Decreased Neuronal Differentiation of Newly Generated Cells
Underlies Reduced Hippocampal Neurogenesis in Chronic Temporal
Lobe Epilepsy
Bharathi Hattiangady1,2 and Ashok K. Shetty1,2*
ABSTRACT: Hippocampal neurogenesis declines substantially in
chronic temporal lobe epilepsy (TLE). However, it is unclear whether
this decline is linked to altered production of new cells and/or dimin-
ished survival and neuronal fate-choice decision of newly born cells.
We quantified different components of hippocampal neurogenesis in
rats exhibiting chronic TLE. Through intraperitoneal administration of
50-bromodeoxyuridine (BrdU) for 12 days, we measured numbers of
newly born cells in the subgranular zone-granule cell layer (SGZ-GCL)
at 24 h and 2.5 months post-BrdU administration. Furthermore, the dif-
ferentiation of newly added cells into neurons and glia was quantified
via dual immunofluorescence for BrdU and various markers of neurons
and glia. Addition of new cells to the SGZ-GCL over 12 days was com-
parable between the chronically epileptic hippocampus and the age-
matched intact hippocampus. Furthermore, comparison of BrdU1 cells
measured at 24 h and 2.5 months post-BrdU administration revealed
similar survival of newly born cells between the two groups. However,
only 4–5% of newly born cells (i.e., BrdU1 cells) differentiated into
neurons in the chronically epileptic hippocampus, in comparison to 73–
80% of such cells exhibiting neuronal differentiation in the intact hippo-
campus. Moreover, differentiation of newly born cells into S-100b1
astrocytes or NG21 oligodendrocyte progenitors increased to
79% in
the chronically epileptic hippocampus from
25% observed in the
intact hippocampus. Interestingly, the extent of proliferation of astro-
cytes and microglia (identified through Ki-67 and S-100b and Ki-67 and
OX-42 dual immunofluorescence) in the SGZ-GCL was similar between
the chronically epileptic hippocampus and the age-matched intact hip-
pocampus, implying that the proliferation of neural stem/progenitor
cells in the SGZ-GCL of the chronically epileptic hippocampus was not
obscured by an increased division of glia. Thus, severely diminished DG
neurogenesis in chronic TLE is not associated with either decreased pro-
duction of new cells or reduced survival of newly born cells in the SGZ-
GCL. Rather, it is linked to a dramatic decline in the neuronal fate-
choice decision of newly generated cells. Overall, the differentiation of
newly born cells turns mainly into glia with chronic TLE from pre-
dominantly neuronal differentiation seen in control conditions. VC 2009
Wiley-Liss, Inc.
KEY WORDS: adult neurogenesis; dentate neurogenesis; depression;
granule cells; learning and memory; neural stem cells; spontaneous
seizures; stem cell proliferation; stem cell
differentiation; TLE
INTRODUCTION
Over 50 million people suffer from epilepsy in the
world and
40% of patients exhibiting epilepsy have
chronic TLE. A progressive expansion of complex par-
tial seizures arising from the limbic system regions
such as the hippocampus is the characteristic feature
of TLE (French et al., 1993; Engel et al., 2003). Fur-
thermore, most TLE patients also display learning and
memory impairments and depression (Devinsky,
2004; Helmstaedter et al., 2004). While changes due
to TLE are apparent in multiple brain regions, the
most conspicuous changes appear to be in the hippo-
campus based on the examination of brain tissues
from TLE patients (Sutula et al., 1989; French et al.,
1993). Animal prototypes of TLE also exhibit neuro-
degeneration in the hippocampus as well as several
other brain regions but the extent of neurodegenera-
tion varies in different models (Dalby and Mody,
2001; Buckmaster et al., 2002; Brandt et al., 2004;
Rao et al., 2006a; Curia et al., 2008). In the hippo-
campus, significant loss of neurons is seen in the CA1
and CA3 pyramidal cell layer and the dentate hilus
(Rao et al., 2006a). Furthermore, while there is no
consensus regarding the extent of loss of hippocampal
g-amino butyric acid positive (GABA-ergic) interneur-
ons in TLE, some prototypes of TLE exhibit consider-
able decline in their numbers (Sloviter, 1987; Franck
et al., 1988; Shetty and Turner, 2000, 2001; but see
Sloviter et al., 2003). Moreover, though controversial,
a reduced functional inhibition in the hippocampus is
another feature observed in some models of TLE,
which is likely due to reduced afferent excitatory input
onto interneurons (Cornish and Wheal, 1989; Dudek
and Sutula, 2007; however see, Bernard et al., 1998).
The above changes are associated with other morpho-
logical alterations that are proposed to be epilepto-
genic, which include the abnormal sprouting and
synaptic re-organization of dentate granule cell, ento-
rhinal and CA3 axons (Tauck and Nadler, 1985;
1Department of Surgery (Neurosurgery), Duke University Medical Cen-
ter, Durham, North Carolina 27710; 2Medical Research and Surgery
Services, Veterans Affairs Medical Center, Durham, North Carolina
27705
Grant sponsor: National Institute of Neurological Disorders and Stroke;
Grant numbers: NS054780, NS043507 Grant sponsor: Department of
Veterans Affairs (VA Merit Review Award to A.K.S.).
*Correspondence to: Ashok K. Shetty, M.Sc., Ph.D., Professor, Division of
Neurosurgery, Box 3807, Duke University Medical Center, Durham, NC
27710, USA. E-mail: ashok.shetty@duke.edu
Accepted for publication 3 February 2009
DOI 10.1002/hipo.20594
Published online in Wiley InterScience (www.interscience.wiley.com).
HIPPOCAMPUS 00:000–000 (2009)
VC 2009 WILEY-LISS, INC.

Sutula et al., 1989; Shetty, 2002; Shetty et al., 2003, 2005;
Siddiqui and Joseph, 2005; Wozny et al., 2005; but see, Longo
and Mello, 1998; Williams et al., 2002; Sloviter et al., 2006).
Recent studies in animal models have suggested that altered
dentate gyrus (DG) neurogenesis is an additional pathophysiol-
ogy likely contributing to some of the deficits such as learning
and memory impairments and depression observed in TLE.
Interestingly, changes in DG neurogenesis are distinct between
the early and later phases of the disease in animal models of
TLE. The early phase after the initial precipitating injury (IPI)
such as status epilepticus (SE) or hippocampal injury is charac-
terized by an increased DG neurogenesis and abnormal migra-
tion of a substantial fraction of newly generated granule cells
into the dentate hilus (Parent et al., 1997, 2006; Gray and
Sundstrom, 1998; Scharfman et al., 2000, 2002, 2003; Gong
et al., 2007; Scharfman and Gray, 2007; Kuruba et al., 2009).
In contrast, the chronic phase of TLE exhibits substantially
declined DG neurogenesis (Hattiangady et al., 2004; Hattian-
gady and Shetty, 2008a), and is associated with spontaneous
recurrent motor seizures (SRMS), learning and memory impair-
ments and depression (Letty et al., 1995; Schwarcz and Witter,
2002; Rao et al., 2006a, 2007). Because of the perceived func-
tions of DG neurogenesis concerning learning, memory and
mood (Shors et al., 2001; van Praag et al., 2002; Drapeau
et al., 2003; Santarelli et al., 2003; Aimone et al., 2006; Sahay
and Hen, 2007; Dupret et al., 2008; Imayoshi et al., 2008), it
is plausible that decreased DG neurogenesis during chronic epi-
lepsy contributes to impairments in these functions. Therefore,
comprehending the mechanisms underlying decreased DG neu-
rogenesis during chronic epilepsy will be important for devel-
oping strategies that improve DG neurogenesis in chronic
TLE.
It is currently unclear whether the decreased DG neurogene-
sis in chronic TLE is linked to altered production of new cells
and/or diminished survival and neuronal fate-choice decision of
newly born cells. To address these issues, we rigorously quanti-
fied different components of DG neurogenesis in male Fischer
344 (F344) rats exhibiting chronic TLE at 6-months after
kainic acid (KA) induced SE. We chose 6-months post-SE
time-point for analyses of neurogenesis in this study because
our pilot studies in chronically epileptic animals have indicated
that the frequency of SRMS remains stable after this time-point
for at least until one-year post-SE. To determine the produc-
tion and survival of newly born cells in the subgranular zone-
granule cell layer (SGZ-GCL) of the DG, we measured the
numbers of newly born cells in these regions at 24 h and 2.5
months after daily administration of 50-bromodeoxyuridine
(BrdU) for 12 days in rats exhibiting chronic TLE. To ascertain
the neuronal fate-choice decision of newly born cells, we per-
formed BrdU & doublecortin (DCX), BrdU & neuron specific
nuclear antigen (NeuN), and BrdU & b-III tubulin (TuJ-1)
dual immunofluorescence and confocal microscopic analyses.
To elucidate the identity of other newly born cells in the SGZ-
GCL, we also performed phenotypic analyses of BrdU1 cells
with markers of glia and immature neuronal markers. These
comprised analyses of cells positive for mature astrocytes
expressing S-100b, oligodendrocyte progenitors positive for
NG2, and immature neurons (with doublecortin and TuJ-1
antibodies). Additionally, we examined the proliferation of glial
cells such as astrocytes and microglia in the SGZ-GCL via dual
immunofluorescence and confocal microscopic analyses of cells
positive for Ki-67 (an endogenous marker of proliferating cells)
and S-100b (a marker of astrocytes), and Ki-67 and OX-42
(a marker of both resting and activated microglia).
MATERIALS AND METHODS
Animals and Kainic Acid Induced
Status Epilepticus
Young adult (5-months old) F344 rats purchased from Har-
lan Sprague-Dawley (Indianapolis, IN) were used in this study.
All experiments were carried out in accordance with the NIH
guide for the care and use of laboratory animals (NIH Publica-
tions No. 80–23), and all protocols employed in this study
were approved by the Duke University Institutional Animal
Care and Use Committee and animal studies subcommittee of
the Durham Veterans Affairs Medical Center. The methodology
for induction of SE and chronic epilepsy in F344 rats was
adapted from the procedure developed earlier by Hellier et al.
(1998) for Sprague-Dawley rats, and the types of seizures
emerging after KA administration were scored as per the modi-
fied Racine’s scale (Hellier et al., 1998). In all rats, SE was
induced through graded intraperitoneal injections of KA (3.0
mg/Kg b.w./h). Because majority of rats (>90%) exhibited
greater than 10 stages IV-V seizures during the first hour after
the 3rd KA injection, the 4th KA injection was reduced to 1.5
mg/Kg b.w. Thus, each rat received a total KA dose of 10.5
mg/Kg b.w., which is consistent with our previous study (Rao
et al., 2006a). The motor seizures were characterized by unilat-
eral forelimb clonus with lordotic posture (stage III seizures),
bilateral forelimb clonus and rearing (stage IV seizures) and
bilateral forelimb clonus with rearing and falling (stage V seiz-
ures). Only the animals receiving a total KA dose of 10.5 mg/
Kg b.w. and exhibiting >10 stages IV-V seizures during the
3-h observation after the onset of the SE were included in this
study. Stages III-V seizures subsided gradually thereafter and
were not apparent at 6 h after SE. Rats were given moistened
rat chow and subcutaneous injections of lactated Ringer’s solu-
tion (10 ml/day) for 4 days after SE. Animals were housed
individually in an environmentally controlled room (
238C)
thereafter with a 12:12-h light-dark cycle, and were given food
and water ad libitum.
Analyses of Chronic Epilepsy After KA-Induced
Status Epilepticus
From the beginning of 3rd month after SE, the frequency of
SRMS in all KA-treated rats were assessed for a total duration
of three months (i.e., during the 3rd, 4th, and 5th months after
2 HATTIANGADY AND SHETTY
Hippocampus