The use of GABAA receptors expressed in neural

Biosensors & Bioelectronics 16 (2001) 481– 489
www.elsevier.com/locate/bios
The use of GABAA receptors expressed in neural precursor cells
for cell-based assays
Kara M. Shaffer a, Hsingchi J. Lin a, Dragan Maric b, Joseph J. Pancrazio a,
David A. Stenger a, Jeffery L. Barker b, Wu Ma a,*
b
a
Center for Bio/Molecular Science and Engineering, Code 6900, Na6al Research Laboratory, Washington, DC 20375, USA
Laboratory of Neurophysiology, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,
MD 20892, USA
Abstract
GABAA receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since
few neural cell lines express functional GABAA receptors, the capacity to rapidly screen for compounds that affect GABAA
receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABAA
receptors that can be monitored via Ca2 + imaging. This study examined GABAA receptor subunit expression to determine
whether GABAA receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells
isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and
passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant
mRNAs encoding various GABAA receptor subunits. Ca2 + imaging showed that the highly proliferating precursor cells in
passaged cultures maintained expression of functional GABAA receptors. In addition, we showed that trimethylolpropane
phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited
muscimol (GABAA receptor agonist) but not depolarization-induced cytosolic Ca2 + increase. The findings of this study suggest
that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity.
The potential use of neural precursor cells in high-throughput screens for compounds acting on GABAA receptors is discussed.
© 2001 Elsevier Science B.V. All rights reserved.
Keywords: Basic fibroblast growth factor; Biosensor; Ca2 + imaging; GABAA receptors; High-throughput screening; Neural stem cells; Neurotoxicity; RT-PCR
1. Introduction
Cell-based biosensors have seen much interest in
recent years. Unlike other biosensor paradigms, cellbased biosensors are not specific for certain compounds, but are capable of responding to a wide range
of biologically active compounds (Pancrazio et al.,
1999). One approach for cell-based biosensor implementation relies on the use of intact, functional receptors. One such receptor that has widely demonstrated
sensitivity to toxins and modulators is the GABAA
receptor. GABAA receptors are expressed at early
* Corresponding author. Tel.: + 1-202-404-6037; Fax: + 1-202767-9598.
E-mail address: [email protected] (W. Ma).
stages of embryonic brain development (Barker et al.,
1998). For example, GABAA receptor subunits a4, b1,
and g1, as well as the GABA synthesis enzyme GAD67
are expressed in the proliferative zone of the embryonic
central nervous system (Ma and Barker, 1995, 1998).
Previous work has shown that cultured, proliferative
neural precursor cells derived from embryonic day 13
(E13) rat telencephalon express functional GABAA receptors (Ma et al., 1998; Ma et al., 2001). GABAA
receptor function in these cells can be monitored via
GABA- or muscimol-induced elevations in cytosolic
free Ca2 + ([Ca2 + ]C), using the fluorescent indicator
dye, Fura-2 (Ma et al., 1998). Due to the depolarized
equilibrium potential for [Cl−] in neural precursor cells,
a consistent feature of immature neural tissue, GABAA
activation triggers membrane depolarization, high-
0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 1 6 2 - 2
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K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
voltage Ca2 + channel opening, followed by [Ca2 + ]C
elevations (Ma et al., 1998).
It is known that neurotransmitters such as GABA
serve as developmental signals regulating basic cellular
functions, such as cell proliferation, differentiation
and morphogenesis, by receptor-mediated mechanisms
(Lauder and Liu, 1998). These functions could make
the developing nervous system especially vulnerable to
environmental neurotoxins that target neurotransmitter receptors. For example, organochlorine pesticides
(Lauder and Liu, 1998) and polychlorinated biphenyls
(Inglefield and Shafer, 2000) are potent antagonists of
GABAA receptors. In addition, ethanol, which is a
well-known developmental neurotoxicant (for a review, see Mihic, 1999), inhibits GABAA receptor function in neural precursor cells in vivo (Haydar et al.,
2000) and in vitro (Ma et al., 2001).
The GABAA receptors expressed in neural precursor
cells may potentially serve as the basis for a functionbased cellular assay. These receptors could detect and
extract information about the biological activity,
mechanisms of action, and consequences of exposure
to toxic agents. Many previous studies have relied on
tumor-derived cell types as in vitro models for developmental neurotoxicology (Harry et al., 1998). Neural
precursor cells may have the capacity to proliferate in
culture and exhibit stable receptor expression, yielding
a ready supply of ‘normal’ rather than transformed
cell types for in vitro studies. In addition, relatively
few of the transformed neural cell lines express functional GABAA receptors (Hales and Tyndale, 1994),
suggesting that neural precursor cells may be unique.
GABAA receptor function in neural precursor cells
can be monitored via GABAA receptor agonist-induced elevations in cytosolic Ca2 + , [Ca2 + ]C, using the
fluorescent indicator dye, Fura-2 (Ma et al., 1998).
The transient increasing [Ca2 + ]C is associated with
Ca2 + entry through Ca2 + -permeable channels such as
voltage-gated Ca2 + channels (VGCC) since neural
precursor cells exhibit L-type VGCCs (Ma et al.,
2001), and with Ca2 + release from intracellular Ca2 +
stores. Since [Ca2 + ]C serves as an intracellular regulatory signal involving a variety of cell functions,
changes in [Ca2 + ]C may be indicative of cell response
to neurotoxins. In the present study, we examined the
expression and function of GABAA receptors in rat
neural precursor cell culture. Using reverse transcription-polymerase chain reaction (RT-PCR), GABAA receptor subunits a1, a2, a4, b1, g1, and g2 were
observed immediately following isolation of neural
precursor cells. Using Ca2 + imaging, functional
GABAA receptors were observed; this functionality
was preserved with subsequent passages of the cells.
Furthermore, we report that GABAA receptor function in neural precursor cells is inhibited by a model
neurotoxicant trimethylolpropane phosphate (TMPP).
These data indicate that neural precursor cells maintain stable expression and function of GABAA receptors and may offer an alternative to tumor-derived cell
lines for neurotoxicology studies.
2. Methods
2.1. Cell culture
The isolation of rat brain precursor cells and their
expansion were carried out as described previously
(Ma et al., 1998). Briefly, timed pregnant Sprague–
Dawley rats (Taconic Farms, Germantown, NY,
USA) were anesthetized with sodium pentobarbital (40
mg/kg body weight, intraperitoneally). Embryos were
removed from the dams at embryonic day 13 and
placed into Earle’s Balanced Salt solution (EBSS).
Embryonic day 1 was defined as the day of conception established by the presence of a vaginal plug. The
crown-rump length was measured to confirm the embryonic age. The neuroepithelium was dissected from
E13 rat brains according to the atlas of the prenatal
rat brain (Altman and Bayer, 1995). Tissue was dissociated by mechanical trituration in EBSS with a sterile, fire-polished glass Pasteur pipette. The cells were
collected by centrifugation and resuspended in a
serum-free medium consisting of Neurobasal medium
(NB) supplemented with B27 and 0.5 mM L-glutamine
(Brewer et al., 1993), and containing 30 ng/ml recombinant human basic fibroblast growth factor (Intergen,
Purchase, NY, USA). For Ca2 + imaging, cells (12×
103) in basic fibroblast growth factor (bFGF)-containing NB/B27 medium were plated on 35 mm plastic
dishes containing a central coverslip, which was photoetched (for relocation of cell fields after imaging and
recording) and was pre-coated with 15 mg/ml poly-Dlysine (10 mM; BD Bioscience, Bedford, MA, USA)
and 1 mg/ml bovine plasma fibronectin (Gibco BRL,
Gaithersburg, MD, USA).
Cultures were passaged zero (primary) to three
times (p1, p2 and p3). Passaging neural precursor cells
was carried out every 7 days. After growing for 7
days in 35 mm plastic dishes coated with PDL and
fibronectin, the neural progenitor cells were incubated
in 2 ml trypsin-ethylenediamine tetraacetic acid
(EDTA) (0.5% w/v trypsin, 5.3 mM EDTA; Gibco,
Gaithersburg, MD, USA) for 10 min at 37 °C. Next,
0.2 ml trypsin inhibitor (Gibco BRL, Gaithersburg,
MD, USA) was added and the cells were centrifuged
for 10 min at 1000×g. After centrifugation, the cells
were resuspended in 2 ml NB/B27 containing 0.5 mM
L-glutamine and 30 ng/ml bFGF. Viable cells were
counted by trypan blue exclusion. Cells were plated
into new 35 mm plastic bottom dishes coated with
PDL and fibronectin at a density of 30 000 per dish.
K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
2.2. Double-immunostaining for anti-bromodeoxyuridine
incorporation and propidium iodide
b1f
b1r
To detect anti-bromodeoxyuridine (BrdU) incorporation, cultures were exposed to 10 mM BrdU for 4 h and
then fixed with 70% alcohol followed by 2%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
Cells were incubated overnight with BrdU and followed
by incubation with FITC-conjugated donkey antimouse IgG (Jackson Immunological Research, West
Grove, PA, USA) for 45 min. The immunoreacted nuclei were counter-stained for total DNA content by addition of 5 mg/ml propidium iodide (PI) for
10 min. The distributions of BrdU and PI signals
were examined and photographed with a Nikon microscope.
g1f
g1r
g2f
g2r
b actinf
b actinr
483
5%-GTTTG GGGCT TCTCT CTTTT
CCT-3%
5%-AGTTA CTGCT CCCTC TCCTC
CATT-3%
5%-TAGTA ACAAT AAAGG AAAAA
CCACC AGA-3%
5%-CCAGC TTGAA CAAGG CAAAA
GCT-3%
5%-TGGTG ACTAT GTGGT TATGT
CCGTG-3%
5%-AGGTG GGTGG CATTG TTCAT
TT-3%
5%-CTGGCA-CCACACACCTTCTAC-3%
5%-CATCTCTTGCTCGAAGTCC-3%
2.4. Ca 2 + imaging
2.3. Isolation of total RNA and RT-PCR
Neuroepithelial cells isolated from embryonic day 13
brain and cultured in bFGF-containing NB/B27
medium were examined for expression of a panel of
mRNAs encoding various GABAA receptor subunits
a1, a2, a4, b1, g1, and g2. Total RNA was isolated
from cells using TRIZOL (Gibco BRL, Gaithersburg,
MD, USA). As determined by 260/280 OD readings, 10
mg RNA were used and reverse transcribed using Superscript II (Gibco BRL, Gaithersburg, MD, USA)
with 1 mg random hexamers. The resulting cDNA was
then diluted with 1 volume of Tris– EDTA (10 mM Tris
(pH 7.5), 1 mM EDTA) and 1 ml diluted cDNA was
added to 25 ml PCR reaction mixture containing the
following: 0.2 mM primers, 1.5 mM MgCl2, 8%
dimethylsulfoxide, and 1× PCR buffer (Perkin Elmer,
Boston, MA, USA). PCR products were resolved on a
1.2% agarose gel. HT29, a colon epithelial cell line, was
used as a template control expected to not express
GABAA receptor subunits. b-Actin was used as a control for RNA integrity. The primer sequences for each
subunit and corresponding PCR product size are listed
in Ma et al. (1993). The primer sequences for b-actin
are described in Lin et al. (1999).
Primer sequences are indicated in the following.
a1f
a1r
a2f
a2r
a4f
a4r
5%-CATTC TGAGC ACTCT CTCGG
GAAG-3%
5%-GTGAT ACGCA GGAGT TTATT
GGGC-3%
5%-AGGTT GGTGC TGGCT AACAT
CC-3%
5%-AACAG AGTCA GAAGC ATTGT
AAGTC C-3%
5%-CAAAA CCTCC TCCAG AAGTT
CCA-3%
5%-ATGTT AAATG CCCCA AATGT
GACT-3%
The cells were loaded with 2 mM Fura-2 AM (Molecular Probes, Eugene, OR, USA) for 1 h at 37 oC. At
the end of the incubation, the cells were rinsed in
normal physiological medium containing (mM): 145
NaCl, 5 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 HEPES, 10
glucose (pH 7.4; osmolaritym 290 mOsm). Fura-2loaded cells were recorded using the Zeiss Attofluor
Ratio Vision workstation (Atto Instruments, Rockville,
MD, USA) equipped with an Axiovert 135 inverted
microscope (Carl Zeiss, Thornwood, NY, USA) and an
ICCD camera (Atto Instruments, Rockville, MD,
USA). The Fura-2 dye was sequentially excited at 500
ms intervals with a 100 W mercury arc lamp filtered at
3349 5 and 3809 5 nm, and the respective emissions
acquired through a 510 nm dichroic mirror and 520 nm
long-pass filter set. The fluorescence intensities from
each region of interest were digitized with a Matrox
image processing board and plotted as line graphs using
Attograph for Windows analysis software (Atto Instruments, Rockville, MD, USA). Drug and ligand applications were performed using a superfusion system
described previously (Maric et al., 2000). Solutions were
delivered to a 150 ml recording chamber using a gravitydriven perfusion system at a flow rate of 2 ml/min. The
duration of exposure to each test condition was about 5
min. All measurements were performed at room temperature. Fura-2 fluorescence emissions were converted
into estimated [Ca2 + ]C concentrations using the following equation: [Ca2 + ]C = Kd[R− Rmin]/[Rmax − R]Fo/F,
where Kd is the fura– Ca2 + binding constant (225 nM)
(Grynkiewicz et al., 1985; Maric et al., 2000), R is a
ratio of Fura-2 fluorescence at 334 and 380 nm, Rmin
and Rmax are values of R in Ca2 + -free and normal
[Ca2 + ]o medium, respectively, using Fura-2 Penta K+
salt (Molecular Probes, Eugene, OR, USA) as the Ca2 +
indicator, and Fo/F is the ratio of Fura-2 fluorescence
at 380 nm in Ca2 + -free and 1.8 mM [Ca2 + ]o medium.
The data were calibrated on-line using the Attofluor
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K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
Ratio Vision acquisition software. After imaging, the
cells were fixed and phenotyped.
2.5. Data analysis
Where appropriate, data are given as mean9 S.E.M.
and statistical significance determined using the Student’s
t-test with P B0.05 considered significant. Cumulative
histograms, which were used to compare GABAA receptor-mediated transients between cell passages, were
statistically compared using the Kolmogorov– Smirnov
test where PB0.05 was considered to yield a significant
difference.
3. Results
3.1. Neural precursor cell expansion in primary and
passaged cultures
Neuroepithelial cells isolated from embryonic day 13
rat brain were expanded in serum-free medium containing bFGF and passaged three times. The bFGF-responsive neural precursor cells rapidly proliferated. The
robustly growing cells in primary and passaged cultures
had flat, multiple-shaped cell bodies with short processes
(Fig. 1A–D). To assess the extent to which primary and
passaged cultures of neural precursor cells could be
expanded compared with their primary counterparts, cell
counting was carried out at day 7 in primary and
passaged cultures of neural precursor cells. Results from
cell counting have shown that the number of cells
increased by tenfold in passage 1 and by 100-fold in
passage 3. Cells in the passaged cultures exhibited a
similar immature morphology as in the primary cultures
(Fig. 1A–D). To test whether the passaged cells maintain
immature morphology and proliferative properties, immunocytochemistry for nestin, an intermediate filament
protein characteristic for central nervous system precursor cells (Lendahl et al., 1990) and cell proliferation assay
were carried out in primary, passage 1 and 2 cultures.
Nestin staining showed that most cells, if not all, were
nestin+ (Fig. 1E,F). Cell proliferation assay was performed using double-immunostaining for bromodeoxyuridine and propidium iodide, in which PI stained
all nuclei and BrdU incorporated proliferating cells
during DNA synthesis (Fig. 1G,H). The distribution of
BrdU- and PI-labeled cells showed that, similar to
primary cells, over 70% of passaged cells were stained for
BrdU.
3.2. bFGF-Expanded neural precursor cells express a
panel of transcripts encoding GABAA receptor subunits
To assess whether neural precursor cells isolated from
E13 brain and cultured in serum-free medium containing
bFGF express transcripts encoding GABAA receptor
subunits, RT-PCR analysis was carried out for transcripts encoding GABAA receptor a1, a2, a4, b1, g1, and
g2 subunits using total RNA isolated from E13 brain
(day 0) and bFGF-expanded cultures for 2 (day 2) and
5 (day 5) days. HT29, a colon epithelial cell line, was used
as a template control for putative non-expression (Fig.
2E) and b-actin was used as a control for RNA integrity.
While a1 mRNA was barely detectable, levels of transcripts encoding GABAA receptor a2, a4, b1, g1, and g2
subunits were all clearly detected in E13 brain cells (Fig.
2B). All six transcripts for GABAA receptor subunits
appeared at day 2 (Fig. 2C) and this expression continued
through day 5 (Fig. 2D), consistent with their strong
expression in adult brain (Fig. 1A). As expected, the a4,
b1, g1 and g2 subunits were not observed in the colon
epithelial cell line. While expression of the a2 subunit was
detected in the epithelial line, a result not unlike previous
work with kidney fibroblasts (Fuchs et al., 1995), this
expression was much lower than that observed in neural
precursor cells.
3.3. Maintained expression of functional GABAA
receptors in passaged neural precursor cells
Our previous study has shown that GABA and the
GABAA receptor agonist muscimol depolarized proliferating neural precursor cells and elevated [Ca2 + ]C, which
was blocked by the GABAA antagonist bicuculline,
indicating that GABAA receptor/Cl− channels mediated
this process (Ma et al., 1998). To test whether passaged
precursor cells, after expansion with bFGF, exhibit the
similar GABAA receptor-mediated effects on cytosolic
Ca2 + levels as their primary counterparts, the primary
and passaged cells were expanded for 7 days in serumfree medium containing bFGF and loaded with Ca2 +
indicator Fura-2 for digital fluorescence imaging. In 50
cells of primary culture, which were determined to be
BrdU+ after Ca2 + imaging, 10 mM muscimol increased
[Ca2 + ]C in 34 cells from 45–50 to 150–250 nM, and this
response was completely and reversibly abolished by
bicuculline (50 mM) (Fig. 3A). In another 50 precursor
cells taken from passage 2 culture, 10 mM muscimol
triggered a transient increase in cytosolic Ca2 + concentration in 37 cells from 42–55 to 135–245 nM, and this
response was completely and reversibly blocked by 50
mM bicuculline.
The GABAA receptor-mediated [Ca2 + ]C transients
elicited from the primary and passaged precursor cells
were statistically compared using the Kolmogorov–
Smirnov test. Fig. 3B shows a cumulative histogram for
GABAA receptor-mediated [Ca2 + ]C transients recorded
from the primary and passage 2 cells. The shifts in the
histogram were not statistically different, showing the
similarity of the response of the GABAA receptor-mediated [Ca2 + ]C transients between the primary and pas-
K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
saged cells. The comparison of GABAA receptormediated [Ca2 + ]C transients was also made between
primary and passage 1 precursor cells; the shifts in the
histogram were not statistically different (data not
shown).
485
3.4. TMPP inhibits GABAA receptor-mediated [Ca 2 + ]C
ele6ation in proliferating neural precursor cells
Trimethylolpropane phosphate is known to inhibit
GABAA receptor function, as indicated by the blockade
Fig. 1. Neuroepithelial cells isolated from embryonic day 13 brain express nestin, and are incorporated with bromodeoxyuridine in primary and
passaged cultures. Phase-contrast photomicrographs (A –C) show immature morphology of neural precursor cells in primary (p0), passage 1 (p1),
passage 2 (p2) and passage 3 (p3) cultures. Fluorescence photomicrographs (E, F) show that most, if not all, cells are nestin+ in primary (not
shown), p1 and p2 cultures. (G ,H) Double-immunostaining of neural precursor cells in the same field of a passage 2 culture for BrdU (green)
and propidium iodide (red). In the fixed sample, all nuclei are stained for PI and only cells in the S-phase are stained for BrdU. Counting the
number of double-labeled nuclei shows that most cells are incorporated with BrdU. Scale bars: 100 mm (A), 50 mm (E), 100 mm (F), 100 mm (G).
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K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
Fig. 2. Neuroepithelial cells isolated from embryonic day 13 brain and cultured in bFGF-containing NB/B27 medium express a panel of mRNAs
encoding various GABAA receptor subunits. (A –D) RT-PCR using total RNA isolated from adult rat brain, embryonic day 13 brain (day 0), day
2, and day 5 primary cultures, respectively. (E) RT-PCR from HT29, a colon epithelial cell line, which is used as a template control for the
non-expresser. Transcripts encoding six GABAA receptor subunits, a1, a2, a4, b1, g1, and g2, and b-actin are detectable at all timepoints in total
mRNAs isolated from brains and cultures as shown in each respective lane. In HT29, mRNAs for the GABAA receptor subunits are not shown
except a2. b-Actin is used as a control for RNA integrity.
of whole-cell GABA-mediated Cl− current and the
reduction in spontaneous inhibitory postsynaptic currents (Kao et al., 1999). We examined the effect of
TMPP on GABAA receptors by monitoring GABA-induced [Ca2 + ]C elevations in proliferating neural precursor cells. Neural precursor cells (n = 64) were exposed
to 10 mM muscimol, then rinsed in normal physiological medium before adding 50 mM TMPP +10 mM
muscimol, and then rinsed in normal physiological
medium again before repeat exposure of the same dose
of muscimol to test for recovery of the response. TMPP
inhibited the muscimol-induced [Ca2 + ]C elevation in a
dose-dependent manner. The lowest concentration of
TMPP tested (10 mM) reduced muscimol-induced
[Ca2 + ]C elevation by 239 2% (n = 32 cells). TMPP (50
mM) completely and reversibly blocked the muscimolinduced increase in [Ca2 + ]C. Thus, TMPP concentra-
tions in the 10– 50 mM range significantly depressed
[Ca2 + ]C responses to muscimol.
Since the transient increasing [Ca2 + ]C can be related
to Ca2 + entry through voltage-gated Ca2 + channels,
and since it is known that GABA’s activation of
GABAA receptors depolarizes precursor cell membrane
and activates VGCCs (Ma et al., 1998, 2001), we investigated the possibility that the effects of TMPP on the
[Ca2 + ]c response to muscimol involved interactions
with VGCCs. We examined [Ca2 + ]c responses to 50
mM KCl with elevations of [Ca2 + ]c from 35–45 to
100–200 nM range (Fig. 4B). However, these elevations
were not affected by 50 mM TMPP (Fig. 4B). These
results suggest that TMPP blocks GABAergic stimulation of cytosolic Ca2 + levels in proliferating precursors
primarily by interacting with GABAA receptors, rather
than VGCCs.
K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
487
4. Discussion
In this study, we showed that neural precursor cells
can be expanded in serum-free medium containing
bFGF for at least three passages. The neural precursor
cells of the passaged cultures exhibits similar morphology to the cells of the primary culture, and also appears
to maintain functional GABAA receptors as do the cells
in the primary culture. We also showed that the neural
precursor cells responded to the model neurotoxicant
TMPP by a reduced muscimol-induced cytosolic Ca2 + .
Our findings indicate that cultured neural precursor
cells can act as a renewable cell source, which can be
exploited for cell-based assay development. One of the
major challenges faced for in vitro neuronal model cell
types is the requirement for a source of cells that is
both renewable and genetically stable in culture. Tumor-derived cell types, such as PC-12 cells, have been
Fig. 4. Trimethylolpropane phosphate inhibits muscimol-induced, but
not depolarization-induced cytosolic Ca2 + elevation in neural precursor cells. (A) TMPP effectively inhibits muscimol-induced cytosolic
Ca2 + elevation in neural precursor cells. The neural precursor cells
were expanded in serum-free medium containing bFGF for 7 days
and loaded with Ca2 + indicator Fura-2 for Ca2 + imaging. In a cell
determined to be BrdU+ after imaging, the [Ca2 + ]C response to 10
mM muscimol is completely and reversibly inhibited by 50 mM TMPP.
(B) TMPP does not affect KCl-evoked [Ca2 + ]c responses. The depolarization caused by 50 mM KCl induces an increase in [Ca2 + ]c peak
levels from 35 to 120 nM. However, brief exposure to 50 mM
TMPP does not change the KCl-induced [Ca2 + ]c peak levels.
Fig. 3. A similar expression of muscimol-induced cytosolic Ca2 +
elevation in primary versus passaged cultures. The neural precursor
cells were expanded in serum-free medium containing bFGF and
passaged three times. The cells are loaded with Ca2 + indicator
Fura-2 for digital fluorescence imaging. (A) In a cell of primary
culture determined to be BrdU+ after imaging, 10 mM muscimol
triggers a transient increase in cytosolic Ca2 + concentration, which is
completely and reversibly blocked by GABAA antagonist bicuculline
(50 mM), indicating that the muscimol-induced cytosolic Ca2 + elevation is mediated by GABAA receptors. (B) Cumulative histograms of
GABAA receptor-mediated [Ca2 + ]c transients between cell passages.
reported to undergo phenotypic changes with increased
number of passages most likely due to point mutations
(Harry et al., 1998). As we have demonstrated, neural
precursor cells, which have the ability to proliferate in
an undifferentiated state under certain culture conditions, are able to maintain reproducibly functional
GABAA receptors when passaged. As a result, neural
precursor cells may be useful in cell-based sensor applications for detection of GABAA receptor antagonists.
Neural precursor cells bearing GABAA receptors
have the ability to respond to analytes that have biological activity and may offer a physiologically relevant in
vitro model of developmental neurotoxicity. Our results
using RT-PCR show GABAA subunit transcripts are
expressed in the E13 brain and are maintained in vitro
through day 5. Our work is consistent with that of Sah
et al. (1997) who showed that GABA receptors in rat
hippocampal progenitor cells exhibited currents with
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K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
normal kinetics, current– voltage relationships, and selectivities that were maintained after multiple passages.
Previous work has suggested that few neuronal cell
lines express functional GABAA receptors in spite of
the expression of GABAA receptor subunits (Hales and
Tyndale, 1994). The expression of GABAA receptors
was examined in the embryonal carcinoma cell line P19,
a pluripotent cell type, using whole-cell voltage-clamp
recordings, RT-PCR and immunostaining (Reynolds et
al., 1996). This study showed that GABAA receptor
subunit mRNAs and GABA-induced currents were observed only following differentiation of p19 cells into
neuronal phenotype after treatment with retinoic acid.
Likewise, human neuroblastoma IMR-32 cells express
functional GABAA receptors (Anderson et al., 1993);
however, there appears to be some unexpected pharmacological characteristics of GABAA receptor in IMR-32
cells, perhaps due to the neoplastic nature of the cell
model. In contrast, neural precursor cells utilized in the
present study express functional GABAA receptors in
proliferating, non-differentiated cells. While the
GABAA subunit expression in neural precursor cells is
consistent with that expressed in the adult mammalian
brain (Paysan and Fritschy, 1998), further work must
be performed to more completely characterize the pharmacological sensitivity and the subunit composition of
expressed protein of these GABAA receptors.
In addition to maintaining functional GABAA receptors, we demonstrated that these receptors expressed in
the neural precursor cells are associated with neurotoxicity. It is well known that developing neurotransmitter
systems may be especially vulnerable to environmental
neurotoxins including organochlorine pesticides
(Lauder and Liu, 1998; Inglefield and Shafer, 2000). We
demonstrated that the neural precursor cells would
respond to a model neurotoxicant, TMPP, by a reduced
muscimol-induced cytosolic [Ca2 + ]C. These findings are
consistent with evidence for the inhibition of GABAA
receptors by bicyclophosphates, which has been previously suggested using radiolabeled binding assays and
GABA-induced Cl− flux experiments (Squires et al.,
1983; Gant et al., 1987). A prior study using rat brain
vesicles revealed that tri-o-cresyl phosphate, a bicyclophosphate precursor to TMPP, inhibited GABA-induced 36Cl− influx (Gant et al., 1987). More recently,
flux measurements into rat brain microsacs showed that
TMPP also inhibits GABA-mediated 36Cl− influx (Higgins and Gardier, 1990). Work by Kao et al. (1999)
directly demonstrated that TMPP blocks GABAA receptor function underlying inhibitory synaptic
transmission.
It is well known that the GABAA receptor is an
important therapeutic target for several disease states,
in particular epilepsy (Whiting, 1999); however, there
are few means available for conducting high-throughput screening (HTS) for GABAA receptor interaction
(Meldrum, 1997). Receptor immobilization on chromatographic stationary phases to evaluate binding
affinities has been suggested as a basis for HTS (Zhang
et al., 1998; Wainer et al., 1999). In addition, HTS
methods for GABAA receptor compounds that rely on
fluorescence-based intracellular pH changes (Simpson
et al., 2000) or fluorescence ratiometric measures of
intracellular Cl− (Kuner and Augustine, 2000) have
been suggested. The fluorometric imaging plate reader
has been shown to enable high-throughput fluorometric
assays of membrane potential and intracellular Ca2 +
mobilization (Kuntzweiler et al., 1998; Sullivan et al.,
1999). Based on our observations, we suggest that
neural precursor cells may be well suited for HTS
assays of GABAA receptor activity. Note, however,
that the use of GABAA-based depolarization of the
cells to activate voltage-gated calcium channels may be
problematic for HTS. This mechanism involves the
participation of the voltage-gated channels, and possibly participation from calcium-induced calcium release
from intracellular stores. In a primary screen, all compounds that block any part of the pathway may appear
as a positive result. Therefore, future work should
consider the possibility of using a membrane potentialsensitive dye assay, which could directly detect the
membrane depolarization induced by the GABAA receptor agonist addition. Still, these cells may have the
capacity to fill a needed technology void for the rapid
identification of potential GABAA related therapeutics.
In summary, we have demonstrated that neural precursor cells maintain stable expression and function of
GABAA receptors. These cells may offer an alternative
to tumor-derived cell lines for neurotoxicology studies
and offer a means of achieving GABAA receptor cellbased sensor and HTS applications.
Acknowledgements
This work was in part supported by the Office of
Naval Reseach and the Naval Research Laboratory.
H.J.L. was supported by a fellowship from the National Research Council. The opinions and assertions
contained herein are the private ones of the authors and
are not to be construed as official or reflecting the views
of the Department of the Navy.
References
Altman, J., Bayer, S.A., 1995. Atlas of Prenatal Rat Brain Development. CRC Press, Boca Raton, FL.
Anderson, S.M., Se Souza, R.J., Cross, A.J., 1993. The human
neuroblastoma cell line IMR-32 possesses a GABAA receptor
lacking the benzodiazepine modulatory site. Neuropharmacology
32, 455 – 460.
K.M. Shaffer et al. / Biosensors & Bioelectronics 16 (2001) 481–489
Barker, J.L., Behar, T., Li, Y.-X., Liu, Q.-Y., Ma, W., Maric, D.,
Maric, I., Schaffner, A.E., Serafini, R., Smith, S.V., Somogyi, R.,
Vautrin, J.Y., Wen, X.-L., Xian, H., 1998. GABAergic cells and
signals in CNS development. Perspect. Dev. Neurobiol. 5, 305 –
322.
Brewer, G.J., Torricelli, J.R., Price, P.J., 1993. Optimized survival of
hippocampal neurons in B27 supplemented neurobasal. A new
serum-free medium combination. J. Neurosci. Res. 35, 567 – 576.
Fuchs, K., Zezula, J., Slany, A., Sieghart, W., 1995. Endogenous
[3H]flunitrazepam binding in human embryonic kidney cell line
293. Eur. J. Pharmacol. 289, 87 –95.
Gant, D.B., Eldefrawi, M.E., Eldefrawi, A.T., 1987. Action of
organophosphates on GABAA receptor and voltage-dependent
chloride channels. Fundam. Appl. Toxicol. 9, 698 –704.
Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of
Ca2 + indicators with greatly improved fluorescence properties. J.
Biol. Chem. 260, 3440 –3450.
Hales, T.G., Tyndale, R.F., 1994. Few cell lines with GABAA mRNAs have functional receptors. J. Neurosci. 14, 5429 –5436.
Harry, G.J., Billingsley, M., Bruinink, A., Campbell, I.L., Classen,
W., Dorman, D.C., Galli, C., Ray, D., Smith, R.A., Tilson, H.A.,
1998. In 6itro techniques for the assessment of neurotoxicity.
Environ. Health Perspect. 106, 131 –158.
Haydar, T.F., Wang, F., Schwartz, M.L., Rakic, P., 2000. Differential modulation of proliferation in the neocortical ventricular and
subventricular zones. J. Neurosci. 20, 5764 –5774.
Higgins, G.M., Gardier, R.W., 1990. Aminobutyric acid antagonism
produced by an organophosphate-containing combustion
product. Toxicol. Appl. Pharmacol. 105, 103 –112.
Inglefield, J.R., Shafer, T.J., 2000. Perturbation by the PCB mixture
aroclor 1254 of GABAA receptor-mediated calcium and chloride
responses during maturation in vitro of rat neocortical cells.
Toxicol. Appl. Pharmacol. 164, 184 –195.
Kao, W.Y., Liu, Q.-Y., Ma, W., Ritchie, G.D., Lin, J., Nordholm,
A.F., Rossi, J. III, Barker, J.L., Stenger, D.A., Pancrazio, J.J.,
1999. Inhibition of spontaneous GABAergic transmission by
trimethylolpropane phosphate. Neurotoxicology 20, 843 –850.
Kuner, T., Augustine, G.J., 2000. A genetically encoded ratiometric
indicator for chloride: capturing chloride transients in cultured
hippocampal neurons. Neuron 27, 447 –459.
Kuntzweiler, T.A., Arneric, S.P., Donnelly-Roberts, D.L., 1998.
Rapid assessment of ligand actions with nicotinic acetylcholine
receptors using calcium dynamics and FLIPR. Drug Dev. Res. 44,
14 –20.
Lauder, J.M., Liu, J., 1998. Neurotoxic and neurotrophic effects of
GABAergic agents on developing neurotransmitter systems. In:
W. Slikker, L.W. Chang (Eds.), Handbook of Developmental
Neurotoxicology. Academic Press, San Diego, CA, pp. 153 – 155.
Lendahl, U., Zimmerman, L.B., McKay, R.D., 1990. CNS stem cells
express a new class of intermediate filament protein. Cell 60,
585 – 595.
Lin, H., Huber, R., Schlessinger, D., Morin, P.J., 1999. Frequent
silencing of the GPC3 gene in ovarian cancer cell lines. Cancer
Res. 59, 807 – 810.
Ma, W., Barker, J.L., 1995. Complementary expressions of transcripts encoding GAD67 and GABAA receptor a4, b1 and g1
subunits in the proliferative zone of the embryonic rat central
nervous system. J. Neurosci. 15, 2547 –2560.
Ma, W., Barker, J.L., 1998. GABA, GAD and GABAA receptor a4,
489
b1 and g1 subunits are expressed in the late embryonic and early
postnatal neocortical germinal matrix and coincide with gliogenesis. Microsc. Res. Tech. 40, 398 – 407.
Ma, W., Saunders, P.A., Somogyi, R., Poulter, M.O., Barker, J.L.,
1993. Ontogeny of GABAA receptor subunit mRNAs in rat spinal
cord and dorsal root ganglia. Comp. Neurol. 338, 337 –359.
Ma, W., Liu, Q.-Y., Maric, D., Sathanoori, R., Chang, Y., Barker,
J.L., 1998. Basic FGF-responsive telencephalic precursor cells
express functional GABAA receptor/Cl− channels in vitro. J.
Neurobiol. 35, 277 – 286.
Ma, W., Pancrazio, J., Andreadis, J.D., Shaffer, K.M., Stenger, D.A.,
Li, B.-S., Zhang, L., Barker, J.L., Maric D., 2001. Ethanol blocks
cytosolic Ca2 + responses triggered by activation of GABAA
receptor/Cl− channels in cultured proliferating rat neuroepithelial
cells. Neuroscience 104, 913 – 922.
Maric, D., Maric, I., Barker, J.L., 2000. Dual video microscopic
imaging of membrane potential and cyosolic calcium of immunoidentified embryonic rat cortical cells. Methods 21, 335 –347.
Meldrum, B.S., 1997. Identification and preclinical testing of novel
antiepileptic compounds. Epilepsia 38, S7 – S15.
Mihic, S.J., 1999. Acute effects of ethanol on GABAA and glycine
receptor function. Neurochem. Int. 35, 115 – 123.
Pancrazio, J.J., Whelan, J.P., Borkholder, D.A., Ma, W., Stenger,
D.A., 1999. Development and application of cell-based biosensors. Ann. Biomed. Eng. 27, 697 – 711.
Paysan, J., Fritschy, J.M., 1998. GABAA-receptor subtypes in developing brain. Actors or spectators? Perspect. Dev. Neurobiol. 5,
179 – 192.
Reynolds, J.N., Prasad, A., Gillespie, L.L., Paterno, G.D., 1996.
Developmental expression of functional GABAA receptors containing the gamma 2 subunit in neurons derived from embryonal
carcinoma (P19) cells. Brain Res. Mol. Brain Res. 35, 11 –18.
Sah, D.W.Y., Ray, J., Gage, F.H., 1997. Regulation of voltage- and
ligand-gated currents in rat hippocampal progenitor cells in 6itro.
J. Neurobiol. 32, 95 – 110.
Simpson, P.B., Woollacott, A.J., Pillai, G.V., Maubach, K.A., Hadingham, K.L., Martin, K., Choudhury, H.I., Seabrook, G.R.,
2000. Pharmacology of recombinant human GABA(A) receptor
subtypes measured using a novel pH-based high-throughput functional efficacy assay. J. Neurosci. Methods 99, 91 – 100.
Squires, R.F., Casida, J.E., Richardson, M., Saederup, E., 1983.
(35S)-t-butylbicyclophosphorothionate binds with high affinity to
brain specific sites coupled to gamma amino butyric acid A and
ion recognition sites. Mol. Pharmacol. 23, 326 – 336.
Sullivan, E., Tucker, E.M., Dale, I.L., 1999. Measurement of [Ca2 + ]
using the fluorometric imaging plate reader (FLIPR). Methods
Mol. Biol. 114, 125 – 133.
Wainer, I.W., Zhang, Y., Xiao, Y., Kellar, K.J., 1999. Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes,
pH and ionic strength on drug – receptor interactions. J. Chromatogr. B. Biomed. Sci. Appl. 724, 65 – 72.
Whiting, P.J., 1999. The GABAA receptor gene family: new targets
for therapeutic intervention. Neurochem. Int. 34, 387 – 390.
Zhang, Y., Xiao, Y., Killar, K.J., Wainer, I.W., 1998. Immobilized
nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug – receptor affinities. Anal. Biochem.
264, 22 – 25.

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