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The Journal of Neuroscience, April 15, 2002, 22(8):3174-3188
Prolonged Seizures Increase Proliferating Neuroblasts in the
Adult Rat Subventricular Zone-Olfactory Bulb Pathway
Jack M.
Parent1,
Vivian
V.
Valentin2, and
Daniel H.
Lowenstein3
1 Department of Neurology, University of Michigan
Medical Center, Ann Arbor, Michigan 48104, 2 Department of
Psychology, University of California, Santa Barbara, Santa Barbara,
California 93106, and 3 Harvard Medical School and
Department of Neurology, Beth Israel Deaconess Medical Center, Boston,
Massachusetts 02115
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ABSTRACT |
Neuronal precursors in the adult rodent forebrain subventricular
zone (SVZ) proliferate, migrate to the olfactory bulb in a restricted
pathway known as the rostral migratory stream (RMS), and differentiate
into neurons. The effects of injury on this neurogenic region of the
mature brain are poorly understood. To determine whether
seizure-induced injury modulates SVZ neurogenesis, we induced status
epilepticus (SE) in adult rats by systemic chemoconvulsant administration and examined patterns of neuronal precursor
proliferation and migration in the SVZ-olfactory bulb pathway. Within
1-2 weeks after pilocarpine-induced SE, bromodeoxyuridine (BrdU)
labeling and Nissl staining increased in the rostral forebrain SVZ.
These changes were associated with an increase in cells expressing
antigenic markers of SVZ neuroblasts 2-3 weeks after prolonged
seizures. At these same time points the RMS expanded and contained more proliferating cells and immature neurons. BrdU labeling and
stereotactic injections of retroviral reporters into the SVZ showed
that prolonged seizures also increased neuroblast migration to the
olfactory bulb and induced a portion of the neuronal precursors to exit the RMS prematurely. These findings indicate that SE expands the SVZ
neuroblast population and alters neuronal precursor migration in the
adult rat forebrain. Identification of the mechanisms underlying the
response of neural progenitors to seizure-induced injury may help to
advance brain regenerative therapies by using either transplanted or
endogenous neural precursor cells.
Key words:
subventricular zone; olfactory bulb; neurogenesis; pilocarpine; seizures; epilepsy; rostral migratory stream; cell
proliferation; neuronal migration; plasticity
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INTRODUCTION |
The hippocampal dentate gyrus and
forebrain subventricular zone (SVZ) generate neurons well into
adulthood in the mammalian brain. In all mammalian species that have
been studied to date, including humans, dentate granule cells are
generated locally by proliferating precursor cells in the subgranular
zone of the dentate gyrus (Altman and Das, 1965 ; Kaplan and Hinds,
1977; Cameron et al., 1993; Kuhn et al., 1996; Eriksson et al., 1998;
Gould et al., 1998; Kornack and Rakic, 1999). Similarly, neuronal
precursors persist and continue to proliferate in the adult rodent
forebrain SVZ (Hinds, 1968; Altman, 1969; Kaplan and Hinds, 1977; Lois
and Alvarez-Buylla, 1994; Lois et al., 1996; Thomas et al., 1996). However, unlike in the dentate gyrus, SVZ neuronal progenitors migrate
long distances to their final destinations in the olfactory bulb
(Kishi, 1987; Luskin, 1993; Lois and Alvarez-Buylla, 1994; Lois et al.,
1996; Thomas et al., 1996) (see Fig. 3H). The
immature neurons migrate from the SVZ to the olfactory bulb via a
relatively unique form of tangential chain migration (Lois et al.,
1996) in a restricted forebrain pathway known as the rostral migratory stream (RMS) (Altman, 1969; Kishi, 1987). The immature neuronal progeny
in the SVZ and RMS of adult rodents can be identified by their
expression of characteristic markers such as the polysialylated form of
neural cell adhesion molecule (PSA-NCAM), neuron-specific  -tubulin,
doublecortin, and collapsin response mediator protein-4 (CRMP-4)
(Bonfanti and Theodosis, 1994; Doetsch and Alvarez-Buylla, 1996; Thomas
et al., 1996; Gleeson et al., 1999; Nacher et al., 2000) (for review,
see Peretto et al., 1999). Once the neuroblasts reach the subependymal
region of their olfactory bulb target, they disperse radially and
differentiate into granule and periglomerular neurons (Luskin, 1993;
Lois and Alvarez-Buylla, 1994; Lois et al., 1996; Thomas et al.,
1996).
Despite recent advances in understanding this neurogenic pathway, the
regulation of neuronal precursor proliferation and migration in the
SVZ-olfactory bulb pathway remains poorly understood. In vitro, cells taken from the adult forebrain SVZ can proliferate, self-renew, and give rise to neurons, astrocytes, and oligodendrocytes (Reynolds and Weiss, 1992; Richards et al., 1992; Lois and
Alvarez-Buylla, 1993). Proliferation in vitro requires
growth factors such as epidermal growth factor (EGF), basic fibroblast
growth factor (bFGF), or brain-derived neurotrophic factor (BDNF)
(Reynolds and Weiss, 1992; Richards et al., 1992; Lois and
Alvarez-Buylla, 1993; Kirschenbaum and Goldman, 1995; Gritti et al.,
1996). These molecules also appear to modify SVZ precursor
proliferation and cell fate in vivo (Craig et al., 1996;
Kuhn et al., 1997; Zigova et al., 1998; Wagner et al., 1999). Several
molecules that potentially regulate neuroblast migration to the
olfactory bulb include Slit-2 (Hu, 1999; Wu et al., 1999), specific
integrin subunits (Jacques et al., 1998), PSA-NCAM (Tomasiewicz et al.,
1993; Cremer et al., 1994; Ono et al., 1994), and BDNF (Zigova et al.,
1998). Interestingly, loss of the olfactory bulb target via bulbectomy
or RMS transection lesions does not prevent SVZ precursor migration
rostrally into the RMS, although the precursors accumulate in the SVZ
and proximal RMS (Jankovski et al., 1998; Alonso et al., 1999;
Kirschenbaum et al., 1999). Recent in vitro studies also
support a role for glial-derived factors in modulating neuroblast
proliferation and migration in the unlesioned SVZ-olfactory bulb
pathway (Lim and Alvarez-Buylla, 1999; Mason et al., 2001). The
importance of neuroblast interactions with astrocytes is underscored
further by the fact that RMS neuroblast chain migration in
vivo in the adult rodent occurs within tube-like structures
composed of astrocytes (Thomas et al., 1996; Peretto et al., 1997).
Importantly, the presence of ongoing neurogenesis in the mature brain
raises the possibility that endogenous precursor cells could be used
therapeutically for repair of neuronal loss associated with brain
injuries or degenerative disorders (O'Leary, 1993; Lowenstein and
Parent, 1999). However, the response of endogenous neural stem or
precursor cells to cerebral injury and their potential involvement in
neurological disease pathophysiology have received relatively little
attention. Recent studies of the adult rodent dentate gyrus reveal that
neuronal precursors in this region respond to various forms of injury
by increasing neurogenesis (Gould and Tanapat, 1997; Parent et al.,
1997; Liu et al., 1998). For example, chemoconvulsant-induced status
epilepticus (SE) markedly increases dentate granule cell neurogenesis
in the adult rat (Parent et al., 1997; Gray and Sundstrom, 1998). The
granule cells that are generated after seizure-induced injury are found
in both normal and ectopic locations in the dentate gyrus, and they
appear to integrate both normally and aberrantly into existing
networks. Several investigations also describe an increase in precursor cell proliferation in the adult rodent forebrain SVZ after various forms of injury. The types of injury include aspiration or transection lesions of the forebrain (Willis et al., 1976; Szele and Chesselet, 1996; Weinstein et al., 1996) and inflammatory demyelination
(Calzà et al., 1998). However, the cell fates and ultimate
destinations of the neural progenitors that proliferate in response to
these forms of injury have not been well characterized. More recent work suggests that SVZ precursors can give rise to glia, either astrocytes or both astrocytes and oligodendrocytes, after brain injury
that has been induced by mechanical trauma (Holmin et al., 1997) or
chemical demyelination (Nait-Oumesmar et al., 1999).
On the basis of the findings of seizure-induced neurogenesis in the
adult rat dentate gyrus and the effects of injury on forebrain SVZ cell
proliferation, we sought to determine whether chemoconvulsant-induced SE alters neuroblast proliferation and migration in the SVZ-olfactory bulb pathway of the adult rat. We administered pilocarpine systemically to induce limbic SE, which results in damage to various limbic structures and neocortical areas, including regions near the
SVZ-olfactory bulb pathway (Turski et al., 1983). Bromodeoxyuridine
(BrdU) and retroviral reporter labeling then was used to identify the
cell proliferation and migration patterns of neuroblasts after
prolonged seizures. We found that SE in the adult rat expands the
neuronal precursor population of the SVZ-olfactory bulb pathway and
alters neuroblast migration in the injured forebrain.
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MATERIALS AND METHODS |
Seizure induction and cell proliferation assays.
Young adult, male Sprague Dawley rats (180-230 gm) were
pretreated with injections of atropine methylbromide (5 mg/kg, i.p.;
Sigma, St. Louis, MO) and 15 min later were given pilocarpine
hydrochloride (340 mg/kg, i.p.; Sigma) to induce SE. If seizure
activity was not initiated within 1 hr after the initial pilocarpine
hydrochloride dose, an additional dose of 170 mg/kg was given. Seizures
were monitored behaviorally and then terminated after 2 hr of SE with
diazepam (10 mg/kg, i.p.; Elkins-Sinn, Cherry Hill, NJ); additional
doses of 5 mg/kg diazepam were given at 45-60 min intervals if
convulsive seizure activity persisted. Only rats that displayed 2 hr of
continuous convulsive seizure activity (head and forelimb clonus) after
pilocarpine treatment were used in these experiments. Control rats were
treated identically except that 0.9% sodium chloride solution was
substituted for pilocarpine. Experimental procedures were approved by
animal research committees at the University of California, San
Francisco and the University of Michigan.
BrdU (Boehringer Mannheim, Indianapolis, IN) was used to label
proliferating (S-phase) cells according to two protocols (Miller and
Nowakowski, 1988). In the first protocol the rats received two BrdU
injections (50 mg/kg, i.p. in PBS) 1 hr apart on days 1, 4, 7, 14, 21, or 35 after saline or pilocarpine treatment (n = 3-4 per time point for each group) and were perfused 1 hr after the
second BrdU dose. This short survival duration after BrdU was used to
assess proliferation in situ, i.e., without allowing sufficient time for the cells to migrate from their location during BrdU incorporation. In the second protocol the same dose of BrdU was
administered three times over 6 hr on day 7 after pilocarpine or saline
treatment. Animals (n = 6-10 per time point for each group) were killed 2, 4, 7, or 14 d after BrdU injections (9, 11, 14, or 21 d after SE, respectively). In separate experiments the
proliferating cells in the rostral SVZ were labeled specifically by
injection of replication-incompetent retrovirus carrying nuclear localization signal -galactosidase (GPGnlsLZ; a gift of Richard Mulligan, Harvard University, Cambridge, MA) or enhanced green fluorescent protein (LNIT-GFP; a gift of Theo Palmer and Fred Gage, The
Salk Institute, La Jolla, CA) reporters. Vesicular stomatitis virus G (VSV-G) protein pseudotyped retrovirus was generated from 293GPG producer cells, the supernatant was collected, and the virus was
concentrated and titered as described previously (Ory et al., 1996).
Animals were anesthetized with intraperitoneal ketamine (70 mg/kg) and
xylazine (8 mg/kg) and positioned on a stereotactic frame (Kopf
Instruments, Tujunga, CA); a 1 µl mixture of concentrated (5 × 108 cfu) virus stock, 80 µg/ml
Polybrene, and 0.01% trypan blue was injected stereotactically into
the rostral SVZ over 5 min. Injection coordinates relative to bregma
were 1.0 mm posterior and 1.3 mm lateral, at a depth of 3.3 mm from the
brain surface. The injection sites were identified easily in processed
tissue by visualizing the needle track. Adult rats were injected either
2 d before or 10 d after pilocarpine or saline treatment
(n = 6-8 per group), and animals were killed 11, 14, 21, or 35 d after retrovirus injection. So that the degree of
virus diffusion through the SVZ-olfactory bulb pathway could be
assessed after focal retrovirus injections, an additional group of
three naïve adult rats received identical SVZ injections of
GPGnlsLZ retrovirus and were killed 3 d later.
Tissue processing, Nissl stain, and -gal histochemistry.
Rats received an overdose of pentobarbital sodium (Abbott
Laboratories, North Chicago, IL) and were perfused transcardially with
PBS, followed by 2% paraformaldehyde in 0.1 M sodium
acetate, pH 6.5 (80 ml) and then by 2% paraformaldehyde/0.1%
glutaraldehyde in 0.1 M sodium borate, pH 8.5 (360 ml).
After post-fixation in situ overnight, brains were removed,
washed once in PBS, cryoprotected with 30% sucrose in PBS, and frozen
in powdered dry ice. Coronal sections 40 µm thick through the rostral
SVZ (extending anteriorly from Paxinos and Watson coordinate, bregma
+0.2 mm; Paxinos and Watson, 1998), RMS, and olfactory bulb (see areas
a-c in Fig. 3H) were cut with a cryostat;
every sixth or eighth section was processed for Nissl or
immunohistochemical stains (see below). Brains from additional animals
(n = 3-4 per condition) were cut in the sagittal plane
(40-µm-thick sections), and every sixth section of the medial
one-third of each hemisphere was processed for Nissl staining or
immunohistochemistry. For Nissl staining the sections were mounted on
slides (Superfrost-plus, Fisher Scientific, Pittsburgh, PA), dehydrated
and rehydrated in graded ethanols and xylenes, incubated in 1% cresyl
violet for 30 sec, decolorized in acetic acid, and then dehydrated and
coverslipped with Permount (Fisher Scientific). -Gal histochemistry
was performed according to established methods (Cepko et al., 1995).
-Gal expression also was visualized with indirect immunofluorescence
histochemistry for double-labeling (see below), and enhanced GFP (eGFP)
expression was visualized directly with epifluorescence microscopy.
Immunohistochemistry. Diaminobenzidine peroxidase
immunohistochemistry was performed on free-floating tissue sections
with the use of antibodies to BrdU or the cell cycle-related kinase cdc2 (p34cdc2; Okano et al., 1993) as
described previously (Parent et al., 1997). For BrdU immunostaining,
DNA first was denatured by incubating tissue sections in 2N HCl for 30 min at 37°C, followed by a 10 min wash in 0.1 M borate
solution, pH 8.5. Tissue was incubated overnight in primary antibody at
4°C; the primary antibody dilutions that were used included 1:1000
for BrdU (mouse monoclonal; Boehringer Mannheim, Indianapolis, IN) and
1:1000 for cdc2 (mouse monoclonal; Santa Cruz Biotechnology, Santa
Cruz, CA). Single and double-label immunofluorescence histochemistry
was done according to previously described methods (Parent et al.,
1997, 1999). The primary antibody dilutions that were used included
1:100 for BrdU (rat monoclonal; Accurate Chemical, Westbury, NY);
1:1000 for PSA-NCAM (mouse IgM monoclonal; a gift of G. Rougon,
Université Aix-Marseille II, France); 1:1000 for doublecortin
(rabbit polyclonal; a gift of C. Walsh, Harvard University); 1:200 for
GFAP (rat monoclonal; a gift of V. Lee, University of Pennsylvania,
Philadelphia, PA) or 1:500 for GFAP (rabbit polyclonal, Sigma); 1:50
for vimentin (mouse monoclonal, Dako, Carpinteria, CA); 1:10,000 for
CRMP-4 (also known as TUC-4; rabbit polyclonal; a gift of S. Hockfield, Yale University, New Haven, CT); 1:500 for glucose transporter-1 (Glut-1; rabbit polyclonal; Chemicon, Temecula, CA); 1:1000 for -gal
(rabbit polyclonal; 5 Prime-3 Prime, Boulder, CO); and 1:400 for class
III -tubulin (TuJ1 clone, mouse monoclonal; Babco, Berkeley, CA).
Secondary antibodies for immunofluorescence were goat anti-rat IgG
conjugated to fluorescein isothiocyanate (FITC) or Texas Red, goat
anti-mouse IgG conjugated to FITC or Texas Red, goat anti-rabbit IgG
conjugated to FITC or Texas Red, and goat anti-mouse IgM conjugated to
Texas Red (all species cross-adsorbed and obtained from Jackson
ImmunoResearch Laboratories, West Grove, PA) at 1:400 dilutions. Tissue
was incubated in secondary antibody overnight at 4°C. For
double-label immunofluorescence with rat anti-BrdU and a second primary
antibody, free-floating sections were incubated in the second primary
antibody for 24 hr at 4°C, washed with Tris-buffered saline (TBS;
0.15 M NaCl, 0.1 M Tris-HCl, pH 7.6) for 45 min, post-fixed in 4% PFA for 20 min, rinsed three times with TBS, and
denatured with 2N HCl as described above. After three TBS washes and a
1 hr incubation in blocking solution [10% normal goat serum, 0.4%
Triton X-100, 3% (w/v) bovine serum albumin, and 1% (w/v) glycine in
TBS] the sections were incubated in rat anti-BrdU antibody and the
second primary antibody for another 24 hr at 4°C. Immunofluorescence
images were obtained by using a Bio-Rad MRC 1024 confocal laser
microscope (Hercules, CA) as single optical images or
z-series stacks, visualized with NIH Image version 1.61 software (Bethesda, MD), and transferred to Adobe Photoshop for color
merging (Adobe Systems, Mountain View, CA).
Quantification and statistical analyses. Nissl staining and
BrdU immunostaining within or adjacent to the RMS were quantified in
images from every sixth coronal section through the mid-RMS (between
Paxinos and Watson coordinates, bregma +3.0 and +4.5 mm) or midportion
of the olfactory bulb captured at 200× magnification and digitized
with a Spot 2 digital camera (Diagnostic Instruments, Sterling Heights,
MI). For PSA-NCAM immunostaining, images from the same RMS regions were
obtained at 200× magnification with a Bio-Rad MRC 1024 confocal laser
microscope. Then all images were imported into NIH Image version 1.61 software for blinded densitometric analysis of BrdU, PSA-NCAM, and
Nissl staining. The region of interest (RMS in cross section or
rectangular areas centered over the subependymal or granule cell layer
for the olfactory bulb) was selected manually with a wand and drawing
tablet, and measurements were made from one hemisphere by using six
sections per animal from three to six animals per group. For BrdU
immunostaining in the RMS or olfactory bulb the mean area that was
immunostained per section was determined for each animal; then group
means and SEM for each time point were calculated. BrdU immunostaining
adjacent to the RMS was calculated by drawing a square region of
interest centered over the RMS, subtracting out the RMS, and
determining the area immunostained in the remainder of the square. For
Nissl staining and PSA-NCAM immunohistochemistry the cumulative RMS area that was labeled was determined for each animal, and group means
and SD values were calculated. Statistical comparisons between groups
were made by using two-tailed Student's t test with
StatView software (Abacus Concepts, Berkeley, CA).
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RESULTS |
Pilocarpine-induced SE increases proliferating cell numbers in the
rostral SVZ
We first examined the effect of prolonged seizures on cell
proliferation in the adult rat forebrain SVZ. The distribution of
mitotically active cells at different times after pilocarpine-induced SE was assessed by BrdU labeling and detection of the endogenous cell
cycle marker cdc2 (Okano et al., 1993). BrdU was given as two pulses, 1 and 2 hr before perfusion, to label S-phase cells in situ at
multiple time points after seizure induction (see Materials and
Methods). Compared with controls, SVZ BrdU immunoreactivity (IR) was
increased markedly 7 d after pilocarpine treatment (Fig. 1A-D) at rostral
levels of the SVZ (between Paxinos and Watson coordinates, bregma +1.6
and +2.5 mm; labeled a in Fig. 3H). In some animals BrdU labeling at this SVZ level was increased to a lesser
extent at 4 and 14 d after SE (Fig. 1B,D), but
the magnitude was variable and the increase was not present in all
animals. No changes in rostral SVZ BrdU labeling occurred at 1, 21, or 35 d after seizure induction or at any time point at more
posterior levels of the SVZ when compared with controls (between
Paxinos and Watson coordinates, bregma +0.2 and +1.6 mm; data not
shown). To confirm the distribution of proliferating cells obtained
with BrdU labeling after SE, we analyzed the pattern of immunostaining for the cell cycle antigen cdc2, a molecule known to be expressed in
mitotically active cells in the adult rodent brain (Okano et al.,
1993). cdc2 expression in the rostral SVZ also was increased at 7 d after SE (Fig. 1E,F), and the pattern was
nearly identical to that of BrdU labeling (Fig.
1A,C). The distribution of cdc2-expressing cells at
other time points after seizures and in controls also correlated
strongly with that of BrdU-labeled cells. The majority of the
proliferating cells identified by BrdU or cdc2 immunocytochemistry was
located in the dorsal or dorsolateral aspect of the SVZ in both control
and pilocarpine-treated rats (Fig. 1). This location is consistent with
previous studies of cell proliferation in the adult rodent SVZ (Okano
et al., 1993; Peretto et al., 1999).
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Figure 1.
Increased cell proliferation and expansion of the
adult rat rostral SVZ after pilocarpine-induced status epilepticus
(SE). A-D, Coronal sections through the rostral
forebrain SVZ immunostained for BrdU after pulse labeling. Compared
with the baseline level of mitotic activity in a representative control
rat (A), the degree of cell proliferation is
increased markedly 7 d after pilocarpine treatment
(C) and elevated more modestly at 4 d
(B) and 14 d (D) after
seizures. E, F, cdc2 immunostaining shows a similar
pattern of increased SVZ cell proliferation 7 d after SE
(F) compared with a control animal
(E). G, H, Coronal Nissl-stained
sections through the rostral SVZ show that the SVZ is expanded greatly
in a pilocarpine-treated rat 14 d after SE
(H) compared with a typical control animal
(G). Sections in A-H correspond
approximately to level a in Figure 3H.
The asterisk in each panel denotes the lateral
ventricle. Scale bars: A-H (in A), 100 µm. Con, Control; Sz, after
seizure.
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In addition to enhanced cell proliferation, we observed an enlargement
of the dorsolateral portion of the SVZ after SE. Nissl staining
revealed that the maximal expansion of the rostral SVZ in
pilocarpine-treated rats occurred 14 d after SE in the same SVZ
regions as the increase in proliferating cells (Fig.
1G,H). The seizure-induced increase in SVZ Nissl
staining first appeared within 7 d and persisted for at least 3 weeks after seizures. Nissl staining of the SVZ from rats 35 d
after pilocarpine treatment appeared similar to that in controls (data
not shown). No difference in the SVZ cell-packing density between
controls and pilocarpine-treated rats was observed. Although some
temporal overlap of seizure-induced cell proliferation and increased
cellularity was seen, the maximal increase in SVZ mitotic activity
after pilocarpine-induced SE preceded the peak increase in SVZ Nissl
staining. Therefore, the temporal and spatial patterns are consistent
with the idea that the enlargement of the rostral SVZ resulted from the
accumulation of proliferating cells after SE.
SE expands the rostral forebrain SVZ neuroblast population
We next sought to determine whether the seizure-induced SVZ
expansion corresponded to an increase in the number of immature neurons. The SVZ neuroblasts are known to express a variety of immature
neuronal markers even before they enter the migratory pathway
(for review, see Peretto et al., 1999). We first examined the
expression pattern of PSA-NCAM, a molecule necessary for
neuroblast migration to the olfactory bulb (Tomasiewicz et al., 1993;
Cremer et al., 1994; Ono et al., 1994), at various times after
pilocarpine treatment. SE markedly increased immunoreactivity for
PSA-NCAM in the rostral SVZ within 14 d after pilocarpine
treatment compared with controls (Fig.
2A,B). The PSA-NCAM-IR
cells were both more numerous and more intensely labeled in the
pilocarpine-treated animals, although the cellular morphology was
similar between the two groups. The increase in SVZ PSA-NCAM expression
peaked at 14-21 d after seizures, and the effect declined by 35 d
after pilocarpine treatment. Because PSA-NCAM expression is not
entirely specific for neurons (Ben-Hur et al., 1998; Theodosis et al., 1999), we also performed immunostaining for additional markers normally
expressed by neuroblasts in the SVZ. Pilocarpine-treated animals showed
increased expression of neuron-specific class III -tubulin (Fig.
2C,D), doublecortin (Fig. 2E,F),
and CRMP-4 (data not shown) 2-3 weeks after SE. The seizure-induced
increase in rostral SVZ neuronal precursors occurred in the same
dorsolateral location as that of enhanced cell proliferation and also
extended to more caudal levels of the SVZ compared with the increase in BrdU labeling (for example, see Fig. 2F). As
expected, the rise in mitotic activity preceded the expansion of SVZ
neuroblasts in pilocarpine-treated rats. Therefore, newly generated
cells accumulated in the SVZ and expressed markers consistent with a neuronal phenotype.
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Figure 2.
Prolonged seizures increase neuroblast numbers in
the forebrain SVZ. A-F, Confocal images of coronal
sections stained by indirect immunofluorescence with the use of
antibodies that recognize immature neurons in the rostral SVZ of adult
rats 2 weeks after saline (A, C, E) or pilocarpine
(B, D, F) treatment. Both the amount and
intensity of immunostaining are increased after SE. Note that the
neuron-specific class III -tubulin (TuJ1) antibody
also labels differentiated neurons outside the SVZ (C,
D), whereas immunostaining for PSA-NCAM (A, B)
and doublecortin (E, F) is restricted mainly to
the SVZ. G, H, Immunofluorescence staining for the
astrocyte protein GFAP in the rostral SVZ of an adult rat 2 weeks after
pilocarpine treatment (H) is similar to
the control (G), although GFAP-IR is increased
outside the SVZ region (H).
Arrows in C,
D, G, and H outline the
dorsolateral SVZ. Scale bar (in H), 100 µm.
LV, Lateral ventricle; PSA-NCAM,
polysialylated neural cell adhesion molecule; GFAP,
glial fibrillary acidic protein.
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At the light microscopic level the SVZ and RMS cells expressing
PSA-NCAM and other immature neuronal markers had the typical morphology
of neuronal precursors arranged in tightly packed clusters or chains
(see Fig. 4L; data not shown). In the normal adult
rodent SVZ, neuroblasts are associated intimately with surrounding
astrocytes that eventually form tube-like structures through which the
neuroblasts migrate within the RMS (Gates et al., 1995; Doetsch and
Alvarez-Buylla, 1996; Thomas et al., 1996; Peretto et al., 1997). To
determine whether the seizure-induced increase in SVZ cellularity also
involved increases in the astroglial component, we immunostained for
GFAP and vimentin, markers known to be expressed by SVZ astrocytes (Gates et al., 1995; Peretto et al., 1999). Pilocarpine-induced SE did
not increase SVZ GFAP- or vimentin-IR, and no changes in the structural
arrangement of glia in the SVZ were noted (Fig. 2G,H; data
not shown). This contrasted with the marked increase in GFAP- and
vimentin-IR that was found adjacent to the SVZ (Fig. 2H) and in other regions of expected seizure-induced
injury (see Fig. 4H,K). Taken together, these
findings indicate that SE causes a relatively specific expansion of the
proliferating neuroblast population in the adult rat rostral SVZ.
The numbers of RMS neuroblasts increase after SE
The RMS is a cell-dense region that extends through the medial
forebrain from the rostral portion of the SVZ anteriorly to the
olfactory bulb (Fig.
3H). It contains
neuronal precursors that arise from the forebrain SVZ and migrate to
the olfactory bulb (Luskin, 1993; Lois and Alvarez-Buylla, 1994; Lois
et al., 1996; Peretto et al., 1999). On the basis of our finding of
increased numbers of dividing neuroblasts in the rostral SVZ after SE,
we also expected to see more newly generated cells migrating within the
RMS. Nissl staining of the RMS at different time points after seizures
revealed that this pathway markedly increased in size within 11 d
after pilocarpine treatment. The increase was maximal after ~14 d
(Fig. 3A,B) and appeared qualitatively throughout the entire
length of the RMS. We quantified the area of Nissl staining in the
midportion of the RMS (Fig. 3H, region labeled b)
in control and pilocarpine-treated rats. The cross-sectional area of
the RMS of pilocarpine-treated rats was significantly greater than it
was of controls at 14 and 21 d after seizures (Fig.
3G).
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Figure 3.
Status epilepticus expands the RMS. A,
B, Nissl staining of coronal sections (corresponding
approximately to level b in H)
shows a marked enlargement of the RMS 2 weeks after pilocarpine
treatment (B) compared with a control
(A). C-F, BrdU immunostaining of
coronal (C, D) and parasagittal (E,
F) sections from adult rats 2 weeks after pilocarpine
(D, F) or saline (C, E) treatment
and 1 week after BrdU administration. The number of mitotically active
cells in the RMS is increased markedly after seizures (D,
F). The parasagittal sections (E, F;
olfactory bulb out of view to the right) demonstrate
increased BrdU labeling throughout nearly the entire length of RMS.
Note also that many more BrdU-IR cells are scattered outside the RMS of
the pilocarpine-treated rats (D, F).
G, Quantification of RMS area identified by Nissl
staining (left; see Materials and Methods) shows
significant cellular expansion of the RMS at 2 and 3 weeks after SE.
The area of PSA-NCAM immunoreactivity in the RMS (right)
also is increased significantly above control levels at 2 weeks after
SE. *p < 0.05; **p < 0.01. Error bars represent SD. H, Schematic parasagittal view
of the adult rodent brain showing the tangential migratory route of
neuronal precursors from the rostral SVZ to the olfactory bulb.
Dashed pairs of vertical lines labeled
a, b, and c denote
approximate regions from which coronal sections are shown in the
figures (see Results and legends). Scale bar (in
F), 100 µm. Sz, After seizure;
OB, olfactory bulb.
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To determine whether the RMS expansion was attributable to increased
numbers of newly generated cells migrating in this pathway after
seizures, we used BrdU to label S-phase cells and determined their
locations after several different survival periods. The 7 d
after-pilocarpine time point was chosen to administer BrdU because this
was the time of maximal BrdU labeling in the rostral SVZ after SE (Fig.
1). When animals were killed within 1 hr of the last BrdU injection on
day 7, we observed a qualitative increase in BrdU-IR. Quantification of
BrdU labeling in the midportion of the RMS at this time point after SE
showed a trend toward an increase in the area immunostained in
pilocarpine-treated animals that was not statistically significant
(p = 0.08, two-tailed t test). At 1 week after BrdU incorporation (2 weeks after SE) a marked increase in
the number of BrdU-IR cells was found throughout the entire RMS
compared with controls (Fig. 3C-F). The majority of
the BrdU-labeled nuclei had a fusiform appearance on parasagittal sections characteristic of migrating cells. Quantification of coronal
sections through the midportion of the RMS at 2 weeks after SE revealed
significantly more BrdU-IR cells in pilocarpine-treated animals
(p = 0.01, two-tailed t test).
Minimal numbers of BrdU-IR cells were seen in the RMS of pilocarpine-
or saline-treated rats after 3 weeks (2 weeks after BrdU
administration), indicating that most of the proliferating cells had
migrated to the olfactory bulb or failed to survive. cdc2-IR also
increased in the RMS of pilocarpine-treated animals compared with
controls (data not shown). The cdc2 expression in migrating neuroblasts
and BrdU incorporation of RMS cells within 2 hr after BrdU
administration (see Fig. 7) are consistent with existing evidence that
these cells remain mitotically active as they migrate (Kishi, 1987;
Luskin, 1998).
On the basis of our finding of a seizure-induced increase in SVZ
neuroblasts and an expansion of proliferating cells in the RMS, we
expected SE to increase the numbers of migrating neuroblasts similarly
within the RMS. Corresponding to the greater cellularity, the
population of PSA-NCAM-IR neuroblasts also expanded after SE (Fig.
4A,B). Measurement of
PSA-NCAM immunostaining in the RMS at various time points after
pilocarpine treatment revealed a significant increase on day 14 after
SE (Fig. 3G). In addition to PSA-NCAM-IR, prolonged seizure
activity also increased immunostaining for doublecortin,
neuron-specific -tubulin, and CRMP-4 in the RMS (Fig.
4D,E; data not shown). To examine astroglial lineage cells, we immunostained sections from pilocarpine- or saline-treated rats with antibodies to GFAP or vimentin. Although the pattern of GFAP-
and vimentin-IR in the innermost region of the migratory pathway
appeared to maintain the typical tubular architecture, immunostaining
increased markedly adjacent to and well outside the RMS in
pilocarpine-treated rats (Fig. 4G,H,J,K; data not shown). No
cellular colocalization was present on confocal microscopic analysis
when double-label immunofluorescence for PSA-NCAM or doublecortin and
these astrocytic markers was performed (data not shown). To confirm
that the labeled RMS cells were newly generated, we performed
immunofluorescence double labeling for BrdU and immature neuronal
markers. In rats given BrdU injections 7 d after pilocarpine treatment and killed 1 week later, many of the BrdU-labeled cells in
the RMS coexpressed neuron-specific -tubulin or PSA-NCAM (see Fig.
6A,B). The RMS BrdU-IR cells also coexpressed CRMP-4
after SE (see Fig. 8A), but not GFAP or the
endothelial marker GLUT-1 (data not shown). Like controls, cells
expressing immature neuronal markers in the RMS of pilocarpine-treated
animals exhibited morphological features consistent with migrating
neuroblasts (Fig. 4L).
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Figure 4.
Immunostaining to detect immature neurons
and glia in the adult rat RMS after SE. Shown are confocal images of
coronal sections through the RMS immunostained for PSA-NCAM
(MenB; A-C, I), doublecortin
(DCx; D-F), or GFAP (G, H,
J, K). Sections from controls are shown in the
left panels and from pilocarpine-treated rats in the
middle and right panels. A-C,
I, PSA-NCAM-IR in the RMS increased markedly 2 weeks after SE
(B) compared with the saline-treated control
(A), and many labeled cells are seen extending
from the borders of the RMS at both proximal (B,
I) and distal (C) RMS levels that
are not seen in the control (A). The
asterisk in I denotes the edge of the
RMS. D-F, Doublecortin-IR in the RMS (at slightly more
distal levels compared with A, B) also is increased by
SE. F shows two optical images at different levels of a
higher magnification (Figure legend continued.)
z-series through the boxed region in
E. Note that the doublecortin-IR cells extending out
from the RMS have the morphology of migrating neuroblasts. G, H,
J, K, GFAP immunostaining of coronal sections through the RMS
14 d after saline (G, J) or pilocarpine
(H, K) treatment. The astrocytic elements within
the central portion of the RMS maintain a similar tubular architecture
2 weeks after SE (enclosed by white arrowheads in
H, K), but the outer portion of the RMS is
indistinct when compared with controls, and substantial astroglial
proliferation is present outside the RMS after seizures.
L, Sagittal section of CRMP-4-IR, migrating neuroblasts
in the RMS 2 weeks after SE. Note the chain-like structures of labeled
cells extending inferiorly from the RMS (white arrows),
which were not present in controls (data not shown). Scale bars:
A, B, D, E, G, H (in D), 75 µm;
C, J-L (in L), 50 µm;
F, I (in F), 10 µm.
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More neuronal precursors migrate to the olfactory bulb
after SE
Previous reports have shown that the SVZ and RMS expand after
either removal of the olfactory bulb target (Kirschenbaum et al., 1999)
or lesioning of the migratory pathway (Jankovski et al., 1998; Alonso
et al., 1999). This raises the possibility that the SE-induced
expansion of neuronal precursors in the RMS that we observed might
result from their failure to migrate to the olfactory bulb (a
"logjam" effect) rather than from an overall increase in SVZ
neuroblast production or survival. To determine whether SVZ neuroblasts
continue to migrate to the olfactory bulb after seizure-induced injury,
we labeled proliferating cells with BrdU 7 d after SE and
determined the pattern of BrdU labeling in the olfactory bulb after
progressively longer survival durations. When BrdU was administered
7 d after pilocarpine or saline treatment and the animals were
killed 1 hr later, only rare labeled cells were found in the olfactory
bulb of rats in either group (Fig. 5A,B). When animals survived
for 2 d additionally, however, BrdU-IR cells began to appear in
the olfactory bulb subependymal region, and significantly more labeled
cells were found in pilocarpine-treated rats than in controls (Fig.
5C,D,G). By 7 d after BrdU administration (14 d after
treatment) many more labeled cells had reached the olfactory bulb
granule cell and external plexiform layers after SE than in controls
(Fig. 5E-G). Two weeks after BrdU administration (21 d
after SE) immunoreactive cells were present in both the granule and
periglomerular layers of the olfactory bulb, and the pattern of BrdU
labeling was indistinguishable between pilocarpine- and saline-treated
rats (data not shown). These findings suggested that not only were SVZ
precursors able to migrate to the olfactory bulb after SE but that they
did so in greater numbers than in controls. Increased cell death of
olfactory bulb neuroblasts or dilution of nuclear BrdU content below
levels of immunocytochemical detection after SE is likely to explain
the absence of increased labeling at 21 d in the
pilocarpine-treated rats. As expected, BrdU-labeled cells in the
olfactory bulb examined between 9 and 21 d after SE expressed
neuronal markers (Fig. 6C,D;
data not shown).
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Figure 5.
Seizure activity increases neuroblast migration to
the olfactory bulb. A-F, BrdU-immunostained coronal
sections through the olfactory bulb (level c in Fig.
3H) in adult rats receiving BrdU 7 d after
saline (A, C, E) or pilocarpine (B, D,
F) treatment. Only rare BrdU-IR cells are found in the
olfactory bulb in either group within 2 hr of the BrdU injection
(A, B), but 2 d later more labeled cells have
reached the subependymal zone (SEZ) of the olfactory
bulb after SE (D) than in a control
(C). At 1 week after BrdU injections (2 weeks
after SE) more BrdU-IR cells have migrated to the olfactory granule
cell (GCL) and external plexiform layers
(EPL) in a pilocarpine-treated rat
(F) than in the control
(E). G, Quantification of BrdU
labeling in the olfactory SEZ (left) or GCL
(right) in control and treatment groups shows that SE
significantly increases the migration of proliferating cells to the
olfactory bulb. Error bars represent SEM; *p < 0.05. Scale bar (in A), 100 µm.
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Figure 6.
Newly generated neurons in the RMS and olfactory
bulb after pilocarpine-induced SE. A, B, Confocal images
of coronal sections through the RMS immunofluorescently double-labeled
for BrdU (red) and neuron-specific -tubulin
(green in A) or BrdU
(green) and PSA-NCAM (red in
B) in an adult rat 14 d after SE (7 d after BrdU
injections). Nearly all BrdU-IR cells coexpress these neuronal markers.
C, Confocal images of a coronal olfactory bulb section
immunofluorescently double-labeled for BrdU (red) and
neuron-specific -tubulin (green) 21 d
after SE. Many BrdU-labeled cells are seen in the olfactory granule
cell layer at this time point (C).
D, A higher magnification view of the boxed
area in C; yellow arrows point to
clusters of double-labeled cells. E-J, RV--gal
(E, G, H) or RV-GFP (F, I,
J) retroviral reporters were injected stereotactically
into the SVZ either 10 d after SE (RV--gal) or 2 d before
SE (RV-GFP). The presence of reporter-labeled cells in the RMS and
olfactory bulb 2 weeks (E) or 11 d
(F) after RV injections confirms that SVZ
precursors continue to migrate to the olfactory bulb after SE.
F, The RV-GFP reporter clearly fills the cells and shows
the morphology of the migrating neuroblasts in the RMS
(arrow) and more differentiated neurons in the olfactory
bulb (arrowhead). Sections are counterstained with
nuclear fast red (E) or propidium iodide
(F). G, H, RV--gal-labeled
cells in the olfactory bulb coexpress neuron-specific -tubulin
(arrows) 24 d after SE. I,
J, RV-GFP-labeled cells (green)
coexpress the mature neuronal marker NeuN (red;
double-labeled cells are yellow) 21 d after SE and
display the morphology of differentiated olfactory granule
(I) or periglomerular
(J) neurons. Scale bars: A, B (in
B), 25 µm; C, 75 µm;
E, 150 µm; F, 100 µm; D,
G-J (in J), 10 µm. SEZ,
Subependymal zone; GCL, granule cell layer;
EPL, external plexiform layer; GL,
glomerular layer.
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To confirm that the newborn neurons in the olfactory bulb migrated from
the SVZ after SE, we stereotactically injected high-titer, replication-deficient, retrovirus carrying nls-gal (RV--gal) or
eGFP (RV-GFP) reporters into the SVZ of adult rats. Like BrdU, retroviral reporters specifically label S-phase cells and integrate into the DNA so that the reporters are expressed faithfully in the
precursor progeny. When RV--gal was injected into the SVZ 10 d
after SE and the animals were killed 2 weeks later, labeled cells were
found throughout the RMS and olfactory bulb (Fig.
6E). The RV--gal-immunoreactive cells in the
olfactory bulb were identified as neurons by coexpression of
neuron-specific -tubulin (Fig. 6G,H). To determine
the migration patterns of precursors in the SVZ that were proliferating
constitutively before SE, we injected RV-GFP into the SVZ 2 d
before pilocarpine treatment. Then 11 d later GFP-labeled cells
with migratory or more differentiated neuroblast morphology appeared in
the RMS and olfactory bulb, respectively (Fig. 6F).
At 21 d after SE the RV-GFP-labeled cells in the olfactory bulb
had the appearance of differentiated olfactory neurons and expressed
the mature neuronal marker NeuN (Fig. 6I,J). So that retroviral labeling of dividing RMS and olfactory bulb cells by
diffusion of retrovirus from the injection site could be excluded,
three controls received RV--gal injections in the rostral SVZ and
were killed 3 d later. In all of these animals the majority of
labeled cells was seen in the SVZ and adjacent to the injection track.
Rare scattered -gal-expressing cells were seen in the proximal RMS,
consistent with a short-distance migration of these cells from the SVZ.
As expected, however, no labeled cells were found in the distal RMS or
olfactory bulb. These results indicate that SE increases the migration
of forebrain SVZ neural precursors to the olfactory bulb, where the
cells differentiate into neurons.
SE induces immature neurons to migrate ectopically into
the forebrain
At nearly all RMS levels in pilocarpine-treated animals, cells
with migrating neuroblast morphology that expressed immature neuronal
markers extended out from the borders of the RMS or were located well
outside the migratory stream (Fig. 4). Putative newly generated
neuroblasts, identified by doublecortin immunostaining, were also found
in the striatum of pilocarpine-treated rats (see Fig.
8D). Such "ectopic" cells in the striatum and
regions near the RMS were not observed in controls. Moreover, the
distribution of migratory profiles adjacent to the RMS corresponded to
the pattern of increased BrdU labeling in these regions after SE (Fig. 3C-F). These findings suggested that the ectopic
cells were newly generated neurons. To test this theory further, we
performed double-label immunofluorescence for BrdU and cell
type-specific antigens. Newly generated cells that had incorporated
BrdU on day 7 after pilocarpine treatment were found 1 week later to be
located in regions adjacent to, but well outside of, the RMS. These
cells coexpressed the immature neuronal markers CRMP-4, neuron-specific
-tubulin, or PSA-NCAM (see Fig. 8A-C; data not
shown). Many of the cells had the appearance of migrating neuroblasts
extending away from the migratory stream toward cortical regions. Cells
outside the RMS only rarely showed colocalization of BrdU and astrocyte
or endothelial cell markers (data not shown).
To determine the origin of these potentially ectopic newly generated
neurons, we administered BrdU 7 d after pilocarpine or saline
treatment and then measured BrdU labeling in areas adjacent to the RMS
after different survival durations. We found that BrdU-IR cells outside
of the migratory pathway increased in number between 7 and 14 d
after SE (from 1 hr to 7 d after BrdU injections; Fig. 7A-F). This increase
was significant at each time point for pilocarpine-treated rats when
compared with controls (Fig. 7G). These data further suggest
that prolonged seizures induce a portion of newly generated neuroblasts
to exit the RMS prematurely. However, another potential explanation is
that BrdU labeling increases outside of the RMS between 7 and 14 d
after SE because of the continued proliferation of cells that are
already in that region, rather than because of the migration of newly
generated cells from the SVZ-olfactory bulb pathway. To identify the
source of the ectopic neuroblasts more definitively, we examined
whether neuronal precursors labeled by stereotactic SVZ retroviral
reporter injections migrate from the RMS after SE. When RV--gal was
injected into the rostral SVZ of controls, labeled cells in RMS regions
1 week later were confined to the migratory pathway (Fig.
8E), similar to
previous findings in neonatal rats (Luskin, 1993). In
pilocarpine-treated rats receiving SVZ RV--gal injections 7 d
after SE, however, increased numbers of labeled cells were present in
the RMS after 1 week (Fig. 8F). Moreover, cells
outside of the RMS also were labeled with the reporter, confirming
their origin in the SVZ. The neuronal identity of -gal expressing
cells outside the RMS was indicated by their coexpression of
neuron-specific -tubulin (Fig. 8G). In similar
experiments RV-GFP was injected 2 d before pilocarpine or saline
treatment. Three weeks later the GFP-labeled cells were found both
within and outside the RMS after SE (Fig. 8H) but
were restricted to the RMS in controls (data not shown). Of note, rare
GFP-labeled cells with apparent neuronal morphology persisted in
cortical regions up to 35 d after SE (Fig. 8I).
Such cells were scarce compared with the numbers of cells that appeared to exit the RMS at earlier time points after SE (Fig. 4). These findings indicate that prolonged seizures induce a portion of newly
generated SVZ neuroblasts to migrate from the RMS into injured forebrain regions. However, the lack of significant numbers of newly
generated putative neurons in the neocortex after SE suggests that the
majority of neuroblasts migrating ectopically into the forebrain fails
to survive.
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Figure 7.
Seizures increase BrdU labeling adjacent to
the RMS. A-F, BrdU was administered 7 d after
saline (A, C, E) or pilocarpine (B, D,
F) treatment, and animals were killed 1 hr (A,
B), 4 d (C, D), or 7 d (E,
F) later. Note that BrdU labeling outside the RMS is
increased in the pilocarpine-treated rat compared with control at each
time point and that the labeling increases with longer survival
durations after BrdU administration in the seizure group.
G, Quantification of BrdU-immunostained cells adjacent
to the RMS (see Materials and Methods) in controls and
pilocarpine-treated rats. Error bars represent SEM.
*p < 0.05 seizure versus control;
**p < 0.01. Scale bar (in A),
50 µm.
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Figure 8.
Ectopic migration of neuroblasts from the RMS
after SE. A-C, BrdU-IR cells
(green in A, B; red
in C) within and outside the RMS 14 d after SE
coexpress CRMP-4 (red in A, B) or
neuron-specific -tubulin (green in
C) and have the morphology of migrating neuroblasts. A
higher-magnification view of the boxed region in
A is shown in B. The dashed
line in C represents the dorsal edge of the
RMS. Arrowheads point to double-labeled cells
outside of the RMS. D, Doublecortin
(DCx)-IR cells in the striatum 14 d after SE.
E-G, RV--gal injected into the rostral SVZ 10 d
after saline (E) or pilocarpine (F,
G) treatment labels cells in the RMS 14 d later.
RV--gal-labeled cells in the control remain restricted to the RMS
(E). After SE (F) many more
labeled cells are present within the RMS, and some cells have migrated
outside the RMS (arrows). G,
RV--gal-labeled cell outside the RMS coexpresses neuron-specific
-tubulin. H, I, RV-GFP was injected into the rostral
SVZ 2 d before SE, and labeled cells were detected 21 d
(H) or 35 d
(I) after seizures. Although most of the
RV-GFP-labeled cells were confined to the RMS (asterisk
in H), a labeled cell (arrow in
H) is seen outside the RMS. I,
RV-GFP-labeled cell is located in frontal cortex distant from the RMS
(asterisk). The inset shows the
differentiated neuron-like morphology of the cell. Additional confocal
optical sections of the z-series show that the process
inferior to the cell body has an axonal morphology (data not shown).
Scale bars: A, I (in A), 100 µm;
B-D (in C), 50 µm; E, F
(in E), 75 µm; G, 7.5 µm; H, 25 µm;
Inset in I, 10 µm. CC,
Corpus callosum.
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DISCUSSION |
The effects of brain injury on persistent neural precursor cells
in the adult rodent forebrain SVZ are poorly understood. We used both
endogenous and exogenous markers of dividing cells to show that
pilocarpine-induced SE increases proliferating cell numbers in the
adult rat rostral SVZ and RMS. Our results also indicate that prolonged
seizures expand the neuroblast population in the SVZ-olfactory bulb
pathway. The proliferative effect of seizures appears to be highly
selective for neuronal precursors, because relatively little change in
astrocyte lineage cells arose in these neurogenic regions despite the
presence of marked seizure-induced astrocyte proliferation in nearby
areas. Moreover, we found that SE increases neuronal precursor
migration to the olfactory bulb and induces a portion of RMS
neuroblasts to exit the pathway prematurely and migrate aberrantly into
other forebrain regions.
The dynamic nature of SVZ-olfactory bulb neurogenesis complicates the
analysis of how seizure-induced injury alters this neurogenic pathway.
For example, although new cells are produced continually in the rodent
forebrain SVZ, these precursors migrate several millimeters or more
from the SVZ to the olfactory bulb within days and continue to divide
in the RMS while migrating (Lois and Alvarez-Buylla, 1994; Lois et al.,
1996; Luskin, 1998). SE could expand the SVZ-RMS neuroblast population
via a number of mechanisms acting alone or in combination. These
include increasing the rate of neuronal precursor generation, enhancing
their short-term survival, or accelerating neuroblast migration
rostrally from more caudal SVZ locations. The prolonged expansion of
proliferating cells and immature neurons throughout the entire length
of the RMS (Fig. 3F) suggests an increase in the
overall size of the neuroblast population. This is supported further by
our finding of increased immunostaining for immature neurons at more
caudal (as well as rostral) SVZ levels (Fig. 3). However, further study
is needed to determine the relative contribution of increased
neurogenesis, enhanced survival, or altered migration after
seizure-induced injury. Another potential explanation for the
seizure-induced expansion of the proliferating SVZ-RMS neuroblast
population is that precursors accumulate from a failure to migrate to
the olfactory bulb, as has been seen after bulbectomy or transection
lesions of the RMS (Jankovski et al., 1998; Alonso et al., 1999;
Kirschenbaum et al., 1999). We tested this possibility by determining
the extent of olfactory bulb BrdU labeling after progressively longer
survival durations (BrdU pulse-chase) and by using stereotactic SVZ
retroviral reporter injections to track precursor migration. These
experiments demonstrated that SVZ neuroblasts are not impeded from
migrating to the olfactory bulb. Thus, our findings indicate that
pilocarpine-induced SE markedly expands the neuroblast population in
the SVZ and RMS.
Persistent germinative zones in the adult rodent brain appear to
respond to injury in a remarkably similar manner regardless of whether
damage is induced by seizures or other acute insults. We found that
increased SVZ neurogenesis after SE occurs with a latent period and
overall time course that parallel the accelerated neurogenesis in the
adult rat dentate gyrus reported in several different epilepsy models
(Parent et al., 1997; Gray and Sundstrom, 1998). Cell proliferation and
neurogenesis in the adult rodent dentate gyrus also increase after
mechanical and ischemic injury (Gould and Tanapat, 1997; Liu et al.,
1998). Likewise, rostral SVZ cell proliferation is stimulated by a
variety of different forebrain insults, including aspiration or
transection lesions (Willis et al., 1976; Szele and Chesselet, 1996;
Weinstein et al., 1996), inflammation (Calzà et al., 1998), and
chemical demyelination (Nait-Oumesmar et al., 1999). In the latter
injury model the progeny of SVZ precursors appeared to differentiate
into glial cells, whereas the daughter cell fates and final
destinations in the other investigations were not characterized. To our
knowledge, the only other report of increased SVZ neurogenesis induced
by injury is the recent study by Fallon and colleagues (2000). These authors showed in the adult rat 6-hydroxydopamine model of Parkinson's disease that the combination of dopaminergic neuron lesions in the
substantia nigra and transforming growth factor- infusion into the
ipsilateral striatum increased forebrain SVZ cell proliferation and
induced directed neuroblast migration and striatal neurogenesis. Taken
together with our findings of seizure-induced increases in SVZ
neuroblast numbers, these results suggest that persistent germinative
zones in the adult mammalian brain offer a potential source for cell
replacement after injury. Moreover, potentially similar cues induced by
different types of injury appear to expand the endogenous neural
precursor population, including those cells with the potential to form
new neurons.
The signals that lead to increased cell proliferation and neurogenesis
after seizures or other causes of brain injury are poorly understood.
The idea that cell death may be necessary to induce new cell production
is supported by studies that have found increased neurogenesis after
targeted apoptosis in adult mouse neocortex and adult songbird brain
(Magavi et al., 2000; Scharff et al., 2000). This same mechanism also
may operate normally in the adult rodent SVZ-olfactory bulb pathway to
induce cell replacement (Biebl et al., 2000). Because damage occurs to
the main and accessory olfactory bulbs in the pilocarpine
epilepsy model (Turski et al., 1983), the expansion of SVZ neuroblasts
induced by seizures could be a response to this damage. Our data
suggest that the majority of SVZ neural precursors produced after
pilocarpine-induced SE remains in the SVZ-olfactory bulb pathway and
therefore would be capable of potentially replacing lost neurons.
However, it is not yet known whether the olfactory bulb neurons
generated in the epileptic brain survive and integrate functionally
into existing networks. Direct electrical activation during seizures or
more physiological activity, such as long-term potentiation, also has
been proposed to accelerate cell proliferation and neurogenesis in the
adult rodent brain (Parent et al., 1997; Derrick et al., 2000). Such
stimuli have not been dissociated unequivocally from injury, however,
given the finding that cell death can occur even during brief
seizure-like episodes (Bengzon et al., 1997).
A number of specific local molecular cues or cell-autonomous factors
are candidates for mediating seizure-induced increases in proliferating
neuroblasts. Seizure activity is known to increase the expression of
growth or neurotrophic factors that are mitogenic for adult SVZ stem
cells or that can influence their differentiation or survival. These
include bFGF (Riva et al., 1992; Humpel et al., 1993; Gall et al.,
1994), EGF-like molecules (Opanashuk et al., 1999), IGF-1 (Young and
Dragunow, 1995), and BDNF (Ernfors et al., 1991; Isackson et al., 1991;
Dugich-Djordjevic et al., 1992). Seizures also induce a marked
astrocyte proliferation (Figs. 3H, 7D), and there
is evidence that contact-mediated interaction with astrocytes can
stimulate adult rodent SVZ neural precursor proliferation in
vitro (Lim and Alvarez-Buylla, 1999). Other molecules involved in
intercellular contacts, including specific integrins, ephrins, and Eph
receptor tyrosine kinases, also may influence SVZ precursor migration
and proliferation (Jacques et al., 1998; Conover et al., 2000).
Finally, certain bone morphogenetic protein (BMP) family members appear
to act in a cell-autonomous manner to block adult rodent SVZ
neurogenesis, whereas neurogenesis is promoted by the BMP antagonist
Noggin (Lim et al., 2000). The effects of seizures on these various
signaling molecules and their potential role in seizure-induced
neurogenesis remain to be determined.
In addition to increasing the numbers of neuronal precursors migrating
to the olfactory bulb, our results show that SE also modifies the
migration of SVZ neuroblasts. Using stereotactic retroviral reporter
injections, BrdU labeling, and immunostaining for immature neuronal
markers, we found that neuronal precursors arising from the SVZ exited
the SVZ or RMS prematurely and migrated to ectopic locations in the
striatum and cortex. Findings of ectopic neuroblasts also have been
reported in the adult rat dentate gyrus after pilocarpine-induced SE
(Parent et al., 1997; Scharfman et al., 2000). Several recent studies
suggest mechanisms by which SE might induce ectopic migration from the
SVZ/RMS and also stimulate neuronal precursor migration to the
olfactory bulb. Luskin and colleagues found that intraventricular
infusion of BDNF in adult rats increased neuronal precursor migration
to the olfactory bulb and also caused neuroblasts to appear in ectopic
sites such as the striatum, septum, and thalamus (Zigova et al., 1998;
Pencea et al., 2001). As mentioned above, SE increases the expression of a number of neurotrophic factors, including BDNF (Ernfors et al.,
1991; Isackson et al., 1991; Dugich-Djordjevic et al., 1992). Another
recent investigation showed that a glial-derived factor increased the
migration rate of neuroblasts from the neonatal rat SVZ-olfactory bulb
pathway grown as explant cultures (Mason et al., 2001). This finding
fits with the known or suspected importance of glia for various types
of neuronal migration (Hatten, 1999), including that of adult-generated
olfactory bulb neuronal precursors (Thomas et al., 1996).
Interestingly, we found that GFAP immunostaining markedly increased
after SE in regions adjacent to the SVZ and RMS (Figs. 2, 4). This
suggests that astrocytes proliferate or are activated in areas in which
they might exert effects on SVZ-olfactory bulb neuroblast migration,
including the potential induction of aberrant migration. A similar
finding of ectopic neuroblast migration from the RMS and along a
proliferating glial "scar" has been reported after transection
lesions of the migratory stream (Alonso et al., 1999), although in that
instance neuroblast migration to the olfactory bulb was blocked by the
injury. Importantly, the modulation of SVZ neuroblast proliferation and
migration by glia could explain how diverse brain insults exert similar
effects on these processes. Such a mechanism also would have
implications for how endogenous neuronal precursors respond to acute
brain injuries and how this response potentially may be manipulated for
therapeutic purposes.
The consequences of increased forebrain SVZ neurogenesis and altered
neuroblast migration in the epileptic brain are unknown. Recent studies
of seizure-induced dentate gyrus neurogenesis provide evidence that the
accelerated birth of neurons after injury may have maladaptive
consequences. For example, presumptive newly differentiating dentate
granule cell precursors in the epileptic hippocampus have been found to
participate in aberrant axonal remodeling (Parent et al., 1997),
maintain morphological features of immaturity such as basal dendrites
(Spigelman et al., 1998; Buckmaster and Dudek, 1999; Ribak et al.,
2000), and appear in abnormal locations such as the dentate hilus
(Parent et al., 1997; Scharfman et al., 2000; Dashtipour et al., 2001).
Hilar ectopic dentate granule-like cells induced by seizures have been
shown to exhibit abnormal, pro-epileptogenic burst firing in
hippocampal slice preparations (Scharfman et al., 2000). Whether
seizure-induced SVZ neuroblast expansion and ectopic migration result
in similar abnormalities of network integration and excitability
remains speculative. Alternatively, the common thread of increased
neurogenesis from adult forebrain neural precursors after various forms
of injury implies that the mature brain maintains a potential for self-repair (Lowenstein and Parent, 1999). We found that prolonged seizures induced migration of a subset of neuronal precursors from the
normal pathway toward forebrain regions that are not their typical
targets. Although this aberrant neuroblast migration could represent a
low-level reparative phenomenon after injury, very few putative newborn
neurons appeared to survive at least 5 weeks after SE. This suggests
that the local cues necessary for their appropriate differentiation and
survival may not be present, even after injury. This idea is supported
by previous work indicating that only specific forms of injury allow
transplanted or endogenous neuronal precursors to survive and integrate
into the adult rodent neocortex (Snyder et al., 1997; Magavi et al., 2000). Further study of seizure-induced SVZ neurogenesis is necessary to determine whether new neurons generated after injury survive, integrate, and replace lost cells in the mature forebrain. Knowledge of
the mechanisms that regulate endogenous neural precursor cells in
normal and disease states may lead to strategies for their use in
treating brain injury or degeneration.
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FOOTNOTES |
Received May 23, 2001; revised Jan. 29, 2002; accepted Feb. 1, 2002.
This project was sponsored by National Institutes of Health (NIH) Grant
NS02006 to J.M.P. and NIH Grants NS39950 and NS35628 and the March of
Dimes Birth Defects Foundation to D.H.L. We thank Susan Hockfield,
Virginia Lee, Christopher Walsh, and Genevieve Rougon for providing
antibodies and Theo Palmer, Fred Gage, and Richard Mulligan for
providing retroviral reagents.
Correspondence should be addressed to Jack M. Parent, Department of
Neurology, University of Michigan Medical Center, Neuroscience Laboratory Building, 1103 East Huron Street, Ann Arbor, MI 48104-1687. E-mail: parent{at}umich.edu.
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ABSTRACT
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