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The Journal of Neuroscience, April 15, 2003, 23(8):3278
Autocrine/Paracrine Activation of the GABAA Receptor
Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell
Adhesion Molecule-Positive (PSA-NCAM+) Precursor Cells from
Postnatal Striatum
Laurent
Nguyen1,
Brigitte
Malgrange1,
Ingrid
Breuskin1,
Lucien
Bettendorff1,
Gustave
Moonen1, 2,
Shibeshih
Belachew1, 2, *, and
Jean-Michel
Rigo1, *
1 Center for Cellular and Molecular Neurobiology,
University of Liège, B-4020 Liège, Belgium, and
2 Department of Neurology, University of Liège,
C.H.U. Sart Tilman, B-4000 Liège, Belgium
 |
ABSTRACT |
GABA and its type A receptor (GABAAR)
are present in the immature CNS and may function as growth-regulatory
signals during the development of embryonic neural precursor cells. In
the present study, on the basis of their isopycnic properties in a
buoyant density gradient, we developed an isolation procedure that
allowed us to purify proliferative neural precursor cells from early
postnatal rat striatum, which expressed the polysialylated form of the
neural cell adhesion molecule (PSA-NCAM). These postnatal striatal
PSA-NCAM+ cells were shown to proliferate in the
presence of epidermal growth factor (EGF) and formed spheres that
preferentially generated neurons in vitro. We
demonstrated that PSA-NCAM+ neuronal precursors from
postnatal striatum expressed GABAAR subunits in
vitro and in situ. GABA elicited chloride
currents in PSA-NCAM+ cells by activation of
functional GABAAR that displayed a typical pharmacological
profile. GABAAR activation in PSA-NCAM+
cells triggered a complex intracellular signaling combining a tonic
inhibition of the mitogen-activated protein kinase cascade and an
increase of intracellular calcium concentration by opening of
voltage-gated calcium channels. We observed that the activation of
GABAAR in PSA-NCAM+ neuronal precursors
from postnatal striatum inhibited cell cycle progression both in
neurospheres and in organotypic slices. Furthermore, postnatal
PSA-NCAM+ striatal cells synthesized and released
GABA, thus creating an autocrine/paracrine mechanism that controls
their proliferation. We showed that EGF modulated this
autocrine/paracrine loop by decreasing GABA production in
PSA-NCAM+ cells. This demonstration of GABA
synthesis and GABAAR function in striatal
PSA-NCAM+ cells may shed new light on the
understanding of key extrinsic cues that regulate the developmental
potential of postnatal neuronal precursors in the CNS.
Key words:
GABAA receptors; newborn rat striata; proliferation; PSA-NCAM; whole-cell patch-clamp; RT-PCR; HPLC; immunocytochemistry
 |
Introduction |
During CNS development, all
types of neurons and glial cells are derived from primordial neural
stem cells (NSCs) (Edlund and Jessell, 1999
) and emerge, according to a
precise time schedule, through a complex sequence of intermediate
precursors. Although the conventional view of the adult CNS used to be
a structurally constant organ, recent experimental evidence determined
that cells are regularly added de novo to several CNS areas
during adulthood (for review, see Gross, 2000
). NSCs are defined by
their ability to self-renew and to generate the main cell lineages of
the CNS (McKay, 1997
). NSCs have been isolated from embryonic and
newborn CNS as well as from specific restricted regions of the adult
mammalian CNS, including the subventricular zone [(SVZ) postnatally
termed the subependymal zone] and the dentate gyrus of the hippocampus (for review, see Weissman et al., 2001
). At early stages of CNS cell
fate determination, NSCs give rise to progenitors that express the
polysialylated form of the neural cell adhesion molecule (PSA-NCAM) (Doetsch et al., 1999
). Many tissues expressing PSA-NCAM during development show a progressive loss of PSA carbohydrate residues, but
PSA-NCAM+ cells persist in several adult
brain regions in which neuronal plasticity and sustained formation of
new neurons occur (Bonfanti et al., 1992
; Seki and Arai, 1993
; Doetsch
et al., 1997
). PSA-NCAM has been shown to be involved in changes of
cell morphology that are necessary for motility, axonal guidance,
synapse formation, and functional plasticity in the CNS (for review,
see Yoshida et al., 1999
; Bruses and Rutishauser, 2001
).
Although they are already restricted to either a glial (Trotter et al.,
1989
; Grinspan and Franceschini, 1995
; Ben Hur et al., 1998
; Vitry et
al., 1999
) or a neuronal (Mayer-Proschel et al., 1997
) preferential
fate, cultured PSA-NCAM+ progenitors
preserve a relative degree of pluripotentiality (Marmur et al., 1998
;
Vitry et al., 2001
). Considering that
PSA-NCAM+ cells can be neatly used for
brain repair purposes (Keirstead et al., 1999
; Vitry et al., 2001
),
there is much interest in studying signaling factors that regulate
their development. In this regard, it has been known for many years
that neurotransmitters, which belong to the microenvironment of neural
cells in vivo, regulate morphogenetic events preceding
synaptogenesis such as cell proliferation, migration, differentiation,
and death (for review, see Nguyen et al., 2001
). Along this line,
previous reports have suggested that GABA, the major inhibitory
neurotransmitter in the mammalian brain, exerts trophic roles during
CNS embryonic and postnatal development (Barker et al., 1998
).
To investigate whether GABA may control the proliferation of postnatal
PSA-NCAM+ neural precursors, we first
established an isolation procedure that allows the purification of
PSA-NCAM+ precursors from newborn rat
striata. Using this in vitro preparation together with
postnatal striatal organotypic slices, we report the following: (1)
epidermal growth factor (EGF)-responsive proliferative PSA-NCAM+ precursors generate spheres
committed mostly to a neuronal fate; (2) postnatal
PSA-NCAM+ precursors express functional
type A GABA receptors (GABAARs) and glutamate
decarboxylase (GAD) 65 and GAD 67; (3) proliferation of
PSA-NCAM+ precursors is inhibited by an
EGF-controlled endogenous production of GABA that activates
GABAAR in these cells; and (4)
GABAAR-dependent inhibition of
PSA-NCAM+ cell proliferation is mediated
by a complex intracellular signaling involving notably the inhibition
of the mitogen-activated protein kinase (MAPK) pathway and an increase
of intracellular calcium concentration by opening of voltage-gated
calcium channels.
 |
Materials and Methods |
Sequential purification of
PSA-NCAM+ progenitors. Newborn Wistar
rats (0- to 3-d-old rat pups) were raised from our animal facility.
They were killed following National Institutes of Health animal
welfare guidelines. Briefly, rats were anesthetized and subsequently
decapitated. Striata were dissected out and collected in PBS
solution supplemented with glucose at 4.5 gm/l. Next, isolated striata,
possibly including small parts of subventricular zones, were gently
triturated in PBS-HEPES (25 mM) by passing
through a fire-polished Pasteur pipette before being filtered with a 15 µm nylon mesh. The cell suspension was then layered on top of a
pre-centrifuged (15 min at 26,000 × g) Percoll density
gradient (1.04 gm/ml; Amersham Biosciences, Uppsala,
Sweden) and further ultracentrifuged for 15 min at 26,000 × g. Proliferating PSA-NCAM+
precursor cells were separated from differentiated postmitotic neural
cells and cell debris by collecting the interphase located between the
bands at 1.052 and 1.102 gm/ml as determined by using density marker
beads (Amersham Biosciences) for the calibration of the
Percoll gradient after centrifugation (Maric et al., 1997
) (see Fig.
1A,B). The resulting suspension was
then centrifuged three times (10 min at 400 × g) in
PBS-HEPES to eliminate Percoll. The final pellet was resuspended in
DMEM/F12 (1:1, v/v; Invitrogen, Merelbeke, Belgium) medium
supplemented with 1% (v/v) N2 (25 µg/ml bovine insulin, 100 µg/ml
transferrin, 20 nM progesterone, 60 µM putrescine, 30 nM
sodium selenite), 1% (v/v) B27 (Invitrogen) with or
without EGF (20 ng/ml) supplementation (PeproTech, Rocky Hill, NJ). We refer hereafter to these media as either EGF-containing or EGF-free medium. The final cell suspension was plated either in 50 µl droplets on poly-ornithine-coated (Becton Dickinson, Erembodegem, Belgium) coverslips at a density of
2.106 cells/ml for immunocytochemical
studies or onto uncoated nonadherent T25 culture flasks in 5 ml of
EGF-containing medium at a density of
2.105 cells/ml (Sarstedt,
Newton, NC). Cells grown in uncoated conditions generated floating
spheres (see Fig. 1I). After 3 d in
EGF-containing medium, growing spheres were allowed to attach for 1 hr
on poly-ornithine-coated coverslips before being used further for
patch-clamp recordings or immunocytochemical studies.
Immunostainings. Cultures were fixed with 4% (v/v)
paraformaldehyde for 10 min at room temperature and permeabilized in
0.1% Triton X-100 (v/v) for 15 min during which subsequent
immunostainings were directed toward cytoplasmic epitopes. For
anti-GABAA
subunit staining, cells were fixed
with a methanol/acetic acid (95:5, v/v) mixture for 5 min. For all
immunostainings, nonspecific binding was blocked by a 30 min treatment
in a PBS solution containing nonfat dry milk (15 mg/ml). Cells were
then incubated overnight at 4°C with primary antibodies, i.e., mouse
anti-PSA-NCAM at 1:500 (anti-Men-B antibody; generous gift from G. Rougon, Université de la Méditerranée, Marseille,
France), rabbit anti-nestin at 1:400 (generous gift from Prof. J. Eriksson, University of Turku, Turku, Finland), mouse anti-A2B5 at
1:100 (Boehringer Mannheim, Mannheim. Germany), mouse
anti-
III tubulin at 1:1500 (clone Tuj1, Babco,
Richmond, CA), mouse anti-MAP2ab at 1:100 (clone AP20, Boehringer
Mannheim), mouse monoclonal anti-O4 at 1:150
(Chemicon, Temecula, CA), rabbit anti-glial fibrillary
acidic protein (GFAP) at 1:1500 (Dako, Prosan, Belgium),
rabbit anti-NF-M at 1:350 (Chemicon), mouse
anti-synaptophysin at 1:200 (Sigma-Aldrich, Bornem,
Belgium), goat anti-GABAA
subunits
(
1-
3,
5) at 1:40 (clone C-20, Santa Cruz
Biotechnology, Santa Cruz, CA), goat
anti-GABAA
1-3 subunits
at 1:50 (clone N-19, Santa Cruz Biotechnology), goat anti-GABAA
1-4 subunits
antibody at 1:50 (clone M-20, Santa Cruz Biotechnology),
rabbit anti-GABA at 1:400 (Incstar, Stillwater, MN),
rabbit anti-GAD (GAD 67) at 1:500 (Biogenesis,
Poole, UK), and rabbit anti-GAD 65 at 1:50 (clone H-95, Santa
Cruz Biotechnology). Secondary antibodies were diluted in PBS
solution and applied for 45 min at room temperature. These included
Cy5-, FITC-, or TRITC-conjugated anti-rabbit Ig antibodies (1:500),
Cy5-, FITC-, or TRITC-conjugated anti-mouse IgG (1:500), and Cy5-,
FITC-, or TRITC-conjugated anti-mouse IgM (all from Jackson
ImmunoResearch Laboratory, West Grove, PA), or FITC- or
TRITC-conjugated anti-mouse IgG2a (ImTec
Diagnostics, Antwerp, Belgium). Three rinses in PBS were performed
between different steps. Preparations were mounted in Fluoprep
(Biomerieux). Images were acquired using a laser scanning confocal microscope (MRC1024, Bio-Rad, Hertfordshire, UK).
For quantitative immunostainings, before immunocytochemical procedure,
spheres were mechanically dissociated and further plated onto
poly-ornithine-coated coverslips. Cells were allowed to attach for 1 hr
before fixation. For counting, cells were counterstained with the
nuclear dye ethidium homodimer-1 (Etd1) (applied at
6.10
7
M for 7 min; Molecular Probes, Leiden, The Netherlands) or
Hoescht 33258 (0.4 µg/ml for 15 min). Ten nonoverlapping microscopic
fields (±50 cells per field) (Axiovert 135 fluorescence microscope,
40× objective; Zeiss) were counted for each coverslip in
a minimum of two or three separate experiments.
Frozen 30 µm tissue sections were prepared as described previously
(Yuan et al., 2002
). Immunohistochemical stainings were processed
following a procedure identical to that of cultured cells.
Electrophysiological recordings. For patch-clamp recordings,
Cell-Tak (Becton Dickinson)-coated coverslips containing
1-3 hr adhesive spheres were transferred to the stage of a
Zeiss interferential contrast microscope equipped with
fluorescence. Coverslips were maintained at 37°C in a recording
chamber that was perfused continuously with a saline solution
containing (in mM): 116.0 NaCl, 11.1 D-glucose, 5.4 KCl, 5.4, 1.8 CaCl2·2H2O, 2.0 MgCl2·6H2O, 10.0 HEPES,
pH 7.2. Cs+-containing solutions were
composed as follows (in mM): 116.0 NaCl, 5.4 CsCl, 11.1 D-glucose, 1.8 CaCl2·2H2O, 2.0 MgCl2·6H2O, 9.0 HCl, 5.0 HEPES, 26.2 NaHCO3, 5.0 BaCl2·2H2O, pH 7.2. Low
chloride solution contained (in mM): 8.0 NaCl,
108.0 Na-gluconate, 5.4 CsCl, 5.4, 11.1 D-glucose, 1.8 CaCl2·2H2O, 2.0 MgCl2·6H2O, 17.0 HCl, 5.0 HEPES, 26.2 NaHCO3, 5.0 BaCl2·2H2O, pH 7.4. All
drugs were applied by a microperfusion system (SPS-8, List Medical). Borosilicate recording electrodes (15-20 M
) were made using a Flaming-Brown microelectrode puller (P97, Sutter
Instruments Novato, CA). Micropipettes were filled with an
intracellular-like solution containing (in mM):
130.0 KCl, 1.0 CaCl2·2H2O, 11.1 D-glucose, 10.0 EGTA, 2.5 Na2-ATP, 2.5 Mg-ATP, 10.0 HEPES, pH 7.4. In
Cs+-containing pipettes, used for the
establishment of the GABA-evoked current-voltage relationship, KCl was
equimolarly replaced by CsCl and
BaCl2·2H2O was added at 5 mM to block K+
channels. Current-voltage relationships were obtained using a series
of voltage steps (ranging from
140 to +100 mV) before, during, and
after application of GABA. The current-voltage curve was established
by fitting experimental data to the Goldman-Hodgkin and Katz
equation:
|
|
where Is corresponds to the current
generated (ampere), Ps the membrane
permeability, Zs the valence,
[S]i the intracellular concentration
(M·l
1),
and [S]o the extracellular
concentration
(M·l
1)
of the ion S, respectively. E corresponds to the
membrane potential, F is the Faraday's constant,
R is the ideal gas constant, and T is the
absolute temperature. Electrophysiological recordings were performed
with a patch-clamp amplifier (RK400, Bio-Logic, Claix,
France) using the whole-cell configuration of the patch-clamp recording
technique (Hamill et al., 1981
). Cells were injected with Lucifer
yellow (Molecular Probes) (1 µg/ml Lucifer yellow solution in the recording pipette) during voltage-clamp recordings to
allow their post hoc immunocytochemical characterization.
Series resistances (10-20
) were electronically compensated
(80-85%), and current traces were filtered at 3 kHz, acquired and
digitized at 0.5 kHz, and stored on an personal computer system.
Control of drug application, data acquisition, and data analysis was
achieved using an ITC-16 acquisition board (Instrutech
Corporation, Great Neck, NY) and the TIDA for Windows software
(HEKA Elektronik Lombrecht/Pfolz, Germany).
RT-PCR. Total RNAs from adult Wistar rat brains and from
PSA-NCAM+ spheres derived from postnatal
day 0 (P0)-P3 Wistar rat striata were extracted and purified using the
RNAgents Total RNA Isolation System kit (Promega, Leiden,
The Netherlands). One microgram of total RNA was reverse transcribed
using primers with oligo-dT and 200 U of reverse transcriptase (Kit
Superscript 1, Life Technologies). Two microliters
resulting from the RT reaction were used as template and added to 50 µl of PCR reaction mixture containing 0.2 µM
of both forward and reverse primers synthesized by
Eurogentec (Seraing, Belgium) (see Table 1), 0.2 mM of each dNTP, 1.5 mM of
MgCl2, and 5 U of Taq Polymerase
(Promega). The PCR program was run with an MJ
Research PTC 200 instrument. The thermal cycling protocol started with a 2 min preincubation at 94°C followed by 35 cycles made
(1) 30 sec at 94°C, (2) 30 sec at 60°C, and (3) 30 sec at 72°C.
The protocol was finally completed by an extension step at 72°C for 7 min. We used 64°C for the annealing of
3
primers and 55°C for
3, GAD 65, and GAD 67 primers. Ten microliters of the PCR reaction were analyzed in a 1.4%
agarose gel in Tris-acetic acid-EDTA (TAE) buffer.
Bromodeoxyuridine and
[3H]thymidine incorporation assays.
After 2 d of growth in EGF-containing medium (as described
previously), PSA-NCAM+ spheres were
harvested, centrifuged (10 min at 200 × g), and rinsed
three times in the EGF-free medium before being transferred into
uncoated nonadherent T25 culture flasks (Sarstedt) in
mitogen-free medium. After 24 hr in this medium, bromodeoxyuridine
(BrdU) (20 µM; Sigma), which is a
S-phase marker, was added to the cultures for 18 hr before fixation and
staining. All treatments were performed simultaneously with the
addition of BrdU. PSA-NCAM,
III tubulin, O4, and GFAP
immunolabelings were performed as described above. Coverslips were then
postfixed for 10 min in 4% (v/v) paraformaldehyde, permeabilized in
0.1% Triton X-100 for 10 min, incubated in 0.07N NaOH for 10 min, and
finally postfixed again for 10 min before incubation with an anti-BrdU
FITC-conjugated antibody for 45 min (1:3, v/v; Becton-Dickinson). The
preparations were mounted in Fluoprep and imaged using a
Bio-Rad MRC1024 laser scanning confocal microscope. The
fraction of cells that incorporated BrdU was determined by counting 10 nonoverlapping microscopic fields (±50 cells per field) (Axiovert 135 fluorescence microscope, 40× objective, Zeiss) for each
coverslip in at least three separate experiments.
In similar culture conditions, the proliferation of
PSA-NCAM+ spheres was also quantified by
measuring the incorporation of [3H]thymidine (Amersham
Biosciences, Roosendaal, The Netherlands). All treatments were
performed simultaneously with the addition of
[3H]thymidine (2 µCi/ml) to the medium
for 18 hr. Cultures were washed three time with PBS and digested with
NaOH (0.1N), and the radioactivity was counted in a liquid
scintillation counter (Wallac WinSpectral 1414 liquid
scintillation counter, Turku, Finland). The
[3H]thymidine incorporation was
normalized for cellular protein concentration measured by the Bradford
technique (Bradford, 1976
) and expressed as disintegrations per minute
of [3H]thymidine incorporated per
milligram of protein. Results from the treated conditions were then
expressed as percentages of control values. We always performed three
separate experiments in triplicate wells for each condition.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling assay. To assess apoptosis occurring in our cultures, spheres were gently dissociated after treatments, and cells were plated
for 15-30 min on Cell-Tak (Becton Dickinson)-coated
coverslips at a density of 2.106 cells/ml.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) staining was then performed according to the method of Gavrieli
et al.(1992)
, using the ApopTag fluorescent detection kit
(Oncor, Gaithersburg, MD). Cultures were fixed with 4%
paraformaldehyde for 10 min at room temperature. Equilibration buffer
was then applied for 30 min at 20°C. Cultures were incubated with
working strength stop/wash buffer, washed, and further incubated with
anti-digoxigenin-FITC. For cell counting, cultures were counterstained with ethidium homodimer-1 (Molecular Probes; applied at
6.10
7
M for 7 min). Ten nonoverlapping microscopic
fields (±50 cells per field) (Axiovert 135 fluorescence microscope,
40× objective, Zeiss) were counted for each coverslip in
a minimum of three separate experiments.
HPLC procedure. We used an adaptation of a procedure
described previously (Bettendorff et al., 1996
). Cultures of
PSA-NCAM+ spheres (25 mg) were homogenized
in 1 ml of an 80% ethanol solution at 0°C in a glass-glass
homogenizer (Potter-Elvehjem device). Homogenates were centrifuged (30 min at 5000 × g), and the supernatants were saved. The
pellets were resuspended in 1 ml of a 60% ethanol solution,
homogenized, and centrifuged as described above. The second supernatant
was pooled with the first, and the liquids were evaporated under a
stream of nitrogen. The residue was dissolved in 300 µl of water and
centrifuged (30 min, 5000 × g). An aliquot of 100 µl
from the supernatant was added to 100 µl of
LiCO3 (80 mM, pH 8.5)
before dansylation by addition of 100 µl dansyl chloride (1.5 mg/ml
in acetonitrile). The mixture was incubated in the dark for 35 min at
25°C, and the reaction was stopped with 10 µl of 2% ethylamine.
We used a Bio-SiL C18 HL column (5 µm, 150 × 4.6 mm;
Bio-Rad Laboratories, Nazareth-Eke, Belgium) heated at
50°C. After injection of the dansylated solution (20 µl), GABA was
eluted by means of a linear gradient at a flow rate of 1.5 ml/min. The
column was equilibrated in 85% solvent A (3% tetrahydrofuran, 0.57%
acetic acid, 0.088% triethylamine in water) and 15% solvent B (3%
tetrahydrofuran, 0.57% acetic acid, 0.088% triethylamine, 70%
methanol in water). After injection, the percentage of solvent B was
increased linearly to reach 100% after 40 min. Initial conditions were
restored within 2 min, and the next sample was injected after a
reequilibration period of 5 min. A fluorescent spectrometer (LS-4,
Perkin-Elmer, Norwalk, CT) was used with the wavelengths
set at 334 nm for excitation and 522 nm for emission. A reference
standard, composed of a GABA solution (0.1 mM) in water,
was dansylated simultaneously with samples.
Calcium imaging. PSA-NCAM+
cells were loaded with the calcium indicator dye fluo-3 AM (6 µM) (Molecular Probes) by bath
application for 30 min at 37°C. Fluo-3 AM is a non-ratiometric
indicator dye that triggers an increase of cell fluorescence intensity
when the intracellular calcium concentration increases. After fluo-3 loading, cells were washed three times in Locke solution containing (in
mM): 154 NaCl, 5.6 KCl, 5.6 glucose 5.6, 2.3 CaCl2·2H2O, 10.0 HEPES,
pH 7.2. Calcium responses were recorded as digitized images acquired
with a Bio-Rad MRC 1000 laser scanning confocal system coupled to a Zeiss Axiovert 135 microscope with a
plan-NEOFLUAR objective (40×, 1.3 numerical aperture, oil
immersion). The Time Course Software Module program
(Bio-Rad) was used to control the confocal microscope to
acquire a series of images at intervals from 2 to 5 sec. The different
reagents diluted in Locke solution were applied by a microperfusion
system (SPS-8, List-Medical). The series of digitized fluorescence
images were analyzed by a program that determined the average level of
fluorescence above the background level of each cell for every time
point sampled. The recorded areas were delimited by placing rectangular
boxes around every cell in a field. A "background" box was also
defined in a noncellular area of each scanned image. The averaged
intensity of the pixels within a boxed cellular region was calculated
by the program, and the averaged intensity of the pixels within the "background" box defined for the image was subtracted from this value. To compensate for variable dye loading between cells, these background-corrected values were normalized by conversion to percentage changes relative to a baseline measurement for each boxed cellular region at the start of a time series
(Ft/F0).
Organotypic slice cultures. We used a technique adapted from
Yuan et al. (1998)
. Briefly, whole brains were dissected from P1
Sprague Dawley rats and placed in oxygenated (carbogen,
95% O2/5% CO2) artificial
CSF containing (in mM): 120 NaCl, 25 NaHCO3, 3.3 KCl, 1.2 NaH2PO4, 1.8 CaCl2, 2.4 MgSO4, 10 glucose, pH 7.2. Brains were then sliced coronally (400 µm) using a
vibratome. The SVZ and striatum (as depicted in Fig.
10A1) were separately microdissected to allow a
distinct assessment of SVZ and striatal cells. SVZ and striatal slices
were placed into sterile Millicell (Millipore, Bedford,
MA) in six-well plates (Falcon) containing 1 ml of EGF-free medium (as
described previously). Treatments with drugs began 4 hr after the
slicing procedure. The medium was replaced by EGF-free medium
containing simultaneously BrdU (20 µM;
Sigma) and drugs for 18 hr. Cell viability was assessed in
each experiment at the end of the BrdU incorporation time frame by
using a LIVE/DEAD viability/cytotoxicity kit (L-3224,
Molecular Probes). Slices were then gently mechanically
dissociated, and cells were plated for 1 hr on poly-ornithine-coated
coverslips before fixation and immunocytochemical analysis.
Drugs. GABA, muscimol, bicuculline, picrotoxin,
pentobarbital, baclofen, saclofen, SR-95531, U0126, and nifedipine were
obtained from Sigma-Aldrich, and clonazepam was purchased
from Roche Diagnostics Belgium (Brussels, Belgium).
Data analysis. For electrophysiological recordings,
n represented the number of recorded cells. Peak currents in
the different experimental conditions were measured and subsequently
normalized to the initial response (100%) in control conditions.
Agonist concentration-response profiles were fitted to the following
equation: I/Imax = 1/(1+(EC50/[agonist])nh),
where I and Imax, respectively,
represent the normalized agonist-induced current at a given
concentration and the maximum current induced by a saturating
concentration of the agonist. EC50 is the
half-maximal effective agonist concentration, and nh is the
Hill slope. The concentration-response of modulations was fitted by a
similar procedure, except for clonazepam, where a polynomial curve was used.
The quantitative results of
[3H]thymidine incorporation assays and
immunocytochemical experiments were expressed as mean ± SEM
values arising from a minimum of three independent experiments (n).
For all experiments, a statistical analysis was performed either using
unpaired two-tailed Student's t test between control and
experimental conditions or using a one-way ANOVA (ANOVA-1) followed by
a Dunnett's post-test for multiple comparisons (GraphPad Prism software, version 2.04 a, San Diego, CA). The level of
significance was expressed as follows: *p < 0.05, **p < 0.01, and ***p < 0.0001.
 |
Results |
Purification and characterization of PSA-NCAM+
progenitors acutely isolated from newborn rat striata
To obtain a highly enriched population of
PSA-NCAM+ progenitors, newborn rat
(P0-P3) striata were first dissociated as described previously for
neural stem cell cultures (Reynolds and Weiss, 1992
). The cell
suspension was then layered on top of a buoyant density Percoll
gradient and further ultracentrifuged (Fig.
1A). This isopycnic
centrifugation allowed cells to sediment in an equilibrium position
equivalent to their own natural buoyant density. We demonstrated that
viable, small (7 ± 1 µm in diameter), round cells were
separated from more differentiated cells and cell debris by collecting
the interphase located in the range of densities between 1.052 and
1.102 gm/ml (Fig. 1B). The final cell suspension harvested according to these density criteria was resuspended in
EGF-containing medium.

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|
Figure 1.
Purification and in vitro
amplification of proliferative and neurogenic
PSA-NCAM+ progenitors from early postnatal striatum.
A, Bands of color-coded density marker beads in
ultracentrifuged Percoll gradient. According to their isopycnic buoyant
densities, living PSA-NCAM+ cells
(B) were separated from differentiated neural
cells and cell debris in a continuous Percoll gradient. Cells were
collected in the interphase located between the ranges of density:
1052-1102 gm/ml as determined by a tube containing control density
beads that was ultracentrifuged simultaneously. C, D,
Confocal images of acutely dissociated cell suspension from newborn rat
striatum before (C) and after
(D) selection by centrifugation in a Percoll
density gradient. Cells were immunostained for PSA-NCAM (green) and
counterstained with the nuclear dye Etd1 (red). Cells acutely purified
from early postnatal striatum (1 hr) or dissociated from 3-DIV-old
spheres were allowed to adhere onto poly-ornithine-coated coverslips
and were assessed by immunostaining. E, Histogram
comparing the percentage of total cells expressing various markers 1 hr
after purification (blue bars) and after 3 d of growth in
vitro in EGF-containing medium (red bars). F-H,
Confocal images of representative fields showing acutely purified cells
immunostained for markers of neuronal commitment: F,
Tuj1 (green); G, MAP2ab (green); H, NF-M
(green), and F-H, counterstaining with
Etd1 (red). Purified cells cultured in EGF-containing medium for 3 d in uncoated conditions formed spheres
(I) that were composed almost exclusively
of PSA-NCAM+ cells. J, PSA-NCAM in
green and Etd1 in red. K, Confocal optical section of a
3-DIV sphere immunostained for BrdU after 18 hr of BrdU incorporation
assay in EGF-containing medium (PSA-NCAM in green and BrdU in red).
L, Histograms representing the percentage of total cells
that incorporated BrdU (20 µM) for each immunophenotype
(left panel) and the percentage of total BrdU+ cells
that expressed a given immunophenotype (right panel), respectively, in
the presence (black bars) or absence (open bar) of EGF (20 ng/ml).
M-O, Confocal optical section of 3-DIV spheres
expressing markers of neuron commitment: M, Tuj1
(green); N, MAP2ab (green); O, NF-M
(green) and counterstaining with Etd1 (red). Scale bars:
B-D, 10 µm;
F-K, 25 µm; M-O, 20 µm.
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To validate our protocol of purification, we assessed
PSA-NCAM+ cells before and after the
Percoll centrifugation. The isopycnic centrifugation of cell suspension
in a buoyant density gradient allowed us to increase the percentage of
PSA-NCAM+ cells from 62.5 ± 15.1%
(n = 3) to 94 ± 1.0% of total cells
(n = 6) (Fig. 1C-E). After
purification by the Percoll centrifugation step, the phenotype of
PSA-NCAM+ cell suspension was
characterized more extensively. Nestin was observed in 74.9 ± 8.3% (n = 4) of total cells, and all
nestin+ cells also expressed PSA-NCAM
(Fig. 1E). We observed that 47.6 ± 4.5% of
total cells (n = 2) were
A2B5+ (Fig. 1E).
Neuronal phenotypes were investigated by studying the expression of
neuron-specific antigens. We found that 74.6 ± 1.4%
(n = 4) of total acutely purified cells expressed
III-tubulin (i.e., Tuj1+) (Fig.
1E,F), 4.5 ± 2.6%
(n = 2) expressed type 2a,b microtubule-associated protein (i.e., MAP2ab+) (Fig.
1E,G), and 2.6 ± 1.6%
(n = 2) were neurofilament 145 kDa-positive (i.e.,
NF-M+) (Fig.
1E,H). Importantly, we never
found cells that were immunoreactive for synaptophysin, which is a
marker of synapse formation (Fig. 1E). Finally, we
found a low expression of oligodendrocyte (O4) or astrocyte (GFAP)
specific markers. Respectively, 2.8 ± 1.2% (n = 4) of total cells were O4+ and 4.4 ± 0.8% of total cells (n = 4) were
GFAP+ (Fig. 1E). These
results provide evidence that purified
PSA-NCAM+ cells from early postnatal rat
striatum mostly show antigenic features of neuron-committed progenitor cells.
Purified proliferative PSA-NCAM+ cells form
spheres that preferentially generate neurons
After 3 d in vitro (DIV) in EGF-containing medium,
PSA-NCAM+ progenitor cells proliferated
and formed spheres with a mean diameter of 61.8 ± 7.3 µM (n = 4) (Fig.
1I). A vast majority of cells within 3-DIV-old
spheres remained PSA-NCAM+ (89.6 ± 4.7% of total cells; n = 6), and
Tuj1+ (57.3 ± 1.2% of total cells;
n = 4) (Fig.
1J,M). Interestingly, we
observed that all Tuj1+ cells were still
PSA-NCAM+ in 3-DIV spheres (data not
shown). To quantify cell proliferation within
PSA-NCAM+ spheres, we performed a BrdU
incorporation assay (18 hr) at 3 DIV. The BrdU incorporation index
(BrdU+ cells per total cells) was
17.2 ± 4.0% in the presence of EGF (20 ng/ml) (n = 2) (Fig. 1K,L). By double
immunostaining, we observed that proliferating cells were mostly
PSA-NCAM+ because 16.0 ± 4.1% of
total cells (n = 3) were immunoreactive for both
PSA-NCAM and BrdU (Fig. 1K,L, left
panel). The other way around, we showed that 92.9 ± 4.1% of the
total BrdU+ cells were
PSA-NCAM+ (Fig. 1L,
right panel). Conversely, cells expressing markers of lineage
commitment were weakly involved in the overall BrdU incorporation index
because only 3% of Tuj1+ cells, 1% of
O4+ cells, and 2% of
GFAP+ cells were also
BrdU+ (Fig. 1L, left
panel). Interestingly, cultured PSA-NCAM+
cells generated predominantly neuron-committed cells after 3 DIV in
EGF-containing medium (Fig.
1E,M-O). As compared
with acutely purified cells, we observed a fourfold and a fivefold
increase, respectively, of the relative percentages of
MAP2ab+ and
NF-M+ cells in 3-DIV spheres, whereas no
change was observed for O4+ or
GFAP+ cells. Furthermore, with respect to
the calculated 1.8-fold increase of the total cell number during the
3-DIV growth of PSA-NCAM+ spheres (data
not shown), the absolute number of cells expressing mature neuronal
antigens MAP2ab and NF-M were increased by seven- and eightfold,
respectively (Figs. 1N,O).
PSA-NCAM+ spheres express type A
GABA receptors
Given that previous works reported the expression of
GABAAR in early postnatal neuronal progenitor
cells, notably in the anterior subventricular zone, we sought to
investigate the presence of these receptors in striatal
PSA-NCAM+ neuronal precursors (Stewart et
al., 2002
). To characterize GABAAR subunit
transcripts expressed in PSA-NCAM+
progenitors, total RNAs extracted from 3-DIV spheres were reverse transcribed, and the subsequent cDNAs were amplified by PCR using specific sets of primers aimed at detecting transcripts for
1-5,
1-3,
1-3, and
GABAAR
subunit genes (Table 1). Experiments were
replicated three times and consistently yielded bands with the
appropriate amplicon size for
2 (549 bp),
4 (532 bp),
5 (300 bp),
1 (578 bp),
3
(587 bp),
1 (296 bp),
2 (423 bp), and
3
(336 bp) transcripts, respectively (Fig.
2A,B).
RNAs isolated from total adult rat brains were used as positive
control.

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Figure 2.
GABAA receptors are expressed by
PSA-NCAM+ progenitors from early postnatal striatum.
A, B, RT-PCR amplification of
GABAAR 1-5, 1-3,
1-3, and subunit transcripts using RNA extracted
from 3-DIV PSA-NCAM+ spheres
(A) and adult rat brain tissue
(B). Bands corresponding to 2 (549 bp), 4 (532 bp), 5 (300 bp),
1 (578 bp), 3 (587 bp), 1
(296 bp), and 3 (336 bp) were detected (+, with RT; ,
without RT). Left margins indicate migration of standard DNA markers
with size indicated in base pairs. C, Z-series confocal
image of 3-DIV PSA-NCAM+ cells immunoreactive for
GABAAR subunits (green). D, E, Confocal
images of 3-DIV PSA-NCAM+ spheres showing
GABAAR + cells (D, green),
and GABAAR + cells (E,
green), respectively. All cultures were counterstained with Etd1 (red).
Scale bars: C-E, 10 µm.
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The expression of GABAAR subunit proteins was
analyzed by immunocytochemistry in
PSA-NCAM+ spheres. We used three
polyclonal antibodies directed against
1-3,5,
1-3, and
1-4
subunits, respectively, of GABAAR. As illustrated
in Figure 2C-E, 70.6 ± 13.8% of total cells (counted after mechanical dissociation of spheres) were immunoreactive for GABAAR
subunit proteins
(i.e.,
1-3,5; n = 2) (Fig.
2C), 65.6 ± 4.3% of total cells were immunoreactive for GABAAR
subunits (i.e.,
1-3; n = 2) (Fig.
2D), and 66.6 ± 6.2% of total cells expressed
GABAAR
subunits (i.e.,
1-4; n = 3) (Fig.
2E).
GABA triggers whole-cell currents in PSA-NCAM+
spheres by GABAA receptor activation
We wanted to ascertain by electrophysiological recordings whether
PSA-NCAM+ cells expressed functional
GABAA receptors. We therefore recorded cells
within PSA-NCAM+ spheres using the
whole-cell patch-clamp technique. Occasionally, the Lucifer yellow
fluorescent dye was added to the intracellular solution and allowed to
diffuse in the recorded cell for post hoc immunostainings.
All recorded cells filled with Lucifer yellow were
PSA-NCAM+ in 3-DIV spheres (Fig.
3A). We selectively assessed
cells that were located at the accessible periphery of the spheres. The
mean membrane potential recorded in current-clamp configuration was
52.9 ± 1.9 mV (n = 110 cells). All recordings
were performed in the presence of 1 µM
strychnine to avoid cross-activation of ionotropic glycine receptors.
In voltage-clamp mode (the holding potential was kept at
70 mV), bath
application of 1 mM GABA, a concentration that
saturates GABAARs (Fig. 3C), elicited
inward currents in 94.6% of total cells with a peak current displaying a mean maximum amplitude of 408.9 ± 46.8 pA (n = 53 cells) (Fig. 3B).

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Figure 3.
GABAA receptor activation
triggers chloride-mediated inward currents in
PSA-NCAM+ progenitor cells. A,
Confocal image showing a GABA-responsive cell injected with Lucifer
yellow (green) and expressing PSA-NCAM (red). B,
Histogram representing the mean maximum current induced by GABA 1 mM and the percentage of responding cells in the total
recorded population of 3 DIV PSA-NCAM+ cells,
respectively. C, Concentration-response curve obtained
from GABA-responsive PSA-NCAM+ progenitors.
E, The specific GABAAR agonist muscimol also
induced concentration-dependent currents in
PSA-NCAM+ cells. D, F, Traces
illustrating inward currents elicited by different concentrations of
GABA (D) and muscimol (D).
G-J, Reversal potential of GABA-induced currents
(EGABA). I,
J, Current-voltage relationship of GABA-evoked currents
was studied by applying voltage steps ranging from 140 to +100 mV
repetitively every 5 sec before, during, and after GABA (100 µM) application. Mean control currents (before and after
GABA application) were subtracted from the currents recorded at the
peak of the GABA response. G-I, Using the currents
obtained in I, we constructed a current-voltage curve
reversing at +5.89 mV (n = 4 cells), which is close
to the calculated Nernst chloride equilibrium potential ( 1.1 mV)
(left panel). H-J, When extracellular chloride
concentration was lowered (J), the reversal
potential shifted to +30.63 mV (n = 5 cells), which
again is close to the expected chloride equilibrium potential in this
condition (+29.00 mV).
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In GABA-responsive PSA-NCAM+ progenitors,
the EC50 value (i.e., the concentration that
yielded an inward current of half-maximum amplitude) calculated from
the sigmoidal concentration-response curve was 6.2 ± 1.1 µM, with a Hill coefficient
(nh) of 0.7 ± 0.2 (n = 11 cells) (Fig.
3C,D). To confirm that GABA-elicited currents
were caused specifically by the activation of
GABAARs, we showed that the specific
GABAAR agonist muscimol also induced inward
currents in PSA-NCAM+ cells (Fig.
3F). For muscimol-induced currents, the
concentration-response curve, fitted by the Hill equation, yielded an
EC50 of 6.5 ± 1.1 µM, with a Hill coefficient
(nh) of 0.5 ± 0.2 (n = 5 cells) (Fig. 3E,F).
The current-voltage relationship of GABA-evoked currents was obtained
by applying voltage steps ranging from
140 to +100 mV during GABA
application. As shown in Figure 3, G and I, the resulting current-voltage curve could be fitted by the
Goldman-Hodgkin-Katz relation (see Materials and Methods) and reversed
at +5.9 mV (n = 4 cells), which is close to the
calculated Nernst chloride equilibrium potential (
1.1 mV). When 108 mM of extracellular sodium chloride was replaced
by sodium gluconate, the reversal potential shifted to a more positive
value (+27.9 mV; n = 5 cells) (Fig.
3H,J), consistent with the
predicted shift of the calculated Nernst chloride equilibrium potential
(+29.0 mV).
Pharmacological characterization of functional
GABAARs expressed in striata-derived
PSA-NCAM+ progenitors
In 3-DIV PSA-NCAM+ cells,
GABA-induced currents were completely and reversibly inhibited in a
dose-dependent manner by the competitive antagonists bicuculline
(IC50 = 1.54 ± 1.12 µM;
n = 5 cells) (Fig.
4A,B)
and SR95531 (IC50 = 0.15 ± 0.01 µM; n = 6 cells) (Fig.
4C,D) and by the noncompetitive antagonist
picrotoxin (IC50 = 4.5 ± 1.1 µM; n = 7 cells) (Fig.
4E,F). We also assessed the
effect of benzodiazepines and barbiturates, which are positive allosteric modulators of GABAAR. The effects of
clonazepam and pentobarbital were studied on currents elicited by a low
concentration of GABA (1 µM = EC10 = GABA concentration inducing an inward
current corresponding to 10% of the maximum GABA-evoked current) to
sensitize the detection of a enhancing effect. Our results showed that
clonazepam potentiated GABA currents at concentrations ranging from 10 nM to 100 µM, with a
maximum effect at 1 µM (212% of
IGABA at EC10) (Fig. 4G,H). Pentobarbital triggered a
maximal 5.6-fold increase of the amplitude of GABA-evoked currents in a
concentration-dependent manner (EC50 = 2.1 ± 0.3 µM; n = 11 cells) (Fig.
5I,J).

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Figure 4.
Pharmacological characterization of
GABAAR expressed by PSA-NCAM+
progenitors. A-F, GABA was applied at 10 µM (IGABA 10 µM), a concentration close to its EC50.
GABA-evoked currents were reversibly inhibited by bicuculline
(A, B), SR-95531 (C, D), and picrotoxin
(E, F). G-J, We also tested
positive allosteric modulators of GABAAR. Clonazepam
potentiated GABA-induced currents (GABA at 1 µM,
EC10) in a range of concentrations between 10 nM and 100 µM, with a maximal effect at 1 µM (G, H). I-J,
Pentobarbital also enhanced GABA-evoked currents in a
concentration-dependent manner.
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Figure 5.
GABAAR activation inhibits the
proliferation of PSA-NAM+ cells at both
Tuj1 and Tuj1+ stages. Cells
were incubated simultaneously with drugs and BrdU (20 µM)
for 18 hr in EGF-free medium. The anti-mitotic agent cytosine
arabinoside (AraC, 10 µM) was used as an internal control
condition. (A, D, G). GABAAR agonists (100 µM GABA and 100 µM muscimol) inhibited the
incorporation of BrdU (n = 6; ANOVA-1 followed by a
Dunnett's post-test; *p < 0.05, **p < 0.01, ***p < 0.0001) in
total PSA-NCAM+ cells (A), in
Tuj1+/PSA-NCAM+ cells
(D), and in
Tuj1 /PSA-NCAM+ cells
(G). The effect of muscimol was totally abolished
by SR-95531 (100 µM). Baclofen (100 µM), a
GABABR agonist, had no effect on BrdU incorporation.
(A, D, G). Muscimol (100 µM) significantly
inhibited (n = 4; Student's t test;
*p < 0.05) the mitogenic effect of EGF (20 ng/ml)
(n = 4, Student's t test;
**p < 0.01, ***p < 0.0001) in
total PSA-NCAM+ cells (B) and
in Tuj1+/PSA-NCAM+ cells
(E), but not in
Tuj1 /PSA-NCAM+ cells
(H). C, F, I, Confocal
images of double-immunostaining for PSA-NCAM (red) and BrdU (green)
(C), triple-immunostaining for PSA-NCAM
(red), Tuj1 (blue), and BrdU (green) (F, I),
respectively, showing that both
Tuj1+/PSA-NCAM+ and
Tuj1 /PSA-NCAM+ cells are
proliferative. Because a vast majority of cells constituting 3-DIV
spheres expressed PSA-NCAM, we found similar results on the whole-cell
population, and these results were confirmed in
[3H]thymidine incorporation assay (data not shown,
but see Fig. 8). Scale bars: C, F,
I, 10 µm.
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GABAAR activation inhibits the proliferation of
PSA-NCAM+ progenitors
Because the activation of ionotropic GABAAR
has been reported to affect the proliferation of neural progenitors in
the ventricular and subventricular zones of the embryonic neocortex
(LoTurco et al., 1995
; Haydar et al., 2000
), we decided to analyze the
effect of GABA on proliferation kinetics in striatal early postnatal PSA-NCAM+ progenitor cells. After 48 hr of
growth in EGF-containing medium, spheres were transferred to the same
medium but devoid of EGF for the next 24 hr. This procedure allowed us
to obtain a synchronization of most cells in G0
(Jones and Kazlauskas, 2001
) before starting BrdU or
[3H]thymidine incorporation assays (18 hr). The removal of EGF from the medium did not affect the phenotype of
cells within these 3-DIV spheres (data not shown).
To compare the proliferation rates of the different cell phenotypes
present within 3-DIV spheres, cells were colabeled for BrdU and lineage
markers (i.e., PSA-NCAM, Tuj1, O4, and GFAP) (Fig.
5C,F,I). Cytosine
arabinoside (10 µM) was used as an internal control inhibiting proliferation in all phenotypes. Although agonists and antagonists of GABAAR did not modify the
percentages of
O4+/BrdU+ and
GFAP+/BrdU+
cells (data not shown), treatment with GABAAR
agonists (GABA at 100 µM and muscimol at 100 µM), but not with GABABR
agonist (baclofen 100 µM), significantly
reduced the percentage of PSA-NCAM+ cells
that incorporated BrdU (Fig. 5A) at both the
Tuj1
(Fig. 5G) and
Tuj1+ (Fig. 5D) stages. The
addition of SR-95531 (10 µM) totally abolished the muscimol (100 µM)-induced decrease of
proliferation in PSA-NCAM+ cells (Fig.
5A) at Tuj1
(Fig.
5G) and Tuj1+ stages (Fig.
5D).
EGF (20 ng/ml) increased the proliferation of
PSA-NCAM+ cells (Fig. 5B)
similarly at Tuj1
(Fig.
5H) and Tuj1+ stages
(Fig. 5E) (n = 4; Student's t
test; **p < 0.01, ***p < 0.0001) but
had no effect on O4+ and
GFAP+ cells (data not shown).
Interestingly, muscimol (100 µM) inhibited EGF-induced increase of proliferation (n = 4;
Student's t test; *p < 0.05) in
PSA-NCAM+ cells (Fig. 5B) at
the Tuj1+ stage only (Fig.
5E,H), whereas addition of
SR-95531 (10 µM) totally abolished the effect
of muscimol (Fig. 5B,E). This
discrepancy may reflect developmental differences of
GABAAR expression and/or function between
potentially more immature
Tuj1
/PSA-NCAM+
precursors and their
Tuj1+/PSA-NCAM+
neuroblastic progeny.
The influence of GABAAR modulators assessed by
BrdU incorporation was also analyzed on the whole-cell population using
the [3H]thymidine incorporation assay
with similar results (data not shown, but see Fig. 8).
EGF-controlled GABA-mediated autocrine/paracrine inhibition of
proliferation in cultured PSA-NCAM+ cells and
Tuj1+ neuron-committed precursor cells from early
postnatal striatum
To investigate whether PSA-NCAM+
progenitors were able to synthesize GABA, we performed RT-PCR
experiments with specific primers to detect the enzymes required for
GABA synthesis by decarboxylation of glutamate,: i.e., the 65 kDa (GAD
65) and 67 kDa (GAD 67) glutamate decarboxylases (Table 1).
During brain development, alternative splicing produces three
transcript isoforms for the GAD 67 but not the GAD 65 gene (Szabo et
al., 1994
). With RNAs extracted both from 3-DIV
PSA-NCAM+ spheres and from control adult
total brain, we detected an appropriate 698 bp band by using a specific
set of primers for GAD 65 (Fig. 6A). For GAD 67 RT-PCR,
we used a set of primers aimed at amplifying cDNAs for the three
alternatively spliced isoforms. However, we detected only a 252 bp band
corresponding to the full-length functional isoform of GAD 67 with RNAs
extracted both from PSA-NCAM+ spheres and
control adult total brain (Fig. 6A). We next sought for the presence of GABAergic cells among
PSA-NCAM+ progenitors by
immunocytochemical stainings using antibodies directed against GABA,
GAD 65, and GAD 67. We found that GAD 65+
cells represented 48.9 ± 5.2% (n = 2) (Fig.
6B) of total cells from 3-DIV
PSA-NCAM+ spheres, whereas 61.5 ± 4.1% (n = 3) (Fig. 6C) of the progenitors expressed GAD 67. GABA+ cells represented
19.3 ± 9.8% (n = 3) (Fig. 6D)
of total cells.

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Figure 6.
EGF-dependent production of endogenous GABA
intrinsically inhibits the proliferation of
PSA-NCAM+ precursor cells. A, RT-PCR
amplification of both GAD 65 and GAD 67 transcripts from 3-DIV
PSA-NCAM+ spheres and from control adult rat brain
using specific sets of primers. RT-PCR analysis yielded bands with the
appropriate amplicon size for GAD 65 (698 bp) and for the full-length
functional GAD 67 (252 bp). Left margin indicates migration of standard
DNA markers with size indicated in base pairs. B,
C, Three-DIV-old dissociated
PSA-NCAM+ spheres labeled for GAD 65 (B, green) or GAD 67 (C, green) and
counterstained by Etd1 (red). D, Dissociated 3-DIV
progenitors immunostained for GABA (green) and counterstained with Etd1
(red). Scale bars: B-D, 10 µm.
E, F, Histograms representing the
differences of BrdU incorporation index (BrdU+
cells/total cells, %) between treated and untreated conditions,
respectively, in total PSA-NCAM+
(E) and
Tuj1+/PSA-NCAM+ cells
(F). Antagonists and positive allosteric
modulators of GABAAR were applied on 3-DIV-old synchronized
cells for 18 hr of BrdU incorporation assay. GABAAR
antagonists (10 µM SR-95531, 5 µM
picrotoxin, and 100 µM bicuculline) significantly
increased the percentage of
PSA-NCAM+/BrdU+ cells
(n = 3; ANOVA-1 followed by a Dunnett's post-test,
ns; *p < 0.05)
(E) and
Tuj1+/PSA-NCAM+/BrdU+
cells (n = 3; ANOVA-1 followed by a Dunnett's
post-test; *p < 0.05, **p < 0.01) (F). Conversely,
GABAAR-positive allosteric modulators decreased the
percentage of PSA-NCAM+/BrdU+
cells (E) and
Tuj1+/PSA-NCAM+/BrdU+
cells (F) as compared with control.
Saclofen (10 µM), a GABABR antagonist, had no
effect (E, F). G,
H, In the presence of EGF (20 ng/ml)
(n = 4; Student's t test;
***p < 0.0001), SR-95531 (10 µM)
(n = 4; Student's t test;
**p < 0.01, ***p < 0.0001)
had no effect on proliferation of total PSA-NCAM+
cells (G) and of
Tuj1+/PSA-NCAM+ cells
(H). I, Histogram showing
the concentration of GABA measured by HPLC in synchronized 3-DIV-old
PSA-NCAM+ spheres treated or not with EGF for 18 hr.
EGF-treated spheres contained a lower amount of GABA than that of
untreated cultures (n = 3; Student's
t test; *p < 0.05).
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Given this demonstration of GABA synthesis in striatal
PSA-NCAM+ cells, we next investigated
whether these cells were able to actively secrete GABA, which in turn
could regulate their proliferation level by an autocrine or paracrine
activation of GABAAR. To test this hypothesis of
an endogenous activation of GABAAR within
PSA-NCAM+ spheres, we studied the effects
on proliferation of antagonists (SR-95531 at 10 µM,
picrotoxin at 5 µM, and bicuculline at 100 µM) and positive allosteric modulators (clonazepam at 1 µM and pentobarbital at 10 µM) of
GABAAR in the absence of exogenously added GABA
in our proliferation assay. In the absence of EGF, GABAAR antagonists triggered an increase
(n = 3; ANOVA-1 followed by a Dunnett's post-test;
*p < 0.05, **p < 0.01), whereas
positive allosteric modulators induced a decrease (n = 3; not significant) of proliferation in total
PSA-NCAM+ and
Tuj1+/PSA-NCAM+
cells (Fig. 6E,F). The lower
level of increase of BrdU labeling in total
PSA-NCAM+ cells in comparison with the
Tuj1+ subpopulation in the presence of
GABAAR antagonists underlies the fact that
Tuj1
/PSA-NCAM+
cells were less sensitive to this pharmacological effect (data not
shown). Our data suggest the existence of an endogenous GABA-dependent inhibition of proliferation in PSA-NCAM+ spheres.
Furthermore, in the presence of EGF (20 ng/ml), the GABA antagonist
SR-95531 (10 µM) did not increase the proliferation of total PSA-NCAM+ and
Tuj1+/PSA-NCAM+
cells (Fig. 6G,H). Therefore, using the
HPLC technique, we measured the GABA contents in spheres synchronized
for 24 hr and subsequently grown for 18 hr in DMEM/F12/N2/B27 with or
without EGF (20 ng/ml). We found that EGF-treated spheres contained a
lower amount of GABA when compared with untreated cultures
(n = 3; Student's t test;
*p < 0.05) (Fig. 6I). These results
emphasize that GABA production in
PSA-NCAM+ cells may be controlled by EGF signaling.
GABAAR activation does not interfere with the survival
of PSA-NCAM+ cells
We performed TUNEL bioassays to ascertain that
GABAAR agonists, antagonists, and positive
allosteric modulators did not modify the percentage of
BrdU-incorporating PSA-NCAM+ cells by
interfering with apoptotic cell death. As shown in Figure 7A,
GABAAR agonist (muscimol 100 µM), antagonist (SR-95531 10 µM), or positive allosteric modulators
(pentobarbital and clonazepam) did not influence the apoptotic events
in PSA-NCAM+ cells. Roscovitine at a high
concentration (40 µM) (Ljungman and Paulsen,
2001
) was used as positive control (n = 3; ANOVA-1 followed by a Dunnett's post-test; **p < 0.01) (Fig.
7A,B).

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Figure 7.
GABAAR modulators do not interfere
with PSA-NCAM+ cell survival. A,
Histogram showing a TUNEL bioassay that demonstrated the absence of
effect of GABAAR modulators on apoptotic events in
PSA-NCAM+ cell cultures (3-DIV, 18 hr of treatment
in the different conditions. Roscovitine (40 µM) was used
as a positive control (n = 3; ANOVA-1 followed by a
Dunnett's post-test; **p < 0.01).
B, Confocal images displaying representative fields
comparing the percentage of TUNEL+ cells (green) in
control (top row) versus roscovitine-treated (bottom row)
conditions.
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Intracellular signaling pathways mediating the effects of
GABAAR activation on cell cycle progression in
PSA-NCAM+ progenitors
Because the mitogen-activated protein kinase (MAPK) signaling
pathway has been shown to be involved in the regulation of cell cycle
progression in neuronal progenitor cells (Li et al., 2001
), we studied
the effect of a chemical inhibition of this cascade on
GABAAR-mediated modulation of
PSA-NCAM+ cell proliferation. We used
U0126, a specific inhibitor of the mitogen-activated kinase kinases
MEK1 and MEK2 (Duncia et al., 1998
). U0126 (10 µM) had no
effect on basal proliferation or on muscimol-induced arrest of
proliferation (Fig. 8). Conversely, we
found that U0126 totally abolished the increase of proliferation induced by SR-95531 or EGF (n = 5; Student's
t test; *p < 0.05, **p < 0.01) (Fig. 8).

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Figure 8.
GABAAR activation inhibits
proliferation of PSA-NCAM+ progenitors by blocking
MAPK signaling pathways. Histogram shows that the increase of
[3H]-thymidine incorporation induced by the
GABAAR antagonist SR95531 (10 µM) and by EGF
(20 ng/ml) was significantly blocked by U0126 (10 µM), a
specific inhibitor of the mitogen-activated protein kinase kinases MEK1
and MEK2 (n = 5; Student's t test;
*p < 0.05, **p < 0.01).
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As described (Belachew et al., 2000
), to assess GABA-induced calcium
responses, PSA-NCAM+ cells have been
imaged using confocal microscopy and the calcium indicator dye fluo-3
in Locke standard extracellular solution. In such conditions, we first
tested the presence of voltage-gated calcium channels (VGCCs) in
PSA-NCAM+ cells by studying the effects of
depolarization. The application of a depolarizing solution containing a
high K+ concentration (50 mM)
resulted in a prolonged rise of intracellular calcium concentration
([Ca2+]i) in
28.3% of the cells (28 of 99 cells tested) (Fig.
9A,B). A cell was considered to be a responding cell if it displayed a
sustained increase of its fluorescence intensity that was significantly (at least 20%) above the average baseline fluorescence. A
muscimol-evoked [Ca2+]i increase
was observed in 20.2% of PSA-NCAM+ cells
(20 of 99 cells tested), and all muscimol-responsive cells exhibited
intracellular calcium responses to depolarization induced by high
extracellular K+ (Fig.
9A,B). Muscimol-induced calcium
responses in PSA-NCAM+ cells were
consistently inhibited by the VGCC blocker nifedipine (10 µM; n = 20 cells) (Fig.
9A,B). These data suggest that GABA interferes with
[Ca2+]i
homeostasis in a subpopulation (20%) of postnatal
PSA-NCAM+ cells from striatum by inducing
a sufficient depolarization to open VGCCs.

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Figure 9.
GABAAR activation inhibits the
proliferation of Tuj1+/PSA-NCAM+
progenitors by inducing a rise of intracellular calcium concentration.
A, Time course
(Ft/F0)
of intracellular calcium concentration [Ca]i assessed in
cultured (3 DIV) fluo-3 AM-loaded PSA-NCAM+ cells.
We displayed fluorescence data recordings and fluo-3 AM on the basis of
confocal images from two different cells (i.e., depolarization and
muscimol responsive, open circles in the dot plot; depolarization
responsive and muscimol nonresponsive, black circles in the dot plot)
during successive treatments with solutions containing a high
extracellular K+ concentration (50 mM),
muscimol (100 µM), or muscimol (100 µM) + nifedipine (10 µM). The [Ca]i increase
mediated by muscimol (100 µM) was abolished by the L-type
voltage-gated calcium channel-blocker nifedipine (10 µM)
(open circle-containing curve). B, Histogram
representing the increase of [Ca]i
( Ft/F0,
%) triggered by a high extracellular K+
concentration (50 mM; n = 28) and
muscimol (100 µM; n = 20) (ANOVA-1
followed by a Dunnett's post-test; **p < 0.01).
Moreover, the [Ca]i increase induced by muscimol
(100 µM) was significantly reduced by nifedipine (10 µM; n = 20) (ANOVA-1 followed by a
Dunnett's post-test; **p < 0.01). Examples of
responsive cells are represented on the right in the image series.
Cells that were both depolarization responsive and muscimol responsive
are indicated by open arrows, and cells that were depolarization
responsive but muscimol nonresponsive are indicated by white arrows.
C, Histograms showing that the inhibition of
proliferation induced by muscimol (100 µM) in
Tuj1+/PSA-NCAM+ cells was
completely blocked by nifedipine (10 µM). When applied
alone, nifedipine significantly increased the proliferation of
Tuj1+/PSA-NCAM+ cells
(n = 4-5; Student's t test;
***p < 0.0001). D, In contrast,
nifedipine (10 µM) did not block the inhibition of
proliferation mediated by muscimol (100 µM) in
Tuj1 /PSA-NCAM+ cells and did
not increase the proliferation of these cells when applied alone
(n = 4-5; Student's t test;
*p < 0.05).
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We have also investigated the effect of the VGCC blocker
nifedipine on GABAAR-mediated regulation of cell
cycle progression in PSA-NCAM+ cells.
Therefore, we provided evidence that a
GABAAR-mediated increase of
[Ca2+]i in
striatal PSA-NCAM+ cells was involved in
GABAAR-mediated inhibition of proliferation in
neuron-committed
Tuj1+/PSA-NCAM+
progenitor cells but not in
Tuj1
/PSA-NCAM+
cells (Fig. 9C,D).
GABAAR is expressed in PSA-NCAM+
cells in situ and autocrine/paracrine GABAAR
activation regulates proliferation of postnatal striatal
PSA-NCAM+ cells in organotypic slices
We performed immunostaining in coronal frozen tissue sections (30 µm) from postnatal (P1) rat brains. We were able to demonstrate that
PSA-NCAM+ cells from striatum as well as
from the adjacent SVZ were immunoreactive for
GABAAR
subunits (Fig.
10A-C).
GABA- expressing cells were also detectable in the postnatal striatum
and adjacent SVZ regions (Fig.
10E,F). Finally, we wanted
to assess cell proliferation, as described previously (Yuan et al.,
1998
), in organotypic slice (P1, 400 µm thick) cultures to gain more
insights from a cytoarchitecturally intact postnatal striatum (Fig.
10G), closer to the in vivo situation. To
restrict our analysis to the postnatal striatum, SVZ regions (as
defined in Fig. 10A1) were microdissected out and
assessed similarly but separately. BrdU incorporation was performed
during the first 24 hr of culture, i.e., between +4 and +22 hr after dissection. Slices next were mechanically dissociated and plated onto
poly-ornithine-coated coverslips to attach for 1 hr before fixation and
immunostaining (Fig. 10H). We ascertained the
viability of our slice culture system by running LIVE/DEAD cytotoxicity assays just before fixation after each experiment, yielding to values
of 88.6 ± 0.6 living cells and 11.4 ± 0.6 dead cells
(percentage of total cells; mean ± SEM; n = 3 independent experiments).