mV of membrane hyperpolarization when multiplied by the averaged daytime input resistance of 2.19 ± 0.15 G. At night, a maximum pump current of 3.5 pA and an averaged input resistance of 1.49 ± 0.12 G would yield 5 mV of membrane hyperpolarization. Further work will certainly be needed to better address this issue. The function is less clear, however, for the daily changes in the ability of strophanthidin to block the Na+/K+-ATPase. Nevertheless, accumulating evidence indicates that endogenous ouabain is present in the hypothalamus (Kawamura et al. 1999) and the adrenals (Schneider et al. 1998) and could be stimulated to release under physiological and pathological conditions (see Schoner 2002).
In conclusion, our results demonstrate that the vSCN neurons exhibit a diurnal rhythm in the Na+/K+-ATPase, the pumping activity of which is higher during the day when the firing activity and intracellular calcium levels are also higher in these neurons. As such, the vSCN clock neurons could alter their pumping activity to meet the daily change in demands for restoring ion gradients and maintaining calcium homeostasis.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R.-C. Huang, Dept. of Physiology, Chang Gung University School of Medicine, 259 Wen-Hwa 1st Rd., Kwei-San, Tao-Yuan, Taiwan (E-mail: rongchi{at}mail.cgu.edu.tw).
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REFERENCES |
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) and night (
). Each data point represents the means ± SE of 7 (
) or 15 (
) neurons
