Functional integration of new hippocampal neurons following insults to the adult
brain is determined by characteristics of pathological environment
James C. Wooda,d,1, Johanna S. Jacksona,d,1, Katherine Jakubsa,d,2, Katie Z. Chapmana,d,
Christine T. Ekdahla,d,e, Zaal Kokaiac,d, Merab Kokaiab, Olle Lindvalla,d,⁎
aLaboratory of Neurogenesis and Cell Therapy, Lund University Hospital, SE-221 84 Lund, Sweden
bExperimental Epilepsy Group, Wallenberg Neuroscience Center, Lund University Hospital, SE-221 84 Lund, Sweden
cLaboratory of Neural Stem Cell Biology and Therapy, Lund University Hospital, SE-221 84 Lund, Sweden
dLund Stem Cell Center, Lund University Hospital, SE-221 84 Lund, Sweden
eDivision of Clinical Neurophysiology, Lund University Hospital, SE-221 84 Lund, Sweden
a b s t r a c ta r t i c l e i n f o
Received 27 October 2010
Revised 14 February 2011
Accepted 24 March 2011
Available online 1 April 2011
We have previously shown that following severe brain insults, chronic inflammation induced by
lipopolysaccharide (LPS) injection, and status epilepticus, new dentate granule cells exhibit changes of
excitatory and inhibitory synaptic drive indicating that they may mitigate the abnormal brain function. Major
inflammatory changes in the environment encountering the new neurons were a common feature of these
insults. Here, we have asked how the morphology and electrophysiology of new neurons are affected by a
comparably mild pathology: repetitive seizures causing hyperexcitability but not inflammation. Rats were
subjected to rapid kindling, i.e., 40 rapidly recurring, electrically-induced seizures, and subsequently exposed
to stimulus-evoked seizures twice weekly. New granule cells were labeled 1 week after the initial insult with a
retroviral vector encoding green fluorescent protein. After 6–8 weeks, new neurons were analyzed using
confocal microscopy and whole-cell patch-clamp recordings. The new neurons exposed to the pathological
environment exhibited only subtle changes in their location, orientation, dendritic arborizations, and spine
morphology. In contrast to the more severe insults, the new neurons exposed to rapid kindling and stimulus-
evoked seizures exhibited enhanced afferent excitatory synaptic drive which could suggest that the cells that
had developed in this environment contributed to hyperexcitability. However, the new neurons showed
concomitant reduction of intrinsic excitability which may counteract the propagation of this excitability to the
target cells. This study provides further evidence that following insults to the adult brain, the pattern of
synaptic alterations at afferent inputs to newly generated neurons is dependent on the characteristics of the
© 2011 Elsevier Inc. All rights reserved.
Neural stem/progenitor cells in the adult dentate subgranular zone
(SGZ) continuously generate new granule cells (Zhao et al., 2008)
which develop functional inputs from the entorhinal cortex (van
Praag et al., 2002) and outputs to the hilus and CA3 region (Toni et al.,
2008). Synaptic integration of adult-born hippocampal neurons in the
intact brain closely resembles that during development (Laplagne
et al., 2006) and is conserved throughout life and in old age
(Morgenstern et al., 2008). Pathological changes in the stem cell
niche and environment encountered by the new neurons influence
adult neurogenesis. For example, seizures and cerebral ischemia
enhance hippocampal progenitor proliferation and neurogenesis
(Bengzon et al., 1997; Parent et al., 1997; Liu et al., 1998) and
epileptic insults can lead to aberrant migration of new granule cells
(Parent et al., 1997; Parent, 2005). Inflammation is detrimental for the
survival of new neurons early after they have been born (Ekdahl et al.,
2003; Monje et al., 2003) and pathologies such as Alzheimer's can
impair neurogenesis and maturation of new neurons in mice (Biscaro
et al., 2009).
How new neurons integrate into existing neural circuitries will
determine their action in the diseased brain. Recent experimental
evidence indicates that pathological environments influence the
morphological and functional integration of adult-born hippocampal
neurons. Following kainate-induced status epilepticus (SE) in rats,
new granule cells extend abnormal basal dendrites into the hilus and
have more mushroom spines on their apical dendrites (Jessberger
Experimental Neurology 229 (2011) 484–493
⁎ Corresponding author at: Laboratory of Neurogenesis and Cell Therapy, Wallenberg
Neuroscience Center, Lund University Hospital, SE-221 84 Lund, Sweden. Fax: +46 46
E-mail address: firstname.lastname@example.org (O. Lindvall).
1These authors contributed equally to this work.
2Present address: National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, MD 20892, USA.
0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yexnr
et al., 2007). We have demonstrated that dentate granule cells born
after electrically-induced SE (eSE) in rats, i.e., into an environment
characterized by neuronal death, spontaneous, recurrent seizures and
inflammation, exhibit more inhibitory and less excitatory synaptic
drive (alterations in frequency and/or amplitude of miniature
postsynaptic currents) compared to new neurons from control
animals (Jakubs et al., 2006). When exposed to lipopolysaccharide
(LPS)-induced inflammation without seizure activity, new neurons
respond with enhanced excitatory and inhibitory drive (Jakubs et al.,
2008). Chronic inflammation also gives rise to larger clusters of the
postsynaptic GABA receptor scaffolding protein gephyrin on dendrites
of new cells (Jakubs et al., 2008). Thus, in pathological environments,
adult-born neurons exhibit a high degree of plasticity at their afferent
synapses, which may act to mitigate abnormal brain function.
Integration of adult-born neurons has so far been analyzed in
pathological environments with pronounced inflammation. How
integration is influenced by less severe insults is unknown. The
objective of the present study was to determine the morphological
and electrophysiological properties of new neurons which developed
in a pathological environment with repeated seizures and minimal
inflammation. Rats were subjected to an epileptic insult and
subsequently exposed to stimulus-evoked seizures twice weekly.
New granule cells were labeled 1 week after the initial insult using a
retroviral (RV) vector encoding green fluorescent protein (GFP). After
6–8 weeks, new neurons were studied using confocal microscopy and
whole-cell patch-clamp recordings. We show that these cells exhibit
only minor differences in morphology. Electrophysiological record-
ings indicate the presence of enhanced afferent excitatory input on
the new cells which may be counteracted by reduced membrane
excitability. Taken together with our previous studies, these findings
indicate that new neurons have mechanisms to counteract or adapt to
pathologies at their afferent synaptic inputs, and that the pattern of
changes is dependent on the characteristics of the environment.
Materials and methods
Animal groups and rapid kindling
All experimental procedures were approved by the Malmö-Lund
Ethical Committee. One hundred and thirty-two male Sprague–Dawley
rats were used, weighing 200–250 g at the beginning of the experi-
ments. Animals were anesthetized with isofluorane (1.5–2%) and
implanted unilaterally with a bipolar stainless steel stimulating/
recording electrode (Plastics One, Roanoke, VA) in the ventral
hippocampal CA1-CA3 region (coordinates: 4.8 mm caudal and
5.2 mm lateral to bregma, 6.3 mm ventral from dura, toothbar at
−3.3 mm) (Paxinos and Watson, 1997). Another electrode was
positioned between the skull and the adjacent muscle to serve as
protocol (40 stimulations, 1 ms square-wave pulses of 400 μA intensity
with100 Hzintratrainfrequencyfor10 severy5 min).Forcomparisons
of the inflammatory response, six rats were subjected to eSE as
described previously (Jakubs et al., 2006). The electroencephalogram
(EEG) was recorded continuously after stimulation until cessation of
focal epileptiform activity (afterdischarge, AD) using Chart 3.6.3
(PowerLab7MacLab, AD Systems). Animals were monitored during
this time and behavioral seizures were characterized according to the
Racine scale (Racine, 1972). Only animals exhibiting grade 2 seizures
and above, and with corresponding ADs were included. Controls were
electrode-implanted but not exposed to electrical stimulation.
Seven days after rapid kindling or corresponding time point in
controls, rats were transcardially perfused with saline and whole
hippocampus contralateral to the electrode was rapidly removed and
frozenon dry ice. Sampleswere homogenized on ice in buffer (pH 7.6)
containingin (mM):50.0Tris–HCl, 150NaCl, 5.0CaCl2, 0.02% NaN3, 1%
Triton X-100, and then centrifuged at 17,000 times gravity for 30 min
at +4 °C. Protein concentration was determined in supernatants by
BCA protein assay (Pierce, USA) and all samples equilibrated to a
concentration of 2 mg/ml total protein. IL-1β, IL-6, TNF-α, IL-10, and
IL-4 concentrations were determined by ELISA (Duoset; R & D
Systems, USA) according to manufacturer's instructions. Results are
presented as means±SEM, and statistical comparisons were per-
formed using Student's unpaired t-test. Level of significance was
Labeling of new neurons
Seven days after the rapid kindling procedure, rats were
anesthetized with isofluorane and injected with a retrovirus contain-
ing the GFP gene (RV-GFP) under the CAG promoter (1.0–1.1
transducing units/ml) (Zhao et al., 2006). Two 1.5 μl retroviral
injections were made in the dorsal hippocampus contralateral to the
electrode (coordinates: 3.6 mm caudal and 2.0 mm lateral to bregma,
and 2.8 mm dorsal to dura; 4.4 mm caudal and 3.0 mm lateral to
bregma, and 3.0 mm dorsal to dura; toothbar at −3.3 mm).
Extra stimulations and assessment of excitability
Starting 2 days after retrovirus injections, animals subjected to
rapid kindling were exposed to stimulus-evoked seizures twice
weekly for 6–8 weeks. Before and after stimulations, EEG was
recorded to determine baseline activity and to observe ADs. Re-
cordings continued until cessation of ADs. Stimulations were
delivered for 1 s at AD threshold, as determined by a 1 s 50 Hz
electrical current, starting at 10 μA and with 10 μA increments until an
AD was registered. At 5 weeks after retrovirus injections, EEG
recordings were made on 4 seizure-exposed and 4 non-stimulated
control animals for 1 h to assess the occurrence of interictal activity.
Mean AD duration (using Chart 3.6.3) and seizure grade were
determined for both rapid kindling and extra stimulations. Mean AD
threshold was assessed for the extra stimulations. Total AD duration,
and mean number and AD duration of partial (grades 1–2) and
generalized (grades 4–5) seizures were also calculated per animal.
Development of seizure threshold and seizure grade in response to
the consecutive extra stimulations was analyzed using linear
regression. Level of significance was pb0.05.
At 6–8 weeks after virus injection, animals received an overdose of
pentobarbital (250 mg/kg, i.p.) and were transcardially perfused with
100 ml saline and 250 ml 4% paraformaldehyde (PFA) in 0.1 M
phosphate-buffered saline (PBS), pH 7.4. Brains were cryoprotected
in 20% sucrose in 0.1 M PBS overnight, cut in 30 μm coronal sections
and stored in cryoprotective solution. For characterization of the
environment, animals were also perfused and their brains sectioned
using the same protocol 1 week after rapid kindling or corresponding
time point in controls. For analysis of gephyrin distribution, rats were
anesthetized and decapitated, brains weredissected and placed in ice-
cold artificial cerebrospinal fluid (aCSF, described below), cut in
300 μm transverse sections and placed in gassed aCSF for 20 min and
then in PFA for 10 min (Jakubs et al., 2008). Sections were
cryoprotected in 20% sucrose in 0.1 M PBS overnight, cut in 12 μm
sections and stored at −20 °C for at least 1 h.
For immunohistochemistry, the following primary antibodies
were used: rabbit anti-Iba1 (1:1000, Wako Chemicals), mouse anti-
ED1 (1:200, Serotec), rabbit anti-GFP (1:10000, Abcam), goat anti-IL-
1β (1:1000, R&D Systems), and mouse anti-gephyrin (1:10000,
Synaptic Systems). Free-floating sections were incubated with the
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
appropriate primary antibody overnight at +4 °C and secondary
antibody for 1 to 2 h at room temperature. Secondary antibodies were
Cy3-conjugated donkey anti-rabbit (1:200, Jackson ImmunoRe-
search), biotinylated horse anti-mouse (1:200, Vector Laboratories),
biotinylated horse anti-goat (1:200, Vector Laboratories), and FITC-
conjugated donkey anti-rabbit (1:200, Jackson ImmunoResearch).
Biotinylated antibodies were visualized using Streptavidin-conjugat-
ed Alexa Fluor-488 (1:200, Invitrogen). Sections were mounted on
gelatin-coated microscope slides and coverslipped. For Fluoro-Jade
staining, mounted sections were pre-treated with 0.06% potassium
permanganate before being agitated for 30 min in 0.001% Fluoro-Jade
(Histochem) in 0.01% acetic acid, immersed in xylene, and cover-
slipped with Pertex mounting medium (Histolab).
Cell counting and morphological analysis were performed ipsilat-
erally to the virus injections in 4 to 6 hippocampal sections by an
observer blind to the treatment conditions as previously described
(Jakubs et al., 2008). The number of Iba1+, Iba+/ED1+, and Fluoro-
Jade+ cells were counted with an Olympus BX61 epifluorescence
microscope in the granule cell layer (GCL) and two cell diameters
below in the SGZ. Iba1/ED1double labelingwasconfirmedby confocal
microscopy. The morphological phenotype of Iba1+ cells in the SGZ
and GCL was classified into four different subtypes, as previously
described (Lehrmann et al., 1997), in 3–4 hippocampal sections. The
relative occurrence of each subtype was expressed as the mean
percentage of the total number of Iba1+ microglia per section.
with IL-1β. GFP+ cells were counted in the GCL, SGZ, dentate hilus,
and molecular layer (ML). For all GFP+ cells, axon exit point, dendrite
exit points, and total number of dendrites leaving the cell soma were
analyzed. Dendritic polarity was determined by classifying the angles
of the dendrites leaving the cell soma as 0–22°, 22.5–67°, or 67.5–90°,
where 90° was perpendicular to the GCL. Location of dendritic
branching was determined by assessing the cumulative number of
branching points of each dendrite from the cell soma in 15 μm
increments. To measure the number of branching points and total
dendrite length, a confocal stack was taken of the whole dendritic tree
of GFP+ cells in 225 μm thick hippocampal sections. Dendrite length
was measured using the NeuronJ plug-in of ImageJ (Meijering et al.,
Spine density (number of spines per micrometer) and morphology
(classified as thin, stubby, filopodia, or mushroom spines) (Zhao et al.,
2006), and gephyrin cluster density (clusters per micrometer) and
size (area in square micrometers) were analyzed by confocal laser
scanning microscopy (Bio-Rad MRC1021UV) using Kr-Ar 488 and
568 nm excitation filters with a 63× objective and 16× digital zoom.
Analysis was carried out on 12 regions-of-interest (ROI, each
221.4 μm2) per animal on proximal and distal dendrites in the inner
and outer ML, respectively. Cluster area was measured using ImageJ
software (Sheffield, 2007).
Results are presented as means±SEM, and analysis was per-
formed using Student's unpaired t-test or one-way ANOVA with
Bonferroni post-hoc test for multiple comparisons. Level of signifi-
cance was pb0.05.
Six to 8 weeks after virus injections, rats were anesthetized with
isofluorane and decapitated. Brains were placed in ice-cold, gassed
(95% O2, 5% CO2) modified-aCSF (pH 7.2–7.4, 295–300 mOsm),
containing (in mM): 225 sucrose, 2.5 KCl, 0.5 CaCl2, 7.0 MgCl2, 28.0
NaHCO3, 1.25 NaH2PO4, 7.0 glucose, 1.0 ascorbate, and 3.0 pyruvate.
Transverse dorsal hippocampal slices (225 μm), cut on a vibratome
(3000 Deluxe, Ted Pella Inc, CA), were placed in an incubation
chamber with gassed (95% O2, 5% CO2) aCSF (pH 7.2–7.4, 295–
300 mOsm) containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 2.5
CaCl2, 26.2 NaHCO3, 1.0 NaH2PO4, and 11.0 glucose, and were allowed
to rest for at least 1 h at room temperature before recordings.
Individual slices were placed in a submerged recording chamber
and perfused with gassed aCSF at +32–34 °C during recordings of
miniature excitatory postsynaptic currents (mEPSCs) to optimize
event frequency for analysis (Jakubs et al., 2006, 2008), or at room
temperature during recordings of miniature inhibitory postsynaptic
currents (mIPSCs) and measurements of intrinsic membrane proper-
ties. Cells for recording were visualized using an Olympus upright
identified under a 40× water immersion lens using fluorescence
microscopy. Infrared light with differential interference contrast was
used for visual approach and acquiring whole-cell recordings.
Recording pipettes with a final tip resistance of 2.5–5.5 MΩ were
filled with pipette solution (pH 7.2–7.4, 295–300 mOsm) containing
the following (in mM): 122.5 K-gluconate, 12.5 KCl, 10.0 KOH-HEPES,
0.2 KOH-EGTA, 2.0 MgATP, 0.3 Na3-GTP, and 8.0 NaCl for current-clamp
recordings of intrinsic properties; 135.0 CsCl, 10.0 CsOH, 0.2 CsOH-
EGTA, 2.0 Mg-ATP, 0.3 Na3-GTP, 8.0 NaCl and 5.0 lidocaine N-ethyl
bromide (QX-314) for voltage-clamp recordings of mIPSCs; or
117.5 Cs-gluconate, 17.5 CsCl, 8.0 NaCl, 10.0 CsOH-HEPES, 0.2
CsOH-EGTA, 2.0 Mg-ATP, 0.3 Na3-GTP, and 5.0 QX-314 for voltage-
clamp recordings of mEPSCs. Biocytin (0.5%, Sigma-Aldrich) was
freshly dissolved in the pipette solution before recordings for post-
hoc identification of recorded cells. Seal resistance was N1 GΩ. For
analysis of intrinsic membrane properties, resting membrane
potential was estimated in current-clamp mode immediately after
breaking the membrane and establishing whole-cell configuration.
For measuring current–voltage relationship, 500 ms hyperpolariz-
ing and depolarizing current pulses were delivered in 30 pA
increments through the whole-cell pipette. Rheobase was deter-
mined by injecting a 300 pA ramp over 1 s. Intrinsic properties
were measured in aCSF containing 50 μM D-AP5 and 5 μM NBQX
(both Tocris) to block NMDA and non-NMDA receptors, respec-
tively, and 100 μM picrotoxin (PTX) (Tocris) to block GABAA
receptor activation. mIPSCs were recorded in aCSF containing
50 μM D-AP5, 5 μM NBQX, and 1 μM TTX (Tocris) to block action
potentials. mEPSCs were recorded with 100 μM PTX and 1 μM TTX
in aCSF. To confirm that recorded cells expressed GFP, fluorescence
microscopy was used to detect GFP in the recording pipette, or
post-hoc immunohistochemical analysis of GFP colocalization with
biocytin was conducted.
Data were filtered at 2.9 kHz and sampled at 10 kHz with an EPC9
patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany).
Miniature postsynaptic currents were detected and analyzed using
MiniAnalysis software (Synaptosoft). Minimum amplitude for detec-
tion was set at 5 times root-mean-square noise level as determined by
the software. Alldetected events werevisually controlled. The 10–90%
rise time of mEPSCs and mIPSCs were analyzed using MiniAnalysis.
Analysis of intrinsic membrane properties was performed using one-
way ANOVA with Bonferroni post-hoc test for multiple comparisons.
Recording duration was 3 minutes and equal numbers of mEPSCs and
mIPSCs from each cell were analyzed to prevent any bias. Group
interevent intervals (IEIs), amplitudes, and 10–90% rise time were
compared using Kolmogorov–Smirnov's statistical test. Mean event
frequency was determined from an equal number of events from each
cell, and analyzed using Student's unpaired t-test. Level of significance
were preincubated for 1 h in 5% serum in 0.25% Triton X-100 in
potassium PBS, and then exposed to rabbit anti-GFP primary antibody
(1:10000, Abcam) overnight at room temperature. Immunoreactivity
was visualized using FITC-conjugated donkey anti-rabbit secondary
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
an Olympus BX61 epifluorescence microscope.
Characteristics of the pathological environment
Animals were subjected to rapid kindling (40 supra-threshold
stimulations over a period of 3 h and 15 min) followed by twiceweekly
to repeated seizures but only to mild, or no inflammation during their
maturation (Fig. 1A). This protocol was based on our previous data
showing that rapid kindling causes AD duration of similar length in the
stimulated and non-stimulated hippocampus (Elmér et al., 1998), and
on a pilot experiment which indicated that the number of activated
microglia (a measure of inflammation) in the dentate gyrus correlated
withthe number of extra stimulations and generalized seizures and the
total AD duration. Using this experimental paradigm, we could address
the role of seizures, without introducing major inflammatory changes,
on the integration of new neurons. Only animals which showed both
behavioral (grade 2 and above) and electroencephalographic seizure
activity (Fig. 1B) during rapid kindling and the extra stimulations were
included in the study (n=36). The rapid kindling paradigm produced
23.4±0.4 partial (grade 1–2) and 2.4±0.2 generalized (grades 4–5)
seizures per animal, the mean AD duration of partial and generalized
seizures was 28.1±3.2 s and 55.7±9.5 s, respectively. The total AD
duration per animal during the rapid kindling protocol was 18.5±
2 min. The extra stimulations gave rise to 17.5±1.5 partial and 11.8±
2.0 generalized seizures and a total AD duration of 9.9±0.9 min per
animal. The mean seizure grade progressively increased, and the
threshold required to produce an AD gradually decreased (Fig. 1C)
with increasing number of extra stimulations, providing evidence for
the development of hyperexcitability. However, we observed no
pathological interictal activity in the EEG of the seizure-exposed group
at 5 weeks after retrovirus injection or during the extra stimulations, or
in the electrode-implanted, non-stimulated group (n=4 rats/group).
These results indicate that the seizure paradigm used here caused
development of hyperexcitability in response to stimulations. The
occurrences of generalized seizures, in combination with data from
previously published studies (Elmér et al., 1998), indicate that the
seizure activity spread to both brain hemispheres.
We next assessed in detail the magnitude of inflammation in the
seizure-exposed group,first by characterizing themicroglialresponse.
At 1 week after rapid kindling, at the time point when the new cells
were born (labeled with RV-GFP), there was a modest, non-significant
change in the number of activated microglia (Iba1+/ED1+ cells) in
the SGZ/GCL (n=4 rats/group×4–6 sections/rat, p=0.06, Fig. 1D–F).
Control animals exhibited 13.8±3.5 Iba1+/ED1+ cells/section
compared with 34.2±8.3 cells/section 1 week after rapid kindling
(148% increase), and 68.1±6.5 cells/section 1 week after eSE (395%
increase, n=6 rats×4–6 sections/rat), prepared as in our previous
study (Jakubs et al., 2006). We then explored whether rapid kindling
gave rise to a change in the morphological phenotype of the microglia
population. The Iba1+ cells in SGZ/GCL were classified into ramified,
intermediate, amoeboid, or round phenotypes using the morpholog-
ical criteria described by Lehrmann et al. (1997). The severity of a
pathological insult determines the degree of microglial activation,
round phenotype signifying the most activated state. We observed no
change of microglia phenotype at 1 week following rapid kindling
(n=4 rats/group×3–4 sections/rat, Fig. 1G), arguing against micro-
glia activation. In contrast, when we assessed the morphology of
microglia in sections from animals at 1 week after eSE, there was a
significant change to a more activated phenotype as compared to
control animals, i.e., a decrease of ramified and an increase of
intermediate microglia in eSE animals (n=6 rats×3–4 sections/rat,
see Supplementary Figure 1). Taken together, these results provide
evidence that rapid kindling causes a mild pathology without the
pronounced microglial activation observed after eSE.
We also assessed the magnitude of hippocampal inflammation at
1 week after rapid kindling by measuring the levels of inflammatory
cytokines using ELISA (Fig. 1A). Consistent with our findings that rapid
kindling did not cause microglial activation, no significant changes in the
levels of IL-1β (control 1188±53 pg/mg; seizures 1254±67 pg/mg),
TNF-α (control 146.2±6.5 pg/mg; seizures 156.2±8.7 pg/mg), IL-4
(control 173.7±8.3 pg/mg; seizures 190.2±9.0 pg/mg), IL-6 (control
1928±52 pg/mg;seizures2002±87 pg/mg),andIL-10(control689.6±
28.5 pg/mg; seizures 719.6±59.3 pg/mg) were detected in seizure-
detect any seizure-induced, increased expression of the pro-inflamma-
tory cytokine IL-1β in Iba1+ microglia in SGZ/GCL using immunohisto-
chemistry (data not shown).
the dentate gyrus of the seizure-exposed animals 1 week after rapid
kindling stimulations (n=4 rats/group×4–6 sections/rat, Fig. 1H, I).
kindling and extra stimulations. Seven weeks after rapid kindling, the
number of Iba1+/ED1+ cells did not differ between seizure-exposed
and control animals (n=4 rats/group×4–6 sections/rat, Fig. 1F). Taken
rapid kindling developed in an environment characterized by repeated
seizures and gradual development of hyperexcitability but without
significant neuronal death or inflammation.
Morphological integration of the new neurons in the pathological
Six to 8 weeks after virus injection, stable GFP expression was
observed in a substantial number of new dentate granule cells in non-
stimulated, electrode-implanted controls and seizure-exposed ani-
mals (Fig. 2A, B). In accordance with previous studies reporting that
seizures enhance neural/stem progenitor cell proliferation (Bengzon
et al., 1997; Parent et al., 1997; Scott et al., 1998), there were
noticeably more new GFP+ cells in seizure-exposed animals
compared to control animals. The distribution of the new cells within
the GCL did not differ between seizure-exposed and non-stimulated
animals, the majority being located within the inner GCL (n=8
control rats, 5 seizure-exposed rats×4–6 sections/rat, Fig. 2C). Very
few aberrant neurons were observed in the hilus in both groups. The
total number of dendrites per new cell (control 1.3±0.07; seizures
1.1±0.08), and the polarity of dendrites leaving the cell soma did not
differ between the groups, most of the dendrites leaving at a 67.5–90°
angle in relation to the GCL (control 72.0±7.1%; seizures 56.7±3.7%)
(n=7 control rats, 6 seizure-exposed rats×4–6 sections/rat). Den-
drite development was similar in seizure-exposed and control new
cells, as no differences were detected in, (i) dendrite length (control
1.87±0.15 mm; seizures 1.95±0.16 mm; pN0.05), (ii) dendrite exit
point from cell soma or number of recurrent basal dendrites (Fig. 2D),
or (iii) number of branching points (control 8.3±1.15 points/cell;
seizures 6.3±0.49 points/cell; pN0.05) or location of dendrite
branches (Fig. 2E). Axons primarily originated from the basal (control
80.0%; seizures 79.2%) and medial soma (control 16.0%; seizures
18.8%) and rarely from the apical side (control 4.0%; seizures 2.0%).
These results indicate that the pathological environment caused by
rapid kindling and repeated extra stimulations did not interfere with
the gross morphological appearance of the new neurons.
We next investigated whether the pathological environment
affected the morphological development of the synaptic inputs on the
new granule cells. Using confocal microscopy and ImageJ we did not
detect any difference in the total spine density in the inner or outer ML
between the seizure-exposed and non-stimulated group (n=8 rats/
group×12 ROI/rat, Fig. 2F).We next examined the individual spine
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
subtypes based on their morphology. Filopodia and stubby spines are
considered immature spine phenotypes whereas thin and mushroom
spines are regarded as more mature (Nimchinsky et al., 2002). There
was no difference in the density of thin and mushroom spines or in
had significantly more stubby spines than the non-stimulated group
(Fig. 2F), indicating that this pathological environment induced subtle
alterations of excitatory synapses. We have previously reported that
chronic inflammation causes increased size of gephyrin clusters on the
scaffolding protein associated with clustering of glycine and GABAA
receptors at inhibitory synapses (Fritschy et al., 2008). Here we found
that the density of gephyrin clusters on the dendrites of the new cells
μm and 0.20±0.02 cluster/μm, respectively, pN0.05, n=5 rats/
group×12 ROI/rat) (Fig. 2G, H). Furthermore, the gephyrin cluster
0.19±0.01 μm2, pN0.05).
Fig. 1. Pathological environment is characterized by repeated seizures and no significant inflammation. A, Schematic representation of experimental timeline. B, EEG recordings from
electrode-implanted animals showing baseline activity (top) before stimulations, high-frequency ictal activity following stimulation during the rapid kindling protocol(middle), and
high-frequency ictal activity following an extra stimulation (bottom). Scale bar is 2 s, 1 mV. C1, Increased seizure grade and (C2) decreased seizure threshold in response to the extra
stimulations (Means±SEM, linear regression). Iba+ (green), ED1+ (red), and Iba1+/ED1+ (yellow, arrowheads, inset) cells 1 week after rapid kindling in control (D) and seizure-
exposed (E) animals (h, hilus; GCL, granule cell layer). F, Minimal increase of activated microglia (Iba1+/ED1+ cells) 1 week after rapid kindling and no difference compared to
control at 7 weeks. G, No differences between seizure-exposed and control animals in morphological phenotype of Iba1+ microglia. Lack of Fluoro-Jade-stained degenerating
neurons in control animals 2 weeks after electrode implantation (H) and in seizure-exposed animals 1 week after rapid kindling (I). Scale bars=10 μm. Means±SEM.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
Functional integration of the new neurons in the pathological
Whole-cell patch-clamp recordings were performed from GFP+
cells (newcells, born at the time of RV-GFPinjection) andneighboring
GFP− cells (mature cells, most likely born before the onset of
RV-GFP injection. Mature, GFP− cells were selected based on their
position within the GCL and their morphology. Immunohistochemistry
performed after recordings revealed development of mature dendrites
on both GFP− and GFP+ neurons. We found that the intrinsic
membrane properties (resting membrane potential, input resistance,
series resistance, action potential threshold, and action potential half-
significantly different compared to new and mature cells in non-
stimulated, electrode-implanted controls (Table 1, Fig. 3A, B). These
properties were also similar to those characteristic of dentate granule
of temporal lobe epilepsy (Young et al., 2009). Interestingly, we
detected an increase in rheobase in the seizure-exposed new cells
compared to all other groups. Furthermore, ramp current injection
elicited fewer action potentials in seizure-exposed compared to control
Fig. 2. New neurons exposed to repeated seizures without inflammation exhibit minor morphological changes. GFP+ cell bodies in the GCL with dendrites extending into the ML
and axons into the hilus (h) 6 weeks after virus injection in control (A) and seizure-exposed (B) animals. Insets show representative images of GFP+ new cells. C, Relative location of
GFP+ cells in inner, middle, or outer GCL (iGCL, mGCL, and oGCL, respectively), or hilus. D, Relative occurrence of apical, basal, or recurrent basal dendrites (RBD) on GFP+ cells.
E, Cumulative number of dendritic branching points at increasing distances from the GFP+ cell body. F, Spine density on GFP+ dendrites from seizure-exposed and control animals
(*, increased density on seizure-exposed compared to control new cells, Student's unpaired t-test, pb0.05). Representative images of gephyrin clusters (arrows) in control (G) and
seizure-exposed (H) animals. Scale bars=50 μm (in A, B) and 1 μm (in G, H). Means±SEM.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
new cells (control 5.6±0.7, n=7 cells from 5 rats; seizures 3.4±0.5,
n=8 cells from 4 rats; pb0.05, Fig 3C).
We then explored whether the environment created by the initial
epileptic insult and the repeated seizures influenced the network-
independent excitatory and inhibitory synaptic input to the new
neurons in the GCL (for recordings of mEPSCs and mIPSCs in mature
cells, see Supplementary Figure 2–3). Whole-cell voltage-clamp
recordings of mEPSCs were carried out in the presence of the GABAA
receptor antagonist PTX and voltage-gated sodium channel blocker
TTX. Cumulative fraction analysis showed that the new cells which
were exposed to seizures throughout development exhibited mEPSCs
of larger amplitude compared to new cells from control animals
(Fig. 4A, E). Moreover, new cells exposed to seizures exhibited
mEPSCs with faster rise times compared to control new cells (Fig. 4B).
Both these changes in mEPSCs are considered to be indicative of
postsynaptic alterations. The mean frequency of mEPSCs tended to be
higher in new cells exposed to seizures but this difference did not
reach statistical significance, possibly due to the low number of cells
accessible for recording (Fig. 4C). Since histograms of IEIs were
skewed, indicating non-normal distribution (data not shown), non-
parametric, cumulative fraction analysis was applied. This analysis
revealed shorter IEIs of mEPSCs in new seizure-exposed cells
compared to new cells from control animals (Fig. 4D), providing
evidence for an increase in mEPSC frequency. Thus, our results
indicate that adult-born granule cells, exposed to repeated seizures
Intrinsic membrane properties of GFP+ and GFP− cells in the dentate granule cell layer.
Control GFP+Control GFP−
Seizures GFP+Seizures GFP−
Resting membrane potential (mV)
Input Resistance (MΩ)
Series Resistance (MΩ)
Action potential Threshold (mV)
Action potential half-width (ms)
Recordings were made from new (GFP+) and mature (GFP−) cells in animals subjected to rapid kindling and repeated seizures or non-stimulated control animals at 6–8 weeks
after RV-GFP injection. Means±SEM. Comparisons using one-way ANOVA with Bonferroni post-hoc test revealed no significant differences (pN0.05) except for rheobase.
*Significantly higher compared to all other groups (pb0.05). Number of recorded cells: new cells-seizures: 8 cells from 4 rats, new cells-control: 7 cells from 5 rats, mature cells-
seizures: 8 cells from 7 rats, mature cells-control: 9 cells from 6 rats.
Fig. 3. New neurons exposed to repeated seizures without inflammation have similar
intrinsic membrane properties but reduced intrinsic excitability compared to new
neurons in control environment. A, Representative traces of action potentials elicited in
a control new cell and a seizure-exposed new cell. Scale bar=100 ms, 20 mV.B,Voltage
responses in control and seizure-exposed new cells. C, Representative traces of
membrane potential responding to ramp current injection showing increased rheobase
and fewer action potentials in seizure-exposed new cells. Scale bar=500 ms, 20 mV.
Fig. 4. Newneuronsexposedtorepeatedseizureswithoutinflammationexhibitenhanced
excitatory input in the absence of network activity. Cumulative fraction curve showing
increased amplitude and faster 10–90% rise time of mEPSCs in new cells from seizure-
exposed compared to new cells from control animals after action potential blockade
with TTX (A, B). No change in mean event frequency (Student's unpaired t-test, pN0.05),
but shorter IEI of mEPSCs in new cells exposed to seizures compared to control new cells
(C,D) (Kolmogorov–Smirnov test).E, Representative traces ofmEPSCs inseizure-exposed
andcontrolcells.1,2 depictrepresentativeevents(left)onanexpandedtime scale (right).
Scale bar=10 pA, 1 s and 10 pA, 5 ms, respectively. Number of recorded cells: new cells-
seizures: 6 cells from 4 rats, new cells-control: 8 cells from 5 rats.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
throughout development, receive enhanced excitatory drive, which is
independent of network-generated action potentials.
We also determined if the new neurons exhibited altered
inhibitory synaptic input when born after rapidly recurring seizures
and subsequently exposed to episodes of seizure activity throughout
their development. Whole-cell voltage-clamp recordings of mIPSCs
were performed while blocking glutamate receptors with NBQX and
D-AP5, and action potentials with TTX. Cumulative fraction analysis
showed that the amplitude of mIPSCs was not different between new
cells born into the seizure environment and new cells from control
animals (Fig. 5A, E). However, mIPSCs from seizure-exposed new cells
exhibited slower 10–90% rise times compared to control new cells
(Fig. 5B), suggesting a relative decrease in the strength of perisomatic
vs. dendritic inhibitory drive (Kobayashi and Buckmaster 2003;
Jakubs et al., 2006; however, see Soltesz et al., 1995). We next looked
at the frequency andIEIsof mIPSCs.mIPSCsoccurredwithlowermean
frequency in seizure-exposed as compared to control new cells, but
the difference was not statistically significant (Fig. 5C). However,
when we used cumulative fraction analysis due to the skewed, non-
normal distribution of IEIs, we detected lengthening of mIPSC IEIs in
seizure-exposed compared to control new cells (Fig. 5D), suggesting a
decreasein mIPSCfrequency. Takentogether, ourresults indicatethat,
in the absence of network-generated action potentials, adult-born
granule cells exposed to repeated seizures throughout their develop-
ment receive reduced perisomatic inhibition compared to new cells
born in control animals.
How neurons, which are born and develop in a pathological
environment, integrate into existing neural circuitries in the adult
brain will determine whether they counteract or contribute to
functional impairments. Here we show that new dentate granule
cells generated following an epileptic insult, comprising 40 rapidly
recurring seizures, and exposed to repeated seizures during their
differentiation exhibited increased overall synaptic excitability com-
paredto newcellswhichhaddevelopedin a normalenvironment. The
increased synaptic excitability of these cells may be counterbalanced,
or even overridden by reduced intrinsic excitability as evidenced by
higher rheobase. In contrast, detailed morphological analysis of the
location, orientation, dendritic arborizations, and spines of these cells
showed only minor differences between the groups.
after seizures is consistent with studies which have reported enhanced
excitability of dentate granule cells after pilocarpine-induced seizures
2001). How seizures influence the inhibition of granule cells is less clear
as there are reports that kainate-induced seizures enhance (Buckmaster
and Dudek, 1997) whereas pilocarpine-induced seizures reduce inhib-
itory input to granule cells (Kobayashi and Buckmaster, 2003).
Furthermore, after eSE, mature granule cells exhibit longer IEIs of sIPSCs
with larger amplitude (Jakubs et al., 2006). Inhibition may be influenced
by changes in zinc distribution. After kindling, granule cells receive
increased inhibitory drive, which may collapse due to zinc released from
aberrantly sprouted mossy fibers interacting with zinc-sensitive GABAA
receptors (Buhl et al., 1996). We found that new granule cells born after
rapid kindling and exposed to repeated seizures exhibited mIPSCs of
similar amplitude but with longer IEIs compared to new cells in control
animals. Taken together, it seems that changes in the inhibitory inputs to
granule cells are dependent on the seizure paradigm and epilepsy model
used, and may be modulated at both pre- and postsynaptic sites.
Two main lines of evidence indicate that the new neurons born
after rapid kindling and exposed to repeated seizures integrate into
hippocampal circuitry in a manner that may contribute to enhanced
synaptic excitability. First, mEPSCs in seizure-exposed new cells had
larger amplitudes and faster rise times compared to mEPSCs recorded
in control new cells. These changes, including excitatory postsynaptic
receptor kinetics, are consistent with alterations of AMPA receptor
subunits (Koike et al., 2000; Liu and Cull-Candy, 2000). Second,
mIPSCs in seizure-exposed new cells display longer rise times of
mIPSCs, which suggest a relative weakening of perisomatic inhibition
in seizure-exposed new cells (Kobayashi and Buckmaster 2003;
Jakubs et al., 2006; however, see Soltesz et al., 1995). If this is the case,
it could lead to less control over action potentials thought to be
generated around the axon hillock, i.e., in the perisomatic area. These
changesin postsynapticreceptor kinetics may also indicate changesin
GABAA receptor subunits (Coulter, 2001). However, in the same
seizure-exposed new cells, we also observed increased rheobase and
fewer action potentials, which may partially, or completely, counter-
act the enhanced synaptic excitability.
The exact source of the presynaptic input to the new neurons and
their postsynaptic targets remain important issues. It is well
established that the entorhinal cortex via the perforant path is the
primarysource of excitatory input to mature dentate granule cells and
also adult-born neurons (Overstreet-Wadiche et al., 2006). However,
there is evidence that after seizures, granule cells can provide
excitatory input to each other due to mossy fiber sprouting (Tauck
and Nadler, 1985; Elmér et al., 1996; Sutula et al., 1989; Represa et al.,
1990). Inhibitory interneurons located throughout the hippocampus
provideGABAergic input togranulecells. Thenumber of interneurons,
Fig. 5. New neurons exposed to repeated seizures without inflammation exhibit altered
inhibitory input in the absence of network activity. Cumulative fraction curves showing
no change in amplitude and slower 10–90% rise time of mIPSCs in new cells from
seizure-exposed compared to control animals after action potential blockade with TTX
(A, B). No change in mean event frequency (Student's unpaired t-test, pN0.05), but
longer IEI of mIPSCs in new cells exposed to seizures compared to control new cells
(C, D). (Kolmogorov–Smirnov test). E, Representative traces of mIPSCs recorded from
seizure-exposed and control cells. 1, 2 depict representative events (left) on an
expanded time scale (right). Scale bar=50 pA, 1 s and 50 pA, 20 ms, respectively.
Number of recorded cells: new cells-seizures: 8 cells from 4 rats, new cells-control: 7
cells from 6 rats.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
their morphology, and development of synapses with granule cells is
influenced by seizures (Wittner et al., 2001; Dinocourt et al., 2003;
Sayin et al., 2003; Zhang and Buckmaster, 2009). To what extent
mossy fiber sprouting or alterations of inhibitory interneurons
influence the integration of the new neurons is not known.
The pre- and postsynaptic changes observed here indicate a net
increase in the excitatory drive onto the seizure-exposed new neurons,
but how this influences their functional output is unclear. Axons of
dentate granule cells (mossy fibers) contact hilar mossy cells, CA3
synapses. The changes in rheobase likely indicate alterations in the
membrane properties of the seizure-exposed cells, particularly changes
input resistance of the seizure-exposed new cells tended to be lower
compared to that of the other recorded cells (although not statistically
significant), which may partially explain the increase in rheobase.
Changes in input resistance suggest alterations in the number,
distribution, or composition of membrane channels in the new cells. It
should be emphasized, finally, that the functional significance at the
network level of the altered afferent synaptic inputs to the new cells,
whether they will counteract or contribute to the development of
their target neurons (Frotscher et al., 2006), an issue that is highly
warranted to address in future studies.
The pattern of alterations in afferent excitatory and inhibitory
synaptic drive on the new cells in the present seizure paradigm differs
from that we have previously reported following eSE (Jakubs et al.,
excitatory and increased inhibitory synaptic drive (Jakubs et al., 2006).
The eSE insult comprised approximately 3 h of seizure activity and the
environment surrounding the new cells was characterized by neuronal
death, chronic inflammation and spontaneous seizures. In contrast, we
the pathologicalenvironment. Thetotal duration of seizuresinduced by
the rapid kindling protocol was much shorter (about 19 min). The eSE
environment was associated with abnormal excitability, as evidenced
by spontaneous behavioral seizures, whereas following rapid kindling,
there was a progressive development of hyperexcitability but no
interictal spikes or spontaneous seizures were detected. The stimulus-
evoked seizures lasted for in total about 10 min. Taken together, the
discrepancy in afferent synaptic connectivity, comparing the present
findings with our previous data (Jakubs et al., 2006), indicates that the
integration of the new cells depends on the type of pathological
environment they encounter.
The characteristics of the pathological environment will also
determine its influences on the morphological development of the
new neurons. Walter et al. (2007) studied new granule cells exposed to
3 h of pilocarpine-induced SE in mice either at 1 week after they had
been generated or when they were born into the pathological
environment 3 weeks after the insult. This insult, which caused
spontaneous seizures and extensive neuronal death in the dentate
hilus and variable cell loss in CA1 and CA3 regions, gave rise to the
formation of basal dendrites projecting into the hilus in 40–50% of new
granule cells. Such dendrites are virtually absent in intact animals. Also,
Jessberger et al. (2007) found basal hilar dendrites in about 20% of new
granule cells born at 1 week following 2–3 h of kainate-induced SE. It is
conceivable that both these severe epileptic insults were associated
total duration of seizure activity was much shorter, resulted in no
significant inflammation or neuronal death, and the occurrence of very
few basal hilar dendrites on the new granule cells. Our findings, that
new neurons which develop in the presence of seizures without
inflammationexhibit nomajormorphological changes indicate thatthe
occurrence of morphological abnormalities is likely dependent on the
severity of pathology in the environment.
We found an increased number of stubby spines on the seizure-
exposed new cells. Stubby spines have been found to be more frequent
on mature hippocampal dendrites in acute slices with blocked synaptic
transmission, which is believed to recapitulate development (Petrak
et al., 2005). Interestingly, application of brain-derived neurotrophic
factor (BDNF) to hippocampal slice cultures under serum-free condi-
tions specifically promoted the formation of stubby spines on mature
CA1 pyramidal neurons (Tyler and Pozzo-Miller, 2003, however, see
Chapleau et al., 2008). These stubby spines may have a role in Ca2+-
dependent synaptic plasticity (Tyler and Pozzo-Miller, 2003). BDNF has
also been proposed to be an important regulator of morphological and
functional hippocampal plasticity in response to seizures (Ernfors et al.,
1991; Kokaia et al., 1995; Binder et al., 2001). Hypothetically, BDNF
signaling may have contributed to the increase of stubby spines
observed here on the new cells.
survival, proliferation,migration, differentiation, and functionalintegra-
tion of the new neurons (reviewed by Ekdahl et al., 2009). LPS-induced
chronic inflammation gave rise to a similar increase of excitatory
synaptic drive in new andmature dentate granule cells, probably due to
drive was increased by inflammation in both new and mature cells but
more enhanced in the new cells. In line with this observation, larger
were found on dendrites of new cells born in the inflammatory
environment. It is conceivable that the larger gephyrin clusters indicate
a more efficacious inhibitory input and contributes to the synapse-
specific enhancement of the afferent inhibitory drive. In contrast, we
found here that the new cells which had been born after rapid kindling
and exposed to repeated seizures did not exhibit any change in mIPSC
amplitude, indicating no postsynaptic alterations. In accordance, we did
not observe any alteration in the density or size of gephyrin clusters at
postsynaptic sites in neuronswhichhaddeveloped in this environment.
The present findings provide further evidence for an important
regulatory role of inflammation for inhibitory synaptic drive on the
new cells. Our data indicate that different pathological environments,
associated with varying magnitude of inflammation, differ with respect
to their ability to induce postsynaptic changes in new cells which will
influence the efficacy of their afferent inputs.
The present findings show that adult-born, new neurons exhibit a
high degree of plasticity at their afferent synapses when developing in
a pathological environment. Our previous data following eSE (Jakubs
et al., 2006) and chronic inflammation (Jakubs et al., 2008) suggested
that the functional integration of the new neurons may act to mitigate
the pathological condition. Here, the new neurons responded to
repeated seizures in an environment without inflammation by overall
more synaptic excitability, which may be counteracted by the reduced
intrinsic excitability compared to control new cells. If this is the case,
the new neurons may have a limited contribution to the hyperexcit-
ability which develops during the course of their maturation or even
counteract the abnormal function. In conclusion, our findings indicate
that the characteristics of the pathological environment, e.g., the
magnitude of inflammation and the seizure paradigm, will play an
important role in determining whether the new neurons will
counteract or contribute to abnormal brain function.
Supplementary materials related to this article can be found online
This work was supported by the Swedish Research Council,
Juvenile Diabetes Research Foundation, and EU project LSHB-2006-
037526 (StemStroke). We thank Dr. Fred H. Gage and Dr. H. van Praag
for RV-GFP, and Dr. Sara Bonde and Dr. Robert E. Iosif for preparation
of animals during the initial part of the study.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493
References Download full-text
Bengzon, J., Kokaia, Z., Elmer, E., Nanobashvili, A., Kokaia, M., Lindvall, O., 1997.
Apoptosis and proliferation of dentate gyrus neurons after single and intermittent
limbic seizures. Proc. Natl. Acad. Sci. USA 94, 10432–10437.
Binder, D.K., Croll, S.D., Gall, C.M., Scharfman, H.E., 2001. BDNF and epilepsy: too much
of a good thing? Trends Neurosci. 24, 47–53.
Biscaro, B., Lindvall, O., Hock, C., Ekdahl, C.T., Nitsch, R.M., 2009. Abeta immunotherapy
protects morphology and survival of adult-born neurons in doubly transgenic APP/
PS1 mice. J. Neurosci. 29, 14108–14119.
Buckmaster, P.S., Dudek, F.E., 1997. Neuron loss, granule cell axon reorganization, and
functional changes in the dentate gyrus of epileptic kainate-treated rats. J. Comp.
Neurol. 385, 385–404.
Buhl, E.H., Otis, T.S., Mody, I., 1996. Zinc-induced collapse of augmented inhibition by
GABA in a temporal lobe epilepsy model. Science 271, 369–373.
Chapleau, C.A., Carlo, M.E., Larimore, J.L., Pozzo-Miller, L., 2008. The actions of BDNF on
dendritic spine density and morphology in organotypic slice cultures depend on
the presence of serum in culture media. J. Neurosci. Methods 169, 182–190.
Coulter, D.A., 2001. Epilepsy-associated plasticity in gamma-aminobutyric acid
receptor expression, function, and inhibitory synaptic properties. Int. Rev.
Neurobiol. 45, 237–252 Review..
Dinocourt, C., Petanjek, Z., Freund, T.F., Ben-Ari, Y., Esclapez, M., 2003. Loss of
interneurons innervating pyramidal cell dendrites and axon initial segments in the
CA1 region of the hippocampus following pilocarpine-induced seizures. J. Comp.
Neurol. 459, 407–425.
Ekdahl, C.T., Kokaia, Z., Lindvall, O., 2009. Brain inflammation and adult neurogenesis:
the dual role of microglia. Neuroscience 158, 1021–1029.
for neurogenesis in adult brain. Proc. Natl. Acad. Sci. USA 100, 13632–13637.
Elmér, E., Kokaia, M., Kokaia, Z., Ferencz, I., Lindvall, O., 1996. Delayed kindling
development after rapidly recurring seizures: relation to mossy fiber sprouting and
neurotrophin, GAP-43 and dynorphin gene expression. Brain Res. 712, 19–34.
Elmér, E., Kokaia, Z., Kokaia, M., Carnahan, J., Nawa, H., Lindvall, O., 1998. Dynamic
changes of brain-derived neurotrophic factor protein levels in the rat forebrain
after single and recurring kindling-induced seizures. Neuroscience 83, 351–362.
Ernfors, P., Bengzon, J., Kokaia, Z., Persson, H., Lindvall, O., 1991. Increased levels of
Neuron 7, 165–176.
Fritschy, J.M., Harvey, R.J., Schwarz, G., 2008. Gephyrin: where do we stand, where do
we go? Trends Neurosci. 31, 257–264.
Frotscher, M., Jonas, P., Sloviter, R.S., 2006. Synapses formed by normal and abnormal
hippocampal mossy fibers. Cell Tissue Res. 326, 361–367.
Jakubs, K., Nanobashvili, A., Bonde, S., Ekdahl, C.T., Kokaia, Z., Kokaia, M., Lindvall, O.,
2006. Environment matters: synaptic properties of neurons born in the epileptic
adult brain develop to reduce excitability. Neuron 52, 1047–1059.
Jakubs, K., Bonde, S., Iosif, R.E., Ekdahl, C.T., Kokaia, Z., Kokaia, M., Lindvall, O., 2008.
Inflammation regulates functional integration of neurons born in adult brain.
J. Neurosci. 28, 12477–12488.
Jessberger, S., Zhao, C., Toni, N., Clemenson Jr., G.D., Li, Y., Gage, F.H., 2007. Seizure-
associated, aberrant neurogenesis in adult rats characterized with retrovirus-
mediated cell labeling. J. Neurosci. 27, 9400–9407.
Kobayashi, M., Buckmaster, P.S., 2003. Reduced inhibition of dentate granule cells in a
model of temporal lobe epilepsy. J. Neurosci. 23, 2440–2452.
of GluR2 AMPA receptor channels by alternative splicing. J. Neurosci. 20, 2166–2174.
Kokaia, M., Ernfors, P., Kokaia, Z., Elmer, E., Jaenisch, R., Lindvall, O., 1995. Suppressed
epileptogenesis in BDNF mutant mice. Exp. Neurol. 133, 215–224.
Laplagne, D.A., Esposito, M.S., Piatti, V.C., Morgenstern, N.A., Zhao, C., van Praag, H.,
Gage, F.H., Schinder, A.F., 2006. Functional convergence of neurons generated in the
developing and adult hippocampus. PLoS Biol. 4, e409.
Lehrmann, E., Christensen, T., Zimmer, J., Diemer, N.H., Finsen, B., 1997. Microglial and
macrophage reactions mark progressive changes and define the penumbra in the
rat neocortex and striatum aftertransient middle cerebral artery occlusion. J.Comp.
Neurol. 386, 461–476.
Liu, J., Solway, K., Messing, R.O., Sharp, F.R., 1998. Increased neurogenesis in the dentate
gyrus after transient global ischemia in gerbils. J. Neurosci. 18, 7768–7778.
Liu, S.Q., Cull-Candy, S.G., 2000. Synaptic activity at calcium-permeable AMPA receptors
induces a switch in receptor subtype. Nature 405, 454–458.
Meijering, E., Jacob, M., Sarria, J.C.F., Steiner, P., Hirling, H., Unser, M., 2004. Design and
validation of a tool for neurite tracing and analysis in fluorescence microscopy
images. Cytometry 2, 167–176.
Monje, M.L., Toda, H., Palmer, T.D., 2003. Inflammatory blockade restores adult
hippocampal neurogenesis. Science 302, 1760–1765.
Morgenstern, N.A., Lombardi, G., Schinder, A.F., 2008. Newborn granule cells in the
ageing dentate gyrus. J. Physiol. 586, 3751–3757.
Nimchinsky, E.A., Sabatini, B.L., Svoboda, K., 2002. Structure and function of dendritic
spines. Annu. Rev. Physiol. 64, 313–353.
Overstreet-Wadiche, L.S., Bromberg, D.A., Bensen, A.L., Westbrook, G.L., 2006. Seizures
accelerate functional integration of adult-generated granule cells. J. Neurosci. 26,
Parent, J.M., 2005. When newborn neurons stray. Epilepsy Curr. 5, 231–233.
Parent, J.M., Yu, T.W., Leibowitz, R.T., Geschwind, D.H., Sloviter, R.S., Lowenstein, D.H.,
1997. Dentate granule cell neurogenesis is increased by seizures and contributes to
aberrant network reorganization in the adult rat hippocampus. J. Neurosci. 17,
Petrak, L.J., Harris, K.M., Kirov, S.A., 2005. Synaptogenesis on mature hippocampal
dendrites occurs via filopodia and immature spines during blocked synaptic
transmission. J. Comp. Neurol. 484, 183–190.
Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor
seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281–294.
Represa, A., Tremblay, E., Ben-Ari, Y., 1990. Sprouting of mossy fibers in the
hippocampus of epileptic human and rat. Adv. Exp. Med. Biol. 268, 419–424.
Sayin, U., Osting, S., Hagen, J., Rutecki, P., Sutula, T., 2003. Spontaneous seizures and loss
of axo-axonic and axo-somatic inhibition induced by repeated brief seizures in
kindled rats. J. Neurosci. 23, 2759–2768.
Scott, B.W., Wang, S., Burnham, W.M., De Boni, U., Wojtowicz, J.M., 1998. Kindling-
induced neurogenesis in the dentate gyrus of the rat. Neurosci. Lett. 248, 73–76.
Sheffield, J.B., 2007. ImageJ, a useful tool for biological image processing and analysis.
Microsc. Microanal. 13, 200–201.
Simmons, M.L., Terman, G.W., Chavkin, C., 1997. Spontaneous excitatory currents and
kappa-opioid receptor inhibition in dentate gyrus are increased in the rat
pilocarpine model of temporal lobe epilepsy. J. Neurophysiol. 78, 1860–1868.
Soltesz, I., Smetters, D.K., Mody, I., 1995. Tonic inhibition originates from synapses close
to the soma. Neuron 14, 1273–1283.
Staley, K.J., Otis, T.S., Mody, I., 1992. Membrane-properties of dentate gyrus granule
cells—comparison of sharp microelectrode and whole-cell recordings. J. Neuro-
physiol. 67, 1346–1358.
Sutula, T., Cascino, G., Cavazos, J., Parada, I., Ramirez, L., 1989. Mossy fiber synaptic
reorganization in the epileptic human temporal lobe. Ann. Neurol. 26, 321–330.
Tauck, D.L., Nadler, J.V., 1985. Evidence of functional mossy fiber sprouting in
hippocampal formation of kainic acid-treated rats. J. Neurosci. 5, 1016–1022.
Toni, N., Laplagne, D.A., Zhao, C., Lombardi, G., Ribak, C.E., Gage, F.H., Schinder, A.F.,
2008. Neurons born in the adult dentate gyrus form functional synapses with target
cells. Nat. Neurosci. 11, 901–907.
Tyler, W.J., Pozzo-Miller, L., 2003. Miniature synaptic transmission and BDNF modulate
van Praag, H., Schinder, A.F., Christie, B.R., Toni, N., Palmer, T.D., Gage, F.H., 2002.
Functional neurogenesis in the adult hippocampus. Nature 415, 1030–1034.
Walter, C., Murphy, B.L., Pun, R.Y.K., Spieles-Engemann, A.L., Danzer, S.C., 2007.
Pilocarpine-induced seizures cause selective time-dependent changes to adult-
generated hippocampal dentate granule cells. J. Neurosci. 27, 7541–7552.
Wittner, L., Maglóczky, Z., Borhegyi, Z., Halász, P., Tóth, S., Eross, L., Szabó, Z., Freund, T.F.,
2001. Preservation of perisomatic inhibitory input of granule cells in the epileptic
human dentate gyrus. Neuroscience 108, 587–600.
Wuarin, J.P., Dudek, F.E., 2001. Excitatory synaptic input to granule cells increases with
time after kainate treatment. J. Neurophysiol. 85, 1067–1077.
Young, C.C., Stegen, M., Bernard, R., Müller, M., Bischofberger, J., Veh, R.W., Haas, C.A.,
Wolfart, J., 2009. Upregulation of inward rectifier K+(Kir2) channels in dentate
gyrus granule cells in temporal lobe epilepsy. J. Physiol. 587, 4213–4233.
Zhang, W., Buckmaster, P.S., 2009. Dysfunction of the dentate basket cell circuit in a rat
model of temporal lobe epilepsy. J. Neurosci. 29, 7846–7856.
Zhao, C.M., Teng, E.M., Summers, R.G., Ming, G.L., Gage, F.H., 2006. Distinct
morphological stages of dentate granule neuron maturation in the adult mouse
hippocampus. J. Neurosci. 26, 3–11.
Zhao, C.M., Deng, W., Gage, F.H., 2008. Mechanisms and functional implications of adult
neurogenesis. Cell 132, 645–660.
J.C. Wood et al. / Experimental Neurology 229 (2011) 484–493