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The Journal of Neuroscience, June 15, 1999, 19(12):4921-4937
GABAA Receptor Subunit Composition and Functional
Properties of Cl Channels with Differential Sensitivity
to Zolpidem in Embryonic Rat Hippocampal Cells
Dragan
Maric1,
Irina
Maric1,
Xiling
Wen1,
Jean-Marc
Fritschy2,
Werner
Sieghart3,
Jeffery L.
Barker1, and
Ruggero
Serafini1
1 Laboratory of Neurophysiology, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland 20892, 2 Institute of Pharmacology,
University of Zurich, CH-8057 Zurich, Switzerland, and
3 Department of Biochemical Psychiatry, University Clinic
for Psychiatry, A-1090 Vienna, Austria
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ABSTRACT |
Using flow cytometry in conjunction with a voltage-sensitive
fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem.
Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive
(ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells
as postmitotic, differentiating neurons expressing 2, 4, 5,
1, 2, 3, 1, 2, and 3 GABAA receptor
subunits, whereas the ZI cells were neuroepithelial cells or newly
postmitotic neurons, expressing predominantly 4, 5, 1, and
2 subunits. Fluctuation analyses of macroscopic
Cl currents evoked by GABA revealed three kinetic
components of GABAA receptor/Cl
channel activity in both subpopulations. We focused our study on ZI
cells, which exhibited a limited number of subunits and functional
channels, to directly correlate subunit composition with channel
properties. Biophysical analyses of GABA-activated Cl currents in ZI cells revealed two types of
receptor-coupled channel properties: one comprising short-lasting
openings, high affinity for GABA, and low sensitivity to diazepam, and
the other with long-lasting openings, low affinity for GABA, and high
sensitivity to diazepam. Both types of channel activity were found in
the same cell. Channel kinetics were well modeled by fitting dwell time
distributions to biliganded activation and included two open and five
closed states. We propose that short- and long-lasting openings
correspond to GABAA receptor/Cl
channels containing 4 1 2 and 5 1 2 subunits, respectively.
Key words:
GABAA receptors; zolpidem; oxonol; FACS; development; rat; hippocampus
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INTRODUCTION |
Zolpidem is an imidazopyridine
hypnotic sedative that is thought to produce its effects by interaction
with the benzodiazepine binding site on GABAA
receptor/Cl channels (for review, see Sanger et
al., 1994 ). Zolpidem binds to benzodiazepine sites on the
receptor-channel complex with an affinity that depends on subunit
composition (Ruano et al., 1992 ). In the adult rat brain, three
zolpidem binding sites have been correlated with GABAA
receptor subunit expression: a high-affinity site
(Kd, 10-20 nM) on
1-containing receptors, a low-affinity site
(Kd, 200-300 nM) on 2-
and 3-containing receptors, and a very-low-affinity site
(Kd, 4-l0 µM) on
5-containing receptors (McKernan et al., 1991 ; Ruano et al., 1992 ;
Mertens et al., 1993 ).
During embryonic development, GABAA receptor subunits
emerge throughout the rat CNS, with elevated expressions of 2, 3, 4, and 5 subunit transcripts but quite low levels of the 1 subunit mRNA (Laurie et al., 1992 ; Poulter et al., 1992 ). The emergence
of the 1-containing GABAA receptors during the early postnatal period parallels the appearance of fast inhibitory GABAergic transients in hippocampal and cortical regions, whereas the abundance of other subunits before birth is presumed to be involved in morphogenic events. However, the subunit composition and the functional properties of GABAA receptors expressed during
embryogenesis remain largely unexplored. Ma and Barker (1995) have
proposed that 4, 1, and 1 are among the earliest expressed
subunits, with their transcripts emerging among neuroepithelial cells.
3, 3, and 2 subunit transcripts and proteins have been
reported in differentiating neurons of the cortical plate region (Maric
et al., 1997 ). Unitary properties of GABAA
receptor/Cl channels have been investigated in
several areas of the developing brain, and differential localization of
the 2 and 3 subunit mRNA has been correlated to different channel
kinetics in these areas (Serafini et al., 1998a ).
Here, we have used zolpidem to correlate subunit composition with the
properties of native GABAA receptor/Cl
channels expressed by embryonic hippocampal cells. First, we isolated
subpopulations of cells whose functional GABAA receptors exhibited differential sensitivity to zolpidem using a potentiometric dye and a fluorescence-activated cell sorter (FACS). Sorted cells were
then characterized for their neuronal epitope and GABAA
receptor subunit expressions. Finally, biophysical properties of
GABAA receptors/Cl channels in sorted
cells were inferred using fluctuation analysis of macroscopic currents
or measured directly with single-channel recordings. We focused our
study on zolpidem-insensitive cells, which were newly postmitotic
neurons that expressed 4, 5, 1, and 2 subunits. Channel
properties of these cells were correlated with sensitivity to diazepam
and affinity for GABA. Probable subunit compositions of native
receptors were inferred by correlating the observed properties with
those of recombinant receptors expressing known subunit constructs. The
results imply that short-lasting channels may be composed of
4 1 2 subunits, whereas long-lasting channels may include
5 1 2 subunits.
A preliminary report of this work has been presented in abstract form
(Serafini et al., 1996 ).
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MATERIALS AND METHODS |
Cell dissociation
Timed pregnant embryonic day 19 (E19) Sprague Dawley rats
(Taconic Farms, Germantown, NY) were killed by
CO2-induced anoxia. Embryos were placed in PBS, and a
standard atlas (Paxinos et al., 1991 ) was used to determine the
embryonic age by measuring the crown-rump length. Hippocampi were
dissected out and incubated for 45 min at 37°C in Earle's balanced
salt solution containing 20 U/ml papain (Boehringer Mannheim,
Indianapolis, IN), 0.01% DNase (Boehringer Mannheim), and 0.5 mM EDTA (Sigma, St. Louis, MO). After gentle trituration,
the single-cell suspension was washed three times in a physiological
medium (medium A; in mM: 145 NaCl, 5 KCl, 1.8 CaCl2, 0.8 mM MgCl2,
10 HEPES, pH 7.3, and 10 glucose), supplemented with 1 mg/ml fatty
acid-free bovine serum albumin (Sigma).
Fluorescence-activated cell analysis and sorting
At the beginning of an experiment, the cells were resuspended in
a "low-Cl " medium A in which 145 mM NaCl was substituted with an equimolar concentration of
Na isethionate (Sigma). This reduced extracellular Cl to 20 mM, thereby enhancing the
transmembrane Cl ion gradient and thus amplifying
GABA-induced depolarizing responses by shifting the
Cl ion equilibrium potential. Membrane potential
changes were detected using bis-(1,3-dibutyl barbituric acid)
trimethine oxonol (Molecular Probes, Eugene, OR), which equilibrates
with the cell according to its transmembrane potential and reports
potentiometric signals over an ~10-fold dynamic range (Maric et al.,
1998 ). Oxonol fluorescence (FLOX) intensity was
measured using the FACSTAR+ flow cytometer (Becton
Dickinson, Mountain View, CA). Cells were excited using an argon ion
laser (model 2016; Spectra Physics, Mountain View, CA) operated at 500 mW and tuned to 488 nm, whereas the FLOX emission was
detected with a bandpass filter set at 530 ± 30 nm. All
experiments were performed at room temperature. The cell suspensions
were stained with 200 nM oxonol for 2 min, and baseline
FLOX was recorded in 10,000 cells at 2000 cells/sec. The
cells were then stimulated with 1-10 µM GABA, with and
without 25 nM-10 µM zolpidem, and the
resulting FLOX signals were profiled after 2 min.
FLOX distributions were quantified and illustrated as
single-parameter frequency histograms using the Cell Quest data
acquisition and analysis software (Becton Dickinson), with which modes,
coefficients of variation, and peak amplitudes could be calculated.
FLOX profiles under control and experimental conditions were analyzed either by integration of the area under each peak or by
measuring the relative amplitudes of their respective modes or with
cumulative histogram statistics (Kolmorogov-Smirnov), all of which
gave comparable results. Overlays of the control and experimental
FLOX histograms permitted quantitative analysis of the
potentiometric response. The modal values of FLOX
distributions were calibrated in terms of estimated membrane potential
(Maric et al., 1998 ). Cells without detectable zolpidem-modulated
responses to GABA were sorted from cells whose GABAergic depolarization was intensely potentiated by zolpidem using the electronic gates shown
in Figure 1C. Sorted cells were then washed, restained with oxonol,
restimulated with GABA and zolpidem, and reanalyzed to test for sort
fidelity, which was always 95% (Fig. 2). After sorting, the
viability of the cells remained unchanged, with <5% trypan blue- or
propidium iodide-positive (dead) cells in each sorted subpopulation.
Immunocytochemistry
Sorted cells were plated on poly-D-lysine
(Sigma)-coated eight-well glass microscope slides (Nalge Nunck
International, Naperville, IL) for 2 hr at 37°C and then fixed in 4%
paraformaldehyde (Polysciences, Warrington, PA) for 15 min at room
temperature. The developmental expression of antigens characteristic of
precursor cells and differentiating neurons was enumerated by
immunoreacting cells with the following antibodies: rabbit anti-nestin
(a gift from R. McKay, National Institutes of Health, Bethesda, MD), a
mixture of tetanus toxin fragment C (TnTx) and mouse anti-TnTx
(Boehringer Mannheim), and mouse anti-class III -tubulin (TuJ-1;
Babco, Richmond, CA). Cells were immunoreacted with a mixture of 4 µg/ml TnTx and anti-TnTx (1:2000) or double-immunoreacted with
anti-nestin (1:1000) and TuJ-1 (1:5000) antibodies for 1 hr at room
temperature. The immunoreacted epitopes were then visualized by
incubating the cells with appropriate secondary antibodies conjugated
with FITC or rhodamine (Jackson ImmunoResearch, West Grove, PA). The
percent of immunopositive cells was quantified by counting ~400 cells
in four fields using standard fluorescence microscopy (Axiophot; Carl
Zeiss, Thornwood, NY).
The expression of 11 GABAA receptor subunits in the sorted
cells was investigated using specific antibodies generated against the
peptide sequences depicted in parentheses: guinea pig polyclonal antibodies specific for 2 (1-9 residues) and 3 (1-15 residues) subunits (generated by J.-M.F.) and rabbit polyclonal antibodies specific for 1 (1-9 residues), 4 (379-421 residues), 5
(2-10 residues), 1 (350-404 residues), 2 (351-405 residues),
3 (345-408 residues), 1 (324-366 residues), 2 (319-366
residues), and 3 (322-372 residues) subunits (generated by W.S.).
The characterization and specificity of these antibodies have been
described in detail elsewhere (Benke et al., 1991 , 1997 ; Buchstaller et
al., 1991 ; Marksitzer et al., 1993 ; Mertens et al., 1993 ; Mossier et
al., 1994 ; Todd et al., 1996 ; Sperk et al., 1997 ). The cells were
immunoreacted with guinea pig antibodies diluted 1:1000 or rabbit
antibodies diluted 1:100-300 overnight at room temperature. In control
slides, the primary antibodies were substituted with diluent only.
Biotinylated donkey antibodies against appropriate species (Jackson
ImmunoResearch) were used as secondary reagents. All antibodies were
diluted in PBS containing 10% normal rat serum and 10% normal donkey
serum to prevent nonspecific binding. Finally, the cells were reacted with streptavidin-conjugated horseradish peroxidase (Jackson
ImmunoResearch) and developed in 3-amino-9-ethyl carbazole (AEC)
substrate [25 mg of AEC (Sigma) in 100 ml of acetate buffer] with
0.01% H2O2 for 10-15 min at room temperature.
After the slides were coverslipped, ~400 cells in four fields were
imaged using a transmission light microscope connected to the Macintosh
workstation. The percentage of immunopositive cells in each field was
counted using the thresholding function of the NIH Image software.
In some experiments, E19 rat brains were fixed in 4% paraformaldehyde
for 4 hr, cryoprotected in 30% sucrose for several days, and frozen in
liquid nitrogen-cooled isopentane. Twenty-micrometer-thick coronal
sections were cut using a cryostat, dried at room temperature for 1 hr,
and immunostained with different GABAA receptor
subunit-specific antibodies as described above.
PCR amplification of transcripts for GABAA
receptor subunits
A previously established reverse transcription (RT)-PCR protocol
(Somogyi et al., 1995 ) was used to measure GABAA receptor subunit gene expression in sorted cells. Gene-specific primers were
designed from GenBank sequences using the Oligo software (National
Biosciences, Plymouth, MN) as described (Ma et al., 1993 ). Total RNA
was isolated from 2 × 106 sorted cells using
RNAstat 60 (Tel-Test, Friendswood, TX) and the protocol recommended by
the manufacturer. To confirm the purity of the product RNA, absorbtion
ratios at 260:280 nm were determined to be >1.8 for all samples. RT
and PCR were performed in a Perkin-Elmer 9600 thermal cycler using the
Perkin-Elmer GeneAmp RNA PCR kit (Perkin-Elmer, Norwalk, CT). Two
hundred nanograms of total RNA were used for each 100 µl PCR
reaction. The PCR reaction was initiated using a hot start at 90°C to
avoid mispriming. The thermal cycling protocol included a 90 sec
preincubation at 97°C, followed by 35 cycles of 30 sec at 95°C
(dissociation), 45 sec at 60°C (primer annealing), and 60 sec at
72°C (extension). Amplification was within the exponential range. PCR
product identities were confirmed by restriction enzyme digestion. All
RT and PCR reactions contained control RNA (transcribed from PAW 108 plasmid DNA; Applied Biosystems, Foster City, CA) to eliminate
inefficient reactions from the analysis. PCR products were separated on
15-well 8-16% polyacrylamide gradient gels (Novex, San Diego, CA),
using BioMarker low-DNA size standards (Bio Ventures, Murfreesboro, TN)
as a reference. Gels were stained for 30 min with 0.1% ethidium
bromide solution, illuminated on a UV transilluminator, and documented
with black-and-white instant film.
Electrophysiology
Solutions and recordings. Extracellular solution
contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and
10 HEPES-NaOH. Intracellular pipette solution for whole-cell recording contained (in mM): 145 CsCl, 2 MgCl2,
1.1 EGTA, 0.1 CaCl2, 10 HEPES-CsOH, and 5 MgATP.
Osmolarity and pH of all solutions were adjusted to 300-320
mOsm/kg and 7.2-7.3, respectively. Sorted cells were plated on
plastic 35 mm dishes for 1 hr at 37°C in the extracellular
medium. Pipettes made of thin glass with filament (WPI, Sarasota,
FL) were pulled by a computerized BB-CH-PC puller (Mecanex, Geneva,
Switzerland). Whole-cell patch-clamp recordings were performed at room
temperature. Series resistance was <20 M and was 50-80%
compensated. Pipette currents were monitored via a Ag-AgCl wire and
amplified through an Axopatch 200 amplifier (Axon Instruments, Foster
City, CA) in the resistive head stage mode, displayed on a chart
recorder, and recorded on tape for off-line analysis. Drugs were
applied to the cells via low-level pressure from closely positioned
micropipettes. Stock solutions of 1 mM were diluted with
extracellular medium to obtain final drug concentrations used in the experiments.
Quantitative analysis of GABA-activated Cl
currents and channels. Membrane currents were recorded at a low
gain as a DC signal and then amplified, filtered, and stored on tape.
For the analysis, data were played back, high-pass-filtered at 0.1 Hz
through a homemade filter, low-pass-filtered at 1 kHz through an 8-pole Butterworth filter (901 Frequency Devices, Haverhill, MA), and digitized at 2 kHz through a National Instruments (Austin, TX) LAB PC
acquisition board. Data were analyzed by Strathclyde
Electrophysiological Software (University of Strathclyde, Glasgow,
Scotland). Spectral analysis of GABA-evoked Cl
currents was performed as previously reported (Serafini et al., 1995 ,
1998a ,b ). We have facilitated the study of native channel properties by
FACS-sorting subpopulations of embryonic hippocampal cells with minimal
numbers of GABAA receptor/Cl channels
and then using algorithms to analyze openings, which control for
bandwidth limitations and multiple superimposed channels. The low
numbers of channels together with low levels of baseline noise allowed
recordings of single-channel activities at wide bandwidth (0.5-1 kHz).
Quantitative analysis was performed in two different ways. First, we
have selected stretches of recording from each cell containing only a
few superimposed events and performed analysis of dwell time
distributions as previously reported (Kristiansen et al., 1995 ;
Serafini et al., 1995 ). Dwell time distributions were also analyzed
through algorithms derived for recordings with multiple superimposed
events (Kijima and Kijima, 1987 ) and with bandwidth limitations (Hawkes
et al., 1992 ).
The following equations were derived by Kijima and Kijima (1987) to
study patch recordings with multiple channels. The distribution function of dwell time of m open channels on a membrane with
N number of total channels is ZN,m
(t). The corresponding probability density function is
YN,m (t):
Po is the steady-state open probability;
t is the derivative of the function versus time;
ftr is the transition frequency; and
zssh(t) is the probability that the
channel is in any of the shut states and is kept shut until time
t. zsop(t) is the
probability that the channel is in any of the open states and is kept
open until time t. zsop(t)
and zssh(t) are calculated as
follows:
The shut states are 1, ... , L. The open
states are L + 1, ... , Q. The conditional
probability qI',I(t) [or
qJ,I(t)] is the probability that the
channel that is in the open (or shut) state SI'
(Sj) at t = 0 ends up in
the open (or shut) state again SI"
(SI) at the time t without
shutting (or opening) from t = 0 to time t.
However, the actual qI',I"(t) used in
our calculations is the corresponding value for a recording with
interval omission attributable to bandwidth limitations
[ARi(t)] (Hawkes et
al., 1992 ) and is the probability that the channel starting in an open
state at time 0 remains in an open state at time t, without
any detected shut state between 0 and t. The
following equations were derived by Hawkes et al. (1992) . Briefly,
using the same terminology as Hawkes et al. (1992) :
where is the dead time of the system,
ci and ri are the right
and left eigenvectors of
H(si), corresponding to the
root si, which is also an eigenvalue of
H(si).
QAA, QAS,
and QSA are the matrices of transition rates
between open states, closed states, and open and closed states,
respectively; exp(QSSt) has been
calculated through spectral expansion, as indicated by Cohlqhoun and
Hawkes (1977 ). To calculate the probability that a channel, being in the closed state at t = 0, apparently remains in the
closed state at t, A and S were just inverted. Numerical
calculations were performed using a program written by R. Serafini with
Mathematica software (Wolfram, Champaigne, IL).
We have calculated the effects of activation of GABAA
receptor/Cl channels on the cell membrane
potential using Goldman-Hodgkin-Katz (GHK) equation:
where Erev is the reversal potential (or,
in this case, the peak) of the membrane potential responses to GABA,
PNa, PK,
and PCl are Na, K, and Cl permeabilities, and
R, T, and F have their usual meanings.
We assumed that PNa is very low, so that
PNa [Na] PK [K] + PCl [Cl]. If [K]o = 5 mM, [Cl]o = 20 mM, and assuming that [K]i is ~140 mM (Maric et al., 1998 )
and [Cl]i is ~25 mM (Owens et al.,
1996 ), then the GHK equation may be simplified to:
The input resistance of embryonic cells recorded in the
whole-cell mode is ~10 G or more (Mienville et al., 1994 ). This would correspond, according to the constant field equation, to a
permeability of 1.6 × 10 15 M.
Based on these calculations, we estimated that the opening of even a
single Cl ion channel with the unitary conductance
characteristic for GABAA receptor/Cl
channels quantified in these cells (see Figs. 7-9) would pass ~1 pA
and depolarize an intact cell ~10 mV. The potentiometric measurements using flow cytometry have a resolution in the ~5-10 mV range (Maric et al., 1998 ). Therefore, even if the number of functionally expressed Cl channels was very low, the potentiometric
recordings of intact cells make it likely that all the cells whose
channels were activated by GABA and zolpidem could be detected.
In sum, the depolarizing shift induced in intact cells that have high
input resistance will depend on the absolute number of channels whose
gating is determined or enhanced by the ligand(s) and not on the
relative fraction of channels that are sensitive to the modulator.
Zolpidem-sensitive cells, expressing a large number of
zolpidem-sensitive receptor/channels, might also express large numbers
of receptor/channels that are insensitive to the ligand. In fact, the
fraction of zolpidem-sensitive constructs may not necessarily be high
in zolpidem-sensitive cells. Because the opening of individual channels
may provide enough current to depolarize cells, zolpidem-insensitive
cells should be truly deprived of zolpidem-sensitive receptor/channels.
The zolpidem-insensitive population should therefore be composed of (1)
cells with no GABA-evoked response, (2) cells with truly
zolpidem-insensitive responses, and (3) cells with zolpidem-sensitive
receptor/channels at very low density and/or very low
Popen of each individual channel and/or a low
unitary conductance, which together are insufficient to depolarize cells.
Experimental observations are mostly in agreement with these
predictions (see Results). There was a marked potentiation by zolpidem
of the GABA-evoked current in the zolpidem-sensitive cells. However,
RT-PCR of subunit transcripts and subunit immunoreactivity revealed the
presence of high levels of 5 subunits in the zolpidem-sensitive population, implying the coexistence of both zolpidem-sensitive and
-insensitive constructs on these cells. The zolpidem-insensitive cells
indeed corresponded to cells exhibiting voltage-clamp responses with no
sensitivity to zolpidem. In agreement with theoretical considerations,
we also found that not all cells were responding to GABA. However, we
did not find evidence for cells expressing zolpidem-sensitive channels
with low Popen and low conductance.
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RESULTS |
Zolpidem-sensitive and zolpidem-insensitive GABAA
receptor Cl ion channels are expressed by
embryonic hippocampal cells
GABA depolarized hippocampal cells in a dose-dependent manner over
1-10 µM, with 10 µM GABA depolarizing
~85% of the cells close to 20 mV from a resting potential of
approximately 75 mV (Fig.
1A). One micromolar
GABA induced modest but detectable depolarization (~10 mV) in ~25%
of the cells. Zolpidem was added to 1 µM GABA to test for
potentiating effects. Twenty-five nanomolar zolpidem had no effect,
whereas 500 nM depolarized ~52% and 10 µM
depolarized 67% of the cells, with 20% depolarizing close to 0 mV
(Fig. 1B). Higher zolpidem concentrations had no
further effects (data not shown).

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Figure 1.
Zolpidem potentiates GABA-induced depolarization
in many embryonic hippocampal cells. Data are frequency histograms of
the oxonol fluorescence (FLOX) intensity
distribution of 10,000 cells compiled in 5 sec using flow cytometry.
Potentiometric profiles of FLOX were acquired under resting
conditions and at the peak of the response after exposure to GABA, with
and without different concentrations of zolpidem (ZOLP).
Gramicidin (GRAM) was added afterward to reveal
the FLOX distribution equivalent to 0 mV. A,
Within 2 min of stimulation, 1 µM GABA induces a modest
depolarization in 25-30% of the cells, whereas 10 µM
GABA depolarizes ~85% of the cells, with a well defined
FLOX mode corresponding to 20 mV. B,
Inclusion of 25 nM zolpidem with 1 µM GABA
does not alter the cells depolarized by GABA, whereas additions of 500 nm and 10 µM zolpidem depolarize ~52 and ~67% of the
cells, respectively, with ~20% of maximally potentiated cells
depolarizing to ~0 mV (open arrow). C,
The FLOX distribution of cells in the presence of 1 µM GABA and 10 µM zolpidem is displayed to
illustrate the sorting gates (shaded areas) used to
separate cells without zolpidem modulation (Zolpidem Insensitive
Sort) from cells with maximal zolpidem potentiation
(Zolpidem Sensitive Sort).
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Cells whose depolarizing responses to GABA were insensitive to 10 µM zolpidem and cells with GABA-induced depolarizations to ~0 mV in zolpidem were physically sorted with the
FACSTAR+ flow cytometer using the electronic gates
shown in Figure 1C. GABA-induced depolarizations of sorted
zolpidem-insensitive cells were not affected by 10 µM
zolpidem (Fig. 2A). All
sorted zolpidem-sensitive cells exhibited GABA-induced depolarizations
that were potentiated by zolpidem (Fig. 2B),
testifying to the high fidelity of the sort. Because the
zolpidem-insensitive population also contained cells without
depolarizing responses to 1 µM GABA, we added 10 µM GABA to discover the size of the population expressing
functional GABAA receptors and found that ~60% responded
(Fig. 2C). As expected, >97% of the cells in the
zolpidem-sensitive subpopulation depolarized to 10 µM
GABA (Fig. 2D). Depolarizing responses to GABA in
both sorted subpopulations were Cl ion-dependent
and completely blocked by preincubation of cells with 50 µM bicuculline (data not shown), identifying the
involvement of functional GABAA
receptor/Cl channels in both cell types.

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Figure 2.
Reanalysis of zolpidem-sorted cells reveals
high fidelity in the sorting. Potentiometry was performed as outlined
in Figure 1. A, 1 µM GABA induces a
just-detectable depolarization of zolpidem-insensitive cells that is
not affected by 10 µM zolpidem (ZOLP).
B, 1 µM GABA evokes a moderate
depolarization of zolpidem-sensitive cells, all of which depolarize
further in the presence of 10 µM zolpidem.
C, 10 µM GABA depolarizes ~60% of the
zolpidem-insensitive population. D, 10 µM
GABA depolarizes >97% of the zolpidem-sensitive cells, with a well
defined FLOX mode corresponding to approximately
20 mV. GRAM, Gramicidin.
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Zolpidem-sensitive cells are differentiating neurons, whereas
zolpidem-insensitive cells are undifferentiated, immature cells
Sorted cells were immunostained for specific epitopes. Nestin, an
intermediate filament protein of neuroepithelium-derived progenitor
cells (Hockfield and McKay, 1985 ), labeled 75% of the zolpidem-insensitive cells and only 3% of the zolpidem-sensitive cells
(Fig. 3A). TnTx, a marker of
postmitotic, differentiating embryonic neurons (Koulakoff et al., 1983 ;
Maric et al., 1997 ), labeled only 5% of the zolpidem-insensitive cells
and ~90% of the zolpidem-sensitive cells. Thus, the
zolpidem-insensitive population was almost entirely composed of
relatively immature, undifferentiated cells, whereas the great majority
of zolpidem-sensitive cells were differentiating neurons.
Immunostaining with the TuJ-1 antibody, which reacts with
neuron-specific tubulin III cytoskeletal protein (Lee et al., 1990 ;
Menezes and Luskin, 1994 ), confirmed the neuronal phenotype of
zolpidem-sensitive cells, because >90% of these cells were
TuJ-1-positive. However, ~35% of the zolpidem-insensitive cells were
also TuJ-1+, indicating the presence of newly
committed neurons in this subpopulation. Double labeling with
anti-nestin and anti-TuJ-1 antibodies demonstrated many double-positive
cells in the zolpidem-insensitive subpopulation (Fig. 3B),
consistent with the relative immaturity of zolpidem-insensitive TuJ-1+ neurons. In contrast,
TuJ-1+ neurons in the zolpidem-sensitive population
were almost all nestin , characteristic of a more
mature stage in neuronal differentiation. Morphologically,
zolpidem-sensitive cells were visibly larger in diameter, and most
exhibited processes, whereas zolpidem-insensitive cells were mostly
spherical and smaller in diameter with few or no processes (Fig.
3B). Some of the heavily labeled TuJ-1 cells in the
zolpidem-insensitive subpopulation exhibited processes, consistent with
their neuronal lineage.

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Figure 3.
Zolpidem-sensitive cells are differentiating
neurons, whereas the zolpidem-insensitive population mostly comprises
immature cells. After sorting, zolpidem-sensitive and
zolpidem-insensitive cells were plated and immunostained with
antibodies against nestin, TnTx binding sites and TuJ-1.
A, Virtually all of the zolpidem-sensitive cells are
nestin , TnTx+, and
TuJ-1+, whereas the zolpidem-insensitive cells are
predominantly nestin+ and TnTx ,
with a fraction (~35%) of them also expressing TuJ-1.
B, Double immunostaining with anti-nestin
(green) and TuJ-1 (red) antibodies
demonstrates the abundance of double-positive young neurons
(yellow, arrows) in the zolpidem-insensitive
(ZI) but not in the zolpidem-sensitive
(ZS) population. Data in A represent
mean ± SE for three to five independent determinations.
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Contrasting GABAA receptor subunit expressions of
zolpidem-sorted cells
The expressions of 11 different GABAA receptor
subunits at protein and transcript levels in sorted cells were
investigated using immunocytochemistry and RT-PCR. In the
zolpidem-sensitive population, ~70-90% of cells immunostained with
antibodies against the 4, 5, 1, 2, 3, 2, and 3
subunits (Fig. 4A).
Approximately 40% of the cells were 2+, and 25%
were 1+. The 3 subunit was expressed in
<10%, whereas the 1 subunit was not detected. Thus,
zolpidem-sensitive neurons expressed a wide variety of subunits, which
may form different GABAA receptor constructs on the same
cell.

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Figure 4.
Immunocytochemical and RT-PCR analyses of 11 GABAA receptor subunits reveal different expression
patterns in zolpidem-sensitive and zolpidem-insensitive populations.
A, Eleven GABAA receptor subunit proteins in
sorted populations were quantified immunocytochemically in terms of
percent (mean ± SD) immunopositive cells. More than 70% of the
zolpidem-sensitive cells immunostain for either 4, 5, 1, 2,
3, 2, or 3 subunits, whereas ~40% are
2+ and ~25% are 1+. Less
than 10% are 3+, and none express 1. In
contrast, only four subunits are well expressed in the
zolpidem-insensitive population: 4, 5, 1, and 2.
B, Ethidium bromide-stained polyacrylamide gels of
RT-PCR products derived from the total RNA of the same number of
zolpidem-sensitive and -insensitive cells are displayed together with
the 149 bp product of amplified control RNA, which serves as an
internal standard. The most intensely stained PCR products among
zolpidem-sensitive cells correspond to the subunits expressed by the
highest percentages of cells (see A). Fainter bands of
other products parallel their more restricted cellular distributions.
The 1 transcript in zolpidem-sensitive cells can be detected in the
absence of the protein. None of the bands derived from RT-PCR of
zolpidem-insensitive cells is as intense as those found in analysis of
zolpidem-sensitive cells, and the most intensely stained bands
correspond to the four subunits, which are detected at the protein
level in 40-50% of the cells. The 1 transcripts are not detected
in zolpidem-insensitive cells. Note that the bands of 1-5 and 1
transcripts correspond to DNA size standards in the left-most
lane of each panel, whereas the bands of 2, 3, and
1-3 transcripts correspond to DNA size standards in the
right-most lane of each panel.
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In contrast, 40-50% of the cells in the zolpidem-insensitive
population immunostained for 4, 5, 1, or 2 subunits (Fig. 4A). The 2, 2, 1, and 3 subunits were
present in 5% of the cells, whereas the 1, 3, and 3
subunits were virtually undetected. Because the potentiometric
experiments (Fig. 2C) demonstrated that up to 60% of these
cells were depolarized by GABA, most of these cells likely express or
coexpress either 4 1 2 and/or 5 1 2 subunit constructs.
RT-PCR analysis revealed an abundance of subunit transcripts in
zolpidem-sensitive cells with polyacrylamide gels showing high-intensity bands for 4, 5, 1, 2, 3, 2, and 3
(Fig. 4B), paralleling the subunit proteins most
widely expressed (Fig. 4A). Low-intensity bands for
4, 5, 1, and 2 were the most evident in the
zolpidem-insensitive cells, corresponding to those proteins detected by
immunocytochemistry. 1 subunit transcripts were detected only in the
zolpidem-sensitive population, although 1 protein was not.
In immunocytochemical studies of intact hippocampal sections we
verified the presence of the expressed subunit proteins (results to be
published elsewhere). The 1 and 3 subunits were not detected, whereas the 2 and 3 subunits were present only in differentiating regions. All remaining subunits were detected in both proliferative and
differentiating regions.
Cl currents evoked by GABA in sorted
subpopulations differ in sensitivity to zolpidem and absolute
density
We performed patch-clamp recordings of sorted cells within hours
of plating to characterize GABAA
receptor/Cl channel properties. GABA evoked inward
current responses in all 22 zolpidem-sensitive neurons (Fig.
5A; n = 5 sorting experiments; mean ± SE of interexperiment variability,
92 ± 10%). The currents reversed polarity at the equilibrium
potential for Cl , which was ~0 mV (data not
shown). The peak amplitude of the current response evoked by 2 µM GABA averaged 186 ± 49 pA (n = 6). Clear enhancement of the current response to GABA by 5-10 µM zolpidem was found in all six cells tested, without
significant differences between the effects of 5 and 10 µM zolpidem (Fig. 5A1). Zolpidem potentiation
of the GABA-evoked current response averaged 284 ± 52% of
control. In zolpidem-insensitive cells, inward current responses to 2 µM GABA were highly variable in amplitude and decay,
reversed at ECl (data not shown), and were detected in 115 of 143 cells tested (Fig. 5B; n = 13 sorting
experiments; mean ± SE of interexperiment variability, 85 ± 6%). The current response in some cells did not involve sufficiently
sustained summation of channel openings to generate a steady current.
Instead, unitary channel activity could be recorded in whole-cell mode at DC to ~1 kHz bandwidth (see below). When sustained, peak currents in zolpidem-insensitive cells averaged only 18.8 ± 4.8 pA
(n = 74) and 36.5 ± 20.3 pA (n = 17) for responses evoked by 2 and 10 µM GABA,
respectively. As expected, zolpidem (5-10 µM) had little, if any, detectable effects on the peak amplitudes of
GABA-evoked responses (115 ± 9% of control) in these cells (Fig.
5B).

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Figure 5.
GABA-evoked Cl currents in
sorted cells are differentially sensitive to zolpidem and diazepam.
After sorting, cells were cultured for ~2 hr and then recorded in
whole-cell mode and voltage-clamped at 60 mV before application of
GABA, with and without zolpidem or diazepam. A, GABA
evokes Cl currents of ~150 and ~170 pA in two
different cells, which are reversibly potentiated approximately
two-fold by zolpidem (A1) and by diazepam
(A2). B, GABA evokes a low-amplitude
current (<10 pA), which is not affected by zolpidem. C,
FACS potentiometry reveals the presence of a subpopulation whose
GABA-induced depolarization is zolpidem
(ZOLP)-insensitive but is potentiated by diazepam
(DZP), with some cells depolarizing to 0 mV, which is
identified using gramicidin (GRAM).
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The ~10-fold greater amplitude of Cl currents
evoked by 2 µM GABA in zolpidem-sensitive neurons could
be related in part to differences in cell size. Capacitance values were
used to index cell size. They averaged 4 ± 1 pF in four
zolpidem-insensitive cells and 9 ± 2 pF in four
zolpidem-sensitive neurons. Thus, zolpidem-sensitive neurons exhibited
approximately twice the surface area, which is consistent with their
larger cell diameters and the presence of processes. These results
also demonstrate that zolpidem-sensitive neurons expressed a fivefold
greater density of macroscopic GABA-activated Cl current.
Sorted subpopulations were also tested for their sensitivity to the
benzodiazepine diazepam. Five micromolar diazepam potentiated responses to 2 µM GABA in three zolpidem-sensitive cells
by 291 ± 42% (Fig. 5A2), mimicking the effects of
zolpidem. Diazepam-induced potentiation was also present among
zolpidem-insensitive cells, ranging from no effect to up to 300%. On
average, the GABA-evoked current in diazepam-sensitive cells was
increased to 212 ± 31% of control (n = 9 using 2 µM GABA; n = 2 using 10 µM
GABA). Potentiometric measurements confirmed the presence of
diazepam-modulated depolarizing responses to GABA among cells in the
zolpidem-insensitive subpopulation (Fig. 5C). Thus, some
zolpidem-insensitive cells exhibited GABAA receptor/Cl channels that were sensitive to diazepam.
Spectral analysis of Cl current responses
evoked by GABA reveals complex channel kinetics
We used fluctuation analysis techniques to estimate the unitary
properties of GABA-activated Cl channels
underlying sum-mated current responses. Three components of
exponentially distributed activity accounted for all of the variance in
the signals (summarized in Table 1). On
average, there was detectably more (~62 vs 48%) contribution of the
low-frequency (LF) component to current fluctuations induced by GABA on
zolpidem-sensitive cells, which exhibited ~25% shorter time
constants associated with this component. In contrast, there was
considerably more contribution of intermediate-frequency (IF) activity
(37 vs 22%) to GABA-induced currents evoked on zolpidem-insensitive
cells, which exhibited a time constant similar to that inferred on
zolpidem-sensitive cells. The residual, high-frequency (HF)
contribution (~15% of the variance) was similar in both
subpopulations, as was the estimated elementary conductance (~17-19
pS). More detailed analyses were performed on currents evoked by GABA
in zolpidem-insensitive cells, because these cells only expressed 4,
5, 1, and 2 subunits, thus increasing the possibility of
correlating channel properties with subunit expression.
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Table 1.
Elementary GABAA receptor/Cl
channel properties and relative contributions estimated from spectral
analyses of GABA-induced Cl currents in
zolpidem-sensitive and zolpidem-insensitive cells
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Heterogenous GABAA receptor/Cl
channel properties in zolpidem-insensitive cells
There were no significant differences between estimated time
constant or unitary conductance values for Cl
channels underlying current responses evoked by 2 and 10 µM GABA in five cells (data not shown). On average, the
LF component in spectral density plots accounted for ~50-60%, the
IF activity accounted for ~20-30%, and the HF fluctuations
accounted for the remaining ~10-20% of the total variance (Fig.
6A3,B3,C3,D3). Two distinctive sets of channel properties were quantified. The power density spectra of some cells were characterized by a relative abundance of power in the IF range (Fig. 6A3,B3),
indicating the presence of one or more populations of exponentially
distributed events with intermediate durations (~2-32 msec). The
time constant of the LF component ( LF) ranged
from 50 to 150 msec, whereas IF values were 3-8 msec,
and HF estimates ranged over 0.2-0.6 msec. Cells with a
pronounced contribution of the IF component (e.g., Fig.
6A3,B3) exhibited a relatively modest increase in current amplitude (40-200%) when 100 µM GABA was
applied (Fig. 6A1,A2), whereas those with less IF but
proportionally more LF activity (e.g., Fig. 6C3,D3)
exhibited a dramatic ~7-8 fold increase in response to 100 µM GABA (Fig. 6C1,C2). The relative increase in current amplitude evoked by 100 µM GABA
(I100
µM/I10
µM) correlated directly with the relative
contribution of the LF component and inversely with that of the IF
component (Table 2). Similarly, diazepam
potentiation of GABA-evoked current correlated directly with the
relative area of the LF component and inversely with the contribution
of the IF component (Fig. 6B1,B2,D1,D2, Table 2).

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Figure 6.
Heterogeneity of Cl current
responses to GABA, with and without diazepam, and GABAA
receptor/Cl channel properties among sorted
zolpidem-insensitive cells. Four different cells in the
zolpidem-insensitive population were clamped at 60 mV and then
exposed to GABA, with and without diazepam (DZP).
A1, A2, B1, B2, 10 µM GABA activates
low-amplitude, fading currents (~20 pA) that are modestly enhanced in
peak amplitude by 100 µM GABA (A2) or 1 µM DZP (B2). C1, C2, D1,
D2, 10 µM GABA evokes larger-amplitude currents,
which are more sustained and associated with dramatically more current
enhancement when exposed to 100 µM GABA
(C2) or 1 µM DZP (D2).
A3, B3, C3, D3, Spectral density plots calculated for
the fluctuations triggered by GABA in each cell show that power is
exponentially distributed among three components whose corner
frequencies (fc) are
identified with downward arrowheads. There is a relative
abundance of power distributed in the IF range (A3, B3),
which accounts for 50% of the variance, considerably more than
in C3 and D3 (~30% contribution).
Conversely, the LF component accounts for ~30% of the variance in
A3 and B3 but ~70% in
C3 and D3. The
fc values indicate estimated
LF values of ~199 msec (A3), ~159
msec (B3), ~80 msec (C3), and ~72
msec (D3) and IF values of ~4 msec
(A3), 5.3 msec (B3), 3.9 msec
(C3), and 3.7 msec (D3). The
corresponding estimates for the high-frequency components are 0.2-0.5
msec.
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Table 2.
Dose-dependent increase and diazepam-induced potentiation
of GABA-evoked Cl currents correlate with the relative
contributions of different components rather than with estimated
kinetics and conductance
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These results reveal at least two distinct types of channel activity:
one with a pronounced contribution of events intermediate in duration,
which are nearly saturated at 10 µM GABA
(EC50, <10 µM) and relatively
insensitive to diazepam; and the other with a prevalence of
low-frequency (long-lasting) events (<5 Hz), an EC50 >10
µM, and an ~200% increase in amplitude promoted by diazepam.
Elementary properties of unitary GABAA
receptor/Cl channels recorded whole-cell
GABA evoked single channel levels of activity in a subpopulation
of zolpidem-insensitive cells (Fig. 7).
Most of these recordings revealed approximately two to five channels
having the same main conductance state (~26-30 pS). Kinetic analyses
were performed on stretches with predominantly single-channel levels of
activity. Open-time distributions of individual cells (derived from
180-6167 transitions) indicated at least two components (S, short; and L, long) with values of ~1 msec ( S) and
3-18 msec ( L), respectively (Fig. 7). In five of
12 cells exposed to 2 µM GABA, L was <5 msec (4.7, 1.6, 2.0, 2.6, and 3.2 msec), whereas in four others L was >7 msec (8.1, 10.1, 8.0, 7.3, and 7.4 msec), and
in the remaining three L exhibited intermediate values
(6.7, 5.6, and 5.7 msec). In three cells exposed to 10 µM
GABA, L was >7 msec (9.7, 13.0, and 13.2 msec), whereas
in the remaining three it was <7 msec (4.5, 3.0, and 3.9 msec). In two
cells exposed to 20 µM GABA, L was 4.9 and
4.7 msec. Arbitrarily, we have identified openings whose
L values were 5 msec as "fast channels" and those with L of >7 msec as "slow channels."

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Figure 7.
Single-channel activities recorded in the
whole-cell configuration. A, B, Stretches of
single-channel activity recorded in the whole-cell configuration (at
500 Hz bandwidth) in cells clamped at 60 mV in response to 10 µM GABA. Open-time, closed-time, and burst length
distributions obtained from the recordings in A and
B are shown in the bottom panels. Kinetic
analyses were performed on the desensitized state and/or on those
stretches of openings with few multiple superimposed events.
A, The open-time distribution of the channel activity
(2144 events) is fitted by two components with values of 0.39 ± 0.09 msec (77 ± 9%) and 4.5 ± 0.14 msec (23 ± 1%). The closed-time distribution (1730 events) can be fitted by three
components with values of 0.28 ± 0.04 msec (80 ± 10%),
3.6 ± 0.08 msec (12 ± 2%), and 107 ± 0.1 msec
(8 ± 2%). The burst length distribution (597 events) can be
fitted by two components with values of 0.89 ± 0.14 (53 ± 20%) and 23.98 ± 0.09 (48 ± 9%). B, The
open-time distribution (819 events) contains two components with values of 0.81 ± 0.16 msec (75 ± 30%) and 12.98 ± 0.30 msec (25 ± 16%). The closed-time distribution (575 events)
can be fitted by two components with values of 1.15 ± 0.1 msec (58 ± 15%) and 89.2 ± 0.09 msec (42 ± 8%). The
burst length distribution (597 events) comprises three components with
values of 0.29 ± 0.22 msec (91 ± 86%), 4.13 ± 0.42 msec (7 ± 7%), and 108.6 ± 1.1 msec (2 ± 5%).
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Closed-time distributions (derived from 125-2076 events) could be
fitted by three components with values of ~1, 5-10, and 30-400
msec (Fig. 7). A clear pattern of closed-time distributions was evident
in cells exposed to 2 µM GABA. Fast channels
exhibited L values of <30 msec (14, 29.3, 7.7, 6.1, and
10.4 msec), whereas slow channels manifested exceptionally long
L values (138.1, 104, 38.7, 39.6, and 153.6 msec).
However, when 10 µM GABA was used, these differences
between fast and slow channels disappeared, and L was
always >87 msec.
We arbitrarily defined bursts of channel activity as groups of openings
clustered by closures shorter than 10 msec. Clear differences in
kinetics were recorded at 10 µM GABA, with fast channels
exhibiting only short-lasting burst length durations (23.9, 20.4, and
22.7 msec) and slow channels only expressing long-lasting ones (75.1, 109, and 74.2 msec; Fig. 7). A few recordings suggested that both types
of channel activity might be present on the same cell (data not shown).
In contrast, no clear differences between the two types of channel
activities were evident when 2 µM GABA was used;
L was always <25 msec. Although the time constants
defined by the curve fitting and analysis are expected to be distorted
by the presence of multiple channels, we resolved at least two distinct
channel activities. Two channel activities were also detected from the
cumulative probability distribution of the L of the open
times, which exhibited distinct plateaus at ~3 and ~9 msec (data
not shown). Channels whose activity resulted in an open-time
L of 3 msec exhibited a burst length distribution with a
long component (20-30 msec) together with a very high frequency of
openings. The upper plateau of the probability distribution of the
open-time L indicated the presence of a second, distinct channel activity with markedly different properties.
Kinetic analyses of multiple slow and fast channel activities
recorded at limited bandwidth
Because the groups of openings characterized by the cluster
analysis were likely to correspond to distinct channel activities, we
quantified their kinetics. For the fast channels, we analyzed only
activity in response to 2 µM GABA, because it rapidly
faded with 10 µM GABA. In contrast, desensitization was
slow or absent in slow channels, even when 10 µM GABA was
used, and there was no apparent time-dependent drift in the
Popen. We estimated the number of channels
(N) in the cell using three different methods: (1)
evaluation of the maximal number of channels observed in the cell, (2)
jackknife, and (3) bayesian GC estimator (Horn, 1991 ). The three
methods gave similar results. In some cells, GC gave a slightly higher
estimate [n = 5 of (1) vs n = 6 of
(3)]. We used the latter method because (1) is the biased estimate of
N. We collected distributions of the dwell times
corresponding to different current levels (Figs.
8, 9),
which indicated biliganded activation with at least two open and three
closed states (Fig. 8B). Other nonconducting states
connected to the closed-liganded state would prolong the duration of
the activated states (Jones and Westbrook, 1996 ). We found that five
closed states and two open states were actually required to fit the
spectral properties. To simplify calculations, we did not take into
account slowly developing "nonconducting" desensitized states. For
each vector of kinetic parameters tested, we calculated the expected
probability density functions (pdf) at different current levels. The
curves of the pdf fitting distribution histograms were calculated for the omission of intervals shorter than the dead time
(td = 0.4 msec). The pdf of an individual
channel is a piecewise function of intervals, which are multiples of
td. The pdf of each interval is the sum of
exponentials with coefficients represented by polynomials. The
complexity of calculations increases for intervals longer than
td. For t
td, an approximate solution can be used.
To simplify, we have limited the evaluations of the approximate
solution to t td. Therefore, the
fitting curve of the estimated pdf is likely to adequately interpolate
the dwell time distributions only at time intervals at least three to
four times longer than the dead time, and therefore the quickest rate
constants may possibly have less accurate estimates. The parameters
finally chosen provided a good fit of dwell time distributions in
different cells of the same hierarchical cluster (Figs.
8A2-A4,C2-C5, 9A2-A5,B2-B5). For the
fast channels, we estimated k1 = 13 × 106 mol 1
sec 1; k1a = 35 × 106 mol 1
sec 1; k2 = 125 sec 1; k2a = 180 sec 1; 1 = 982 sec 1; 1 = 1080 sec 1; 2 = 725 sec 1; 2 = 155 sec 1; '1 = 600 sec 1; '1 = 80 sec 1; '2 = 705 sec 1; and '2 = 95 sec 1. For the slow channels, we
estimated k1= 1.05 × 106 mol 1
s 1; k1a = 50 × 106 mol 1
s 1; k2 = 202 sec 1; k2a = 220 sec 1; 1 = 3480 sec 1; 1 = 3780 sec 1; 2 = 385 sec 1; 2 = 46 sec 1; '1 = 500 sec 1; '1 = 33 sec 1; '2 = 255 sec 1; and '2 = 380 sec 1. t values of the longest lasting
component of the closed-time distribution were sensitive to the number
of channels estimated and to the product [GABA]
k1. In contrast, the weight of the latter component was mainly affected by the values of
k2 and k2a. Therefore, the estimated values for these parameters were specifically linked to
the features of the observed dwell time distribution. The absence of
desensitization with 10 µM GABA in the slow channels
allowed us to test the consistency of the estimated values at different concentrations. The latter kinetic parameters predict Lorentzians with
corner frequencies of 30 and 1.5 Hz for fast and slow channels, respectively, which for slow channels predict spectra similar to those
actually calculated in many recordings. Furthermore, the parameters
also account for the relative abundance of power in the 10-50 Hz
frequency range characteristic of fast channels, but they do not
describe adequately the LF component. The LF component might be
explained by the coexistence of slow and fast channels on the same cell
or by the clustering of openings between desensitized states of long
durations. Finally, estimated kinetic parameters predicted
EC50 values of ~4 and ~40 µM for the fast
and slow channels, respectively, which are consistent with the results.
As recently reported (Jones et al., 1998 ), on-rate binding constants
were below the diffusion limits (10 8
M 1 sec 1).

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Figure 8.
Kinetic analysis of multiple slow channel
activities recorded at limited bandwidth. Current activities induced by
2 and 10 µM GABA on the same cell are shown in
A1 and C1, respectively.
A1, 2 µM GABA triggers activity with long
closed intervals that are interrupted by many brief openings. Only a
few of the openings superimpose. C1, 10 µM
GABA induces intense activity (5 openings superimpose from time to
time), and only short times are spent in the closed state.
B, Kinetic scheme formulated to calculate theoretical
pdf to fit dwell time distributions. A2-A4, Dwell time
distributions corresponding to level 0 (closed state;
A2), level 1 (one channel open at a time;
A3), and level 2 [two channels open at a time; A4]. Histograms illustrate
the prevalence of the events in levels 0 and 1. Lines
superimposed on the frequency histograms define the theoretical pdf
expected for the rate constants reported in Results.
C2-C5, Dwell time distributions corresponding to level
0 (closed state; C2), level 1 (one channel open at a
time; C3), level 2 (two channels open at a time;
C4), and level 3 (three channels open at a time;
C5). Histograms illustrate the prevalence of events in
levels 1-3. D, Dose-response plot corresponding to the
same kinetic scheme as in B and rate constant values
fitting the dwell time distributions of A and
C. The model predicts an EC50 of ~30-40
mM GABA and maximal Popen of
~0.8. The current increase between 10 and 100 µM GABA
is approximately sevenfold. E, Theoretical spectral
density plot expected for scheme B and calculated for
rate constants fitting dwell time distributions of
A and C. Dots
correspond to the spectral density derived from actual current
fluctuations. The model closely approximates the observed experimental
data.
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Figure 9.
Kinetic analysis of multiple fast channel
activities recorded at limited bandwidth. Current activity evoked by 2 mM GABA in two cells is shown in A1 and
B1. Both recordings exhibit many characteristic
short-lasting openings. A2-A5, B2-B5, Dwell time
distributions corresponding to level 0 (closed state;
A2, B2), level 1 (one channel open at a
time; A3, B3), level 2 (two channels open
at a time; A4, B4), and level 3 (three channels open at a time; A5, B5).
Histograms illustrate the prevalence of events in the levels 0 and 1. Lines superimposed on frequency histograms define the
theoretical expected for the rate constants reported in Results. Scheme
and rate constants are identical for both recordings. C,
Dose-response plot corresponding to the same kinetic scheme (as in
B) and rate constant values fitting dwell time
distributions of A and B. The model
predicts an EC50 of 5 µM GABA and maximal
Popen of ~0.35. The current increase
between 10 and 100 µM is ~40%. D,
Theoretical spectral density plot (bottom continuous
line). The top continuous line outlines the sum
of long- and short-lasting components (290 slow and 950 fast channels).
Dots correspond to the spectral density derived from
actual current fluctuations. The model provides an adequate account for
the relative abundance of power in the 10-50 Hz range observed in many
spectra. The LF (0.5-10 Hz) component in the spectrum calculated from
actual current fluctuations could be explained by the coexistence of
the two types of channel kinetics activated in the same cell.
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Interestingly, isomerization rate constants ( and ) were larger
than those related to the binding step
(k1, k2,
k1a, and k2a). In a simple kinetic scheme with
monoliganded activation, variance analysis underestimates the unitary
amplitude by a factor equal to the fraction of time the activated
channel is in the open state. In both the slow and fast channels, this
value is ~60%. If a similar effect is assumed for the biliganded
kinetic scheme, then, for the 27 pS channel, the unitary conductance
inferred by variance analysis should be ~18 pS, which is in excellent
agreement with the estimates from variance analysis (Table
1).
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DISCUSSION |
Salient findings
Embryonic hippocampal cells were sorted according to the zolpidem
sensitivity of their functional GABAA
receptor/Cl channels. Zolpidem-sensitive cells
were differentiating neurons, whereas zolpidem-insensitive cells were
immature or newly committed neurons. More than 70% of the
zolpidem-sensitive neurons expressed 4, 5, 1, 2, 3,
2, or 3 subunits, whereas ~50% of the zolpidem-insensitive cells exhibited 4, 5, 1, or 2 subunits. GABA activated
Cl ion channels with the same unitary conductance
but variable, yet stereotypical patterns of kinetics in both
populations. Zolpidem-insensitive cells expressed minimal to moderate
numbers of channels whose activation by GABA and modulation by diazepam
correlated directly with short- or long-lasting openings. These
biophysical and pharmacological properties resemble those reported for
specific constructs expressed in recombinant studies. Collectively, the
results lead us to conclude that 4, 1, and 2 subunits may
compose short-lasting channels, whereas 5, 1, and 2 subunits
may compose long-lasting channels.
Zolpidem-sorted subpopulations express different GABAA
receptor subunits
Cells from the E19 embryonic hippocampus were sorted according to
the sensitivity of their GABAA
receptor/Cl channels to zolpidem. The
zolpidem-sensitive cells were larger in diameter, process-bearing,
nestin , and TnTx+, indicative
of differentiating neurons. The zolpidem-insensitive cells were
smaller, spherical, nestin+, and
TnTx , characteristics of undifferentiated cells.
TuJ-1 antibody immunostained almost all zolpidem-sensitive cells,
confirming their neuronal phenotype (Menezes and Luskin, 1994 ).
Approximately 35% of the zolpidem-insensitive cells were
TuJ-1+, half of them costaining with nestin, thus
identifying them as newly postmitotic neurons. More cells depolarized
to GABA (~60%) than were TuJ-1+ (~35%),
indicating that some cells not yet expressing this neuronal epitope
already have functional GABAA receptors, which could
modulate proliferation (LoTurco et al., 1995 ).
Subunit expression patterns among sorted cells differed. Most of the
zolpidem-sensitive neurons immunostained with most of the
subunit-specific antibodies, indicating the probable coexistence of
receptor/channel constructs comprising different subunit combinations in the majority of these neurons. Most of the zolpidem-insensitive cells expressed only 4, 5, 1, and 2 subunits. These cells exhibited GABAA receptor/Cl channels
that were insensitive to zolpidem, despite the putative presence of a
functional 5 subunit. It is possible that the absence of other
subunits (such as 2, 3, or 3) accounted for this
insensitivity. Our findings are similar to those of Mertens et al.
(1993) , who showed that zolpidem displayed striking differences in
affinity for constructs when the 5 subunit was coexpressed with
different and subunit combinations, indicating a role of other
subunits in determining zolpidem affinity.
RT-PCR analysis of sorted subpopulations was in general agreement with
the results of immunocytochemical studies, demonstrating marked
abundance of GABAA receptor subunit mRNAs in
zolpidem-sensitive neurons compared with zolpidem-insensitive cells.
PCR amplification also revealed several subunit transcripts in the
zolpidem-insensitive population that were not detected using
immunocytochemistry, again suggesting that these cells are more
immature, with transcript synthesis preceding expression of the encoded
subunit proteins. This was observed as well for 1 subunit in the
zolpidem-sensitive population, which was expressed at transcript but
not protein levels.
Short- and long-lasting channels in zolpidem-insensitive cells may
correspond to the expression of 4 1 2 and 5 1 2 subunit
constructs
Zolpidem-insensitive cells expressed at least two types of channel
activity: (1) fast, with short clusters of openings, high affinity for
GABA, and modest potentiation by diazepam; and (2) slow, with long
clusters of openings, low affinity for GABA, and severalfold
potentiation by diazepam. Single-channel activity recorded whole-cell
revealed the coexistence of short- and long-lasting openings on the
same cell. We have observed a continuity in the distribution of the
values of diazepam potentiation, and because there was no evidence for
two distinct distributions in the values of diazepam-induced
potentiation, different zolpidem-insensitive cells are likely to
express variable proportions of constructs with high and low
sensitivity to benzodiazepines.
It is possible that fast, short-lasting and slow, long-lasting channels
are composed of 4 1 2 and 5 1 2 subunit constructs, respectively. The 5 subunit is frequently associated with the 1
subunit in the adult (Laurie et al., 1992 ). Studies on recombinant receptors indicate that the 5-containing constructs have a
relatively high EC50 for GABA when they are associated with
the 1 subunit (Burgard et al., 1996 ). The EC50 for
GABA-gated currents flowing through recombinant 5 1 2 receptors
(36 µM) is close to that estimated for the long-lasting
channels observed in the present study. In constructs containing the
5 subunit, diazepam has a potentiating effect of 100% (200% of the
GABA-activated current in control) (Puia et al., 1991 ; Burgard et al.,
1996 ), which is in the range of values described in the present study.
It has also been demonstrated that the duration of the LF component
expressed by embryonic GABAA
receptor/Cl channels correlates with the relative
expression of the 5 mRNA quantified in different regions (Serafini
et al., 1998a ). Taken together, these properties indicate that
long-lasting channels most likely contain 5 1 2 subunit
constructs. Alternatively, these channels might contain 2, 3,
1-3, and 3 subunits, because these subunits were also
immunoidentified. However, with the exception of 1, these subunits
were expressed in <5% of the cells, and thus few cells would be
expected to express channels composed of these subunits. Channels
containing 2 and 3 subunits exhibit a significant degree of
potentiation by diazepam (Puia et al., 1991 ), as well as relatively low
affinity for GABA (Ebert et al., 1994 ) and long-lasting open times
(Verdoorn, 1994 ).
Because the other most abundant subunit in zolpidem-insensitive
cells was 4, and the constructs containing the 4 subunit are
expected to be insensitive to either zolpidem (Scholze et al., 1996 ) or
diazepam modulation (Benke et al., 1997 ), the channels with
short-lasting openings, high affinity for GABA, and insensitivity to
zolpidem or diazepam might be composed of the 4 1 2 subunit constructs. GABAA receptor/Cl channels
in hippocampal cells sensitive to furosemide (a drug acting on
constructs containing 4 subunits) have time constants corresponding
to those characterized for the short-lasting channels described in the
present study (Banks et al., 1998 ). Recombinant GABAA
receptors expressing the 2-subunits are diazepam-insensitive when
associated with 4 (Benke et al., 1997 ). Recombinant receptors without any subunit have also been reported to have poor
sensitivity to zolpidem. However, it is unlikely that receptor
constructs without subunits were expressed by zolpidem-insensitive
cells, because recombinant receptors without subunits have a main
conductance state of 13 pS (Angelotti and Macdonald, 1993 ), whereas the
main conductance state in our whole-cell recordings of single-channel activity was 27 pS.
Time constant values quantified in the present study may be compared
with those previously described for the
GABAA/Cl channels expressed by
hippocampal neurons. Spectral density plots of GABA-evoked
Cl currents in embryonic hippocampal neurons
cultured for ~3-4 weeks were fitted by one component ( = 23 msec)
in an early study by Segal and Barker (1984) and two components with
values of ~5 and ~51 msec by Ozawa and Yuzaki (1984) .
Nonstationary fluctuation analysis of IPSCs in the granule cell layer
of adult rats showed a spectral density that was fitted by three
components with values of 25, 1.7, and 0.3 msec, respectively (De
Koninck and Mody, 1994 ). IPSCs recorded in adult rat CA1 neurons were
fitted by one exponential with a value of either 11 msec
(Collingridge et al., 1984 ) or 25 msec (Ropert et al., 1990 ). Pearce et
al. (1995) have shown that evoked IPSCs were fitted by short ( = 3-8 msec) or long-lasting exponentials ( = 30-70 msec), depending on the site of stimulation in CA1. The long-lasting channel durations of ~100 msec recorded in embryonic cells are comparable with the decay of the longest-lasting IPSCs and might correspond to the continued expression of such channels in the adult. Thus, some channels
with very-long-lasting openings might contain 5, 1, and 2
subunits, both in the embryonic and adult period. This receptor channel
type would be of particular pharmacological and clinical relevance,
because 5 subunit-containing constructs are thought to play roles in
epilepsy (Houser et al., 1995 ) and in tolerance to diazepam (Zhao et
al., 1994 ).
In sum, the advent of functional GABAA
receptor/Cl channels among embryonic hippocampal
neurons in the earliest stages of differentiation includes assembly of
two channel types, with clusters of short- and long-lasting openings of
~30 and ~100 msec, which may be composed of 4 1 2 and
5 1 2 subunit constructs, respectively. The long-lasting
openings would be expected to induce significant depolarizing effects,
whereas clusters of short-lasting openings would likely be less effective.
 |
FOOTNOTES |
Received Dec. 4, 1998; revised April 1, 1999; accepted April 6, 1999.
Correspondence should be addressed to Dr. D. Maric, Laboratory of
Neurophysiology, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Building 36, Room 2C-02, Bethesda, MD 20892.
Dr. Serafini's present address: Department of Anesthesiology Research
Unit, Washington University School of Medicine, St. Louis, MO 63110.
 |
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