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The Journal of Neuroscience, October 15, 2000, 20(20):7531-7538
Abnormal GABAA Receptor-Mediated Currents in Dorsal
Root Ganglion Neurons Isolated from Na-K-2Cl Cotransporter Null
Mice
Ki-Wug
Sung1, 3,
Michael
Kirby2,
Michael P.
McDonald2,
David M.
Lovinger1, 2, 3, and
Eric
Delpire1, 3
Departments of 1 Anesthesiology,
2 Pharmacology, and 3 Molecular Physiology and
Biophysics, Center for Molecular Neuroscience and Kennedy Center for
Research on Human Development, Vanderbilt University Medical Center,
Nashville, Tennessee 37232
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ABSTRACT |
We have recently disrupted Slc12a2, the gene
encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1)
(Delpire et al., 1999 ). Gramicidin perforated-patch and whole-cell
recordings were performed to study GABA-induced currents in dorsal root
ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1
knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward
current was observed at the resting membrane potential, suggesting
active accumulation of Cl in these cells. This
GABA-induced current was blocked by picrotoxin, a GABAA
receptor blocker. The strong Cl accumulation that
gives rise to depolarizing GABA responses is caused by Na-K-2Cl
cotransport because reduction of external Cl or
application of bumetanide induced a decrease in
[Cl]i, whereas an increase in
external K+ caused an apparent
[Cl]i accumulation. In contrast to
control neurons, little or no net current was observed at the resting
membrane potential in homozygote NKCC1 mutant DRG neurons.
EGABA was significantly more negative,
demonstrating the absence of Cl accumulation in
these cells. Application of bumetanide induced a positive shift of
EGABA, suggesting the presence of an
outward Cl transport mechanism. In agreement with
an absence of GABA depolarization in DRG neurons, behavioral analysis
revealed significant alterations in locomotion and pain perception in
the knock-out mouse. Our results clearly demonstrate that the
Na-K-2Cl cotransporter is responsible for
[Cl]i accumulation in DRG neurons
and that via regulation of intracellular Cl, the
Na-K-2Cl cotransporter participates in the modulation of GABA
neurotransmission and sensory perception.
Key words:
Na-K-2Cl cotransporter; knock-out mouse; dorsal root
ganglion; bumetanide; chloride; GABA; nociception
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INTRODUCTION |
GABA, via
hyperpolarization of postsynaptic membranes, acts as an
inhibitory neurotransmitter in the adult CNS. However, in several adult sensory neurons, GABA elicits depolarizing
responses. GABAergic terminals, mostly from interneurons, are
presynaptic to central terminals of muscle and nociceptive afferent
fibers ending in the dorsal columns of the spinal cord. The release of GABA by interneurons depolarizes the terminals of these primary afferent fibers leading to presynaptic inhibition. Presynaptic inhibition is thought to reduce or eliminate sensory "noise"
originating from second-order neurons, enhancing sensory contrast and
allowing only the passage of more potent sensory inputs (Willis, 1999). Thus, GABA via primary afferent depolarization and presynaptic inhibition participates in the modulation of excitability of the sensory terminals of both large (muscle) and fine (nociceptive, touch,
and temperature) fibers.
Microelectrode and gramicidin perforated-patch studies in amphibian
dorsal root ganglion (Alvarez-Leefmans et al., 1988) and spinal cord
(Rohrbough and Spitzer, 1996) demonstrated a high intracellular
Cl concentration in primary sensory
neurons. High neuronal Cl concentration
is consistent with GABA depolarization and presynaptic inhibition (for
review, see Alvarez-Leefmans et al., 1998). Accumulation of
Cl in these cells was shown to be
Na+ and K+
dependent and sensitive to the loop diuretic bumetanide, three hallmarks of Na-K-2Cl cotransport.
Electrically silent coupled movement of
Na+, K+, and
Cl across biological membranes occurs
via two isoforms of the Na-K-2Cl cotransporter: NKCC1
(Slc12a2 gene) and NKCC2 (Slc12a1 gene). NKCC2 is
exclusively expressed in the kidney, whereas NKCC1 exhibits a wide
pattern of expression that ranges from
Cl-secreting epithelia to red blood
cells, myocytes, and neurons (for review, see Haas and Forbush,
1998; Mount et al., 1998).
Using a polyclonal antibody directed against a C-terminal region of the
NKCC1 protein (Kaplan et al., 1996), we demonstrated high levels of
cotransporter expression in rat dorsal root ganglion neurons (Plotkin
et al., 1997a), suggesting an important role for the
cotransporter in accumulating
[Cl]i in
mammalian sensory neurons.
In this study, we take advantage of a mouse knock-out model of the
Na-K-2Cl cotransporter that we developed recently (Delpire et al.,
1999) to examine the role of the cotransporter in regulating [Cl]i in dorsal
root ganglion neurons. We show that absence of the Na-K-2Cl
cotransporter dramatically affects the reversal potential of
GABA-mediated responses via a significant reduction in basal Cl concentration. The strong
depolarizing responses elicited by the GABAA
receptor in control mice turned into mild depolarizing or even
hyperpolarizing responses in the NKCC1 knock-out mice. In agreement
with these abnormal GABA responses in sensory neurons, we show that
NKCC1 knock-out mice display both abnormal gait and locomotion as well
as impaired nociception phenotypes. These experiments clearly
demonstrate that the Na-K-2Cl cotransporter accumulates Cl in mouse dorsal root ganglion
(DRG) neurons and that no other Cl transport pathways are present in
these cells to achieve this function. Cl
accumulation by the Na-K-2Cl cotransporter is a key component of GABA
depolarization and modulation of sensory input to the spinal cord.
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MATERIALS AND METHODS |
Na-K-2Cl cotransporter knock-out mice.
Slc12a2/
(NKCC1/) mice were obtained by
deletion of exon 9 of the Na-K-2Cl cotransporter gene and replacement
with a  -galactosidase/neomycin fusion cassette (Delpire et al.,
1999). The disrupted gene was integrated by recombination in L129
embryonic stem cells and propagated into C57b6 mice. Homozygote animals
survive and exhibit multiple phenotypes. They are smaller in size, are
hypotensive, present impaired epithelial
Cl secretion, and present inner ear
defects resulting in significant impairment of locomotion as well as
deafness (Delpire et al., 1999; Flagella et al., 1999). Animals used in
this study were 6-12 weeks old.
Immunofluorescence. Dorsal root ganglia, isolated from
control and homozygote mice, were fixed overnight at 4°C with 4%
paraformaldehyde, washed with PBS, and cryoprotected with 30% sucrose.
For indirect immunofluorescence, 7-µm-thick frozen sections were thaw
mounted on Superfrost Plus slides. The sections were treated with 1%
SDS and 8% 2-mercaptoethanol for 5 min and then washed in PBS and 1%
BSA at room temperature for 30 min, followed by incubation with
affinity-purified antibody (Kaplan et al., 1996; Plotkin et al., 1997a,
b) at a dilution of 1:200 in PBS and 1% BSA overnight at 4°C.
After washes in PBS, slides were incubated with
indocarbocyanine-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch, West Grove, PA) diluted 1:800 in PBS and 1% BSA for 1 hr at room temperature, washed with PBS, and mounted with
Vectashield (Vector Laboratories, Burlingame, CA). Sections were
analyzed with a Nikon Eclipse E800 microscope equipped with an
Optronics DEI-750 color CCD camera (Optronics Engineering, Goleta, CA)
coupled to an IBM-compatible computer. Photographs were obtained with a
color Tektronix Phaser 450 printer (Tektronics, Wilsonwill, OR).
DRG neuron isolation. Wild-type and homozygote mice were
killed by cervical dislocation. DRGs were dissected from the lower thoracic to midlumbar regions of the vertebral column and placed in
oxygenated DMEM (Life Technologies, Gaithersburg, MD). The connective tissue sheath around the ganglia was removed, and DRGs were
minced two to three times with iridectomy scissors. The minced DRGs
were then placed in a flask containing 5 ml of DMEM containing 1 mg/ml
collagenase D (Boehringer Mannheim, Indianapolis, IN), 0.6 mg/ml
deoxyribonuclease I (DNase; Sigma, Saint Louis, MO), and 0.6 mg/ml
trypsin (Sigma). DRGs were incubated at 37°C in a shaking water bath
for 30-40 min. At the end of the incubation period, 1 mg/ml soybean
trypsin inhibitor (Sigma) was added, and individual neurons were
isolated from the ganglia by vigorous shaking. Neurons were kept at
room temperature (20-22°C) in the DMEM solution and were aliquoted
as needed to 35 mm culture dishes.
Recording. Electrophysiological responses were recorded
using the gramicidin perforated-patch whole-cell recording technique with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) and
pClamp 6 software (Axon Instruments). DRG neurons were allowed to
settle in a 35 mm culture dish placed onto an inverted microscope
(Zeiss). After attachment to the dish (30 min), an external solution
was perfused at a rate of 1.5-2.0 ml/min. The solution contained 150 mM NaCl, 5 mM KCl, 0.5 mM
CaCl2, 1 mM
MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4. Osmolarity was adjusted to 340 mmol/kg
with sucrose. Gramicidin perforated whole-cell voltage-clamp recordings
were obtained from DRG neurons with capacitances ranging from 15 to 45 pF. Patch pipettes with resistances of 2-4 M were made from
borosilicate glass capillaries. The pipette tip was initially filled
with gramicidin-free pipette solution by brief immersion, and the
remainder of the pipette was back-filled with a 310 mOsm internal
solution containing gramicidin plus 140 mM CsCl (or 140 mM KCl, when indicated), 5 mM EGTA, and 10 mM HEPES, pH 7.4. A 50 mg/ml stock solution of gramicidin
(Calbiochem, La Jolla, CA) was prepared in dimethylsulfoxide (DMSO;
Sigma). Gramicidin was diluted into the pipette solution to a final
concentration of 100 µg/ml before use.
The junction potential between the patch pipette and the bath solutions
was nulled before the gigaohm seal was formed. After the formation of a
tight seal, the progress of gramicidin perforation was evaluated by
monitoring the capacitative current transient produced by a 10 msec
hyperpolarizing voltage step (5 mV) from the holding potential ( 50
mV) every 30 sec. With 100 µg/ml gramicidin in the pipette, the
access resistance dropped to <20 M within 20 min after seal
formation. GABA and other drugs were applied by gravity-driven
perfusion as described previously (Choi and Lovinger, 1996). Drugs were
dissolved in external solution and delivered from a linear array of
microcapillary tubes (0.32 mm inner diameter). The tips of the drug
application pipettes were placed within 100 µm of the neurons. For
pretreatment with bumetanide or high potassium, solutions were
delivered continuously over the cells for 10 min before application of
GABA. GABA (100 µM to 1 mM) was applied with
the membrane potential held at a series of set values.
The peak amplitude of GABA-activated current was calculated by taking
the difference between the current amplitude during the predrug
application baseline period and the amplitude at the maximal negativity
during drug application. Amplitude measurements were performed by the
use of the cursor-based system in the pClamp software. Peak current
responses for each voltage were plotted three times, and the data were
fit by the use of linear regression analysis. The reversal potential of
GABA-activated current (EGABA) was
extracted from the x-intercept value of the fit. All
averaged values are expressed as the mean ± SE. Statistical
significance was determined by the use of Student's t test
with the criterion set at p < 0.05. The intracellular
Cl concentration was estimated by
solving for
[Cl]i in the
Nernst equation: ECl = RT/F
ln
[Cl]i/[Cl]o,
using the measured EGABA to estimate
ECl, the usual value of
RT/F, and the
[Cl]o
concentration set in the experiment.
Behavioral testing. Subjects were 51 mice (22 NKCC1+/+, 15 NKCC1+/, and 14 NKCC1/). Mice were group-housed, three
to five per cage, and separated by gender. Food and water were
available ad libitum. The colony room was maintained on a
12:12 hr light/dark cycle, with lights on at 6 A.M. All procedures were
approved by the Vanderbilt University Medical Center Institutional
Animal Care and Use Committee.
For the general neurological screen, a subset of tests from the Irwin
Gross Neurological Screen (Irwin, 1968) was performed. First, physical
characteristics such as coat color and consistency, skin color,
appearance of fur, presence of whiskers, and wounds are examined. Then,
the mice are weighed, and their body temperature is measured by the use
of a rectal probe. The mice are then placed in an empty cage for 3 min,
and the responses to the novel environment, such as wild running,
excessive grooming or freezing, abnormal gait or postures,
piloerection, palpebral closure, tail elevation, urination, and
defecation, are observed and noted. For touch sensitivity, mice are
stroked with a finger from above, starting light and getting firmer,
and the intensity at which mice try to escape is recorded. Then,
whisker, righting, reaching, and corneal reflexes are examined.
Abdominal and limb tone, as well as pupillary contraction and responses
to tail pinch and toe pinch, is also recorded. The Irwin test battery
consists of assessments of body temperature and spontaneous behavior in
a novel environment, as well as gross measures of vision, hearing, and
nociception. Reflexes such as righting, touch escape, trunk curl,
reaching, vibrissal, and corneal were also assessed.
The ability to maintain balance on a rotating cylinder was measured
with a standard rotorod apparatus (model 7650; Ugo Basile, Varese,
Italy). The cylinder was 3.2 cm in diameter and was covered with scored
plastic. Mice were confined to a section of the cylinder 6.0 cm long by
gray Plexiglas dividers. Each mouse was placed on the cylinder, which
increased rotation speed over a 5 min period from 4 to 40 rpm. The
latency at which the mouse fell off the rotating cylinder was measured.
Mice that fell in <15 sec were given a second trial. Mice that did not
fall during the 300 sec trial period were removed and given a score of
300 sec. Mice were tested three times daily for 3 d.
For nociception threshold measurement, each mouse was placed on a hot
plate maintained at 52°C (model 35D; IITC Corporation, Woodland
Hills, CA). A plastic cylinder 15 cm in diameter and 12.5 cm high
confined the mouse to the surface of the hot plate. The time it took
the mouse to jump or lick its paw was measured with a stop watch. Mice
were then immediately removed from the hot plate. Because NKCC1
knock-out mice are spastic and may not be able to lick their paws as
deftly as wild-type mice, changes in other behaviors thought to be
indicative of a nociceptive response were also recorded. Shaking or
lifting paws, increasing motor activity, and jumping were all
considered responses to the thermal stimulus for all three genotypes.
Analyses of the independence of observations of the hot plate behaviors
were performed using  2 and Cramer's
 , a transformation of 2 that results
in a coefficient ranging from 0 to 1 representing the strength of
association (Welkowitz et al., 1982). Hot plate latencies were analyzed
by the use of ANOVA. Planned follow-up comparisons were
conducted with Fisher's protected least significant difference (PLSD)
statistic after a significant omnibus F ratio. Analysis of
rotorod data was conducted by the use of repeated-measures ANOVA
(RMANOVA). Follow-up analyses of rotorod data were made with two-group
RMANOVA. Degrees of freedom for RMANOVAs were adjusted for sphericity
by the use of the Greenhouse-Geisser  .
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RESULTS |
Gramicidin perforated-patch (PP) recordings were successfully
performed in a total of 48 DRG neurons isolated from 15 wild-type mice
and 48 neurons isolated from 9 homozygote NKCC1 knock-out mice. The
whole-cell capacitance of these neurons ranged from 13.2 to 57 pF, with
several neurons from both wild-type and
NKCC1/ mice having capacitances in the
15-20, 20-30, and 30-50 pF ranges, indicating that the cells we
examined spanned the entire range of sizes of primary sensory neurons.
There was no significant difference in the whole-cell capacitances when
comparing the wild-type and NKCC1/
mice [average capacitance = 29.5 ± 1.1 pF for wild-type
mouse neurons (n = 39) and 26.9 ± 1.5 pF for
NKCC1/ mice (n = 39);
p = 0.15, unpaired t test].
Application of 1 mM GABA to the neurons during recordings
from wild-type mice elicited inward current when the membrane potential was voltage-clamped at 50 mV in the large majority of cells (Fig. 1A). Inward current was
also observed after GABA application at 30 mV, but current reversed
to an outward direction when the membrane potential was clamped at 10
mV in all DRG neurons tested (Fig. 1A). When 10 µM bumetanide was applied before and during GABA application, outward current was elicited after GABA exposure at
30 mV (Fig. 1B). Similar reversal potentials and
effects of bumetanide were observed in 12 neurons from eight wild-type
mice.
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Figure 1.
Bumetanide sensitivity of GABAA
receptor-mediated current in mouse DRG neurons. A,
Current activated by GABA application to an isolated DRG neuron from a
wild-type mouse during gramicidin PP recording. B,
Recording from the same neuron shown in A after a 10 min
application of 10 µM bumetanide. C,
Recording from the same neuron beginning 5 min after initiation of
whole-cell recording after completion of the PP recording.
D, Current-voltage relationship for GABA-activated
responses under the three recording conditions shown in
A-C. Note the hyperpolarizing shift in
EGABA in the presence of bumetanide and the
shift of EGABA to near 0 mV during
whole-cell recording. E, Current activated by GABA in a
second neuron in the absence (top) and presence
(bottom) of picrotoxin during gramicidin PP recording.
The strong inhibition of the response by picrotoxin indicates that the
current is GABAA receptor-mediated.
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Subsequent to completion of recordings in the PP mode, we applied
additional negative pressure to the recording pipette to rupture the
cell membrane for whole-cell recording in the same DRG neurons. The
intracellular solution contained a high CsCl concentration (140 mM). We allowed at least 5 min after rupture of the
membrane before initiating GABA application in these whole-cell recordings to allow the high CsCl-containing solution to permeate the
cell. We obtained successful whole-cell recordings in nine neurons in
this experiment, and GABA-activated current was inward at all negative
membrane potentials and outward at positive membrane potentials (Fig.
1C). Figure 1D presents the
current-voltage (I/V) relationship for responses to
1 mM GABA under PP and whole-cell recording
conditions for the recordings from the same neuron that generated the
responses shown in Figure 1A-C. It can be seen that the reversal potential for GABA-activated current is shifted to a
slightly more negative value in the presence of bumetanide relative to
that in the bumetanide-free condition (control = 31.3 ± 3 mV; bumetanide = 37.2 ± 2.9 mV; n = 12 cells per group; p < 0.05, unpaired t
test). However, it should be noted that the amplitude of GABA-activated
current was reduced in the presence of bumetanide even at membrane
potentials at which the current should be larger (e.g., 10 mV) if the
only effect of the drug was to alter the reversal potential for the
GABA-gated channel. This decrease in the amplitude of GABA-activated
current at all membrane potentials reflects a change in the whole-cell
GABAA receptor-channel conductance (Fig.
2). Similar direct effects on
GABAA receptor function have been observed
previously in the presence of high concentrations of the loop diuretic
furosemide (Nicoll, 1978; Inomata et al., 1988; Pearce, 1993; Korpi and
Luddens, 1997). It can also be seen that the reversal potential moved
to ~0 mV during whole-cell recording in this cell. The reversal
potential for GABA-activated current averaged 31.3 ± 3 mV
(n = 12) during gramicidin PP recordings, 37.2 ± 3 mV (n = 12) during gramicidin PP recordings in the
presence of bumetanide, and 0.6 ± 3 mV (n = 9)
during whole-cell recordings in a subset of this group of neurons.
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Figure 2.
Bumetanide inhibition of GABAA
receptor function. Plot of GABA-activated whole-cell conductance before
bumetanide treatment (left) and after 2 min of exposure
to 10 µM bumetanide (right;
n = 12 per group). The asterisk
indicates a significant decrease (p < 0.05, paired t test).
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When 100 µM picrotoxin was applied before and during GABA
application in gramicidin PP experiments, a large reduction in the amplitude of GABA-gated current was observed (Fig.
1E). Similar results were observed in five neurons,
and the average reduction in current amplitude in the presence of
50-100 µM picrotoxin was 65 ± 11%.
Picrotoxin alone did not elicit any current.
The observation that bumetanide and other loop diuretics have direct
actions on GABAA receptors raises some concern
about the interpretation of experimental results generated with these reagents. Furthermore, these inhibitors only selectively block Na-K-2Cl cotransport over a limited concentration range and may have
actions on other Cl transporters at drug
concentrations that are required to block the cotransporter completely
(Russell, 2000). Thus, it is important to use alternative
experimental approaches to determine the role of the Na-K-2Cl
cotransporter in setting the Cl
equilibrium potential in neurons.
An experimental approach that would provide a strong test of the
hypothesis that the cotransporter plays a role in setting the
transmembrane Cl gradient in DRG neurons
would be to remove expression of the transporter altogether. This was
accomplished by disrupting the cotransporter gene and thus generating
the NKCC1 knock-out mouse (Delpire et al., 1999). Using a rabbit
polyclonal antibody directed against a specific C-terminal epitope of
the cotransporter (Kaplan et al., 1996), we demonstrated high
expression levels of the cotransporter in wild-type DRG neurons (Fig.
3A) and confirmed the absence
of cotransporter expression in the NKCC1 homozygote mutants
(Fig. 3B).
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Figure 3.
Immunofluorescence staining shows the absence of
Na-K-2Cl cotransporter expression in DRG neurons from homozygote
mutant mice. The rabbit polyclonal antibody is directed against a 74 amino acid peptide located in the C terminal of NKCC1 (Kaplan et al.,
1996). A, Control mouse DRG neurons highly express the
NKCC1 protein. The signal is predominantly located at the cell membrane
(arrows). Note the presence of a few neurons with a
minimal amount of cotransporter expression (arrowhead).
B, Identical view of DRG neurons from a homozygote
mutant animal demonstrates the complete absence of NKCC1 expression.
Scale bar, 20 µm.
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We then performed gramicidin perforated-patch recording experiments in
DRG neurons isolated from wild-type and NKCC1 knock-out mice. We first
wanted to determine whether the depolarizing effect of GABA normally
observed in DRG neurons would be impaired in the NKCC1 knock-out mouse
neurons. Using our standard CsCl/gramicidin PP recording conditions, we
observed that EGABA was consistently more depolarized in DRG neurons from wild-type compared with neurons from NKCC1 knock-out mice. Figure 4
illustrates representative current traces (Fig. 4A,B)
and representative current-voltage (I/V)
relationships (Fig. 4C) for GABA-activated current in
wild-type and NKCC1 knock-out mouse DRG neurons. When recording under
our initial gramicidin PP conditions (5 mM
[K]o and Cs-based internal solution), the
reversal potential averaged 37 ± 2.7 mV in wild-type mouse
neurons (n = 35 cells) and 53 ± 2 mV in NKCC1
knock-out mice (n = 39 cells), which correspond to
intracellular Cl concentrations of
46 ± 4 and 24 ± 2 mM, respectively.
Whole-cell capacitance and EGABA were
not significantly correlated in neurons from either wild-type or
NKCC1/ mice. We also measured the
reversal potential of GABA-activated current during whole-cell
recordings performed subsequent to completing gramicidin PP recordings
in the wild-type and knock-out mouse neurons. Reversal potentials were
near 0 mV for neurons from both groups of mice, and no significant
difference between the two groups was observed [wild type = 2 ± 1.3 mV (n = 24); knock-out = 1 ± 1.3 mV (n = 26); p = 0.082, unpaired
t test]. This finding supports the idea that the difference
in reversal potentials observed under the gramicidin PP condition was
caused by a difference in the transmembrane
Cl gradient and not caused by a
difference in the ion permeability of the GABAA
receptor-linked channel. The density of GABA-activated current in NKCC1
knock-out mouse neurons was similar to that measured in neurons from
wild-type mice [8.01 + 1.16 pA/pF (n = 26 cells, 9 mice) for homozygote vs 10.22 + 1.49 pA/pF (n = 26 cells, 12 mice) for wild type; p > 0.2, t test, NS). Furthermore, the whole-cell GABA-activated
conductance was similar in wild-type and knock-out mice [2.9 ± 0.83 nS (n = 12) vs 2.98 ± 0.67 nS
(n = 11); p = 0.93, unpaired
t test]. Thus, elimination of the transporter does not appear to have altered expression or function of
GABAA receptor channels.
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Figure 4.
Knock-out of the NKCC1 gene produces a
hyperpolarizing shift in EGABA. A,
B, Current activated by GABA in a single wild-type mouse neuron
(A) and a single NKCC1 knock-out mouse neuron
(B). Note that the current reverses polarity at
approximately 35 mV in the wild-type mouse neuron but at
approximately 50 mV in the NKCC1 knock-out mouse cell.
C, Current-voltage relationship for GABA-activated
current in single Cs-filled wild-type and NKCC1 knock-out mouse
neurons. Peak current amplitudes were calculated from the
traces in A and B. Note
that EGABA is at a more depolarized value in
the wild-type mouse neuron than in the knock-out mouse neuron.
D, Data for six neurons from wild-type and six from
NKCC1 knock-out mice recorded with a potassium-based intracellular
solution. This graph shows the difference between the RMP and
the reversal potential of GABA-activated current
(EGABA), as well as the RMP, for each
neuron. Positive values indicate that
EGABA is depolarized relative to the RMP,
whereas negative values indicate that
EGABA is hyperpolarized relative to rest.
Inset, A plot of the mean ± SEM values for all of
the wild-type and NKCC1 knock-out neurons. Note that
EGABA is more depolarized in wild-type than
in NKCC1 knock-out mouse neurons. The asterisk indicates
a significant decrease (p < 0.05, paired
t test).
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To determine whether application of GABA would produce depolarization
or hyperpolarization at the resting membrane potential, we recorded the
resting potential (0 current level) just after the initiation of PP
recordings using a KCl-based intracellular solution (see Materials and
Methods). We then voltage-clamped the membrane potential at the level
of the measured resting potential and applied 1 mM GABA to
activate the receptors. Application of GABA at the resting potential in
DRG neurons from wild-type mice produced a substantial inward current,
whereas GABA application at the resting membrane potential in DRG
neurons from NKCC1 knock-out mice produced little or no net current
flow, on average. We observed inward current at the resting potential
in all of the six neurons from wild-type mice, but neurons from NKCC1
knock-out mice exhibited small-amplitude inward current at the resting
potential in four of the six neurons examined, whereas the two
remaining neurons responded to GABA with outward current. We determined
the reversal potential for GABA-induced current in these neurons and
compared this value with the resting membrane potential measured in the same neurons. These data are summarized in Figure 4D.
This figure plots the difference between
EGABA and the resting membrane
potential (RMP) with the resting membrane potential for each cell shown on the x-axis. It can be seen from this graph that
EGABA was considerably positive to the
RMP in almost all of the neurons from wild-type mice, whereas
EGABA was observed at membrane
potentials either slightly depolarized or slightly hyperpolarized
relative to the RMP in NKCC1 knock-out mouse neurons. The averaged
values for all of these neurons are shown in Figure
4D, inset. This figure also shows that the
RMP values for the different neurons did not differ systematically
between wild-type and NKCC1 knock-out mouse neurons. The average
resting membrane potential did not differ significantly in knock-out
mouse neurons in comparison with wild-type mouse neurons [RMP = 68 ± 2 mV in wild-type mouse cells (n = 6) and
62 ± 3.3 mV in cells lacking NKCC1 (n = 6);
p = 0.144, unpaired t test].
Elimination of NKCC1 expression should eliminate the bumetanide-induced
hyperpolarizing shift in EGABA by
eliminating the major diuretic-sensitive mechanism for
Cl accumulation in the neuron. Thus, we
examined the effect of application of 10 µM
bumetanide on current activated by GABA and the reversal potential of
this current in DRG neurons from wild-type and NKCC1 mice. Figure
5A shows that the reversal
potential for GABA-gated Cl current was
actually shifted to a more depolarized potential during bumetanide
application to DRG neurons from NKCC1 knock-out mice (control = 55.5 ± 3.7 mV; bumetanide = 47 ± 2.5 mV;
p < 0.05, unpaired t test). This is the
opposite of what is observed in neurons from wild-type mice as shown in
Figures 1 and 5A. Thus, a bumetanide-sensitive mechanism for
decreasing intracellular Cl likely
exists in DRG neurons. This mechanism is normally overshadowed by the
activity of the Na-K-2Cl cotransporter but is unmasked when the
inward cotransport mechanism is removed. The decrease in the
GABA-activated conductance in the presence of bumetanide was observed
even in the NKCC1 knock-out mouse neurons [2.98 ± 0.67 nS
(n = 11) for untreated knock-out cells and 1.67 ± 0.33 nS (n = 11) for bumetanide-treated knock-out
cells; p < 0.05], and this decrease was similar in
magnitude to that observed in wild-type mice. This observation further
supports a direct effect of the diuretic on the
GABAA receptor complex.
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Figure 5.
Pharmacological properties and ion
sensitivity of the reversal potential of GABA-gated current in
wild-type and NKCC1 knock-out mouse DRG neurons. A, Plot
of the change in EGABA during exposure to
bumetanide in the wild-type and knock-out mouse neurons
(n = 12 wild-type mouse neurons and 11 knock-out
mouse neurons). Note the opposite effects of bumetanide in the two sets
of neurons. B, Plot of the change in
EGABA after exposure to increased
[K+]o in wild-type and knock-out mouse
neurons (n = 6 wild-type mouse neurons and 8 knock-out mouse neurons). C, Plot of the change in
[Cl]i after exposure to low
[Cl]o in the wild-type and NKCC1
knock-out mouse neurons (n = 9 wild-type mouse
neurons and 6 knock-out mouse neurons). Intracellular
Cl was estimated by the use of
EGABA and the known
[Cl]o as described in the text.
Single and double asterisks refer to p < 0.05 and p < 0.005, respectively.
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Because cation-Cl cotransporters are
sensitive to extracellular cation concentrations, we examined the
effect of altering extracellular K+ on the
reversal potential of GABA-activated current to determine whether this
manipulation produced a differential effect in wild-type versus NKCC1
knock-out mice. We observed that increasing the
K+ concentration from 5 to 10 mM caused a shift in EGABA
to more depolarized potentials that was approximately equivalent in
both wild-type and knock-out mouse DRG neurons (Figure 5B).
This finding indicates that a K-sensitive transporter is acting to
lower ECl in the absence or presence
of NKCC1. This observation also supports the idea that DRG neurons
contain cation-Cl cotransporters other
than NKCC1.
We also lowered the extracellular Cl
concentration by reducing the NaCl concentration in our extracellular
solution from 150 to 100 mM, substituting the remaining 50 mM with Na-gluconate. This treatment should reduce the
inward transport mediated by NKCC1 and result in a reduction in
intracellular Cl concentration. The
EGABA values were shifted to slightly
more depolarized values in both the wild-type and NKCC1 knock-out mouse neurons (from 34 ± 2 to 29 ± 2 mV in wild-type neurons
and from 52 ± 5 to 45 ± 5 mV in NKCC1 knock-out
neurons) after reducing [Cl]o. In both
sets of mice these shifts in EGABA
were statistically significant (p < 0.05, repeated measures t test). This is to be expected because of
the change in Cl-driving force produced
by this treatment. Using these measured EGABA values, we were then able to
calculate the
[Cl]i value by
the use of the Nernst equation (see Materials and Methods). Figure
5C shows that the reduction in
[Cl]o produced a
significant decrease in
[Cl]i in DRG
neurons from wild-type but not knock-out mice (p < 0.05 for wild-type data, p > 0.2 for NKCC1
knock-out data, repeated measures t test). This finding is
consistent with the idea that the intracellular
Cl concentration in DRG neurons is
sensitive to
[Cl]o only when
NKCC1 is present. Figure 5C also shows that
[Cl]i was lower
in the NKCC1 knock-out mouse neurons in comparison with wild-type mouse
neurons under both the normal and reduced [Cl]o
conditions, consistent with the outcome of previous experiments indicating that loss of NKCC1 reduces the
Cl concentration in DRG neurons. As
expected, [Cl]i
measured during whole-cell recording in the reduced
[Cl]o condition
did not differ in wild-type compared with knock-out mouse neurons (data
not shown).
To relate these abnormal GABAergic responses in DRG neurons to
proprioception, we subjected the mice to a series of behavioral tests,
starting with a gross behavioral analysis. To assess gross neurological
function in the knock-out mice, a subset of tests from the Irwin (1968)
screen was performed. The Irwin screen is a comprehensive set of
assessment tools for detecting neurological and behavioral
abnormalities in mice. The Irwin screen is the standard basic test
battery used to control for procedural abilities (e.g., walking)
required in more complex behavioral tests (e.g., memory) in both
behavioral pharmacology and behavioral genetics (Irwin, 1968;
Banfi et al., 1982; Hunter et al., 2000). The Irwin screen has been
incorporated into or is the basis for a number of alternate behavioral
test batteries (Crawley and Paylor, 1997; Rogers et al., 1997).
Perhaps the most salient behavior observed during the gross
neurological screen was the spontaneous retropulsive response to a
novel environment on the part of the knock-out mice. In this test, mice
were placed in a clean mouse tub cage and observed for a period of 3 min. Retropulsion was immediate after placement into the tub cage and
was characterized by rapidly walking backward until reaching the edge
of the cage and continuing until the rear legs were propped up on the
side of the cage. The mice then maintained a vertical, head-down
position, although spastic movements continued. After a short period in
this vertical position, knock-out mice began moving again and exploring
the rest of the cage. In addition to this behavior, knock-out mice
appeared to struggle more when touched or restrained and when held
aloft by the tail, although this may be a manifestation of their
spasticity. Other behaviors assessed during the gross neurological
screen were normal, including all reflexes tested, toe and tail pinch,
and muscle tone.
The rotorod performance of the knock-out mice was severely impaired
[F(2,48) = 53.0; p < 0.0001], as depicted in Figure 6. Follow-up analyses demonstrate that the knock-out mice fell off the
rotorod much sooner than did both the wild-type
[F(1,34) = 101.9; p < 0.0001] and heterozygous [F(1,27) = 92.5; p < 0.0001] mice. Interestingly, the rotorod
performance of the knock-out mice improved significantly over the
nine testing sessions [ = 0.282;
F(2,29) = 3.3; p = .0023], increasing from a mean of 15-18 sec on day 1 to 30-39 sec on
day 3. This demonstrates that the knock-out mice retain some ability to
learn motor skills in spite of their debilitating spasticity. The
rotorod performance of wild-type and heterozygous mice did not
differ significantly [F(1,35) = 1.0;
p = 0.335] .
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Figure 6.
Performance on the rotorod test of motor
coordination. Mice were placed on an accelerating rotorod for three
trials per day for 3 d. The latency to fall off the rotorod was
assessed. NKCC1/ mice had significantly impaired
motor coordination compared with that of wild-type or NKCC1
heterozygous mice. Data represent the mean ± SEM.
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The types of behavior that the mice exhibited in response to pain
differed significantly across genotype (df = 6;
2 = 25.1; = 0.646;
p = 0.0003). Wild-type (df = 1;
2 = 11.8; = 0.789;
p = 0.0006) and heterozygous (df = 1;
2 = 12.7; = 0.798;
p = 0.0004) mice were more likely to exhibit guarding
behavior (lifting or shaking the paw), whereas the knock-out mice were
more likely to increase locomotor activity in response to the thermal
stimulus. There was no significant difference across genotypes in the
proportion of mice that exhibited alternate behavior (i.e., other than
licking) after being placed on the hot plate (df = 2;
2 = 2.6; = 0.226;
p = 0.278) or in the topography of nociceptive responding between wild-type and heterozygous mice (df = 2;
2 = 0.9; = 0.2;
p = 0.645).
Knock-out mice exhibited impaired pain perception on the hot
plate, as demonstrated by longer response latencies [Fig.
7; F(2,43) = 4.3; p = 0.0196]. Follow-up analyses showed that the knock-out mice were
significantly less sensitive to the thermal stimulus compared with both
the wild-type (PLSD = 4.72; p = 0.0054) and
heterozygous (PLSD = 5.02; p = 0.0466) groups. The
mean response latency of the heterozygous mice did not differ from that
of the wild-type mice (PLSD = 4.16; p = 0.3974).
Four knock-out mice were excluded from the analysis of hot plate
latencies because they exhibited spontaneous retropulsion after being
placed on the hot plate. Because this retropulsive response was
immediate and occurred long before the typical latencies of other
nociceptive responses in any of the mice, it was impossible to
distinguish whether the retropulsion was in response to the thermal
stimulus or in response to a novel environment, as observed in the
gross neurological screen.
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Figure 7.
Performance on the hot plate test of
nociception. Mice were placed on the hot plate at a temperature of
52°C. The latency to lick a paw or to emit another
pain-associated behavior was assessed. NKCC1/
mice were significantly (asterisk, p < 0.05)
less sensitive to the thermal stimulus than were wild-type or
NKCC1+/ heterozygous mice. Data represent the
mean ± SEM.
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In spite of differences in behaviors in response to the thermal
stimulus, all but five mice (three wild types and two knock-outs) also
licked their paws when exposed to the high temperature, including two
of the four knock-out mice that retropulsed and whose rear paws were in
contact with the acrylic restraining wall and not with the hot plate.
When only latency to lick was considered, knock-out mice were still
significantly impaired relative to wild-type and heterozygous mice
[F(2,43) = 5.9; p = 0.0054].
| |
DISCUSSION |
Removal of NKCC1 reduces
[Cl]i in mouse
DRG neurons (Fig. 4), supporting the idea that this cotransporter plays
a major role in determining the transmembrane
Cl concentration gradient in these
cells. The experiments involving bumetanide application and
manipulation of ion concentrations further support this idea. The
predominant effect of bumetanide in wild-type DRG neurons is to shift
EGABA to more hyperpolarized potentials, indicating a decrease in
[Cl]i. In
addition, reducing
[Cl]o reduces
[Cl]i in
wild-type neurons, consistent with a reduction in activity of an inward
Cl transporter such as NKCC1. Both of
these effects are lost in the NKCC1 knock-out mouse, strongly
implicating this transporter in concentrating
Cl inside mouse DRG neurons under normal
conditions. These data are consistent with those obtained from frog
dorsal root ganglion neurons by the use of ion-selective
microelectrodes or from Amphiuma Rohon-Beard spinal neurons
by the use of the gramicidin perforated-patch method, which clearly
indicated accumulation of Cl via a
bumetanide-sensitive, Na+- and
K+-dependent Na-K-2Cl cotransporter
(Alvarez-Leefmans et al., 1988; Rohrbough and Spitzer, 1996)
Our findings also suggest that another transporter, in addition to
NKCC1, regulates intracellular Cl
concentration in mouse DRG neurons. We observed a bumetanide-induced depolarizing shift in EGABA in the
NKCC1 knock-out mouse neurons that was not observed in neurons from
wild-type animals. This suggests that bumetanide inhibits a
transporter, other than NKCC1, that mediates
Cl extrusion from these neurons. Further
support for involvement of another transporter comes from the
observation that altering [K+]o alters
EGABA, and thus
[Cl]i, to a
similar extent in both wild-type and knock-out mouse neurons. One
likely candidate transporter that could mediate this action is a K-Cl
cotransporter. Indeed, the effect of increasing [K+]o is
consistent with a role for this transporter because this treatment
would be expected to increase
[Cl]i if this
transporter is present, and our results indicate that such an increase
actually takes place. Four isoforms of the K-Cl cotransporter have
been cloned with one, KCC2, being expressed exclusively in neurons.
Using PCR and immunofluorescence, we have demonstrated previously the
presence of KCC2 in DRG neurons (Lu et al., 1999). Other isoforms of
the K-Cl cotransporter might also be expressed in these cells. This
K-Cl cotransporter may function in wild-type DRG, and its action would
tend to oppose that of NKCC1.
Interestingly, Ehrlich et al. (1999) have observed a positive shift in
Eglycine induced by furosemide in
postnatal day 8 (P8)-P10 lateral superior olive neurons, instead of
the negative shift observed in younger P2-P4 neurons. The positive
shift in mature neurons suggests inhibition of a net outward
Cl transport mechanism, likely KCC2,
whereas the negative shift in glycine reversal potential observed in
immature neurons indicates inhibition of a net inward
Cl transport system, likely to be the
Na-K-2Cl cotransporter. Unlike the case in CNS neurons, it appears
that the function of NKCC1 dominates Cl
transport in wild-type DRG neurons because bumetanide produces a
hyperpolarizing shift in EGABA when
NKCC1 is present and EGABA is shifted
to more hyperpolarized values when NKCC1 is removed. This strong NKCC1
inward Cl transport may overshadow a
smaller bumetanide-sensitive component of transport.
The observed actions of bumetanide in the absence of NKCC1 suggest that
some caution should be exercised in interpreting results from
experiments examining the effect of this drug and other loop diuretics
on transmembrane Cl concentration and
the physiological roles of distribution of this ion. The limitations of
the usefulness of loop diuretics in studies examining
GABAA receptor function and transmembrane Cl distribution are further demonstrated
by the past and present findings that these compounds inhibit the
conductance of GABAA receptor channels (Figs.
1D, 2) (Nicoll, 1978; Inomata et al., 1988; Pearce,
1993; Korpi and Luddens, 1997). These problems with the experimental
use of bumetanide (or furosemide) highlight the importance of the
genetic model that we have used in the present study. Selective
knock-out of the NKCC1 gene provides strong support, independent of
pharmacological approaches, for a role of this transporter in
regulation of the neuronal Cl gradient.
Using this method, we have now firmly established that NKCC1 activity
strongly contributes to the depolarizing effects of GABA in primary
sensory neurons.
As alluded to previously, the functional data obtained in sensory DRG
neurons can very likely be extended to immature CNS neurons (cortical
and hippocampal) that have intracellular
Cl concentrations higher than the
electrochemical potential equilibrium and exhibit GABA- or
glycine-depolarizing responses (Ben-Ari et al., 1989; Owens et al.,
1996; Ehrlich et al., 1999). These GABA- or glycine-depolarizing
responses observed in immature neurons coincide with high NKCC1
expression (Plotkin et al., 1997b). Hyperpolarizing GABA or
glycine responses develop shortly after birth in concert with a
decrease in intracellular Cl
concentration because of a simultaneous decrease of NKCC1 expression (Plotkin et al., 1997b) and increase in KCC2 expression (Clayton et al., 1998; Lu et al., 1999).
This study did not reveal the existence of alternative pathways for
Cl accumulation. However, our
experiments were performed in the absence of bicarbonate. Thus, we
cannot exclude the possibility that, in the homozygote, bicarbonate
could compensate and affect the direction and extent of GABA
responses in vivo. It is important to note, however,
that in CNS neurons such as GABAergic sensorimotor cortical neurons
(Owens et al., 1996) and glycinergic lateral superior olive
neurons (Ehrlich et al., 1999), furosemide induced a marked negative
shift in the GABA or glycine reversal potential in the presence of
bicarbonate or CO2, suggesting that
Cl accumulation via the cotransporter is
indeed the basis for depolarizing GABA or glycine responses in immature
neurons and that inhibition of this pathway does not result in any
compensation by bicarbonate. Furthermore, we have shown that the
absence of depolarizing GABA currents in homozygote NKCC1 mutant DRG
neurons results in sensory perception defects, indicating one potential
role of Cl regulation via the Na-K-2Cl
cotransporter in the somatosensory system.
GABAA-mediated primary afferent depolarization
and presynaptic inhibition are known to regulate the excitability of
sensory terminals of both large (muscle) and fine (pain, temperature, and touch receptors) afferent fibers. Abnormal synaptic transmission of
type 1A afferent fibers to spinal cord motor neurons is likely to
result in gait and movement coordination phenotypes. Spasticity and
locomotion phenotypes definitely exist in the homozygote NKCC1 mutants.
Knock-out mice were unable to remain on the rotorod for a substantial
period of time because of motor uncoordination and spasticity (Fig. 6).
Despite these differences, the knock-out mice made significant
improvements over the course of the nine rotorod sessions. These
results are consistent with the sensory neuron Na-K-2Cl cotransporter
playing an important role in motor coordination, but other factors are
involved as well. For instance, it is difficult at this stage to sort
out the contribution of the spinal cord and of the inner ear in the
uncoordination phenotype. We have reported previously that disruption
of the secretory Na-K-2Cl cotransporter gene results in
anatomical defects of the inner ear associated with an absence of
K+ secretion. These defects result in
deafness and abnormal control of balance phenotypes (Delpire et al.,
1999) that could contribute to poor rotorod performance.
Type A and type C fibers are both involved in the perception of
pain. Abnormal synaptic transmission in these fibers is likely to
affect pain threshold. Because of the wide range of capacitance values
for the neurons from which we recorded, it is likely that we examined
somata of A and C type neurons. Using the standard hot plate test to
evaluate the pain threshold, we have shown that homozygote NKCC1 mutant
mice display a significantly higher pain threshold when compared with
their control or heterozygote littermates (Fig. 7). This result cannot
be attributed to a complete absence of sensory perception or to a
general apathy and lack of reflexes because the mice passed the general
tests involved in the gross neurological screen. Therefore, this result
clearly demonstrates that elimination of the Na-K-2Cl cotransporter
results in alteration of the processing of sensory signals. On the
basis of the premise that primary afferent depolarization and
presynaptic inhibition filter sensory noise, we propose that NKCC1
knock-out animals likely experience decreased sensory contrast
resulting in a higher pain threshold. Our findings do not provide
evidence of a strong causal link between the change in the sensory
neuron transmembrane Cl gradient and the
increase in pain threshold. However, these observations indicate that
the NKCC1 knock-out mice do experience alterations in pain perception
that are consistent with what would be expected after alteration of
GABAergic effects at primary afferent synapses. The change in pain
thresholds that we observed is not caused by generalized hypokinesia or
hypoflexia because the animals show vigorous retropulsive movement and
reflexive response patterns. Interestingly, our data differ somewhat
from experiments showing that intrathecal administration of GABA and
GABAA receptor agonists increases the nociceptive
threshold in rats (Hammond and Drower, 1984; Roberts et al.,
1986) whereas bicuculline, a GABAA
receptor antagonist, results in profound agitation. However, these
studies examine the short-term effect of applying pharmacological
agents in the spinal cord that can affect both postsynaptic and
presynaptic neurons, and it is not clear whether the NKCC1 knock-out
affects postsynaptic GABAergic responses in the spinal cord. In
agreement with our data, these studies stress the important role of
GABAA-mediated transmission in peripheral
sensation. In light of the nociception phenotype, it will be important
to examine in future studies presynaptic inhibition in the NKCC1
knock-out mouse.
| |
FOOTNOTES |
Received May 23, 2000; revised July 13, 2000; accepted Aug. 9, 2000.
This work was supported in part by the National Institute of
Neurological Disorders and Stroke Grant NS 36758, by a Vanderbilt University Medical Center Intramural Discovery Grant to E.D., and by
the National Institute on Alcohol Abuse and Alcoholism Grant AA 08986 to D.M.L. K.-W.S. was supported by a Korean Science and
Engineering Foundation (KOSEF) grant. E.D. is an Established Investigator of the American Heart Association.
Correspondence should be addressed to Dr. Eric Delpire,
Anesthesiology Research Division, Department of Anesthesiology,
Vanderbilt University, T-4202, Medical Center North, Nashville, TN
37232-2520. E-mail: eric.delpire{at}mcmail.vanderbilt.edu.
| |
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T. A. Simeone, S. D. Donevan, and J. M. Rho
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N. T. Blair and B. P. Bean
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R. A. DeFazio, S. Heger, S. R. Ojeda, and S. M. Moenter
Activation of A-Type {gamma}-Aminobutyric Acid Receptors Excites Gonadotropin-Releasing Hormone Neurons
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S. Uchida, E. Noda, Y. Kakazu, Y. Mizoguchi, N. Akaike, and J. Nabekura
Allopregnanolone enhancement of GABAergic transmission in rat medial preoptic area neurons
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D. Billups and D. Attwell
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J. Nabekura, T. Ueno, A. Okabe, A. Furuta, T. Iwaki, C. Shimizu-Okabe, A. Fukuda, and N. Akaike
Reduction of KCC2 Expression and GABAA Receptor-Mediated Excitation after In Vivo Axonal Injury
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T. Ueno, A. Okabe, N. Akaike, A. Fukuda, and J. Nabekura
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S. Wagner, N. Sagiv, and Y. Yarom
GABA-induced current and circadian regulation of chloride in neurones of the rat suprachiasmatic nucleus
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S. L. Schomberg, G. Su, R. A. Haworth, and D. Sun
Stimulation of Na-K-2Cl Cotransporter in Neurons by Activation of Non-NMDA Ionotropic Receptor and Group-I mGluRs
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G. Su, D. B. Kintner, and D. Sun
Contribution of Na+-K+-Cl- cotransporter to high-[K+]o- induced swelling and EAA release in astrocytes
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G. Su, D. B. Kintner, M. Flagella, G. E. Shull, and D. Sun
Astrocytes from Na+-K+-Cl- cotransporter-null mice exhibit absence of swelling and decrease in EAA release
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TOP
ABSTRACT
INTRODUCTION
