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
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ABSTRACT |
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A link between the
circadian rhythm and the function of Cl-permeable
-aminobutyric acid (GABA) type A (GABAA) receptors on suprachiasmatic nucleus (SCN) neurons was studied by measuring intracellular activity of Cl
(aCl
-aminobutyric acid;
-aminobutyric acid type A receptor; gramicidin-perforated patch clamp; rat
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INTRODUCTION |
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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
-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
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
To test this hypothesis in a quantitative manner, we measured
aCl current through GABAA
receptors without disrupting aCl
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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 M. 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 M
. All experiments were carried out at
room temperature (23-25°C).
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
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.
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.
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RESULTS |
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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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IGABA recorded with nystatin-perforated patch method.
When the nystatin-perforated patch-clamp technique was used,
application of 3 × 105 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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IGABA recorded with gramicidin-perforated patch
method.
Gramicidin forms Cl-impermeable cation channels that
allow ionic current measurement without disrupting
aCl
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
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(1) |
Relationship between EGABA, aCl
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Lack of relationship between aCl
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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
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(2) |
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(3) |
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(4) |
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(5) |
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(6) |
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Lack of correlation of chord conductance with measured
aCl
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DISCUSSION |
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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
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
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
aCl47 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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aCl across neuronal plasma membrane. Several transporters
control these processes. Major transporters that increase
aCl
cotransporter and
the Cl
-HCO
cotransporter, Cl
-ATPase,
and the Na+-dependent
Cl
-HCO
cotransporter and the K+-Cl
cotransporter
merit further consideration. Developmental change in
aCl
-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
The present study does not preclude the possibility that
aCl 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
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FOOTNOTES |
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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.
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