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Subunit
1 Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, California 90095; 2 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095; 3 Department of Anesthesiology/Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260
Submitted 13 November 2002; accepted in final form 8 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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subunit of the 
-aminobutyric acid (A) receptor
(GABAAR) is expressed postnatally mostly in the cerebellum,
thalamus, and dentate gyrus. Previous studies in mice with a targeted
disruption of the subunit revealed a considerable attenuation of
behavioral responses to neuroactive steroids but not to other neuromodulatory
drugs. Here we show that subunit loss leads to a concomitant reduction
in hippocampal 
4 subunit levels. These changes were accompanied by
faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate
granule neurons of –/– mutants (decay 
= 25 ms) compared with
+/+ controls ( = 50 ms). Furthermore, the GABAAR-mediated
miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in
-mutants ( = 6.3 ms) than controls ( = 7.2 ms) and had
decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of
mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1–10 µM),
was smaller in -mutants (at 10 µM, 65% increase) compared with +/+
littermates (308% increase). In competition binding experiments, alphaxalone
(0.03–1 µM) modulation of
[35S]t-butylbicyclophosphorothionate binding was reduced in
-mutant brain homogenates, indicating that the decreased alphaxalone
effects on mIPSCs were due to changes in the GABAAR protein. Faster
decay of evoked IPSPs and mIPSCs in -mutants suggests presence of the
subunit at both synaptic and extrasynaptic GABAARs.
Decreased synaptic and extrasynaptic inhibition likely contributes to the
pro-epileptic phenotype of -mutants. Reduced neurosteroid sensitivity
might also contribute to seizure susceptibility. While the simplest
explanation is that subunit-containing GABAARs represent
the actual target of neurosteroids, it is possible that the behavioral and
physiological sensitivity to neuroactive steroids is indirectly altered in the
–/– mice. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-Aminobutyric acid type A receptors (GABAARs) mediate the
bulk of fast inhibitory synaptic transmission in the brain and have been
implicated in responses to sedative/hypnotic, anesthetic, convulsant, and
anticonvulsant drugs (Olsen and Homanics
2000
). Mammalian GABAARs are a family of
heteropentamers formed from a family of 
19 related subunits, which confer
on the resultant GABAARs different sensitivities to GABA and to
modulatory drugs. The subunit of the GABAAR is expressed
exclusively postnatally, mostly in the cerebellum, thalamus, and dentate gyrus
(Persohn et al. 1992). The
subunit has been shown to predominantly co-assemble with the 4
and 6 subunits, the latter limited to cerebellar granule cells
(Huh et al. 1996;
Jechlinger et al. 1998;
Mertens et al. 1993;
Quirk et al. 1995).
Genetically altered mice with a knockout of the 6 subunit exhibit a
dramatic reduction in the subunit polypeptide in the cerebellum, which
is further evidence for co-assembly of these two subunits
(Jones et al. 1997). The
presence of either 4 or 6 subunits in the GABAAR
confers a relative insensitivity to classical benzodiazepine agonists
(Benke et al. 1997;
Khan et al. 1996;
Quirk et al. 1994).
Mice with a targeted disruption of the subunit gene
(Mihalek et al. 1999) produced
heterozygotes (+/–) and null mutants (–/–) that had reduced
levels of [3H]muscimol binding and reduced inhibitory synaptic
transmission. They were observed to have spontaneous convulsive seizures,
increased susceptibility to pentylenetetrazol (PTZ)-induced seizures, and
epileptic electroencephalogram recordings
(Banerjee et al. 2000;
Olsen et al. 1997).
Furthermore, in vivo behavioral testing revealed a strikingly selective
attenuation of responses to neuroactive steroids but not to other
neuromodulatory drugs and anesthetics
(Mihalek et al. 1999). This is
consistent with recent reports that 4-containing
GABAARs are more sensitive to neurosteroids in recombinant
expression studies than other subunit combinations
(Brown et al. 2002;
Wohlfarth et al. 2002);
however, others reported decreased neurosteroid sensitivity of
6-containing GABAARs
(Zhu et al. 1996). The present
study was designed to further explore the physiological mechanisms by which
mutant phenotype expression may occur. A preliminary account of some
experimental results has been made (Li et
al. 1998; Spigelman et al.
2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Mice were produced and genotyped as described previously
(Mihalek et al. 1999). All
mice were of a mixed C57BL/6J x Strain 129Sv/SvJ genetic background and
were derived from heterozygote matings. Experiments were performed on male or
female mice at 2–4 mo of age.
Membrane preparation and Western blot
Individual hippocampi were dissected on ice from each mouse brain, and P2
membrane fractions were prepared by homogenization, low-speed centrifugation
in 0.32 M sucrose and centrifugation of the supernatant at 12,000 g
for 20 min. The pellet was resuspended and washed in 20 volumes of
phosphate-buffered saline (PBS: 150 mM NaCl, 10 mM
Na2HPO4/NaH2PO4, pH 7.4). The
final pellet was resuspended in 5 volumes of PBS, and protein concentration
was detected by BCA protein assay system (Pierce Rockford USA). Aliquots of 20
µg protein from each sample and 5 ml of Rainbow colored protein molecular
weight marker (Amersham Life Science) were separated on 10% SDS-polyacrylamide
gel electrophoresis under reducing conditions using the Biorad
Mini–Protean 3 Cell electrophoresis system. Proteins were transferred to
PVDF membranes (Hybond-P Amersham Pharmacia Biotech, Buckinghamshire England)
with LKB2117 Multiphor II Electrophoresis system (Pharmacia LKB Biotechnology
Uppsala, Sweden). Blots were probed either with anti-4
(aa379–421) antibody or anti- (aa1–44) antibody, 1 mg/ml
final concentration, followed by horseradish peroxidase-conjugated secondary
antibody, and bands were detected by ECL detection kit (Amersham Pharmacia
UK), apposed to X-ray film under nonsaturating conditions. W. Sieghart and
colleagues (Vienna) kindly provided the antibody. The bands from different
+/+ (n = 8) and –/– (n = 8)
corresponding to the appropriate molecular weight for 4 subunit were
analyzed, and absorbance values were compared by densitometric measurements
using C.IMAGING image analysis systems (Complix, Cramberry Township, PA) and
Simple 32 software application program. Data analysis was conducted by
t-test and the difference was expressed as % of control peptide
levels ± SE. P values <0.05 were considered statistically
significant.
Electrophysiology
Transverse slices (400 µm thick) of dorsal hippocampus were obtained
using standard techniques (Spigelman et
al. 1992). Recordings were obtained from cells located in the
upper blade of the dentate gyrus at 32°C during perfusion with artificial
cerebrospinal fluid (ACSF) composed of (in mM) 125 NaCl, 2.5 KCl, 2
CaCl2, 2 MgCl2, 26 NaHCO3, and 10
D-glucose. The ACSF was continuously bubbled with a 95%
O2-5% CO2 mixture to ensure adequate oxygenation of
slices and a pH of 7.4. Intracellular sharp microelectrodes contained 3 M
potassium methylsulfate (pH = 7.25). All membrane potentials were corrected
with respect to the null potentials measured after withdrawal from the cell.
Patch pipettes contained (in mM) 135 cesium gluconate, 2 MgCl2, 1
CaCl2, 11 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 10
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2
K2ATP, and 0.2 Na2GTP; pH adjusted to 7.25 with CsOH.
GABAAR-mediated mIPSCs were pharmacologically isolated by adding
tetrodotoxin (TTX, 0.5 µM),
D(–)-2-amino-5-phosphonopentanoate (APV, 40 µM),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), and CGP 35348 (500
µM) or CGP 54,626 (1 µM) to the ACSF from stock solutions. Stock
solutions of CNQX and alphaxalone were made with pure dimethyl sulfoxide
(DMSO). Final concentration of DMSO did not exceed 42 µM in the recording
chamber. Signals were recorded in voltage-clamp mode with an amplifier
(Axoclamp 2B, Axon Instruments). Whole cell access resistances were in the
range of 8–15 M
before electrical compensation by 
70%.
During voltage-clamp recordings, access resistance was monitored by measuring
the size of the capacitative transient in response to a 5-mV step command, and
experiments were abandoned if changes >20% were encountered. At least 10
min was allowed for equilibration of the pipette solution with the
intracellular milieu before commencing miniature inhibitory postsynaptic
current (mIPSC) recordings. All mIPSC recordings were 60 s in duration in the
continuous voltage-clamp mode at 0-mV holding potential. Data were acquired
with pClamp 7 or 8 software (Axon Instruments), digitized at 20 kHz (Digidata
1200B, Axon Instruments) and analyzed using the Clampfit software (Axon
Instruments) and the Mini Analysis Program (version 5.4.8, Synaptosoft).
Detection and analysis of mIPSCs
The recordings were low-pass filtered off-line (Clampfit software) at 2
kHz. The mIPSCs were detected (Mini Analysis Program) with threshold criteria
of 5-pA amplitude and 20-pA*ms area. Frequency of mIPSCs was determined from
all automatically detected events in the 60-s recording period. For kinetic
analysis, only single-event mIPSCs were chosen during visual inspection of the
recording trace. This included mIPSCs with a stable baseline, sharp rising
phase, and exponential decay. Double and multiple peak mIPSCs were excluded.
The mIPSC kinetics were obtained from analysis of the averaged chosen single
events (29–182 events/60-s recording period) aligned with half rise-time
in each cell. We also excluded from final analysis all recordings where the
rise time (10–90%) of the averaged mIPSCs was 1 ms in any single
recording period. We invariably found these recordings to be of poor quality
such that the averaged mIPSC amplitudes were very small, had greatly prolonged
decay times, and most likely represented electrotonically filtered events from
distal synapses. Decay time constants were obtained by fitting a single
exponential to the falling phase of the averaged mIPSC in each neuron. The
investigators who performed the recordings and analysis were blind to the
phenotype of the mice.
[35S]bicyclophosphorothionate binding
The binding of the GABAA receptor channel ligand
t-butyl [35S]bicyclophosphorothionate (TBPS) was measured
as previously described (Srinivasan et al.
1999; Turner et al.
1989) using thoroughly washed total rat brain homogenates
(mitochondrial plus microsomal fraction). Osmotically shocked, frozen, and
thawed membranes were incubated at ca. 0.5 mg/ml protein in 0.1 M KCl, 10 mM
potassium phosphate buffer, pH 7.5. Incubation was at room temperature
(21°C) for 90 min, with [35S]TBPS (6 nM; ca. 100 Ci/mmol,
DuPont-New England Nuclear, Boston, MA; correct specific radioactivity was
calculated on the day of assay) and various concentrations of alphaxalone. A
duplicate set of assay tubes in quadruplicate contained 100 µM picrotoxinin
(Sigma, St. Louis, MO) for estimation of nondisplaceable background, which was
subtracted from total binding to calculate specific binding. After
equilibration, the samples were vacuum filtered through Whatman GF/B filters
using a Brandel Cell Harvester (Gaithersburg, MD) and counted in
Cytoscint-toluene scintillation cocktail (ICN, Irvine, CA). Specific binding
was >90% (Sapp et al.
1992).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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4 subunit levels in hippocampi of knockout
mice
The 4 antibody recognized a 67-kDa band in the membrane fractions
from +/+ and –/– mice.
Figure 1A shows that
the 4 subunit was decreased significantly (-25 ± 5.5%) in
–/– mice (n = 8) compared with +/+ mice
(n = 8). Figure
1B shows representative Western blots of GABAA
receptor (top) and 4 (bottom) subunit peptide
levels. The staining for the 54-kDa GABAA protein present
in +/+ is completely absent from –/– lanes.
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Membrane properties and IPSPs of granule cells from knockout
mice
Initially, sharp electrode recordings revealed that the intrinsic membrane
properties of dentate granule neurons (Fig.
2) did not differ between the wild-type and mutant mice
(Table 1). However, differences
were observed when the properties of evoked inhibitory postsynaptic potentials
(IPSPs) were compared between the three groups of mice
(Fig. 3). The IPSPs in
–/– mutants had decay rates ( = 25 ± 3.5 ms)
twice as fast as those of wild-type controls ( = 51 ± 5.2 ms),
whereas heterozygotes had intermediate decay rates ( = 45 ± 6.3
ms). These differences were observed in the absence of changes in the reversal
potential of the IPSPs among the three groups of mice
(Fig. 3).
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Altered kinetics of mIPSCs in subunit knockouts
The decay rates of evoked IPSPs are determined partly by kinetics of GABA
release as well as activation of both synaptic and extrasynaptic GABA
receptors. However, the decay rates of miniature GABAAR-mediated
synaptic currents (mIPSCs) predominantly reflect activation of synaptic
GABAARs. Therefore we recorded mIPSCs to investigate directly the
relaxation time course of postsynaptic GABAAR currents. The average
amplitude and rise time of mIPSCs recorded at 0 mV did not differ between
mutants and controls (Fig. 4).
However, the decay of mIPSCs from –/– mice was significantly
faster (6.34 ± 0.18 ms, n = 19) than that of +/+ controls
(7.21 ± 0.22 ms, n = 18). The total charge transfer measured
as the area under the averaged mIPSCs was also significantly different
(P = 0.028) between +/+ controls (174.8 ± 10.4 pA · ms)
and –/– mice (146.6 ± 6.6 pA · ms), suggesting
reduced synaptic inhibition in mutant mice. Furthermore, the frequency of
mIPSCs in granule cells from –/– mutants (6.6 ± 0.7
Hz) was significantly smaller than in +/+ controls (10.5 ± 1.3 Hz),
suggesting a reduction in the number of GABAergic synapses in
mutants.
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Decreased alphaxalone potentiation of mIPSCs in knockout
mice
The potentiation of mIPSCs by bath application of the neuroactive steroid,
alphaxalone (1–10 µM), was examined in wild-type controls and
mutants. In granule cells from both groups of mice, alphaxalone had no
significant effect on the rise time, amplitude, or frequency of mIPSCs. The
main concentration-dependent effect of alphaxalone was to prolong the decay
time course of mIPSCs (Fig. 5).
Partial reversal of this effect was obtained on wash in some recordings (data
not shown). After application of 3 µM alphaxalone, this effect appeared
considerably smaller in –/– mice (30 ± 12%
increase, n = 6) compared with wild-type littermates (99 ± 27%
increase, n = 9) but did not reach statistical significance
(P = 0.057). However, at 10 µM alphaxalone the differences were
highly significant (P = 0.0002). Thus decay of mIPSCs from
–/– mice was increased by only 64 ± 27% (n = 5),
whereas the wild-type decay was increased by 308 ± 30%
(n = 6).
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Decreased alphaxalone modulation of [35S]TBPS binding in
knockout mice
To explore the mechanism behind the reduced alphaxalone effects on mIPSCs
in mutants, we studied the ability of alphaxalone to allosterically
modulate the binding of a selective GABAAR ligand in brain
homogenates. Alphaxalone has previously been shown to modulate three specific
binding sites on the GABAAR complex: the GABA site, the
benzodiazepine site, and the TBPS site
(Hawkinson et al. 1994;
Lambert et al. 2001).
Alphaxalone at higher concentrations (0.3–1 µM) was capable of
complete allosteric inhibition of [35S]TBPS binding in brain
homogenates from +/+ mice, whereas <37% displacement was achieved by 1
µM alphaxalone in homogenates from –/– mice
(Fig. 6). Thus the reduced
behavioral effect of neurosteroids on –/– mice is also
reflected in vitro at the level of GABAAR function in hippocampal
slices and at the level of receptor protein in binding assays.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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subunit is
accompanied by a reduction in 4 subunit levels in the hippocampus, the
dentate granule cells of mice deficient in the GABAAR
subunit exhibit faster decay of evoked IPSPs and spontaneous mIPSCs, the
mIPSCs of granule cells in subunit knockouts exhibit greatly decreased
responses to the neuroactive steroid, alphaxalone, and alphaxalone modulation
of [35S]TBPS binding was reduced in mutant brain homogenates.
Previously, subunit knockouts were observed to have spontaneous
convulsive seizures and epileptic electroencephalogram recordings
(Olsen et al. 1997) as well as
increased susceptibility to PTZ-induced seizures
(Banerjee et al. 2000). The
most plausible explanation for these is that deletion of the subunit
results in decreased GABAAR-mediated inhibitory neurotransmission.
This explanation is now fully supported by the results of our
electrophysiological recordings in which the evoked IPSPs and, to a lesser
extent, the spontaneous mIPSCs in granule cells from the subunit
mutants, exhibit faster decay compared with their wild-type controls. In the
absence of differences in amplitude and rise times, faster mIPSC decay and
reduced total charge transfer through the GABAAR-activated chloride
channels represents reduced synaptic inhibition. This is consistent with the
reduction in subunit-containing GABAARs, whether or not
they are replaced by other subunits with different properties. In addition,
the observed decreased frequency of mIPSCs suggests a reduction in the number
of GABAergic synapses in mutants.
The mIPSCs from wild-type controls recorded with CsGluconate-based whole
cell patch pipettes in the presence of TTX (0.5 µM), APV (40 µM), CNQX
(10 µM), and CGP 54,626 (1 µM) had kinetics
(Fig. 4) that were slower than
those reported previously for rat dentate granule neurons
(Buhl et al. 1996;
De Koninck and Mody 1996;
Poisbeau et al. 1997). The
slower kinetics could be explained by using lower than physiological
temperature (32°C), by use of higher tip resistance recording electrodes
(8–15 M) and incomplete series resistance compensation (70%)
during our recordings. However, comparison among our experimental groups is
validated through the use of blinded acquisition and analysis of the data in
our studies.
Others have shown with the aid of subunit-specific antibodies that
GABAARs with different subunit composition may localize to
different anatomical locations on neurons
(Fritschy et al. 1998;
Nusser et al. 1996). For
example, in the cerebellum, the subunit was reported to localize
predominantly in extra-synaptic GABAARs
(Nusser et al. 1998). Evoked
IPSPs in granule cells, and other CNS sites, activate both synaptic and
extrasynaptic GABAARs (Mody et
al. 1994; Rossi and Hamann
1998; Williams et al.
1998). Thus deletion of the subunit could affect evoked
IPSP decay rates even if the subunit was located exclusively at
extrasynaptic GABAARs. Indeed, the evoked IPSPs from
–/– mice decayed on the average 50% faster than in +/+ mice.
However, our data show that the spontaneous mIPSCs in granule cells from
subunit knockout mice also have faster decay rates than controls,
although the difference is only 12.5%. Miniature IPSCs measured at the soma of
granule cells result almost exclusively from the release of GABA at synapses
located at or near the soma (Soltesz et
al. 1995). Therefore the subunit-containing
GABAARs likely make a small but significant contribution to normal
synaptic transmission in the dentate gyrus of wild-type mice.
The differences between alphaxalone effects on mIPSCs of subunit
knockouts and wild-type mice provide a physiological substrate for the
decreased behavioral effect of synthetic neuroactive steroids demonstrated in
our previous in vivo studies (Mihalek et
al. 1999). Furthermore, we show that the decreased effect of
alphaxalone seen in electrophysiological recordings is most likely due to
decreased alphaxalone binding at the GABAA receptors rather than a
potential downstream effect on second messengers. By contrast, early studies
on recombinant receptors reported decreased neurosteroid sensitivity of cells
transfected with the subunit (Zhu
et al. 1996). However, our data are consistent with recent reports
that 4-containing GABAARs are more sensitive to
neurosteroids in recombinant expression studies than other subunit
combinations (Brown et al.
2002; Wohlfarth et al.
2002).
The adult rat subunit has a low abundance and is highly
concentrated in a few brain regions, namely, dentate gyrus, cerebellar granule
layer, thalamus, and, to a lesser extent, the cortex
(Persohn et al. 1992). Recent
studies indicate that the distribution of various GABAAR subunits
in the mouse brain closely resembles that of the rat
(Peng et al. 2002;
Tretter et al. 2001). Despite
the low abundance of subunit in wild-type animals, subunit
deletion in knockout mice leads to a considerable decrease in the response of
these mice to neuroactive steroids such as ganaxalone
(Mihalek et al. 1999). This
suggests that the brain regions containing high concentrations of the
subunit may be particularly important to the actions of exogenously
administered neuroactive steroids. However, the decreases in alphaxalone
sensitivity of mIPSCs in brain slices were more robust than the previously
observed decreases in hypnotic potency of alphaxalone in subunit
mutants, suggesting that areas of the brain other than the dentate gyrus
(e.g., cortex) may be more important in mediating the hypnotic effects of
neurosteroids.
There is also indirect evidence that endogenous neurosteroids may play a
role as CNS depressants with anxiolytic and anticonvulsant activity. For
example, it has been shown that the time course of the increase in brain
concentrations of endogenous neurosteroids in response to stress coincides
with the restoration of GABAergic tone that was reduced by stress
(Barbaccia et al. 1998). Also,
kainic acid-induced seizure activity is increased under conditions
characterized by a decline in endogenous levels of the neurosteroid
5-pregnan-3-ol-20-one (Frye
and Bayon 1998). If the actions of endogenous neurosteroids temper
the proconvulsant state via activation of GABAARs, then the
subunit knockouts would be expected to be more susceptible to seizures as a
result of the reduced GABAAR sensitivity to neurosteroids. Provided
that responses to endogenous neurosteroids are attenuated similarly to the
responses of exogenous synthetic neurosteroids, the subunit knockout
mice will be very useful for elucidating the physiological roles of these
compounds.
It must be underscored that our data do not preclude the possibility that
any of the observed changes in the GABAAR function of
subunit knockout mice may be due to compensatory changes rather than reflect
directly the loss of the subunit. It is possible or even likely that
other subunit or subunits may substitute for the subunit, thereby
conferring the kind of kinetic and pharmacological characteristics on the
GABAAR-chloride channel complex observed in our experiments. Some
subunits like 4 and 6 may be especially affected by
subunit loss because they normally appear to co-assemble in the same pentamer
(Huh et al. 1996;
Jechlinger et al. 1998;
Quirk et al. 1995). Indeed,
the 4 subunit was significantly reduced in our study and was
demonstrated to be consistently reduced in specific brain regions that express
the subunit (Peng et al.
2002). This could result in other heteropentamers being elevated
to higher than normal levels in compensation. The –/– mice
were previously observed to have a small decrease in GABA/muscimol binding
sites and an increase in benzodiazepine binding
(Mihalek et al. 1999),
consistent with increased expression of the 2 subunit. Recently, an
upregulation of 2 subunit was demonstrated in cerebellum
(Tretter et al. 2001) and also
in thalamus and hippocampal formation
(Peng et al. 2002) of
subunit knockout mice.
Compensation for the loss of the subunit may be different for
GABAARs in different regions of the brain or even for different
regions of the dentate granule cells. In addition to postsynaptic changes, we
showed that the frequency of mIPSCs is reduced in dentate granule cells,
implying potential presynaptic changes in GABAergic interneurons or reduced
number of GABAergic synapses. In cerebellar granule cells, concurrent loss of
6 and subunits in 6 knockout mice results in a complete
loss of tonic GABAergic inhibition mediated via extrasynaptic
GABAARs (Brickley et al.
2001). Interestingly, excitability of cerebellar granule cells and
motor control does not appear to be affected in 6 knockout mice because
of an adaptive upregulation in activity of a voltage-independent
K+-conductance (Brickley et al.
2001). This implies that tonic background inhibition is important
and perhaps ubiquitously necessary for controlling neuronal excitability. If
so, then it would be of interest to determine if the subunit knockouts
may also exhibit adaptive changes in activity of ion channels other than the
GABAAR-activated Cl– channels.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: I. Spigelman, UCLA School of Dentistry, 10833 Le Conte Ave., 63-050 (CHS), Los Angeles, CA 90095-1668 (E-mail: igor{at}ucla.edu).
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) and
) brain preparations (n = 4 brains from
–/– mice, n = 2 brains from +/+ mice). The lines are fits
to the 4 parameter logistic curves of the form y =
ymin + (ymax –
ymin/1 + 10(log EC50–x)Hillslope), where
y is the [35S]TBPS binding and x is the
alphaxalone concentration. Note the greatly decreased alphaxalone displacement
of [35S]TBPS binding at all concentrations of alphaxalone in the