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Next Article 
The Journal of Neuroscience, 2000, 20:RC111:1-5
RAPID COMMUNICATION
Induction and Activation by Zinc of NADPH Oxidase in
Cultured Cortical Neurons and Astrocytes
Kyung-Min
Noh and
Jae-Young
Koh
National Creative Research Initiative Center for the Study
of CNS Zinc and Department of Neurology, University of Ulsan College of
Medicine, Seoul 138-736, Korea
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ABSTRACT |
Zinc overload may be a key mechanism of neuronal death in acute
brain injury. We have demonstrated previously that zinc overload neurotoxicity involves protein kinase C (PKC)-dependent rises in
intracellular levels of reactive oxygen species (ROS). However, the
cascade linking PKC activation to ROS generation in cultured cortical
neurons has been unknown. A recent study has demonstrated that
ROS-generating NADPH oxidase is present in sympathetic neurons and
contributes to NGF deprivation-induced cell death. Because NADPH
oxidase is activated by PKC, in the present study, we examined the
possibility that NADPH oxidase is the effector for oxidative stress in
zinc-overloaded cortical cells.
Reverse transcription-PCR and Western blot analyses revealed that naive
cultured cortical cells express subunits of NADPH oxidase at low
levels. Exposure to zinc substantially increased levels of NADPH
oxidase subunits in both neurons and astrocytes. In addition, zinc
exposure induced translocation of the p47PHOX and
p67PHOX subunits to the membrane, a signature event
for NADPH oxidase activation. Addition of a selective PKC inhibitor,
GF109203X, blocked both the induction and the membrane translocation of
NADPH oxidase by zinc. Supporting the role for NADPH oxidase in
zinc-triggered oxidative injury, NADPH oxidase inhibitors attenuated
ROS production and cortical neuronal death induced by zinc. In
addition, Cu/Zn-superoxide dismutase and catalase attenuated
zinc-induced cortical neuronal death.
Our results have demonstrated that zinc overload induces and activates
NADPH oxidase in cortical neurons and astrocytes in a PKC-dependent
manner. Thus, NADPH oxidase may be an enzyme contributing to ROS
generation in zinc-overloaded cortical neurons and astrocytes.
Key words:
oxidative stress; protein kinase C; superoxide; astrocyte; calcium; neuronal death
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INTRODUCTION |
Recent
evidence indicates that endogenous zinc may play a key role in neuronal
death after acute brain insults such as ischemia, seizures, and trauma
(Frederickson et al., 1989 ; Tonder et al., 1990; Koh et al., 1996; Choi
and Koh, 1998; Suh et al., 2000). In these brain injury paradigms,
intracellular accumulation of zinc correlates well with neuronal death
at the single cell level. Furthermore, chelation of zinc prevents both
intracellular zinc accumulation and neuronal death in those brain
injury models. Cytotoxic mechanisms of intracellular zinc overload may
involve diverse processes such as mitochondrial damage, nicotinamide
adenine dinucleotide (NAD)-positive (NAD+)
degradation, ATP depletion, and caspase activation (Lobner et al.,
1997; Manev et al., 1997; Sheline et al., 2000). In addition, evidences
suggest that oxidative stress plays a significant role in zinc
neurotoxicity in cortical culture. First, zinc influx increases the
levels of reactive oxygen species (ROS) in neurons (Kim et al., 1999;
Sensi et al., 1999). Second, various neuroprotective measures effective
against zinc toxicity, such as protein kinase C (PKC) inhibitors,
attenuate zinc-induced increases in ROS in parallel (Noh et al., 1999).
Finally, antioxidants attenuate zinc neurotoxicity (Kim et al., 1999).
Although the evidence indicates that oxidative stress is a significant
mechanism of zinc-induced neuronal death, the effector protein(s)
directly responsible for the generation of ROS in zinc-injured cells is unknown.
NADPH oxidase is a superoxide-producing enzyme
consisting of the membrane (gp91PHOX and
p22PHOX) and the cytosolic
(p47PHOX,
p67PHOX, and
p40PHOX) components (DeLeo and Quinn,
1996; Babior, 1999). In addition, small G-proteins such as rac1, as
well as kinases including PKC, regulate its activity (Heinecke et al.,
1990; Benna et al., 1997; Reeves et al., 1999; Ozaki et al., 2000).
Although NADPH oxidase is mainly expressed in phagocytic cells, an
increasing body of evidence suggests that various subunits of NADPH
oxidase are also expressed in nonphagocytic cells such as mesangial
cells, endothelial cells, vascular smooth muscles, and fibroblasts
(Fukui et al., 1995; Jones et al., 1995, 1996; Thannickal and Fanburg,
1995). However, functions of NADPH oxidase subunits in nonphagocytic cells are not yet clearly delineated. Recently, all of the subunits of
NADPH oxidase are to be expressed by sympathetic ganglion neurons and
to contribute to NGF-deprivation-induced ROS generation and cell death,
suggesting the possibility that neurons in general might express NADPH
oxidase (Tammariello et al., 2000). Because NADPH oxidase can be
activated by PKC and because PKC activation appears to be a key step in
zinc-induced oxidative injury, we hypothesized that NADPH oxidase may
be the effector enzyme mediating the PKC-dependent oxidative injury in
zinc-overloaded cultured cortical neurons. This possibility was
examined in the present study.
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MATERIALS AND METHODS |
Cortical cell culture and assessment of cell death.
Mixed mouse cortical cultures containing both neurons and astrocytes, and pure astrocyte cultures, were prepared from fetal (15 d of gestation) and neonatal (1-3 postnatal days) mice, respectively, as
described previously (Kim et al., 1999). Near-pure neuronal cultures
were prepared from fetal mice (Sheline and Choi, 1998). Immunocytochemical staining with anti-Macrophage (Mac-1-antigen) antibody (Boehringer Mannheim, Mannheim, Germany) revealed that <3%
of cells were microglia in all three types of cultures.
Brief (15 min) exposure of cortical cultures (10-13 d in
vitro) to zinc was performed in serum-free HBSS at room
temperature. After exposure, cultures were incubated in serum-free MEM
and placed back into the incubator. In addition to morphological
assessment under a phase-contrast microscope, in most experiments,
overall neuronal cell injury was quantitatively assessed by the
measurement of lactate dehydrogenase (LDH) (Koh and Choi, 1987)
released by damaged cells into the bathing medium 24 hr after exposure
to zinc. Each LDH value, after subtracting background LDH value in sham-washed controls, was scaled to the maximal neuronal LDH release (of 100) produced by 24 hr exposure of sister cultures to 300 µM NMDA, which produces near-complete neuronal
death with no glial damage.
Reverse transcription-PCR. RNA was prepared with TRIZOL
(Life Technologies, Gaithersburg, MD) and reverse-transcribed to cDNA using oligo-dT 14 primers (Promega, Madison, WI). Specific cDNAs were
amplified for 30 cycles. Signals for amplified cDNAs, after separation
on 1.7% agarose gels, were visualized with ethidium bromide and quantitatively measured with an image analyzer (Alpha Imager 2000; Alpha Innotech, San Leandro, CA). Primer sequences were as
follows: 5'-CTTGGATGATAGCACTGCAC-3' and 5'-CTTCATCTGAAGCTCAATGG-3' for p91PHOX (626 bp);
5'-AGAGCGACTTTGAGCAGCTT-3' and 5'-TGTGGAGACACACCCTTGAT-3' for
p40PHOX (460 bp);
5'-AGCCTGAGACATACCTGGTG-3' and 5'-AGACTTCTGCAGATACATGG-3' for
p47PHOX (446 bp);
5'-CAAGGCTACGGTTGTAGCAT-3' and 5'-ACCTTGAGCATGTAAGGCAT-3' for
p67PHOX (465 bp); and
5'-CTCAAGATTGTCAGCAATGC-3' and 5'-CACTGAGGACCAGGTTGTCT-3' for
glyceraldehyde-3-phosphate dehydrogenase (415 bp).
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Cell lysis, fractionation, and
immunoblotting |
Cortical cells were lysed in lysis buffer and centrifuged at
14,000 rpm for 20 min. The pellet was discarded, and the supernatant was used for protein quantification. Fractionation of cytosol and
membrane was performed as described previously (Noh et al., 1999).
Equal amounts of protein from total cell lysates or membrane and
cytosolic fraction were electrophoresed on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were then incubated with respective primary antibodies (anti-p67PHOX, anti-p47PHOX,
rac-1, and anti-gp91) overnight at 4°C The enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) protocol was used
to visualize the immunoreactive bands.
Immunohistochemistry. After fixation (4% paraformaldehyde)
and blocking, cells were double-labeled with
anti-p67PHOX antibody (Transduction
Laboratories, San Diego, CA) and either anti-glial fibrillary acidic
protein (GFAP) or anti-microtubule-associated protein-2 (MAP2) antibody
(Sigma, St. Louis, MO). After washes, signals were visualized by
appropriate secondary antibodies coupled to either fluorescein
isothiocyanate (FITC) or rhodamine (Jackson ImmunoResearch, West Grove, PA).
Measurement of ROS generation. Two independent methods were
used to measure ROS production in cortical cultures.
2,7-Dichlorodihydrofluorescein diacetate (DCF) fluorescence was
used to visualize intracellular superoxides (Greenlund et al., 1995).
H2O2 accumulation in media was measured using the Amplex Red
(10-acetyl-3,7-dihydroxyphenoxazine) Hydrogen Peroxide Assay kit
(Molecular Probes, Eugene, OR).
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RESULTS |
Expression and induction of NADPH oxidase in cultured
cortical cells
Subunits of NADPH oxidase were examined at the mRNA level by
reverse transcription (RT)-PCR analysis. Whereas cortical cultures expressed low levels of mRNA for NADPH oxidase subunits (Fig. 1A), 15 min exposure to
400 µM zinc substantially increased the mRNA
levels for p47PHOX,
p67PHOX, and
gp91PHOX in cortical cultures with little
change in that for p40PHOX (Fig.
1A). Western blot assay confirmed that zinc exposure
increased the expression of both p47PHOX
and p67PHOX, beginning ~30 min to 2 hr
after the exposure (Fig. 1B). Because mixed cortical
cultures contain both neurons and astrocytes, we examined in which cell
type NADPH oxidase expression increases in response to zinc exposure.
Immunocytochemical staining of neuron-rich cultures with
neuron-specific anti-MAP2 antibody revealed that >90% of cells were
indeed neurons (92.8 ± 1.2% of Hoechst
33342+ cells; n = 4) (Fig.
1C,E). In control cultures, immunoreactivity to
p67PHOX was present at low level (Fig.
1D). However, 4 hr after 15 min zinc exposure,
immunoreactivity to p67PHOX markedly
increased in the majority of MAP2+ neurons
(Fig. 1F). On the other hand, in astrocyte-rich
cultures, almost all cells exhibited immunoreactivity to anti-GFAP
antibody (97.8 ± 1.4% of Hoechst
33342+ cells; n = 4) (Fig.
1G,I). Again, baseline expression of
p67PHOX appeared quite low in astrocytes
(Fig. 1H). However, exposure to zinc substantially
increased p67PHOX levels in most
astrocytes (Fig. 1J). Hence, zinc exposure increased p67PHOX expression in both neurons and
astrocytes.
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Figure 1.
Induction of NADPH oxidase subunits in cortical
cells exposed to zinc. A, RT-PCR for
p40PHOX, p47PHOX,
p67PHOX, and gp91PHOX in
sham-washed control cultures (CTRL) and in sister
cultures 2 hr after 15 min exposure to 400 µM zinc
(representative of 3 experiments). Bars denote densitometer readings of
RT-PCR signals expressed as folds of respective control values
(mean ± SEM; n = 3). Asterisks
denote difference from respective controls
(p < 0.05; two-tailed t
test). B, Western blots for p67PHOX
and p47PHOX. Fifteen minute exposure to 400 µM zinc increased both p67PHOX and
p47PHOX protein levels in cortical culture beginning
0.5-2 hr after zinc exposure. C-F, Fluorescent
photomicrographs of near-pure neuronal cultures, sham-washed control
(C, D), or 4 hr after 15 min exposure to
400 µM zinc (E, F),
stained with anti-MAP2 antibody (C, E,
FITC) and then with anti-p67PHOX antibody
(D, F, rhodamine). Arrows
and arrowheads denote identical landmark cells in
matched sets. G-J, Fluorescent microphotographs of
astrocyte-rich cultures, sham-washed control (G,
H), or 4 hr after 15 min exposure to 400 µM zinc (I, J),
stained with anti-GFAP antibody (G,
I) and anti-p67PHOX antibody
(H, J). Scale bar, 50 µm.
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PKC mediates induction and membrane translocation of
NADPH oxidase
Next, we examined whether zinc exposure leads to the functional
activation of NADPH oxidase. The necessary step for NADPH oxidase
activation is the translocation of cytosolic subunits, including
p47PHOX and
p67PHOX, to the membrane (Leusen et al.,
1996; Han et al., 1998; Johnson et al., 1998). Consistent with the
membrane translocation, at 2 and 4 hr after 15 min zinc exposure,
levels of p47PHOX and
p67PHOX in the membrane fraction markedly
increased (Fig. 2A).
Because p47PHOX was barely detectable in
the cytosol in both control and zinc-exposed cultures (Fig.
2A), markedly increased
p47PHOX in membrane of zinc-exposed cells
may signify the fairly rapid recruitment of newly synthesized protein
to the membrane. It is well known in phagocytic cells that levels and
activity of NADPH oxidase are regulated by protein kinase C (Heinecke
et al., 1990; Benna et al., 1997; Reeves et al., 1999). Because zinc
activates PKC (Murakami et al., 1987; Csermely et al., 1988; Noh et
al., 1999), we examined whether the effect of zinc on NADPH oxidase induction and activation is mediated by PKC. Indeed, a selective PKC
inhibitor, GF109203X, completely blocked the induction of p67PHOX by zinc, whereas a selective
inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), did not (Fig.
2B). Furthermore, the membrane translocation of
p67PHOX by zinc was also inhibited by
GF109203X (Fig. 2C). Conversely, addition of a PKC
activator, PMA, alone was sufficient for the induction and
translocation of p67PHOX in cortical cultures
(Fig. 2D,E). Induction of NADPH
oxidase by PMA was blocked by GF109203X but not by DPI (Fig.
2D).
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Figure 2.
Mediation by PKC of translocation and induction of
NADPH oxidase. A, Western blots for indicated subunits
of NADPH oxidase in the cytosolic fraction and the membrane fraction of
cortical cultures, 2 or 4 hr after sham wash or 15 min zinc
exposure (representative of 5 experiments). Whereas levels of rac1
in each fraction did not change much with zinc exposure, levels of
p67PHOX appeared to be decreased in the cytosolic
fraction and to be increased in the membrane fraction. Also, the
membrane levels of p47PHOX markedly increased upon
zinc exposure. The level of the membrane-anchored subunit
gp91PHOX did not change much with zinc exposure.
B, GF109203X (GFX) blocks NADPH
oxidase induction by zinc. Western blots show levels of
p67PHOX in cortical cultures 4 hr after sham wash
(CTRL) or 15 min exposure to 400 µM zinc
without (Zinc) or with addition of DPI (100 nM) or GF109203X (3 µM) during and
continuously after the zinc exposure (representative of 4 experiments). GF109203X markedly attenuated the induction of
p67PHOX by zinc, whereas DPI had no
effect. C, GF109203X blocks NADPH oxidase activation by
zinc. Western blots of the membrane fractions with
anti-p67PHOX antibody after 4 hr exposure to 400 µM zinc (representative of 5 experiments). Addition of 3 µM GF109203X during and after the zinc exposure decreased
membrane translocation of p67PHOX by zinc.
D, PMA increases p67PHOX levels.
Western blots show levels of p67PHOX in sham-washed
control or cultures after 8 hr exposure to 200 nM PMA
(representative of 4 experiments). The increase in
p67PHOX levels by PMA was completely blocked by 3 µM GF109203X, whereas 100 nM DPI had little
effect. E, PMA induces translocation of
p67PHOX. Membrane
p67PHOX levels markedly increased with 8 hr exposure
to 200 nM PMA (P8) (representative of 4 experiments) compared with cultures 8 hr after sham wash
(C8).
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NADPH oxidase contributes to ROS generation induced by zinc
We have shown previously that zinc exposure leads to the
activation of PKC, which leads to increases in superoxide generation in
cultured cortical neurons (Noh et al., 1999). Having demonstrated that
PKC mediates the induction and activation of NADPH oxidase by zinc, we
examined whether NADPH oxidase really contributes to superoxide
generation after zinc exposure. Exposure of mixed cortical cultures to
zinc markedly increased cellular DCF fluorescence (Fig.
3B), an indicator for
superoxide, compared with controls (Fig. 3A). NADPH oxidase
inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and DPI
(Diatchuk et al., 1997; Li and Trush, 1998) markedly reduced the DCF
fluorescence (Fig. 3C,D). This finding was
confirmed independently by the Amplex Red fluorimetry assay; gradual
H2O2 accumulation in the
media was observed at 6 and 12 hr after zinc exposure. NADPH oxidase
inhibitors AEBSF and DPI markedly reduced
H2O2 buildup in the media
of zinc-exposed cultures at both times (Fig. 3E).
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Figure 3.
NADPH oxidase inhibitors attenuate ROS increases
by zinc. A-D, DCF fluorescence in cortical cultures 3 hr after sham wash (A) or 15 min exposure to 400 µM zinc without (B) or with
(C) addition of 50 µM AEBSF or 500 nM DPI (D) during and after the zinc
exposure. E, Levels of H2O2 in
the bathing media of cortical cultures (mean ± SEM;
n = 3 cultures) as estimated by the Amplex Red
fluorimetry (arbitrary units of fluorescence). Media samples were
obtained from sham-washed control cultures (CTRL) or
from cultures 6 hr (left bars) or 12 hr (right
bars) after 15 min exposure to 400 µM zinc
without (Zinc) or with addition of AEBSF (50 µM) or DPI (100 or 500 nM).
Asterisks denote difference from Zinc
(p < 0.05; two-tailed t test
with Bonferroni correction for 3 comparisons). Scale bar, 100 µm.
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NADPH oxidase contributes to oxidative neuronal death induced
by zinc
The next question was whether ROS generated by NADPH oxidase
contributes to zinc-induced neuronal death. Neurons exhibited marked
cell body swelling several hours after 15 min zinc exposure, followed
by release of LDH into the bathing medium (Fig.
4), as described previously (Kim et al.,
1999). Addition of AEBSF significantly reduced neuronal death and
resultant LDH release induced by the zinc exposure (Fig. 4); we could
not use another NADPH oxidase inhibitor, DPI, because even at low
concentrations (100 nM), it had significant cytotoxicity
(data not shown). Addition of Cu/Zn-superoxide dismutase (SOD), which
lowers superoxide levels, also attenuated zinc-mediated neuronal death
(Fig. 4).
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Figure 4.
An NADPH oxidase inhibitor as well as Cu/Zn-SOD
and catalase attenuate zinc neurotoxicity. LDH release in cortical
cultures (mean ± SEM; n = 3) 24 hr after 15 min exposure to 400 µM zinc without (Zinc)
or with addition of 50 µM AEBSF, catalase (300 mU/ml), or
Cu/Zn-SOD (50 U/ml). Asterisks denote difference from
Zinc (p < 0.05; two-tailed
t test with Bonferroni correction for 3 comparisons).
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DISCUSSION |
Although oxidative stress has been proposed as one of the major
mechanisms of ischemic brain injury (Uyama et al., 1992; Chan et al.,
1998), detailed information about the cascades connecting ischemic
events to increased ROS generation is currently lacking. Combined with
the evidence that zinc overload is a key mechanism of ischemic neuronal
death (Choi and Koh, 1998), the present study suggests that the
induction and activation of NADPH oxidase in intrinsic brain cells by
zinc overload may play such a role in ischemic brain injury.
Although the presence of NADPH oxidase in sympathetic ganglion neurons
has raised the possibility that other neurons may also express NADPH
oxidase (Tammariello et al., 2000), functional NADPH oxidase expression
in central neurons and astrocytes has not been directly documented. The
present study has demonstrated that NADPH oxidase subunits, albeit at
low levels, are indeed expressed in central neurons and astrocytes.
More interestingly, certain subunits of NADPH oxidase were rapidly
induced after injurious zinc exposure. Furthermore, in addition to
being induced, NADPH oxidase appears to be functionally activated after
zinc exposure.
The next question was which event links between zinc overload and NADPH
oxidase induction-activation. Whereas several known activators of
NADPH oxidase are known, we focused on PKC in this study, because zinc
has been shown to directly activate PKC (Noh et al., 1999). Supporting
this possibility, addition of a selective PKC inhibitor blocked the
induction and membrane translocation of
p67PHOX after zinc exposure. Conversely,
addition of a PKC activator (PMA) was sufficient to induce and activate
NADPH oxidase. Together, these results suggest a critical role for PKC
in both induction and activation of NADPH oxidase by zinc.
In the present study done in cortical cultures, the contribution by
NADPH oxidase to zinc-induced oxidative injury was directly supported
by the findings that inhibitors of NADPH oxidase attenuate both
superoxide generation and neuronal death after zinc exposure. This
again supports the idea that oxidative stress is a significant mechanism of zinc overload neurotoxicity. Consistently, Cu/Zn-SOD and
catalase, enzymes reducing free radical levels, significantly attenuated zinc-induced neuronal death. Together, the present results
suggest that, in brain injury conditions in which zinc neurotoxicity
contributes, PKC activation and the resultant NADPH oxidase
induction-activation may play a significant role in causing oxidative
neuronal injury. Consistent with this idea, the PKC inhibitor
staurosporine and antioxidative measures have been shown to attenuate
the death of CA1 neurons after transient ischemia (Hara et al., 1990;
Tagami et al., 1999; Sheng et al., 2000) in which zinc neurotoxicity
may play a significant role (Koh et al., 1996).
The present study, for the first time, has demonstrated the expression
and activation of NAPDH oxidase in cultured cortical neurons and
astrocytes are under control of PKC, which is activated by zinc influx.
Additional studies seem warranted to elucidate the potential role for
NADPH oxidase in oxidative injury in vivo as well, because
oxidative stress is likely a common mechanism for cellular damage in
various neurological diseases.
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FOOTNOTES |
Received June 26, 2000; revised Aug. 21, 2000; accepted Sept. 26, 2000.
This study was supported by the National Creative Research Initiatives
of Korean Ministry of Science and Technology (J.-Y.K.).
Correspondence should be addressed to Dr. Jae-Young Koh, National
Creative Research Initiative Center for the Study of CNS Zinc, and
Department of Neurology, University of Ulsan College of Medicine, 388-1 Poongnap-Dong Songpa-Gu Seoul 138-736, Korea. E-mail:
jkko{at}www.amc.seoul.kr.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2000, 20:RC111 (1-5). The
publication date is the date of posting online at
www.jneurosci.org.
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ABSTRACT
INTRODUCTION
