Circadian rhythm in intracellular Clminus activity of acutely dissociated neurons of suprachiasmatic nucleus

Masahiko Shimura1, Norio Akaike2, and Nobutoshi Harata3

1 Department of Ophthalmology, Tohoku University School of Medicine, Sendai 980-8574, Japan; 2 Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and 3 Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5345


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A link between the circadian rhythm and the function of Cl--permeable gamma -aminobutyric acid (GABA) type A (GABAA) receptors on suprachiasmatic nucleus (SCN) neurons was studied by measuring intracellular activity of Cl- (aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>) at different times during a circadian cycle in SCN neurons acutely dissociated from rat brains. To measure aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, the voltage-clamp mode of the gramicidin-perforated patch-clamp technique was used, and reversal potential of GABA-induced currents (EGABA) was converted to aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. Measured aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was significantly higher at around noon (20.1 ± 1.4 mM) than at three other time zones of a circadian cycle (means ranging from 11.6 to 14.3 mM). Chord conductance of GABA-induced currents showed no circadian changes, indicating a lack of circadian changes in the number or single-channel conductance of GABAA receptors. These results suggest that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> participates in modulating GABAA receptor functions on SCN neurons during the circadian rhythm.

gamma -aminobutyric acid; gamma -aminobutyric acid type A receptor; gramicidin-perforated patch clamp; rat


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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RECENT DEVELOPMENT OF MOLECULAR biological techniques has identified numerous genes involved in controlling circadian rhythms. An emerging view involves an intracellular transcriptional/translational feedback loop. Two clock proteins CLOCK and BMAL1 bind to DNA to enhance transcription of period (per) and timeless (tim) genes, increasing the amount of PER and TIM proteins. After a lag, PER and TIM feed back to negate the activation of CLOCK and BMAL1, reducing the amount of per and tim mRNA. Eventually the level of PER and TIM is reduced, and the loop is completed (6, 9). Some of the clock-controlled genes were also shown to be regulated by this feedback loop (15). Clock genes and proteins are expressed in suprachiasmatic nucleus (SCN), and they control the firing rate of individual SCN neurons (13), suggesting that the circadian mechanism is operational at the individual cellular level. However, outputs of this loop and, therefore, the functional correlates of changes in clock genes and proteins remain unknown.

Physiologically, evidence has been building in support of gamma -aminobutyric acid (GABA) as a neurotransmitter that controls circadian rhythms in mammals by its action on Cl--permeable GABA type A (GABAA) receptors. Administration of GABA or its modulators effectively changes the phases of circadian rhythms. For example, application of muscimol, a specific GABAA receptor agonist, in SCN in vivo induced phase shifts in locomotor activity of rodents, and the effect was blocked by a competitive GABAA receptor antagonist, bicuculline, or a Cl- channel blocker, picrotoxin (36). The circadian rhythm of locomotor activity was modified by intraperitoneal injection of diazepam, an allosteric GABAA receptor modulator (29). SCN neurons in vitro showed circadian rhythm in firing rate (10, 11, 24, 33). Muscimol inhibited the spontaneous firing of SCN neurons in slices (21), leading to a phase-resetting effect (39). Furthermore, picrotoxin produced phase advances in the firing rate of SCN neurons in slices (30). We also showed (34) the presence of GABAA receptors on individual SCN neurons in rats by acutely isolating the neurons from surrounding tissue and monitoring the effects of GABA and related modulators by a patch-clamp technique.

One possible link between the circadian rhythm and the function of GABAA receptor on SCN neurons is changes in intracellular Cl- activity (aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>). The GABAA receptor forms a Cl- channel (14). Therefore, the amplitude of the inhibitory postsynaptic current or the inhibitory postsynaptic potential mediated by GABAA receptors is directly modified by changes in Cl- equilibrium potential (ECl), which is determined by the ratio of Cl- activities inside and outside of neurons (aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> and aCl<UP><SUB>o</SUB><SUP>−</SUP></UP>). Recently, aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> has been shown to be a variable parameter, not a fixed constant, during neuronal development (17, 27, 38). There is also an indication that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> differs between day and night in SCN (43). Circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> of SCN neurons would modify GABAA receptor activity by changing ECl and could contribute to generating and/or modifying circadian rhythm in vivo.

To test this hypothesis in a quantitative manner, we measured aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> in SCN neurons acutely dissociated from the rat brain at different times during the circadian cycle. To measure aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, we used the voltage-clamp mode of the gramicidin-perforated patch-clamp technique, which allows recording of Cl- current through GABAA receptors without disrupting aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> (7).


    MATERIALS AND METHODS
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Preparations. The isolation technique used here was similar to those previously described (34). Briefly, 11- and 12-day-old Wistar rats of either sex were maintained under a 12:12-h light-dark cycle (lights on 0700-1900). They were decapitated, and the brains were quickly removed from the skulls and sliced coronally at 400-µm thickness. The slices were preincubated in an incubation solution well saturated with 95% O2-5% CO2 for 30-60 min at room temperature (23-25°C). Thereafter, the slices were treated in an incubation solution containing 1 mg pronase/6 ml at 37°C for 25 min. The SCN, ~400 µm in diameter, was punched out by a needle (150 µm in diameter) and transferred into a 35-mm culture dish (Primaria 3801-Falcon, Becton Dickinson, Franklin Lakes, NJ) filled with a standard external solution. The SCN neurons were mechanically isolated with fire-polished Pasteur pipettes with tip diameters of 100-450 µm.

Electrophysiological study. The electrophysiological measurements were performed using either nystatin- or gramicidin-perforated patch recording under voltage-clamp conditions (1, 7). The patch pipettes were pulled from glass capillaries (outer diameter 1.5 mm). The resistance of the patch pipette was 3-7 MOmega . A liquid junction potential of 4 mV was used to calibrate the holding potential. Ionic currents were measured with a patch-clamp amplifier (Axopatch-1C, Axon Instruments, Foster City, CA). After formation of a gigaohm seal, GABA (3 × 10-5 M) was repetitively applied at 5-min intervals until two consecutive stable currents were obtained. The number of applications was minimized by delaying the first application by ~10 min. Once a stable response was obtained, ramp voltage commands were given before and during application of GABA, to examine the current-voltage (I-V) relationship. After this measurement, GABA was applied again to confirm the stability of the response amplitude. Series resistance was <30 MOmega . All experiments were carried out at room temperature (23-25°C).

For temporal analysis of circadian changes, the following three time parameters were used: 1) time of death of the animals, 2) "recording time" (when the I-V relationship was examined in a neuron), and 3) "lapse time" (time between death and recording). Midnight was defined as 0000. Time of death and recording time were grouped into four time zones of equal duration (6 h).

Fluorescent Nissl staining. Localization of SCN was confirmed in our slices by fluorescent Nissl staining. Rat pups were anesthetized with ether and transcardially perfused with cold standard external solution. Brain slices through SCN were obtained with 400-µm thickness and immediately fixed in 4% paraformaldehyde in Dulbecco's modified phosphate-buffered saline (PBS; D-5652, Sigma, St. Louis, MO) at 4°C for 1 h. The slices were rinsed in PBS containing 100 mM glycine for 3 h, rinsed in PBS containing 0.1% sodium borohydride for 10 min, rinsed in PBS for 1 h, permeabilized by 0.1% Triton X-100 in PBS for 20 min, and rinsed in PBS for 20 min. They were then treated with 5% Neurotrace red fluorescent Nissl stain (Molecular Probes, Eugene, OR) in PBS for 40 min. They were treated with 0.1% Triton X-100 in PBS for 10 min, rinsed in PBS for 6 h, and mounted in Citifluor (Ted Pella, Redding, CA). Except where otherwise stated, every treatment was done at room temperature. Fluorescence was detected with a Texas red filter set (excitation 560/55, dichroic 595, emission 645/75 nm).

Solutions. The ionic composition of the incubation solution was (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 24 NaHCO3, and 10 glucose. pH was adjusted to 7.4 with 95% O2-5% CO2 gas. The composition of the standard external solution was (in mM) 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. pH was adjusted to 7.4 by adding Tris-base. aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> corresponding to a Cl- concentration of 161 mM was reported to be 114.5 mM in the same solution used in this study (18). During recording, 10-7 M tetrodotoxin and 10-4 M CdCl2 were added to the standard external solution to block voltage-dependent Na+ and Ca2+ channels.

The composition of the patch pipette (internal) solution was (in mM) 150 CsCl and 10 HEPES. pH was adjusted to 7.2 with Tris-base. Nystatin and gramicidin were first dissolved in methanol at a concentration of 10 mg/ml and then further diluted in the internal solution to give a final concentration of 200 µg/ml. A Cs+-based internal solution was necessary for suppression of K+ channels and accurate I-V measurements of GABA-induced currents (IGABA). Although known to suppress K+-Cl- cotransport (18), internal Cs+ did not bring aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> near or above the passively distributed value (23.4 mM; see Fig. 3B), indicating either that blockade of the cotransport was not complete or, more likely, that different mechanisms were operative in SCN neurons.

Drugs. The following drugs were used: paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) and bicuculline, GABA, glycine, gramicidin D, nystatin, pronase, sodium borohydride, tetrodotoxin, and Triton X-100 (Sigma, St. Louis, MO). GABA and bicuculline were directly dissolved in the standard external solution and were applied to neurons by the "Y tube" technique (26, 34).

Statistics. Numerical values are given as means ± SE. Excel (Microsoft, Redmond, WA) was used to assess statistical significance with the t-test.


    RESULTS
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ABSTRACT
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MATERIALS AND METHODS
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SCN neurons in slices and after acute dissociation. For acute dissociation of SCN neurons in electrophysiological studies, we punched out the paramedian region that lies above the optic chiasm. Localization of SCN was confirmed in our preparation with fluorescent Nissl staining. Although SCN was not clearly identifiable under phase-contrast optics (data not shown), it was visualized by fluorescence microscope as a paired ovoid structure lying dorsal to the optic chiasm (Fig. 1A). SCN demonstrated the highest density of neurons in the hypothalamus, as reported previously in studies with conventional Nissl staining (25, 41). Within SCN, neuronal density was highest in the dorsomedial region (Fig. 1A), as reported previously (41).


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Fig. 1.   Suprachiasmatic nucleus (SCN) in slice (A) and acutely dissociated SCN neurons (Ba-d). A: a coronal brain slice through SCN was chemically fixed by 4% paraformaldehyde and stained with the fluorescent Nissl method. Arrowheads, bilateral SCN that lie along 3rd ventricle and above optic chiasm. Ba-d: representative phase-contrast images of acutely dissociated SCN neurons. The neurons were dissociated from a nonfixed SCN slice. Scale bar in Bd applies to all 4 panels in B.

Dissociated SCN neurons are shown in four images in Fig. 1B. They were monopolar (Fig. 1Ba), bipolar (Fig. 1Bb and Bc), or tripolar (Fig. 1Bd). These morphologies were similar to those of monopolar, bipolar, and radial multipolar neurons found in SCN in situ (41), indicating that the acute dissociation was performed properly.

IGABA recorded with nystatin-perforated patch method. When the nystatin-perforated patch-clamp technique was used, application of 3 × 10-5 M GABA to acutely dissociated SCN neurons induced an inward current at a holding potential (VH) of -40 mV (Fig. 2A, left). The current was completely blocked by pretreating the cell with 10-4 M bicuculline (Fig. 2A, right), a competitive antagonist of GABAA receptor. This indicates that the IGABA was mediated by GABAA receptor. To measure the reversal potentials, a ramp voltage command was applied before and during the application of 3 × 10-5 M GABA (Fig. 2A, left). The ramp command was linearly changed from a VH of -40 to +20 mV, then to -100 mV, and back to -40 mV during a 1-s period (Fig. 2B, inset). The reversal potential of IGABA (EGABA) was measured as the voltage at which I-V curves before and during IGABA intersect (Fig. 2, B and D). EGABA measured with the nystatin method was -3.8 ± 0.5 mV (n = 20). This was close to the theoretical ECl of -4.1 mV calculated with the Nernst equation. These results confirm that GABAA receptor mainly allows permeation of Cl- and give credence to the assumption that EGABA = ECl in acutely dissociated SCN neurons under the current experimental conditions.


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Fig. 2.   gamma -Aminobutyric acid (GABA)-induced currents recorded by nystatin (A and B)- and gramicidin (C and D)-perforated patch methods. A: application of 3 × 10-5 M GABA induced an inward current (IGABA) at a holding potential (VH) of -40 mV. IGABA was completely blocked by treating the cell with 10-4 M bicuculline. B: current-voltage (I-V) relationship of IGABA recorded with the nystatin-perforated patch method. Ramp voltage commands were applied before (control, C) and during (IGABA) application of GABA. Protocol of ramp command is shown in inset. Arrow, reversal potential of IGABA (EGABA). MP, membrane potential. C: IGABA recorded by gramicidin-perforated patch method was outward at the same VH. IGABA was completely blocked by 10-4 M bicuculline. D: I-V relationship for control (C) and IGABA recorded with the gramicidin-perforated patch method.

IGABA recorded with gramicidin-perforated patch method. Gramicidin forms Cl--impermeable cation channels that allow ionic current measurement without disrupting aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> (7). When the gramicidin-perforated patch method was used, the application of 3 × 10-5 M GABA induced an outward current at the same VH (Fig. 2C, left). The current was blocked by 10-4 M bicuculline (Fig. 2C, right), as with the nystatin-perforated patch method (Fig. 2A). These results indicate that IGABA was still mediated by GABAA receptor and that EGABA had shifted to a potential more hyperpolarized than VH. This result was confirmed by analyzing the I-V relationship (Fig. 2D). With the same ramp voltage command as in Fig. 2B, EGABA with the gramicidin method was measured as -54.3 ± 1.1 mV (n = 44).

Conversion of EGABA to aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. With the measured EGABA (gramicidin method) and the given aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> of 114.5 mM in our solution, it is possible to calculate aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, using a Nernst equation
aCl<SUP>−</SUP><SUB>i</SUB><IT>=a</IT>Cl<SUP>−</SUP><SUB>o</SUB><IT>×e</IT><SUP>(<IT>zF×</IT>E<SUB>GABA</SUB>/<IT>RT</IT>)</SUP><IT>=</IT>114.5<IT>×</IT>10<SUP>(<IT>E</IT><SUB>GABA</SUB><IT>/</IT>58)</SUP> (1)
where R is the gas constant, T is temperature, Z is valence, and F is the Faraday constant. This calculation assumes that the GABAA receptor is exclusively permeable to Cl- in our nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free external solution. The measured EGABA ranged between -37 and -64 mV, and the calculated aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> ranged between 9.0 and 26.4 mM (n = 44).

Relationship between EGABA, aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, and time of death. To examine a circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, the measured EGABA and calculated aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> were plotted against time of death (Fig. 3). With division of 24 h into four time zones (Fig. 3B), the 1200 zone showed aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> of 20.1 ± 1.4 mM (n = 13). The values were significantly higher than in the three other zones. aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was 11.9 ± 0.8 mM (n = 12) for the 0000 zone (P < 0.0001), 11.6 ± 0.5 mM (n = 9) for the 0600 zone (P < 0.0001), and 14.3 ± 1.3 mM (n = 10) for the 1800 zone (P < 0.01). These results clearly indicate that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> showed a circadian change.


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Fig. 3.   Relationship between EGABA, intracellular Cl- activity (aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>), and time of death. A: the measured EGABA was plotted against the time at which the animal was killed; n = 44 neurons. B: aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was calculated from the data set in A using the Nernst equation (see text) and was plotted against time of death. Time of death was divided into four 6-h time zones (shown by different shadings). *aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> in 1200 zone was significantly higher than in other time zones. Broken horizontal line (23.4 mM) indicates the level of aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> assuming a passive equilibrium at the given VH.

Lack of relationship between aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, recording time, and lapse time. In addition to time of death, we analyzed the effects of different recording times on aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. The plot of aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> against the recording time of individual neurons did not show a significant difference among the four time zones (P > 0.1; Fig. 4A).


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Fig. 4.   A: relationship between aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> and recording time (time when EGABA was measured). There were no significant differences in mean aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> of different time zones. B: relationship between aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> and lapse time. The lapse time was defined as the elapsed time from time of death to recording time. There was no systematic change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. A straight line represents a linear regression line with r = 0.071 (P > 0.1, n = 44). Mean lapse time in the present experiment was 4 h 58 min.

Another temporal parameter is lapse time. If dissociated neurons showed time-dependent deterioration, it is possible that neurons with longer lapse time could not sustain a constant level of aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> lower than passively acquired aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. In this case, aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> would become higher as lapse time prolonged. However, aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> did not show positive correlation with lapse time (r = 0.071, P > 0.1, n = 44) (Fig. 4B). This finding excludes the possibility that the observed change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was induced by artifactual deterioration of neurons.

Chord conductance of GABAA receptor-Cl- channel complex. We used another set of analyses to address whether there was a circadian change in the number or single-channel conductance of GABAA receptors. The effect of open GABAA receptors is determined by a simple equation
I=N×g×(E<SUB>GABA</SUB><IT>−E</IT><SUB>rest</SUB>) (2)
where I is the amplitude of IGABA from the whole neuronal surface, N is the number of channels on the neuron, g is the single-channel conductance, and Erest is the resting membrane potential. The first two factors on the right-hand side of Eq. 2 can be lumped into a single parameter, chord conductance (G)
G=N×g (3)
changing Eq. 2 to
I=G×(E<SUB>GABA</SUB><IT>−E</IT><SUB>rest</SUB>) (4)
Under the voltage-clamp condition used in this study
E<SUB>rest</SUB><IT>=</IT>V<SUB>H</SUB> (5)
Therefore, Eq. 4 is rewritten as
I=G×(E<SUB>GABA</SUB><IT>−</IT>V<SUB>H</SUB>) (6)
We measured the transient peak amplitude of IGABA as I and used Eq. 6 to calculate G for each recorded neuron. Figure 5 shows the plots of G against time of death (Fig. 5A), recording time (Fig. 5B), and lapse time (Fig. 5C). The three plots showed no time zone-dependent changes (P > 0.1, n = 44). This result demonstrates that there were no time-dependent changes in the lumped parameter representing a number and/or a single-channel conductance of GABAA receptors.


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Fig. 5.   Lack of correlation of chord conductance (G) with time of death (A), recording time (B), or lapse time (C). Linear regression line was drawn with r = 0.116 (P > 0.1, n = 44).

Lack of correlation of chord conductance with measured aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. One general drawback of the patch-clamp technique is that high series resistance results in more depolarized reversal potentials (closer to 0 mV). This is especially true for perforated patch methods if ionophores (gramicidin channels) are not functioning well. This artifact can be excluded by examining G because G takes a smaller value when ionophores are closed. However, the plot of G against aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> showed a lack of correlation (r = 0.175, P > 0.1, n = 44; Fig. 6). These results ensure that the circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was accurately measured.


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Fig. 6.   Lack of correlation of chord conductance with measured aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>; r = 0.175 (P > 0.1, n = 44).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown the circadian change in EGABA in acutely dissociated SCN neurons of rats. In our previous work (34), we showed that GABAA receptors on SCN neurons are mainly permeable to Cl- under the present experimental conditions, demonstrating that EGABA is equivalent to ECl. Because aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> is assumed to be constant as a result of rapid perfusion of extracellular solution around dissociated neurons (19), a circadian change in EGABA (or ECl) indicates a circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>. In contrast, conductance of GABAA receptors in each neuron remained constant. These findings indicate that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> alone is responsible for circadian change in GABAA receptor functions in the present preparation.

GABA is not a static inhibitory transmitter. Response to GABA changes under various conditions. GABA is depolarizing in immature neurons, whereas it changes to hyperpolarizing in more mature neurons (3, 5). To support this view, several groups measured the neuronal aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> using gramicidin patch. It was assessed in rat hypoglossal motoneurons with reversal potentials of glycine currents, with a reduction from 33 to 8 mM during the first weeks of postnatal development (35). This corresponded to a shift from depolarizing to hyperpolarizing effects. In rat cerebral cortical neurons, it was 37 mM at embryonic day 16 and was gradually reduced to 12 mM at postnatal day 16 (28). In acutely dissociated Meynert neurons, aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> showed a similar decrease: 33.4, 21.1, and 11.3 mM in 0- to 1-day-old, 2-wk-old, and 6-mo-old rats, respectively (31). In addition to these developmental changes in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> that span days to weeks, a change within a shorter time was reported. Normal aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> in gerbil inferior colliculus neurons was 8 mM, but it was increased to 22 mM after 1 day of afferent denervation (40). Even faster change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was suggested, although not quantitatively, in cultured hippocampal neurons, in which activity-dependent synaptic plasticity induced a change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> over a time scale of minutes (8). Our quantitative analysis showed that average aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> in SCN neurons ranged between 11.6 and 20.1 mM in four time zones and that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> at noon (20.1 mM) was 1.4- to 1.7-fold higher than in the other time zones (Fig. 3). Thus a circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> was within the reported ranges of concentration and time.

The effect of GABA on the activity of SCN neurons has been documented by several groups. They showed inhibitory effects of GABA on spontaneous firing rate during day and night (20, 22, 23). One possible reason why these reports did not observe circadian changes between day and night (enhancement during day and suppression during night) might be the use of nonphysiological pipette solutions for extracellular recording. These solutions were either 0.5 M NaCl (22, 23) or 0.5 M Na acetate (20). Leakage of the pipette solutions into extracellular space would change aCl<UP><SUB>o</SUB><SUP>−</SUP></UP>, shifting EGABA to a different level. Depending on the variable degrees of leakage and variable distances of pipettes to recorded cells, there could have been cell-to-cell differences in aCl<UP><SUB>o</SUB><SUP>−</SUP></UP>, obscuring circadian changes in EGABA. Furthermore, the effects of GABA on spontaneous firing rate are influenced by multiple factors, including the driving force determined by EGABA and Erest, the properties of Na+ and K+ channels, and shunting inhibition, to name a few, rendering the interpretation of the results more difficult. Another factor is the age of the animals (not given in Refs. 20, 22, and 23). In one study, suppressing effects of GABA on the firing rate were reported in rats over 3 wk of age, using 1) multiple-unit extracellular recording with a platinum-iridium wire electrode, 2) cell-attached recording, and 3) gramicidin-perforated patch recording (12). In light of the developmental change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>, assessment of circadian changes in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> would not be straightforward if assessment was done using a crude factor, firing rate, and especially if the reported Erest was depolarized (-47 mV) (Ref. 12; see below).

To reconcile discrepancies among previous reports, we present here a hypothesis on circadian changes in EGABA (ECl) in SCN neurons (Fig. 7). EGABA shows a developmental, hyperpolarizing change described by a single exponential function (3). Circadian change in EGABA overlies this downward trend. Assuming Erest to be from -55 to -60 mV (4, 42) and to be constant throughout development, the effect of GABAergic synaptic transmission could be uniformly depolarizing during early development, mixed (depolarizing-hyperpolarizing) during middle stages of development, and uniformly hyperpolarizing late during development. The subtle differences and/or degrees of depolarization or hyperpolarization induced by GABA might have been difficult to assess by firing rate alone, because the effect of GABA on membrane potential is a continuous function of ECl and Erest, whereas the firing of Na+ action potentials is a step function with a certain threshold. Differential GABA effects are clearly demonstrated only with voltage-clamp experiments as in this study.


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Fig. 7.   Hypothesis on circadian changes in EGABA. Developmental change in EGABA (ECl) has been shown to be exponential (3). Erest, resting membrane potential of SCN neurons.

aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> is balanced by influx and efflux of Cl- across neuronal plasma membrane. Several transporters control these processes. Major transporters that increase aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> under physiological conditions are the Na+-K+-Cl- cotransporter and the Cl--HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. Transporters that decrease aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> are the K+-Cl- cotransporter, Cl--ATPase, and the Na+-dependent Cl--HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (16). Of these transporters, the Na+-K+-Cl- cotransporter and the K+-Cl- cotransporter merit further consideration. Developmental change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> of hippocampal pyramidal neurons was mediated by increased expression levels of the Cl--extruding K+-Cl- cotransporter KCC2. Blockade of the transporter by antisense RNA led to a dramatic reduction in the hyperpolarizing GABA responses in mature neurons, which normally exhibited hyperpolarization in response to GABA (32). A similar change in lateral superior olive neurons was made possible by an interplay of the K+-Cl- cotransporter and the Na+-K+-Cl- cotransporter (17). On the basis of these reports, we propose that the activity of one (or some) of the transporter proteins that controls aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> is actively involved in circadian rhythm in SCN neurons.

The present study does not preclude the possibility that aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> also shows circadian changes in situ. The microenvironment of a narrow synaptic cleft has been shown to drastically change on synaptic stimulation. For example, extracellular Ca2+ concentration in the synaptic cleft was reduced by at least one-third when pre- or postsynaptic neurons were activated and there was a large Ca2+ influx into neurons (2, 37). This opens a possibility that aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> in the synaptic cleft might also show a circadian change, although small in effect, if neuronal firing rate shows a circadian change and Cl- influx/efflux is altered due to variable postsynaptic GABAA receptor activation by released GABA.

We found that circadian rhythm was not evident in recording time or lapse time, whereas it was evident in time of death. These results indicate that aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> in neurons was frozen after death of the animals and dissociation of neurons. The lack of changes in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> during lapse time further suggests that there was little deterioration of the preparation, because deterioration would lead to lessening of the difference between aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> and aCl<UP><SUB>o</SUB><SUP>−</SUP></UP> and therefore a higher aCl<UP><SUB>i</SUB><SUP>−</SUP></UP> and more depolarized EGABA. The freezing effect could be brought about by the lack of synaptic inputs in our preparation. The acute dissociation procedure removed most of the synapses that had formed close to maturation level on postnatal days 11-12 (25). Although the circadian change in firing rate of cultured SCN neurons was shown to be independent of the presence of synaptic inputs (44), further investigation is needed to elucidate the effect of synaptic inputs on the circadian change in aCl<UP><SUB>i</SUB><SUP>−</SUP></UP>.


    FOOTNOTES

Address for reprint requests and other correspondence: N. Harata, Dept. of Molecular and Cellular Physiology, Beckman Center B103, Stanford Univ. School of Medicine, Stanford, CA 94305-5345 (E-mail: harata{at}stanford.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00187.2000

Received 17 April 2000; accepted in final form 26 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akaike, N, and Harata N. Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Jpn J Physiol 44: 433-473, 1994[ISI][Medline].

2.   Borst, JGG, and Sakmann B. Depletion of calcium in the synaptic cleft of a calyx-type synapse in the rat brainstem. J Physiol (Lond) 521: 123-133, 1999[Abstract/Free Full Text].

3.   Chen, G, Trombley PQ, and van den Pol AN. Excitatory actions of GABA in developing rat hypothalamic neurones. J Physiol (Lond) 494: 451-464, 1996[Abstract].

4.   Chen, G, and van den Pol AN. Multiple NPY receptors coexist in pre- and postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons. J Neurosci 16: 7711-7724, 1996[Abstract/Free Full Text].

5.   Cherubini, E, Gaiarsa JL, and Ben-Ari Y. GABA: an excitatory transmitter in early postnatal life. Trends Neurosci 14: 515-519, 1991[ISI][Medline].

6.   Darlington, TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS, and Kay SA. Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280: 1599-1603, 1998[Abstract/Free Full Text].

7.   Ebihara, S, Shirato K, Harata N, and Akaike N. Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. J Physiol (Lond) 484: 77-86, 1995[Abstract].

8.   Ganguly, K, Bi GQ, Schinder AF, Berninger B, and Poo MM. Activity-dependent associative modifcation of GABAergic synapses. Soc Neurosci Abstr 25 (291): 6, 1999.

9.   Gekakis, N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, and Weitz CJ. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280: 1564-1569, 1998[Abstract/Free Full Text].

10.   Gillette, MU. The suprachiasmatic nuclei: circadian phase-shifts induced at the time of hypothalamic slice preparation are preserved in vitro. Brain Res 379: 176-181, 1986[ISI][Medline].

11.   Gillette, MU, and Prosser RA. Circadian rhythm of the rat suprachiasmatic brain slice is rapidly reset by daytime application of cAMP analogs. Brain Res 474: 348-352, 1988[ISI][Medline].

12.   Gribkoff, VK, Pieschl RL, Wisialowski TA, Park WK, Strecker GJ, de Jeu MT, Pennartz CM, and Dudek FE. A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus. J Biol Rhythms 14: 126-130, 1999[Abstract/Free Full Text].

13.   Herzog, ED, Takahashi JS, and Block GD. Clock controls circadian period in isolated suprachiasmatic nucleus neurons. Nature Neurosci 1: 708-713, 1998[ISI][Medline].

14.   Hevers, W, and Luddens H. The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes. Mol Neurobiol 18: 35-86, 1998[ISI][Medline].

15.   Jin, X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, and Reppert SM. A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96: 57-68, 1999[ISI][Medline].

16.   Kaila, K. Ionic basis of GABAA receptor channel function in the nervous system. Prog Neurobiol 42: 489-537, 1994[ISI][Medline].

17.   Kakazu, Y, Akaike N, Komiyama S, and Nabekura J. Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. J Neurosci 19: 2843-2851, 1999[Abstract/Free Full Text].

18.   Kakazu, Y, Uchida S, Nakagawa T, Akaike N, and Nabekura J. Reversibility and cation selectivity of the K+-Cl- cotransport in rat central neurons. J Neurophysiol 84: 281-288, 2000[Abstract/Free Full Text].

19.   Kira, T, Harata N, Sakata T, and Akaike N. Kinetics of sevoflurane action on GABA- and glycine-induced currents in acutely dissociated rat hippocampal neurons. Neuroscience 85: 383-394, 1998[ISI][Medline].

20.   Liou, SY, and Albers HE. Single unit response of neurons within the hamster suprachiasmatic nucleus to GABA and low chloride perfusate during the day and night. Brain Res Bull 25: 93-98, 1990[ISI][Medline].

21.   Liou, SY, Shibata S, Albers HE, and Ueki S. Effects of GABA and anxiolytics on the single unit discharge of suprachiasmatic neurons in rat hypothalamic slices. Brain Res Bull 25: 103-107, 1990[ISI][Medline].

22.   Mason, R. Circadian variation in sensitivity of suprachiasmatic and lateral geniculate neurones to 5-hydroxytryptamine in the rat. J Physiol (Lond) 377: 1-13, 1986[Abstract].

23.   Mason, R, Biello SM, and Harrington ME. The effects of GABA and benzodiazepines on neurones in the suprachiasmatic nucleus (SCN) of Syrian hamsters. Brain Res 552: 53-57, 1991[ISI][Medline].

24.   Medanic, M, and Gillette MU. Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day. J Physiol (Lond) 450: 629-642, 1992[Abstract].

25.   Moore, RY, and Bernstein ME. Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin I immunoreactivity. J Neurosci 9: 2151-2162, 1989[Abstract].

26.   Murase, K, Randic M, Shirasaki T, Nakagawa T, and Akaike N. Serotonin suppresses N-methyl-D-aspartate responses in acutely isolated spinal dorsal horn neurons of the rat. Brain Res 525: 84-91, 1990[ISI][Medline].

27.   Obrietan, K, and Van den pol AN. GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing. J Neurosci 15: 5065-5077, 1995[Abstract].

28.   Owens, DF, Boyce LH, Davis MBE, and Kriegstein AR. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16: 6414-6423, 1996[Abstract/Free Full Text].

29.   Ralph, MR, and Menaker M. Effects of diazepam on circadian phase advances and delays. Brain Res 372: 405-408, 1986[ISI][Medline].

30.   Rangarajan, R, Heller HC, and Miller JD. Chloride channel block phase advances the single-unit activity rhythm in the SCN. Brain Res Bull 34: 69-72, 1994[ISI][Medline].

31.   Rhee, JS, Jin YH, and Akaike N. Developmental changes of GABAA receptor-chloride channels in rat Meynert neurons. Brain Res 779: 9-16, 1998[ISI][Medline].

32.   Rivera, C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, and Kaila K. The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251-255, 1999[ISI][Medline].

33.   Shibata, S, Oomura Y, Kita H, and Hattori K. Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice. Brain Res 247: 154-158, 1982[ISI][Medline].

34.   Shimura, M, Harata N, Tamai M, and Akaike N. Allosteric modulation of GABAA receptors in acutely dissociated neurons of the suprachiasmatic nucleus. Am J Physiol Cell Physiol 270: C1726-C1734, 1996[Abstract/Free Full Text].

35.   Singer, JH, Talley EM, Bayliss DA, and Berger AJ. Development of glycinergic synaptic transmission to rat brain stem motoneurons. J Neurophysiol 80: 2608-2620, 1998[Abstract/Free Full Text].

36.   Smith, RD, Turek FW, and Slater NT. Bicuculline and picrotoxin block phase advances induced by GABA agonists in the circadian rhythm of locomotor activity in the golden hamster by a phaclofen-insensitive mechanism. Brain Res 530: 275-282, 1990[ISI][Medline].

37.   Stanley, EF. Presynaptic calcium channels and the depletion of synaptic cleft calcium ions. J Neurophysiol 83: 477-482, 2000[Abstract/Free Full Text].

38.   Sun, D, and Murali SG. Na+-K+-2Cl- cotransporter in immature cortical neurons: a role in intracellular Cl- regulation. J Neurophysiol 81: 1939-1948, 1999[Abstract/Free Full Text].

39.   Tominaga, K, Shibata S, Hamada T, and Watanabe S. GABAA receptor agonist muscimol can reset the phase of neural activity rhythm in the rat suprachiasmatic nucleus in vitro. Neurosci Lett 166: 81-84, 1994[ISI][Medline].

40.   Vale, C, and Sanes DH. Afferent regulation of inhibitory synaptic transmission in the developing auditory midbrain. J Neurosci 20: 1912-1921, 2000[Abstract/Free Full Text].

41.   Van den Pol, AN. The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy. J Comp Neurol 191: 661-702, 1980[ISI][Medline].

42.   Van den Pol, AN, Obrietan K, and Chen G. Excitatory actions of GABA after neuronal trauma. J Neurosci 16: 4283-4292, 1996[Abstract/Free Full Text].

43.   Wagner, S, Castel M, Gainer H, and Yarom Y. GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity. Nature 387: 598-603, 1997[ISI][Medline].

44.   Welsh, DK, Logothetis DE, Meister M, and Reppert SM. Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14: 697-706, 1995[ISI][Medline].


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