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The Journal of Neuroscience, August 1, 2000, 20(15):5764-5774
Differential Modulation of Proliferation in the Neocortical
Ventricular and Subventricular Zones
Tarik F.
Haydar,
Feng
Wang,
Michael L.
Schwartz, and
Pasko
Rakic
Section of Neurobiology, Yale University School of Medicine,
New Haven, Connecticut 06510
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ABSTRACT |
Recent studies have implicated the classical neurotransmitters
GABA and glutamate in the regulation of neural progenitor
proliferation. We now show that GABA and glutamate have opposite
effects on the two neural progenitor populations in the ventricular
zones (VZs) and subventricular zones (SVZs) of the embryonic cerebrum.
Application of either molecule to organotypic slice cultures
dramatically increases proliferation in the VZ by shortening the cell
cycle, whereas proliferation in the SVZ is decreased. These disparate effects, measured both by bromodeoxyuridine uptake and the expansion of
retrovirally labeled progenitor clones, are mimicked by the application
of specific GABA and glutamate agonists and are blocked by antagonists.
Thus, the relative contributions of the VZ and SVZ to neocortical
growth may be regulated by differential responsiveness to GABA and glutamate.
Key words:
neurogenesis; neurotransmitter; progenitor; cell cycle; cerebral cortex; corticogenesis
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INTRODUCTION |
Differences in neural progenitor
cell proliferation may underlie the variations in cortical size between
species as well as some of the neocortical malformations that are
present in various neuropathological conditions (Rakic, 1988 ; Haydar et
al., 1999b; Walsh, 1999). Both cell-intrinsic and -extrinsic factors
contribute to changes in cell production and affect cerebral cortical
growth. Recently, several extracellular molecules, such as growth
factors and neurotransmitters, have been implicated in the extrinsic
regulation of cell proliferation in the developing telencephalon (for
review, see Cameron et al., 1998). For example, basic fibroblast
growth factor (bFGF), when added either to cultured cells or
microinjected into embryonic brains, prolongs the proliferation of
cortical progenitors, leading to increases in the numbers of cortical
neurons (Gensburger et al., 1987; Cattaneo and McKay, 1990; Ghosh and Greenberg, 1995; Vaccarino et al., 1995; Cavanagh et al., 1997; Vaccarino et al., 1999). In contrast, the neurotransmitters GABA and
glutamate reportedly reduce the number of proliferating cells in
dissociated or organotypic cultures of the neocortex (LoTurco et al.,
1995). Furthermore, GABA can partially block the bFGF-induced increase
in proliferation (Antonopoulos et al., 1997). However, GABA was also
shown to promote cell proliferation in cultures of cerebellar
progenitors (Fiszman et al., 1999). Thus, it is unclear whether GABA
and/or glutamate affect all neural progenitor cells in a similar manner
or if these modulatory molecules affect cell proliferation differently
in various brain regions. It is also unknown whether specific
progenitor subpopulations in the same region are differentially
affected during neurogenesis.
We have addressed these conceptually and practically important
questions in the mammalian neocortex because it develops from two
distinct proliferative populations, the ventricular zone (VZ) and
subventricular zone (SVZ). The VZ lines the lateral ventricles and
forms first, followed by the SVZ, which emerges superficial to the VZ
(Boulder Committee, 1970). These two zones also differ in the behavior
of their constituent cells. Progenitors within the VZ proliferate in a
stereotypical manner termed interkinetic nuclear migration, in which
DNA is replicated deep within the VZ, whereas cell division always
occurs at the surface of the lateral ventricle. In contrast, SVZ cells
do not exhibit movements as they divide but proliferate in
situ without nuclear translocation (for review, see Sidman and
Rakic, 1973; Takahashi et al., 1995b). Differences between these two
proliferative populations are further underscored by eventual fate. The
VZ is a transient embryonic structure that is ultimately replaced at
the end of neurogenesis by ependymal cells with limited proliferative
capacity in adulthood. Conversely, the SVZ [postnatally termed the
subependymal zone (SEZ)] persists as a proliferative population
throughout the remaining life span (Smart, 1961). Finally, although the
VZ and SVZ intermingle at the most superficial extent of the VZ during
prenatal development, the generative potential of these two populations
is thought to be different, with progenitors in the VZ generating
mainly neurons (Sidman et al., 1959) and progenitors in the SVZ/SEZ
predominantly generating glial cells and a limited repertoire of
neurons (Altman, 1969; Reynolds and Weiss, 1992; Doetsch et al.,
1999).
Despite the cytological, functional, and developmental differences
between the VZ and SVZ progenitors, little is known about the controls
of cell proliferation in these two compartments and how they contribute
to cortical growth. In the present study, we have used an organotypic
slice culture that maintains the spatial separation between the VZ and
SVZ to examine how GABA and glutamate affect the proliferative behavior
of cells in these two zones. The results reveal inherent differences
between VZ and SVZ progenitors in their physiological response to the
same molecules.
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MATERIALS AND METHODS |
Generation of neocortical organotypic slices.
Embryonic day 13 (E13) and E14 ICR strain (Harlan Sprague
Dawley) mouse fetuses were used for all slice experiments. Slices were
prepared as described previously (Haydar et al., 1999a). Briefly,
brains were dissected and collected in cold HEPES-buffered MEM (Life
Technologies, Gaithersburg, MD). The brains were sliced into 300 µm coronal slices on a McIlwain tissue chopper and then transferred
back into MEM media, in which slices were separated with forceps under
a dissecting microscope. Intact coronal slices at the level of
the future sensorimotor cortex were hemisected and then transferred
to collagen-coated tissue culture inserts (Corning Costar
Transwell; catalog #3494) containing Neurobasal medium
supplemented with B27, L-glutamine, and N2 (Life
Technologies). Slices were arranged flat, the level of media was
lowered to form a meniscus just above the slices, and the slices were
then cultured in an incubator at 37°C in 5% CO2. After 4 hr incubation to allow for recovery,
5-bromo-2'-deoxyuridine (BrdU) (0.25% final
concentration; Sigma) was added to the culture medium and was present
for the remainder of the experiment. The pharmacological agents (in
µM): 30 GABA, 50 glutamate, 30 muscimol, 150 kainate, 10 bicuculline (BMI) (Sigma, St. Louis, MO), and 10 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX) (Research
Biochemicals, Natick MA) were added to the media before addition of the
slices at the start of the 4 hr recovery period. The concentrations
were used based on dose-response curves published previously (LoTurco et al., 1995).
After culturing for variable time periods, slices were fixed overnight
in 4% paraformaldehyde and cryoprotected in 30% sucrose. Slices were
then resectioned in 20 µm increments in the coronal plane with a
cryostat. The middle three frozen sections of each slice were stained
(see below). The central 10 µm of these sections was then optically
sectioned in the coronal plane into a stack of 10 × 1 µm images
using a Zeiss LSM 510 confocal microscope. Image stacks were assembled
to construct an optical dissector in which nuclei contained within the
depth of the stack were counted with a 100 × 125 µm (width × height) box (Fig.
1B). This box was horizontally transected at a height of 70 µm. VZ nuclei were counted within the bottom 70 µm, and SVZ cells were counted within the top 55 µm (Fig. 1B). The boundary between the VZ and SVZ
was determined both by the position of the deepest abventricular
mitotic figure and the bottom of the band of SVZ cells after BrdU pulse
chase experiments analyzed when the labeled VZ cells had migrated to the ventricular surface (data not shown). This position was at 70 µm
above the ventricular surface throughout all experiments. Cell counts
were analyzed in a double-blind fashion with respect to culture
conditions. Counts of neocortical slices were consistently made midway
between the medial and lateral angles of the lateral ventricle at the
level of the future sensorimotor cortex (for example, see Schambra et
al., 1992, their plate GD14, COR. 5).
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Figure 1.
VZ cells in the organotypic slices undergo
interkinetic migration as they progress through the cell cycle.
A, Cells in S-phase form an abventricular
BrdU+ band in the VZ after 1 hr labeling with BrdU
(surface of lateral ventricle is at bottom).
B, After 8 hr of cumulative BrdU labeling,
many originally labeled S-phase cells have migrated to the apical
surface of the VZ and are dividing. In addition, as more unlabeled
cells enter S-phase and incorporate BrdU, the VZ begins to fill with
BrdU+ cells. C, E, After 1 hr of BrdU
labeling, flow cytometric analysis shows BrdU+ cells
in S-phase of the cell cycle. Cells in S-phase for the entire period of
labeling have a high DNA content, whereas cells in S-phase for a short
period of time have lower DNA content. D,
F, After 8 hr of labeling,
BrdU+ cells are spread throughout the cell cycle.
Some cells that were labeled at the end of S-phase have progressed
through mitosis and are now in G1/G0 phase.
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BrdU slice proliferation assays. The proliferative
characteristics of slice VZ cells was assayed both by morphology
(mitotic figures) of proliferating cells as well as with BrdU
incorporation into cells in S-phase of the cell cycle. BrdU-labeled
nuclei were visualized using an anti-BrdU antibody (1:77, overnight at
4°C; Becton Dickinson, Mountain View, CA) coupled to an anti-mouse IgG1-FITC (1:200, 1 hr RT; Southern Biotechnology
Associates, Birmingham, AL) secondary antibody. Propidium iodide
(1 × 10 4%; PI)
was used as a counterstain to visualize the DNA of all cells. The
labeling index (LI; percentage of BrdU+
cells of total VZ cells) after variable BrdU exposure times was determined by cell counting as detailed above.
Cell cycle analysis. The duration of the cell cycle in the
slice VZ was estimated as described previously (Takahashi et al., 1995a; Haydar et al., 1999b). Briefly, slices incubated in the presence
of BrdU for increasing periods of time were then processed for BrdU
immunohistochemistry. Labeling indices were plotted (Fig. 2), and the cell cycle parameters were
solved using the equation:
where Tc is the duration of the entire cell cycle,
GF is the growth fraction or maximum number of proliferating
cells in the VZ, and t is the duration of BrdU application.
In Figure 2, the LI plots reach a maximum value over time and then
level off; the time when Ymax is
reached reflects the duration of Tc  Ts, where Ts is the duration of S phase. The magnitude of
Ymax also determines the
GF.
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Figure 2.
GABA and glutamate increase proliferation in the
VZ. A, In slices cultured at E13, the LI rises steadily
in control slices (black lines) as VZ cells progress
through the cell cycle and are labeled with BrdU. In contrast, the LI
curves for GABA (30 µM)- and glutamate (50 µM)-treated slices (red and blue
lines, respectively) rise with a steeper slope, indicating that
the cell cycle is faster in treated slices. B,
Similarly, the cell cycle of GABA- and glutamate-treated slices is
shorter than controls at E14. C, The addition of GABA
and glutamate agonists to E14 slice cultures also increases the rate of
VZ proliferation (compare to black curve in
B). D, Conversely, the GABA and glutamate
antagonists BMI (10 µM) and CNQX (10 µM)
tend to prolong the duration of the cell cycle. Interestingly, addition
of CNQX to the slices increases the duration of the cell cycle when
compared to controls (black curve in
B).
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Flow cytometry assays. The cultures were ended by placing
slices into ice-cold MEM-HEPES. Neocortical areas were dissected out
and collected. After two washes in ice-cold HBSS (Life
Technologies), the samples were digested at 37°C for 20 min with
0.25% trypsin (Life Technologies) dissolved in HBSS. Digestion was
stopped by adding an equal percentage of trypsin inhibitor (Sigma) on
ice, and the samples were triturated with a fire-polished Pasteur
pipette. The cells were fixed in cold 70% ethanol for at least 30 min
and stored at 20°C. Approximately 1 × 106 fixed cells were centrifuged and
resuspended in 2 N HCl containing 0.5% Triton X-100 for 30 min. Cells
were washed in 0.1 M
Na2B4O7 · 10 H2O, pH 8.5, for 10 min to neutralize the
acid, and then incubated with anti-BrdU antibody solution (1:50 in
blocking solution containing PBS, 0.5% Tween 20, and 1% bovine serum
albumin) overnight at 4°C. After a PBS wash, cells were incubated 1 hr in FITC-conjugated sheep anti-mouse IgG (1:25 in blocking solution).
Cells were washed in PBS and incubated at 37°C for 30 min with 1 mg/ml RNase A (Sigma) and then stained with 5 µg/ml PI (Sigma). Flow
cytometric analysis was performed with a FACS Vantage flow
cytometer (Becton Dickinson). The cells were excited at 488 nm, and the
emission was collected simultaneously through two bandpass filters
(530/30 nm and 630/22 nm). A total of 100,000 cells was collected from
each sample. The two-dimensional contour graph (BrdU vs PI) and the
histogram of the BrdU-positive cell population (Fig.
1C-F) were plotted with WinMDI 2.7 software.
Immunohistochemistry. The distributions of GABA and
glutamate were analyzed in fixed 20 µm cryostat sections of
the developing neocortical wall. Guinea pig anti-GABA primary antibody
(Protos Biotech, New York, NY) was applied at 1:1000 to sections from brains fixed in 4% paraformaldehyde and was visualized using a FITC-conjugated secondary antibody (Southern Biotechnology Associates) used at 1:200 dilution. Rabbit anti-glutamate primary antibody (Arnel,
Cherokee Station, NY) was applied at 1:1000 to sections from brains
fixed in 4% paraformaldehyde/0.25% glutaraldehyde, and sections were
then incubated in Cy2-conjugated species-appropriate secondary antibody
(Jackson ImmunoResearch, West Grove, PA) at 1:300. All sections were
counterstained with PI (1 × 104 % in
PBS; Sigma). Images of stained sections were collected with a SPOT2 CCD
camera on a Zeiss Axioplan2 microscope and were standardized with
respect to the level of GABA and glutamate staining of cellular elements and the propidium iodide staining. Confocal images (1 µm
single optical sections) were collected on a Zeiss LSM510 and were
similarly standardized.
Surgical procedures and retroviral labeling. Retroviral DNA
(pLIA) (Bao and Cepko, 1997; Furukawa et al., 1997) encoding the alkaline phosphatase gene was introduced into the Phoenix viral packaging cell line by transfection using calcium phosphate (Pear et
al., 1996). Viral particles were collected from the supernatants of
these cultures, concentrated in Centricon tubes, and were titered at
1.2 × 107 cfu/ml before being stored
in aliquots at 80°C until use. Pregnant dams at 14 d gestation
were anesthetized with ketamine (100 mg/ml) and xylazine (20 mg/ml).
The uterine horns were exposed by midline incision, and embryos were
visualized through the uterine wall by transillumination from a fiber
optic light source. One microliter of pLIA virus/fast green
(0.1% in phosphate buffer) mixture (10:1) was injected into the
lateral ventricles using a picoinjector (Medical Systems Corporation,
Greenvale, NY). The uterus was then replaced into the abdominal cavity,
and the incisions were sutured closed. After 24 hr, neocortical
slices were cultured from each embryo as detailed above. Some slices
were also cultured in the presence of GABA or glutamate for 24 hr, and
then slices were stained for alkaline phosphatase (AP) activity using
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP).
Briefly, slices were fixed in 4% paraformaldehyde overnight and sunk
in 30% sucrose. Serial sections (25 µm) were cut on a cryostat and washed with PBS, pH 7.4, before endogenous phosphatases were
inactivated at 65°C for 30 min. Sections were then incubated in
NBT/BCIP (Boehringer Mannheim, Mannheim, Germany) per manufacturer's
instructions for 24 hr, rinsed for 1 hr in PBS, and then mounted using
30% glycerol in PBS. Labeled clusters, defined as immediately adjacent
cells (the only type of labeled cells), were sparse and widely
separated in infected brains. The number of neocortical clusters
averaged 8 ± 2.36 (mean ± SEM) per brain, with one or two
clusters per individual brain section.
Migration analysis. The migration of neocortical cells out
of the VZ after cell division was determined by measuring the number of
BrdU+ cells present outside of the
germinal zones after increasing periods of time. BrdU was added to
slices for either 24 or 48 hr. The number of
BrdU+ cells that were present superficial
to the VZ and SVZ was then determined to measure how many
postproliferative cells had exited the cell cycle and migrated up into
the neocortical wall. The superficial border of the SVZ was determined
empirically for each slice as a line parallel to the ventricular
surface at the level of the most superficial abventricular mitotic figure.
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RESULTS |
GABA and glutamate increase cell proliferation in
the VZ
To analyze the effect of GABA and glutamate on the proliferation
of different populations of neural progenitors, we used organotypic slices of the embryonic cerebral wall which, when cultured in minimal
defined media, maintained the morphological separation between the VZ
and SVZ as well as the respective behaviors of these populations during
proliferation (Haydar et al., 1999a). In particular, by combining BrdU
labeling (Fig. 1A,B) and flow cytometric analyses
(Fig. 1C-F), it was evident that VZ cells underwent
interkinetic nuclear migration as they progressed through the cell
cycle, whereas SVZ cells did not. In addition, the slice cultures
maintained the separation between the proliferative zones and
differentiated neurons and glia, and cells in the slices survived well
up to 72 hr in culture (Haydar et al., 1999a).
Using cumulative BrdU labeling, we determined that the VZ cell cycle
duration (Tc) for E13 and E14 control slices was 22.4 and 25 hr,
respectively (Fig. 2A,B, Tables
1, 2). As
observed previously (Takahashi et al., 1999), the length of the cell
cycle in vitro was longer than durations reported for
corresponding ages in vivo. Nevertheless Tc from E13-E14
cultured slices was similar to the increase in Tc reported in
vivo for the VZ (Takahashi et al., 1995a). This increase of Tc in
the slice was not attributable to a change in the duration of S-phase
(Ts), which remained relatively constant at ~8.5 hr in controls, but
rather was attributable to the lengthening of the remaining cell cycle
phases (Tc-Ts) (Tables 1, 2). This corresponds to the increase of Tc
in vivo which is attributable to progressive lengthening of
G1 phase (Takahashi et al., 1995a).
To determine the effect of GABA and glutamate on the proliferation of
VZ cells, slices were cultured in the presence of both BrdU and either
of the two neurotransmitters. Both neurotransmitters reduced the cell
cycle duration of VZ cells; exogenously applied GABA (30 µM) reduced Tc in the E13 VZ to 8.8 hr, whereas Tc at E14
was 10 hr. Similarly, glutamate (50 µM) also shortened Tc in the E13 VZ to 8.1 hr whereas the E14 Tc was shortened to 10 hr.
Notably, all phases of the cell cycle in neurotransmitter-treated slices were reduced to between 50 and 80% of control values (Fig. 2A,B, Tables 1, 2).
To determine the specific receptors underlying the effects of GABA and
glutamate on VZ proliferation, we applied pharmacological agonists and
antagonists of GABAA and AMPA/kainate glutamate
receptors to the culture medium in the place of GABA and glutamate.
Like GABA and glutamate themselves, GABA and glutamate
agonists also increased VZ proliferation. Muscimol (30 µM), a GABAA receptor agonist,
decreased the VZ Tc by 62%. A similar decrease was found for other
cell cycle phase durations (Fig. 2D, Table 2). Kainic acid (KA) (150 µM), an AMPA/kainate receptor
agonist, reduced the slice Tc by 48%, whereas Tc-Ts was reduced by
43%, and Ts was reduced by 56% (Fig. 2D, Table 2).
Moreover, BMI (10 µM) and CNQX (10 µM), antagonists of GABAA
and AMPA/kainate glutamate receptors, respectively, did not
significantly decrease Tc. In the case of CNQX, Tc was even longer than
in controls suggesting the presence of endogenous glutamate in the
slice cultures (Fig. 2C, Table 2). Taken together, these
results indicate that exogenously applied GABA and glutamate shorten
the cell cycle of VZ progenitors, and that this effect is mediated by
GABAA or AMPA/kainate receptors.
GABA and glutamate increase the size of VZ clones
The cell cycle experiments described above used a population
approach to measurement of the GABA and glutamate effects on the entire
VZ progenitor pool. To determine how the neurotransmitters influence
the clonal expansion of individual progenitors, the number of VZ cells
in retrovirus-infected progenitor clusters was used as an indicator of
the number of cell divisions over time (Fig.
3). Embryos were injected intracerebrally
with a low concentration of retrovirus containing the reporter gene
alkaline phosphatase (AP) to yield widely separated infected VZ
progenitors. The number of cells per cluster after 24 and 48 hr of
infection in vivo was measured to determine
whether this method is useful for following the number of cell
divisions per cluster. Because retroviral DNA can only integrate during
a mitotic division, with only one daughter cell receiving the viral
DNA, after a single division only one cell should be labeled
(Hajihosseini et al., 1993). The number of
AP+ cells per cluster should then grow
exponentially as the originally labeled cell divides further. As
expected, embryos injected on E13 and analyzed 24 hr later, which
allowed less than two cell cycles to elapse (Takahashi et al., 1995a),
had 1.25 ± 0.25 cells per cluster (mean ± SEM, 15 clusters). In contrast, clusters were either two or four cells 48 hr
after infection (2.84 ± 0.19, 31 clusters), which is enough time
for a maximum of three cell cycles (Fig. 3). Thus, we found that the
number of cells/cluster is consistent with the mitotic history within
the cluster.
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Figure 3.
GABA and glutamate increase VZ cluster size.
A, B, Because progenitors had only enough
time to divide once under control conditions (Table 1), the number of
pLIA-infected cells per cluster 24 hr after retroviral infection of E13
embryonic brains was one cell per cluster. Image in A
shows a retroviral-infected VZ progenitor dividing at the surface of
the lateral ventricle. C, D, By 48 hr after infection
in vivo, infected cells cultured under control
conditions had divided two or three times yielding two or four cells
per cluster. Inset in C shows the cells
in this cluster at higher magnification. E, F, In
control slice cultures made 24 hr after in vivo
retroviral infection on E13 and then cultured for an additional 24 hr,
there were one or two cells per cluster. G,
H, GABA or (I and
J) glutamate application during the slice
incubation caused VZ progenitors to divide more quickly, increasing the
number of VZ cells/cluster to two or four cells.
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To determine the effects of GABA and glutamate on
AP+ cluster size, E13 embryos were
infected with pLIA for 24 hr, and neocortical slices were then cultured
in the presence or absence of GABA and glutamate. After 24 hr of
additional in vitro incubation, the VZ cluster size in
control slices was still only one or two cells (1.42 ± 0.23, 24 clusters) (Fig. 3E). This is likely attributable to the
length of Tc in E14 control slices (~25 hr, Table 2). Thus, the one
AP+ cell resulting after the initial 24 hr
in vivo incubation (Fig. 3B) would be unable to
complete another division during the 24 hr of in vitro
incubation. In contrast, VZ clusters in GABA and glutamate-treated
slices consisted of either two or four cells after the second 24 hr of
incubation in vitro (3.13 ± 0.41, 26 clusters and
2.76 ± 0.67, 75 clusters, respectively) (Fig. 3F,G). Thus, the number of VZ cells per cluster was significantly larger in
GABA and glutamate-treated slices, further indicating that GABA and
glutamate shorten the cell cycle of individual VZ progenitors resulting
in more divisions compared to controls.
GABA and glutamate decrease cell proliferation in
the SVZ
To determine how GABA and glutamate affect proliferation in the
SVZ, we examined cell proliferation in the same slices used for the VZ
analysis. Using cumulative BrdU labeling, the LI (percentage of
BrdU+ cells) in the SVZ
(LISVZ) steadily increased over time as
proliferating cells replicated their DNA and incorporated BrdU (Fig.
4A, dashed line). GABA
(purple line), glutamate (light red line),
muscimol (dark green line), and KA (dark red
line) all significantly decreased the number of
BrdU+ SVZ cells over time so that the
LISVZ did not rise normally. In contrast, BMI and
CNQX application resulted in a positive rise in
LISVZ over time (Fig. 4A, light
green and yellow lines), although the slope of the
LISVZ was slightly decreased compared to
controls. Thus, surprisingly in contrast to the findings in the VZ,
proliferation in the SVZ is markedly decreased in response to GABA,
glutamate, and their agonists.
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Figure 4.
GABA and glutamate decrease SVZ proliferation.
A, The LI in the E14 control slice SVZ increases
steadily over time as more cells enter S-phase and incorporate BrdU
(dashed line). The GABA and glutamate antagonists BMI
and CNQX also caused positive slopes in the SVZ LI curves
(green and yellow lines,
respectively). In contrast, GABA, glutamate, and their agonists all
cause no increase in the number of BrdU+ SVZ cells
over time, suggesting that SVZ proliferation is inhibited in response
to GABA and glutamate. B, At 16 and 24 hr of cumulative
BrdU labeling in E14 slices, no difference in LI is seen in the VZ
between control, GABA-, glutamate-, and antagonist-treated slices. In
contrast, when the VZ and SVZ LIs at 16 and 24 hr are pooled, GABA and
glutamate decrease the combined LI and BMI and CNQX block this
decrease. Thus, GABA and glutamate have an overall inhibitory affect on
progenitor proliferation when the VZ and SVZ are analyzed
together.
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These data are partly in disagreement with previous studies suggesting
that GABA and glutamate decrease the proliferation of neocortical
progenitors (LoTurco et al., 1995; Antonopoulos et al., 1997). However,
the distinction between the VZ and SVZ was not made in these studies
because all progenitors were analyzed en masse. Therefore, we decided
to examine whether GABA and glutamate caused a general decrease in
proliferation if we treated the neocortical proliferative zones as a
homogenous population. To accomplish this, we pooled the LI data for
the VZ and SVZ together (Fig. 4). When the LIVZ
was analyzed separately at either 16 or 24 hr of cumulative BrdU
labeling, no effect was seen in response to GABA, glutamate, or their
agonists and antagonists (Fig. 4B). This is likely
attributable to the fact that all VZ progenitors had already passed
through S-phase and incorporated BrdU and because the growth fraction
(GF; or maximum number of proliferating VZ cells) was similar in
treated and control conditions (~95-99%) (Fig. 2). In contrast,
because the SVZ was not saturated with BrdU+ cells, when the
LIVZ and LISVZ were pooled
at 16 and 24 hr, significant differences were seen between control and
neurotransmitter-treated groups. Specifically, exogenously applied GABA
and glutamate caused a decrease in the LIVZ+SVZ
that could be blocked by BMI and CNQX, respectively (Fig.
4B). Thus, although the LIVZ
was maximum for all experimental groups at these time points, the
substantial decrease in SVZ proliferation due to GABA and glutamate
overwhelmed the increase in the VZ and caused a general decrease in the
combined LIVZ+SVZ.
GABA and glutamate inhibit generation of
postmitotic cells
To test whether GABA and glutamate affect other parameters of cell
proliferation in addition to modulating the cell cycle duration, we
analyzed cell production and migration from the proliferative zones
into the neocortical wall (Fig. 5). We
previously showed that cells in our slice preparation exit the
proliferative zones when they complete their last cell cycle (Haydar et
al., 1999a). Therefore, during a cumulative BrdU-labeling experiment,
the number of cells that migrate above the proliferative zones can be
used as a measure of the amount of postmitotic cell production over time. After 24 hr cumulative BrdU labeling on control E14 slice cultures, which roughly corresponds to the length of one in
vitro cell cycle (Table 2), very few
BrdU+ cells had migrated above the
overlying SVZ. However, even fewer BrdU+
migratory cells were found in slices treated with GABA for 24 hr,
although this time point corresponded to the length of two full cell
cycles under these conditions (Table 2). Similarly, compared to
controls, fewer BrdU+ migratory cells were
found in slices cultured in the presence of either GABA or glutamate
after 48 hr, although VZ cells in neurotransmitter-treated slices had
divided twice as many times as controls during this period (four vs two
cell divisions) (Table 2). These results indicate that GABA and
glutamate reduce the generation of postmitotic cells. Specifically, the
neurotransmitters may prevent exit from the cell cycle and thus keep VZ
cells proliferating.
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Figure 5.
Postmitotic cell generation is inhibited by GABA
and glutamate. The number of BrdU+ cells from 24 or
48 hr cumulative labeling experiments that had migrated into the
neocortical wall above the SVZ were recorded for slices cultured on
E14. After 24 hr, few BrdU+ cells in control slices
(black bar) had exited the cell cycle and migrated away
from the VZ and SVZ. Similarly, few cells in GABA-treated slices had
exited the proliferative zones even though VZ cells in treated slices
(gray bar) had progressed through two cell cycles
compared to the one cell cycle in controls (Table 2). After 48 hr and
two VZ cell cycles of labeling, many BrdU+ cells had
exited the proliferative zones in controls (black bar).
In contrast, 80% fewer cells had migrated out of the proliferative
zones in GABA- (gray bar) and glutamate-
(striped bar) treated slices over the four cell cycles
under those conditions (Table 2). *p < 0.01.
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Sources of GABA and glutamate in the embryonic
cerebral wall
In light of the previous and present results that GABA and
glutamate can influence progenitor proliferation, we sought to determine the source and distribution of these two molecules. Several
studies have previously identified GABAergic and glutaminergic cells in
the neocortical wall during neurogenesis (Van Eden et al., 1989;
Schwartz and Meinecke, 1992; Yan et al., 1992; Lidow and Rakic, 1995),
and it is possible that these cells release their neurotransmitters
into the proliferative zones. The released transmitter could diffuse
long distances as well as be produced locally. Thus, to identify
whether changes in the extracellular distribution of GABA and glutamate
correlate with known neurogenetic gradients and therefore whether their
diffusion accounts for the changes in neocortical proliferative
kinetics, we used immunofluorescence in developmental series of
neocortical brain sections. Similar to previous work (Yan et al.,
1992), staining for GABA revealed a striking distribution that is
dynamically regulated during the period of neurogenesis (Fig.
6). The first GABAergic cells were observed on E10 and were situated near the pial surface of the neuroepithelial wall. At this age there was diffuse GABA
stainingpresent throughout the neuroepithelium. By E12,
GABA-immunoreactive cells were present in the preplate (cells of the
future subplate and marginal zone), and diffuse GABA staining was still
found throughout the proliferative zones. By E14, GABA staining
was seen in the Cajal-Retzius cells of the marginal (MZ), in the
subplate (SP), and in the intermediate zones (IZ). The diffuse staining
throughout the proliferative zones remained but was decreased compared
to E10 and E12. At E16, many GABAergic cells were found in the MZ, cortical plate (CP), SP, and IZ, but GABA immunoreactivity in the
proliferative zones was markedly reduced. Finally, at postnatal day 0 (P0), many cells in the neocortex were GABAergic, whereas no staining
was detected in the proliferative SEZ.
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Figure 6.
GABA distribution during neurogenesis. The
extracellular distribution of GABA (green) was
assessed by immunohistochemistry throughout prenatal neocortical
development. The presence of diffuse staining in the proliferative
zones is initially high during early neurogenesis (E10-E14), but then
gradually diminishes during the remainder of neurogenesis (E14-P0).
The panel insets are confocal images of propidium iodide
and GABA staining in the VZ at the respective ages. Each section is
counterstained with the DNA stain propidium iodide (red)
to demarcate the layers of the neocortical wall. The ventricular
surface of each section is at the bottom of the image.
Inset for P0 pictures the subependymal zone.
VZ, Ventricular zone; SVZ, subventricular
zone; IZ, intermediate zone; SP,
subplate; CP, cortical plate; MZ,
marginal zone. Scale bar, 50 µm.
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Staining for glutamate in the neuroepithelium followed a similar
course it was high during early neurogenesis and diminished as
neurogenesis progressed (Fig. 7). At E12,
diffuse glutamate staining was observed throughout the neocortical
wall. However, on E14 and after, staining for glutamate gradually
diminished in the proliferative epithelium. Thus, diffuse staining for
GABA and glutamate was observed in the proliferative zones early during neurogenesis and then diminished as neurogenesis proceeds, even though
the number of GABA and glutaminergic neurons in the cerebral wall
increased.
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Figure 7.
Glutamate distribution during neurogenesis. The
distribution of glutamate (green) during the
period of neocortical neurogenesis was examined using
immunohistochemistry. Diffuse staining throughout the neocortical wall
was present at E12 and E14, but the comparative levels of glutamate
staining in the proliferative zones subsided thereafter. On E16 and
E18, high amounts of staining were only present in the marginal zone,
subplate, and intermediate zone. Each section was counterstained with
propidium iodide (red) to elucidate the layers of the
neocortical wall. The ventricular surface of each section is at the
bottom of each image. Scale bar, 50 µm.
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DISCUSSION |
Species-specific differences in laminar thickness and the relative
contribution of neurons from the VZ and SVZ are well documented and are
presumed to have been important for evolution. However, the genetic and
molecular differences between the proliferative zones that generate
this diversity are not well understood. It has been suggested that
changes in cortical size and shape are attributable to intrinsic
differences between progenitor cells in the proliferative zones
(McConnell, 1988; Parnavelas et al., 1991; Luskin et al., 1993; Kornack
and Rakic, 1998). Here we propose that differential regulation
of cell production in the VZ and SVZ by GABA and glutamate may both
elucidate and exploit these intrinsic differences.
GABA and glutamate differentially modulate cell
proliferation in the VZ and SVZ
Both the morphological separation between VZ and SVZ and the
interkinetic nuclear migration particular to VZ progenitors were maintained in the neocortical slices, enabling us to analyze the proliferation of these two progenitor populations separately. In
contrast to the stimulating effect of GABA and glutamate on proliferation in the VZ, these molecules substantially decreased proliferation in the SVZ. In addition, the effects of specific agonists
and antagonists on VZ and SVZ progenitor proliferation indicate that
GABA and glutamate act via GABAA and AMPA/kainate receptors as reported previously (LoTurco et al., 1995). Nevertheless, molecular differences between VZ and SVZ progenitors result in divergent proliferative behavior in response to GABA and glutamate.
In an attempt to explain the apparent discrepancy between the above
results and those published previously that suggested that GABA and
glutamate decrease neocortical proliferation (LoTurco et al., 1995;
Antonopoulos et al., 1997), we analyzed the cumulative BrdU LI in the
VZ and SVZ both together and separately. Because data presented
previously are derived either from dissociated neocortical cells or
counts from cultured slices without distinction between the
proliferative zones, we reasoned that any differences between the VZ
and SVZ proliferation in response to GABA and glutamate may have been
masked when both progenitor populations were pooled. Indeed, consistent
with previous reports, when the LI of both the VZ and SVZ are pooled
after 16 or 24 hr cumulative BrdU labeling, there is an overall
decrease in LI as a result of the substantial reduction in SVZ
proliferation. However GABA and glutamate cause opposite effects on VZ
and SVZ proliferation when each population is analyzed separately.
Modulation of proliferation during neurogenesis
Whereas some species-specific size and regional differences depend
on the number of founder progenitor cells and are determined early
before the first neurons are born (Rakic, 1995; Haydar et al., 1999c),
here we have focused on the changes that affect neuron production
during the phase of neurogenesis. Many parameters of progenitor
proliferation are dynamically modulated during development of the
mammalian brain (Fig. 8). For example, as
neurogenesis in the cerebral wall proceeds, the VZ cell cycle duration
progressively lengthens, and the proportion of progenitors that
terminally divide to generate neurons also increases (Takahashi et al.,
1996). These terminal divisions tend to decrease the size of the VZ
until it is exhausted by the end of the neurogenetic interval. Also,
whereas only the VZ is present at the start of neurogenesis, the SVZ
appears later and its constituent progenitors still proliferate at the end of neurogenesis when the VZ progenitors cease proliferation.
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Figure 8.
Proliferative gradients during neocortical
histogenesis. Levels of GABA and glutamate (grayscale gradient) in the
proliferative zones are high at the start of neurogenesis when VZ cells
proliferate rapidly (long dashed line) and tend to
reenter the cell cycle rather than become neurons, causing a slow rise
in the amount of early neurogenesis (solid line). The
decrease in GABA and glutamate levels throughout the remainder of the
neurogenetic interval is concomitant with slower VZ proliferation,
increased production of neurons until the VZ is exhausted of
progenitors, and the emergence and predominance of the SVZ as a
distinct proliferative compartment (short dashed
line).
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The present results, which suggest that GABA and glutamate act in
a similar manner to differentially modulate the proliferation of VZ and
SVZ progenitors, are consistent with the depolarizing effects of both
these neurotransmitters early in development (Mueller et al., 1983,
1984; Janigro and Schwartzkroin, 1988; Swann et al., 1989; LoTurco et
al., 1995). In addition, the specific changes in proliferation induced
by the neurotransmitters are concordant with the observed dynamics of
the progenitor populations during neurogenesis (Fig.
8A,B). For example, high amounts of these
neurotransmitters early during neurogenesis would promote both the
rapid cycling of VZ progenitors and their re-entry into the cell cycle
while at the same time inhibiting SVZ proliferation. Later, decreased levels of GABA and glutamate would cause a lengthening of the VZ cell
cycle, promote VZ neurogenesis, and release the inhibition on SVZ
proliferation. Indeed, previous work (Yan et al., 1992), and the
staining for GABA and glutamate in Figures 6 and 7, suggest that
proliferative zone levels of these two amino acids are high at early
stages of neurogenesis and gradually decrease thereafter. Although
precisely what causes this progressive decrease of GABA and glutamate
levels in the proliferative zones during neurogenesis is unknown, the
time at which this decrease occurs correlates with several processes
underway in the developing neocortical wall. It may be significant that
the bulk of cortical efferent and afferent axonal projections undergo
extension through the IZ early in neurogenesis. The number of
projections that reach their target structures then increases during
neurogenesis, after which axons in the IZ become compacted. It may
therefore be this compaction that acts as a barrier to further
diffusion of GABA and glutamate later in neurogenesis. In any
case, the present results which show that GABA and glutamate
differentially regulate progenitor proliferation are consistent
with the observed proliferative dynamics during neurogenesis.
Cell production in the developing cerebral wall
The modulation of VZ and SVZ proliferative kinetics by GABA
and glutamate could lead to changes in the number of postmitotic cells
generated from these two progenitor populations. Because these
neurotransmitters increase VZ progenitor proliferation, the VZ
population would grow, enabling more postmitotic cells to be generated
by the end of neurogenesis (Takahashi et al., 1997). In contrast, less
SVZ proliferation would lead to fewer cells generated from the SVZ
progenitor population. To test whether GABA and glutamate also
influence the decision proliferative cells make after mitosis either to
reenter the cell cycle or to exit and become postmitotic, we took
advantage of the migration of newly generated cells into the
neocortical wall, a process that is maintained in the neocortical
slices (Haydar et al., 1999a). In the presence of either GABA or
glutamate, the number of postmitotic cells that had migrated into the
neocortical wall was dramatically reduced (Fig. 5). These fewer
migrated BrdU+ cells may have been
caused by selective inhibitory effects of GABA and glutamate on
migration or by the elimination of GABA- and glutamate-induced
chemoattractive gradients. However, two lines of evidence support the
notion that fewer migrated cells may instead be attributable to the
proliferative behavior of progenitors. First, rather than inhibiting
migration, both GABA (Behar et al., 1998) and glutamate (Komuro and
Rakic, 1993; Behar et al., 1999) stimulate migration of newly generated
neocortical neurons. Second, because the neurotransmitters were present
for 4 hr before addition of BrdU, if exogenous GABA and glutamate
inhibited the directed migration of newly generated neurons, we would
have expected to see an initial build-up of
BrdU cells in the VZ of treated slices
that would have then decreased the GF in the VZ. Because no change from
controls was seen in the VZ GF in GABA- and glutamate-treated slices
(Fig. 2, Tables 1, 2), exogenous neurotransmitter application is likely
not to block cell cycle progression or to disrupt directed radial migration. Thus, the most parsimonious explanation of the results is
that GABA and glutamate not only increase the kinetics of VZ proliferation, but may also inhibit neurogenesis by promoting symmetrical progenitor divisions and re-entry of daughter cells into
the cell cycle. This inhibition of neurogenesis would cause an
expansion of the VZ progenitor population and have the overall effect
of increasing the final numbers of cells destined for the cerebral cortex.
While the lineal relationship between VZ and SVZ progenitors is not
known, it is likely that the SVZ cells are initially derived from the
VZ because the VZ is present first. Moreover, if the SVZ is being
continually "seeded" by the VZ, our measured effects of GABA and
glutamate on the VZ might affect SVZ size in an indirect manner by
reducing the number of SVZ progenitors over time. Although we cannot
rule out this possibility, the decrease in SVZ labeling index caused by
GABA and glutamate reported here is an immediate and long-lasting
influence on the SVZ cells that are present at the start of the
experiment. Thus, despite the possible contamination of the SVZ by VZ
cells, it is clear that GABA and glutamate differentially affect the
proliferation of VZ and SVZ progenitors.
Regulation of cell cycle dynamics is considered a contributing
mechanism for generating diversity within the neocortex, producing differences in the size of cortical areas and the thickness of cortical
layers (Rakic, 1988; Dehay et al., 1993; Polleux et al., 1997). Recent
studies have shown that although the cell cycle progressively slows as
mouse neocortical neurogenesis proceeds (Takahashi et al., 1995a), the
cell cycle of VZ progenitors in macaque monkey visual cortex
transiently accelerates midway through neurogenesis during the time
when layer IV, a relatively hypercellular cortical layer, is generated
(Kornack and Rakic, 1998). Simultaneously, in other cortical areas,
fewer cells destined for layer IV are produced. These areal differences
during layer generation may be regulated by localized and transient
increases in GABA and glutamate in the progenitor populations at the
correct time during neurogenesis.
The modulation of progenitor proliferation by neurotransmitters
may also explain the pathogenesis of certain neocortical malformations. For example, the number of terminal progenitor divisions during neocortical neurogenesis is decreased in the Trisomy 16 mouse (Haydar
et al., 1999b), a model for Down's syndrome that has three rather than
two copies of GluR5, a gene encoding an AMPA/kainate glutamate receptor
(Reeves et al., 1986; Holtzman and Epstein, 1992). This decreased
neurogenesis leads to expansion of the VZ population and a concomitant
transient delay in neocortical growth (Haydar et al., 1999b). The amino
acids GABA and glutamate are present at the right times and exert
appropriate effects on the progenitor populations to provide a
plausible mechanism for all of these instances of neurogenetic regulation.
Several studies in rodents have shown that the length of S-phase
is relatively conserved throughout neurogenesis (Reznikov and van der
Kooy, 1995; Takahashi et al., 1995a) and that changes in the duration
of G1 phase are mostly responsible for the increase in the overall
cycle duration (Takahashi et al., 1995a). However, there are also
examples of modulation of S-phase duration that are consistent with the
GABA and glutamate-induced changes reported here. For example, there is
a transient threefold increase in S-phase duration during monkey
neurogenesis (Kornack and Rakic, 1998), and exogenous vasoactive
intestinal peptide has been shown to shorten S-phase by 50% when
applied to whole cultured mouse embryos (Gressens et al., 1998). Taken
together, these results suggest that, while normally relatively
constant during mouse neurogenesis, the length of S-phase can be
modulated and that this added control may account for some of the
differences in cortical growth between species.
Several recent studies have indicated that significant
differences exist between neural progenitors during early forebrain development. The lineage-dependent and selective response of progenitor subpopulations to environmental cues (Eagleson et al., 1998) and the
different kinetic and developmental histories of FGF and EGF-responsive neural stem cells (Kuhn et al., 1997; Martens et al., 2000) point to
the emergence of particular molecular traits during early phases of
forebrain development. Similarly, this study suggests that the
differential effects of GABA and glutamate on VZ and SVZ progenitors may be attributable to variations in the signaling mechanisms that
control their proliferation. Because the SVZ appears immediately juxtaposed to the VZ some time after VZ amplification and postmitotic cell generation has already started, this secondary proliferative compartment may in fact be generated from VZ progenitors. The molecular differences between VZ and SVZ cells described here should
therefore be specified soon after these "SVZ-generating" divisions and may act to facilitate the adoption of the distinct role
and new position of SVZ progenitors. Nevertheless, now that distinct
differences between progenitors in the cerebral wall have been
identified mechanistically, the road is paved for elucidation of the
molecular mechanisms controlling proliferation of progenitor groups. An
appreciation of such mechanisms is crucial for understanding normal and
pathological cortical growth and may lead to the development of
strategies for neural stem cell production and utilization as well as
the treatment of diseases affecting higher brain function.
| |
FOOTNOTES |
Received Jan. 11, 2000; revised May 4, 2000; accepted May 5, 2000.
This work was supported by National Institutes of Health Grants
P01 NS22807 and P01 NS354765 and also by Grant F32 NS10729 to T.H.
Correspondence should be addressed to Dr. Pasko Rakic, Section of
Neurobiology, SHM C-303, Yale University School of Medicine, 333 Cedar
Street, New Haven, CT 06510. E-mail: pasko.rakic{at}yale.edu.
| |
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ABSTRACT
INTRODUCTION
