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. Author manuscript; available in PMC: 2018 Jul 1.
Published in final edited form as: Exp Neurol. 2017 Apr 18;293:190–198. doi: 10.1016/j.expneurol.2017.04.005

Mice with conditional NeuroD1 knockout display reduced aberrant hippocampal neurogenesis but no change in epileptic seizures

Rebecca Brulet 1, Jingfei Zhu 1, Mahafuza Aktar 1, Jenny Hsieh 1,*, Kyung-Ok Cho 2,*
PMCID: PMC5503142  NIHMSID: NIHMS871391  PMID: 28427858

Abstract

Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroDl (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroDl+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroDl in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroDl+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroDl cKO mice, suggesting that NeuroDl cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Proxl+ hilar ectopic granule cells were unchanged after NeuroDl cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons.

Keywords: Temporal lobe epilepsy, Seizure, Pilocarpine, NeuroD1, Neurogenesis, Therapy

Introduction

Epilepsy is a set of neurological disorders characterized by spontaneous recurrent seizures (SRS). Mesial Temporal Lobe Epilepsy (mTLE) is one of the most common adult epilepsies, but is often refractory to currently available therapeutics (Jessberger S. & Parent J. 2015). Animal models of mTLE have demonstrated several important cellular changes in the hippocampal formation and surrounding structures. These include astrogliosis, mossy fiber sprouting, hilar basal dendrites, ectopic migration of newborn neurons, neuronal cell death, granule cell layer dispersion, and an increase in adult neurogenesis (Jessberger S. et al. 2005; Shapiro L.A. et al. 2005; Binder D.K. & Steinhauser C. 2006; Noebels J.L. et al. 2012; Jessberger S. & Parent J. 2015). Neurons born after epileptic seizures have been demonstrated to become aberrant, often mis-migrating and forming inappropriate connections with the surrounding hippocampal network (Parent J.M. et al. 1997; Scharfman H.E. 2002). This has led to the hypothesis that these aberrantly integrated adult-generated neurons contribute to the SRS seen in mTLE (Noebels J.L. et al. 2012; Bielefeld P. et al. 2013; Cho K. et al. 2015). Use of genetic ablation or pharmacological approaches to suppress adult neurogenesis resulted in a 40–70% reduction in SRS frequency in rodent models of epilepsy, supporting this hypothesis (Jung K.H. et al. 2004; Jung K.H. et al. 2006; Cho K. et al. 2015). In addition, ablation of neurogenesis normalized many of the epilepsy-associated cognitive deficits indicating that neurogenesis plays a significant contributing role in the development of epilepsy-related comorbidities (Cho K. et al. 2015). In order to translate these findings to the clinic, it would be ideal to identify molecules that specifically promote aberrant adult-generated neurons as they may represent molecular targets to reduce chronic seizures.

To better understand which molecular regulators of neurogenesis contribute to epilepsy, we have decided to study NeuroD1, a bHLH transcription factor expressed in immature neuroblasts in the hippocampus (Pleasure S.J. et al. 2000; Guillemot F. 2007; Gao Z. et al. 2009). Previously, we showed NeuroD1 is essential for the survival and maturation of adult-generated neurons in the hippocampus under physiological conditions (Gao Z. et al. 2009). Furthermore, kainic acid (KA)-induced seizures increased the number of NeuroD1+ immature neuroblasts in the dentate and hilus, while conditional deletion of NeuroD1 in that model reduced the number of YFP+ recombined neuroblasts, suggesting it may play a role in chronic seizure development(Cho K. et al. 2015). Here, we employ the pilocarpine model of epilepsy to investigate the role NeuroD1 plays in aberrant neurogenesis and SRS. Using this approach, deletion of NeuroD1 in mice prior to status epilepticus (SE) led to a significant reduction in the total number of NeuroD1+ neuroblasts, however, this did not affect the overall seizure frequency. These data reinforce the idea that aberrant neurogenesis needs to be reduced below a threshold to impact chronic epilepsy.

Materials and Methods

Animals

All the experiments were performed in compliance with the animal care guidelines issued by the National Institutes of Health and by the Institutional Animal Use and Care Committee (IACUC) at University of Texas Southwestern Medical Center. All mice were bred and housed in the animal facility with a 12-h light, 12-h dark cycle with no more than five mice per cage, food (2916 Global irradiated diet, Teklad Labs), and water ad libitum. To generate the mice used in this study, we crossed homozygous Nestin-CreERT2; NeuroD1loxP/+mice with homozygous R26R-YFP; NeuroD1loxP/+mice(Goebbels S. et al. 2005; Lagace D.C. et al. 2007) to generate male and female Nestin-CreER/R26R-YFP; NeuroD1+/+ (NeuroD1 WT) and Nestin-CreERT2/R26R-YFP; NeuroD1loxP/loxP (NeuroD1 cKO) mice. Mice were genotyped by PCR using genomic DNA and primers for NeuroD1 (5′ GTT TTT GTG AGT TGG GAG TG 3′, 5′ TGA CAG AGC CCA GAT GTA 3′), NestinCreERT2(5′ GGT CGA TGC AAC GAG TGA TGA GG 3′, 5′ GCT AAG TGC CTT CTC TAC ACC TGC G 3′), and R26R-YFP (5′ AAA GTC GCT CTG AGT TGT TAT 3′, 5′ GCG AAG AGT TTG TCC TCA ACC 3′, 5′ GGA GCG GGA GAA ATG GAT ATG 3′). NeuroD1 WT and cKO mice were backcrossed to C57BL/6NHsd mice obtained from Envigo Laboratories (Cat no: 4403F/M) for at least 4–6 generations prior to beginning studies. Male and female NeuroD1 WT and cKO mice at approximately 5.5 weeks of age were administered tamoxifen (TAM) intraperitoneally (i.p.) at 150 mg/kg per day for 5 days prior to SE. TAM was dissolved in 10% EtOH/90% sunflower oil. In Fig. 1 experiments, we used female C57BL/6NHsd (Cat no: 4403F) purchased from Envigo. SNP analysis testing performed for NeuroD1 WT and cKO and C57BL/6NHsd mice showed an average 99.33% genetic similarity between these two lines.

Figure 1. Acute seizures increase the number of NeuroD1-positive cells in adult dentate gyrus.

Figure 1

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A) Experimental Timeline. Female C57BL/6NHsd Envigo animals were subjected to pilocarpine induced status epilepticus (SE) and then euthanized 3 weeks post pilocarpine. B) Animals were euthanized 3 weeks post pilocarpine treatment and brains were stained with NeuroD1 to determine the total cell number in the granule cell layer (GCL) and hilus. (n=6 sham, n=6 pilo) C) There was a significant increase in the NeuroD1+ cells in both the GCL (p=0.0013) and hilus (p=0.0016) in pilocarpine treated animals compared to sham treated animals. D,E) The majority of pilocarpine induced NeuroD1+ cells co-localized with the immature neuronal marker Dcx. (n=8 sham, n=8 pilo). Scale bar in B: 100 um, inset bar in B: 25um, both valid for D.

Chemoconvulsant model of TLE

Male and female NeuroD1 WT and cKO mice at approximately 6 weeks of age were administered scopolamine methyl nitrate (i.p.; 2 mg/kg; Sigma-Aldrich S2250) and terbutaline hemisulfate salt (i.p.; 2 mg/kg; Sigma-Aldrich T2528) to block the peripheral effects of pilocarpine and dilate the respiratory tract, respectively. Thirty minutes later, pilocarpine hydrochloride (i.p.; Sigma-Aldrich P6503) at 220 mg/kg for males and 260 mg/kg for females was injected, and mice were placed in an incubator maintained at 31°C (ThermoCare). Acute seizures were behaviorally monitored using a modified Racine’s scale (Racine R.J. 1972) (stage 1, mouth and facial movement; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing with forelimb clonus; stage 5, rearing and falling with forelimb clonus). Once SE began (defined by continuous tonic-clonic convulsive seizures), mice were placed at room temperature for 3 h and returned to the incubator after seizure activity was reduced using diazepam (10 mg/kg; Sigma-Aldrich D0899). Only mice showing SE for a total of 3 hours were included in EEG recording studies. After SE, mice were administered 5% dextrose solution (i.p.; 1 ml) and saline (i.p.; 1 ml) to facilitate their recovery. Mice were weighed each day during the recovery period and if found to have lost ≥ 2 g from the previous recorded weight were given a single i.p. dose of 1ml 5% dextrose, and moistened chow. At 3 days after SE, mice were returned to their home cage. Male and female NeuroD1 WT and cKO mice included in the sham seizure group were given (TAM) intraperitoneally (i.p.) at 150 mg/kg per day for 5 days prior to sham treatment. All sham animals were administered scopolamine methyl nitrate (i.p.; 2 mg/kg; Sigma-Aldrich S2250) and terbutaline hemisulfate salt (i.p.; 2 mg/kg;Sigma-Aldrich T2528) 30 minutes prior to an i.p. vehicle (saline) injection. In some cases, male and female NeuroD1 WT and cKO mice were given access to Metoclopramide (Henry Schein Animal Health, Cat no. 055411) treated water (1mg/kg) for 5 days prior to SE to help alleviate gastrointestinal symptoms (constipation) associated with use of pilocarpine in this mouse line. EnvigoC57BL/6NHsd female mice were treated the same as above, but did not receive TAM injections, and received a dose of 185 mg/kg pilocarpine to induce SE. For reference we have made available data on the total number of male and female animals used in our experiments, the total number of each that entered SE and survived for a total of 3 hours, and the total number of that made it to EEG recording are represented in Table 1.

Table 1. Animal numbers included in study.

This table details the total number of male and female animals used in our SRS recording experiments and is further broken down into the total number of each sex that entered SE and survived for a total of 3 hours, as well as the total number that survived to the SRS recording phase of the study. The * in the table indicates a number where there were originally 12 animals, but after re-genotyping 1 animal was excluded from the recording phase due to being heterozygous for the NeuroD1 allele.

Received Pilo Entered SE and survived Included in data
Males 87 42 12*
Females 50 21 10
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Video/EEG monitoring

Video/EEG recording was performed between 5–7 weeks after pilocarpine injection. One week before EEG recording, mice were stereotaxically implanted with cortical surface electrodes connected to wireless EEG transmitters placed subcutaneously under the skin on the back (TA11ETAF10, Data Sciences International, St. Paul, MN). Mice were anesthetized using 2–3% isoflurane gas mixed in a 1L/min mixture of 70% nitrous oxide and 30% oxygen. Two cortical electrodes were placed at the coordinates from Bregma AP: +0.1 ML: +0.1 (Reference; R) and AP:−0.2 ML: +0.22 (Left parietal cortex; LPC). Mice received the analgesic buprenorphine (subcutaneously; 0.05 mg/kg) as necessary following surgery. Animals underwent continuous monitoring by video/EEG for a total of two weeks. Video-EEG data was reviewed and quantified by a user blinded to the experimental groups using NeuroScore Software (Version 3.0.7703-0, Data Sciences International). Behavioral seizures were defined by repetitive epileptiform spiking activity (≥3Hz) that persisted for (≥10s) and was confirmed using video recordings. Seizure activity was marked at the beginning and end of each event to account for seizure duration and the number of seizures for each mouse was recorded. After EEG recording was completed, mice were sacrificed and brains harvested for immunohistochemistry.

Immunohistochemistry

Mice were anesthetized using 250mg/kg Avertin injected i.p. and perfused transcardially with cold 4% paraformaldehyde (PFA) in 0.1 M PBS. Brains were removed and post-fixed in 4% PFA overnight, then cryoprotected in 30% sucrose in 0.1 M PBS. Brains were bisected and half-brains were coronally sectioned 30 um thick on a freezing microtome (Leica, SM 2000R). Immunohistochemistry was performed with either tissue mounted on charged slides or free-floating tissue sections. Slides underwent antigen retrieval using 0.01 M citric acid, pH 6.0 at 100 °C for 15 min, followed by 12 min in 1x TBS at room temperature. Staining with free-floating tissue sections was the same except for the antigen retrieval step that was omitted. For Tyramide Plus signal amplification, we removed endogenous peroxidase activity by incubating sections with 0.3% H2O2 for 30 min at room temperature. Nonspecific binding was blocked with 3% normal donkey serum and 0.3% Triton-X-100 or 3% normal donkey serum and 1% Triton X-100 in 1x TBS for 1 h at room temperature. Primary antibodies used in this study were as follows: goat anti-NeuroD1 (1:500, Santa Cruz Biotechnology sc-1084), chicken anti-GFP (1:1,000 for free floating sections, 1:8,000 for Tyramide Plus Amplification; Aves Lab GFP-1020), goat anti-Dcx (1:1,000, Santa Cruz Biotechnology sc-8066), guinea pig anti-Dcx (1:1,000, Millipore AB2253), and mouse anti-NeuN (1:1,000, Millipore MAB377). For double labeling, primary antibodies were simultaneously incubated and further processed for each antibody. Antibodies combined for double immunostains were as follows: GFP/NeuroD1; GFP/Dcx; GFP/Prox1; and NeuroD1/Dcx. For GFP, Dcx, Prox1, and NeuN, a fluorescent-tagged secondary antibody was used (1:100–1:500, Donkey anti-Cy2/Cy3/Cy5 Jackson ImmunoResearch). For GFP and NeuroD1 (slide mounted), primary antibody incubation was followed with an appropriate biotin-tagged secondary antibody (1:200, Jackson ImmunoResearch) for 1 h at room temperature, followed by ABC (Vector Laboratories PK-6100) for 1 h, and Tyramide-Plus signal amplification (1:50, PerkinElmer NEL701001KT) for 1–3 min. For Dcx, after biotin-tagged secondary antibody followed by ABC labeling was completed, sections were visualized with a metal-enhanced DAB substrate kit (Thermo Scientific 34065). Sections visualized with DAB were mounted and dehydrated (70, 80, 90, 95, and 100% EtOH - 3 min each; and 50% EtOH/xylene, and 100% xylene- 3 min each) before coverslipping. Fluorescence stained sections were mounted in a 2.5% PVA-DABCO Media (PVA Sigma-Aldrich D2522, DABCO: Sigma-Aldrich D2522).

Microscopic analysis and quantification

Quantification of cell number was performed by a user blinded to the experimental groups. In fluorescence labeled sections of the hippocampus, quantification was performed using either an upright microscope (BX60; Olympus), or a confocal microscope (LSM700/LSM710; Carl Zeiss Microscopy). In DAB labeled sections of the hippocampus, quantification was performed using a MicroBrightfield scope (BX51; Olympus) equipped with StereoInvestigator software (Version 11.03). Subgranular and hilar zones were defined as the area within and beyond the diameter of one granule cell from the margin of granule cell layer, respectively. Immunoreactive cells were quantified in every twelfth 30-μm coronal section throughout the dentate gyrus. For double- or triple-stained sections, confocal images in Z planes were scanned for quantification. The numbers counted from each section were added and multiplied by 24 to estimate the total number of cells in one mouse brain.

Statistics

All of the data are expressed at mean ± S.E.M. Experimental groups were assigned by simple randomization. No statistical methods were used to pre-determine the sample size in each group, however the sample sizes are similar to those reported in previous publications (Parent J.M. et al. 1997; Pun R. et al. 2012; Cho K. et al. 2015). Data that passed selection criteria were collected blind. Prism software was used to perform statistical analysis (Version 6.0, Graphpad Software, Inc.) Statistical differences were analyzed using two-tailed Student’s t-test for the data with equal variances, or Student’s t-test with Satterwaite’s correction for the data with unequal variance. Figure 4C was analyzed using ANOVA analysis. Values of P<0.05 were considered statistically significant.

Figure 4. NeuroD1 is not required for Prox1-positive ectopic granule neurons.

Figure 4

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A, D) Experimental Timeline. Male and female NeuroD1 WT or cKO animals were given 5 days of tamoxifen injections and then subjected to sham-or pilocarpine treatment. Mice were euthanized 7 weeks post-sham or post-pilocarpine. B, E) Representative images from sham-treated or pilocarpine-treated WT and cKO animals stained with Dcx. C) Deletion of NeuroD1 significantly reduced the number of Dcx+ cells in the GCL (p=0.0226) in sham-treated mice (n=6 WT, n=8 cKO) but not in pilocarpine-treated mice (n=11 WT, n=10 cKO). F: Deletion of NeuroD1 significantly reduced the total number of Dcx+ cells ectopically localized in the hilus (p=0.0362) (n=11 WT, n=10 cKO). G: Deletion of NeuroD1 does not affect the total number of Prox1+ cells in the hilus in pilocarpine-treated mice (n=9 WT, n=10 cKO). Scale bar in B: 100um, inset bar in B: 25um, also valid for E. NS=Not significant. For C; Wildtype (sham) vs. Wildtype (pilo) p=0.2010, and wildtype (pilo) vs. cKO (pilo) p=0.3578.

Results

Pilocarpine-induced SE increases the number of NeuroD1+ cells in the hippocampus

To determine the role of NeuroD1+ cells in chronic epilepsy, we used the cholinergic agonist pilocarpine to induce SE. This model has been shown to faithfully recapitulate much of the pathology seen in human patients with mTLE, and has the benefit of leading to SRS in mice with only a single i.p. injection of pilocarpine (Cavalheiro E.A. et al. 1991; Covolan L. & Mello L.E. 2000; Sarkisian M.A. 2001; Curia G. et al. 2008). Female EnvigoC57BL/6NHsd mice at approximately 6 weeks of age were given a single injection of pilocarpine (185mg/kg) to induce SE, which was allowed to continue for 3 hours before seizures were terminated using diazepam. Mice were euthanized 3 weeks later to determine the total number NeuroD1+ cells found in both the GCL and hilar regions of the hippocampus (Fig 1A). Pilocarpine-treated animals showed a significant increase in the total number of NeuroD1+ cells in both the GCL and hilus as compared to control animals (Fig 1 B,C). In addition, the vast majority (>99%) of NeuroD1+ cells in both the GCL and hilus were found to co-express doublecortin (Dcx) (Fig 1 D, E), a marker of immature neuroblasts (Kempermann G. et al. 2015). These results were consistent with our previous work using a KA-induced mouse model of epilepsy (Cho K. et al. 2015). Therefore, we decided to further investigate the role of NeuroD1 in the cellular and functional outcomes of epileptogenesis with a model showing more robust SRS.

Deletion of NeuroD1 reduces the number of adult-generated neurons in the hippocampus

In order to delete NeuroD1 from the adult neural progenitor cell population found in the hippocampus, we used Nestin-CreERT2/R26R-YFP mice to conditionally delete NeuroD1 in the adult nestin-expressing stem/progenitor cell population (Lagace D.C. et al. 2007). Male and female NeuroD1 WT and cKO mice were given 5 consecutive days of tamoxifen injections to induce Cre-mediated recombination followed by pilocarpine-induced SE (220 mg/kg for males and 260 mg/kg for females), and then sacrificed 3 weeks later to confirm NeuroD1 deletion (Fig 2A). Quantification revealed that the total number of NeuroD1+ cells in the cKO was significantly reduced in the GCL at 3 weeks post-pilo, but unexpectedly not the hilus (Fig 2 B,C). Therefore, we decided to further examine the total number of YFP+ labeled cells expressing NeuroD1, Dcx, and Prox1. Surprisingly, we did not observe a reduction of YFP/NeuroD1+, YFP/Dcx+, or YFP/Prox1+ cells in the GCL or hilus of the cKO compared to the NeuroD1 WT littermates, although there was a reducing trend (Fig 2D–I). This finding could potentially be explained by inefficient recombination of the NeuroD1 allele in our cKO mice for several reasons which we will explore further in our discussion section. Taken together, although the reduction of NeuroD1 in YFP+ cells is lower than expected, the significant reduction of total NeuroD1+ cells prompted us to examine the role of NeuroD1 in epilepsy using the NeuroD1 cKO mice.

Figure 2. NeuroD1 is required for pilocarpine-induced adult-generated neurons.

Figure 2

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A) Experimental Timeline. Male and female NeuroD1 WT or cKO animals were given 5 days of tamoxifen and then subjected to pilocarpine induce status epilepticus (SE). Mice were euthanized 3 weeks post pilocarpine. B,C) Sections were stained with NeuroD1 to assess the total number of remaining NeuroD1 cells after NeuroD1 cKO in the GCL and hilus (p=0.0128). D,E) Sections were stained for YFP and NeuroD1 to confirm reduction of NeuroD1+ amongst the YFP+ population. F–I) Sections were stained with YFP/Dcx to assess reduction of immature neuronal, and YFP/Prox1 to assess reduction of mature neuronal cells in the recombined population in the GCL and hilus. (n=10 WT, n=7 cKO). Scale bar in B: 100um, inset bar in B: 25um, both valid for D,F,H. NS=Not significant.

Deletion of NeuroD1 in adult-generated neurons does not reduce SRS

To examine the role of NeuroD1 in epilepsy, we treated male and female NeuroD1 WT and cKO mice with 5 days of tamoxifen, followed by pilocarpine to induce epilepsy. These animals were then implanted with wireless EEG recording devices at 4 weeks post-pilo (Fig 3 A,B). Two cortical epidural screws were placed, one located over the hippocampus (Left parietal cortex; LPC) for recording generalized seizure activity, and the other over the olfactory bulb (Reference; R) to serve as reference activity (Fig 3 B). Starting at five weeks post-pilocarpine, freely moving animals were video/EEG recorded for a period of two continuous weeks (Fig 3A). The total number of generalized seizures was quantified and confirmed using video. Removal of NeuroDl from adult-generated neurons prior to SE did not significantly alter either the total number of seizures or the total duration of each seizure event (Fig 3 C,D). Together these results suggest that adult neural stem/progenitor-specific knockout of NeuroDl does not significantly impact SRS.

Figure 3. No change in SRS in animals with NeuroD1-deficient adult-generated neurons.

Figure 3

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A) Experimental Timeline. Animals were implanted with wireless EEG recording devices at 4 weeks post pilocarpine (pilo). EEG recording began at 5 weeks post-pilo and continued for a total of 2 weeks. B) Representative images showing where the wireless EEG screws were implanted and how the recording electrodes were attached. R=Reference, LPC=Left parietal cortex C) Deletion of NeuroD1 did not have a significant effect on spontaneous recurrent seizure (SRS) frequency or duration. D) Representative EEG seizure traces from WT and cKO animals. (n=11 wt, n=10 cKO), NS=not significant.

NeuroDl deletion does not alter the number of aberrant adult-generated neurons

To reconcile our data that NeuroDl deletion reduces the total number NeuroDl+ adult-generated neurons in the GCL and the surprising finding that NeuroDl cKO mice had similar levels of SRS compared to WT, we hypothesized that the level of aberrant neurogenesis was still above the threshold to reduce SRS. Thus, we performed histological analysis on mice seven weeks after pilocarpine treatment to determine the total number of immature Dcx+ neurons present in the GCL and hilus, and the total number of Proxl+ mature granule neurons in the hilus during the chronic period of epilepsy (Fig 4 A,D). Sections were stained with the immature neuroblast marker Dcx (Fig 4 B). There was no difference in the total Dcx+ cells within the GCL in NeuroDl cKO mice compared to WT (Fig 4 C). This result is in contrast to sham-treated (non-epileptic) animals sacrificed at the same time point which showed a significant reduction in the total number of Dcx+ cells localized to the GCL (Fig 4 B,C,E). Interestingly, deletion of NeuroDl did however reduce the total number of Dcx+ immature neuroblasts ectopically localized to the hilus in epileptic animals (Fig 4 E,F). However, the total number of Proxl+ mature ectopic granule cells was similar between NeuroDl WT and cKO mice (Fig. 4G). These results suggest that while NeuroDl deletion can remove a population of total NeuroDl+ cells in the GCL three weeks after pilocarpine treatment, this does not translate to a reduction of aberrant Dcx+ immature neurons at seven weeks after pilocarpine. These results provide evidence that suggests persistent levels of aberrant neurogenesis may contribute to SRS.

Discussion

Aberrant adult neurogenesis is one of the key hallmarks of mTLE and has been suggested to contribute significantly in the development of SRS (Parent J.M. et al. 1997; Parent J.M. et al. 1998; Parent J.M. et al. 2006; Walter C. et al. 2007b; Scharfman H.E. & McCloskey D.P. 2009; Myers C.E. et al. 2013; Cho K. et al. 2015). Therefore, it is critical to understand the role of key molecular players involved in controlling aberrant neurogenesis for the development of future therapeutics. NeuroD1 is a bHLH transcription factor that is transiently expressed in late-stage adult neural progenitors and neuroblasts, and is essential for the differentiation and survival of newborn neurons in the hippocampus (Gao Z. et al. 2009). In this study, we have shown that the number of NeuroD1+ immature neuroblasts increase in the hippocampus after SE, and are found in a large population of ectopic adult-generated neurons. Additionally, we demonstrate that deletion of NeuroDl in the pilocarpine model is sufficient to reduce the total number of Dcx+ ectopic neuroblasts by 52%. However, deletion of NeuroD1 from adult-generated neurons did not have any effect on SRS frequency or duration. Several possible explanations exist for why this might be the case, which we will discuss in more detail below.

Previous work from our lab has shown that near complete ablation (>98%) of adult-generated neurons prior to seizure can reduce overall seizure frequency by approximately 40% (Cho K. et al. 2015). Additionally, antimitotic treatment to kill proliferating neural progenitors after acute seizures also reduced the frequency of subsequent spontaneous seizures(Jung K.H. et al. 2004; Jung K.H. et al. 2006). The approaches utilized in prior work all removed a large population of aberrant adult-generated neurons, leaving open the question whether partial ablation would still be effective at reducing seizure frequency. Supporting the idea that a small number of residual aberrant adult-generated neurons may still contribute to epilepsy, previous work has shown that excessive activation of the mTOR signaling pathway in as few at 9% of postnatally generated hippocampal neurons is sufficient to cause spontaneous seizures(Pun R. et al. 2012). Therefore, it is possible that in our current study we failed to reduce the population of aberrant adult-generated neurons below the critical threshold needed to reduce SRS frequency. Indeed, in our work, while we saw a 52% reduction in the number of Dcx+ ectopic neuroblasts in our NeuroDl cKO mice, there was no change in the number of Dcx+ immature neurons in the GCL or Proxl+ ectopic granule cells after pilocarpine treatment. This could be due to a cohort of migratory Proxl+ cells that no longer required NeuroDl at the time of TAM administration, while the Dcx+ cells present still required NeuroDl for their survival and maturation. Regardless of whether it is the presence of Dcx+ cells in the GCT or Proxl+ cells in the hilus, a population of aberrant neurons persisted potentially leading to the lack of reduction in SRS seen in this study.

One limitation of this present study, and another possible reason for why we did not observe a reduction in SRS frequency is that NeuroDl deletion prior to SE removed granule cell progenitors present at the time of SE while failing to remove immature granule cells born weeks before or after the insult. This point is noteworthy because both immature granule cells born up to 5 weeks before and granule cells born after the insult may integrate abnormally and contribute to epilepsy (Jessberger S. et al. 2007; Walter C. et al. 2007a; Kron M.M. et al. 2010; Pun R. et al. 2012). In support of this hypothesis, recent work has shown that ablation of cells directly after SE that were labeled 5 weeks prior can lead to a 50% reduction in SRS suggesting that targeting a more comprehensive population of adult born neurons might be necessary in order to reduce SRS (Hosford B.E. et al. 2016). In the future, it would be interesting to determine whether more extensive administration of tamoxifen to delete NeuroDl+ cells before and after SE might lead to more efficient removal of aberrant adult-generated neurons that may serve as excitatory “hubs” within the hippocampal network. Additionally, emerging work also suggests functional heterogeneity amongst the newly generated granule cells, which may contribute differing, and perhaps even opposing, effects on epileptogenesis. Despite many reports describing that adult-generated granule cells display physiological and morphological changes in epilepsy models consistent with a pro-excitatory function (Esclapez M. et al. 1999; Ribak C.E. et al. 2000; Morgan R.J. et al. 2008; Pun R. et al. 2012), there are also studies suggesting these neurons exhibit decreased excitatory input (Williamson A. et al. 1999; Kobayashi M. & Buckmaster P.S. 2003; Jakubs K. et al. 2006). Moreover, reducing newly generated neurons before acute seizures increases sensitivity to kainic acid, suggesting that newly generated cells may show both increased and decreased excitability (Iyengar S.S. et al. 2015). Since our current work to delete NeuroDl cannot distinguish between different populations of adult neural stem/progenitor subpopulations, future work using longer tamoxifen treatment may allow for more comprehensive labeling and deletion of NeuroD1+ immature neuroblasts within the stem/progenitor population.

Lastly, the possibility remains that efficient Cre-mediated recombination of the NeuroDl allele in our cKO mice was not achieved. Consistent with this idea, there was no change in the number of YFP/NeuroDl+, YFP/Dcx+, and YFP/Proxl+ cells in NeuroDl cKO mice compared to WT, although there was a reducing trend in the GCL. These numbers are in contrast to our previous work showing a significant difference in YFP/Proxl and YFP/Ki67/Dcx at 3 weeks post seizure (Cho K. et al. 2015). Several potential explanations for this exist which we will go into greater detail below. Inefficient recombination could potentially by due to the fundamental differences between the two chemoconvulsant seizure models utilized (Houser C.R. & Esclapez M. 1996; Reddy D.S. & Kuruba R. 2013; Levesque M. et al. 2015). In our previous work, we used the drug kainic acid (KA) to induce acute seizures in our NeuroDl WT or cKO mice, whereas in our current work we utilized the pilocarpine method of chronic seizure formation. Experimental evidence suggests differences between each of these models, and it is possible that use of pilocarpine allows for greater survival of the YFP population of cells at 3 weeks, especially those ectopically localized to the hilus. This could be due to the fact that pilocarpine treatment results in chronic SRS at higher, more reproducible rates in treated animals, a process linked to an increase in neurogenesis. In other words, if YFP labeled cells were stimulated to divide by the action of SRS in the pilocarpine model, the total YFP population and its progeny would increase in number possibly explaining the strong trend, but ultimate lack of statistical significance. Further, the age at which our experimental mice received neurogenic ablation (ablation at 5.5 weeks of age for 5 days in our current pilocarpine model vs. ablation at 4.5 weeks of age for 5 days in our previous kainic acid model) followed by chemoconvulsant treatment differed from that of our previous work. As a result, the mice in each of these studies could have had differing rates of neurogenesis at the time of chemoconvulsant treatment resulting in the lack of efficiency seen in our current work (Cho K. et al. 2015). Additionally, the length of time between tamoxifen administration and chemoconvulsant treatment differed between this and our previous work (24 hrs later in our current pilocarpine model vs. 1 week later in our previous kainic acid model). This could have potentially allowed for more extensive deletion of the YFP labeled cell population in our previous study contributing to the significant difference in YFP/NeuroDl+ cells in our Cho et al. work, but not our current work (Cho K. et al. 2015).

Mesial temporal lobe epilepsy remains a major clinical challenge lacking interventional strategies due to an incomplete understanding of the underlying cellular and molecular mediators. Here in this study, we present data regarding the role of NeuroD1 in adult-generated neurons, and link its requirement in the development of chronic seizures. Our work supports the idea that efficient labeling of adult-generated neurons in combination with targeting key molecules is necessary to achieve complete ablation of pro-epileptic neurogenesis and reduce SRS. Furthermore, additional regulators of each step of seizure-induced adult neurogenesis may allow for more effective targeting of aberrant adult-generated neurons below a threshold to impact SRS. We also suggest that there may be a small population of aberrant adult-generated neurons that contribute to the overall recurring seizure activity. By highlighting these lessons learned, we hope this knowledge will inform future strategies aimed at preventing epilepsy.

Table 2. Summary of Experiments.

This table details the experiments conducted throughout this work and provides an easy reference to the strain of mice, experimental condition, survival, and results of analysis across all figures.

Figure: Mouse Line: Tamoxifen? Treatment: Sacrified: Staining: Result:
1 HC57Bl6 None Pilocarpine or Sham 3 wpp NeuroD1 Significant increased NeuroD1+ cells in GCL and hilus
2 NestinCreERT2;Rosa YFP; NeuroD1 +/+ or −/− 5 dpp Pilocarpine 3 wpp NeuroD1 Significant decrease in total NeuroD1+ in GCL of cKO
2 NestinCreERT2;Rosa YFP; NeuroD1 +/+ or −/− 5 dpp Pilocarpine 3 wpp YFP/NeuroD1; YFP/Dcx; YFP/Prox1 No significant change in GCL or hilus
3 NestinCreERT2;Rosa YFP; NeuroD1 +/+ or −/− 5 dpp Pilocarpine 7 wpp none No significant change in EEG frequency or duration
4 NestinCreERT2;Rosa YFP; NeuroD1 +/+ or −/− 5 dpp Pilocarpine 7 wpp Prox1 No significant change in total Prox1+ cells in hilus of pilocarpine treated animals
4 NestinCreERT2;Rosa YFP; NeuroD1 +/+ or −/− 5 dpp Pilocarpine or Sham 7 wpp Dcx Significant reduction of total Dcx+ cells in GCL of sham treated animals and total Dcx+ cells in hilus of pilocarpine treated animals. No change in total Dcx+ cells in GCL of pilocarpine treated animals
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Highlights.

  • NeuroD1 is upregulated following pilocarpine-induced status epilepticus

  • Conditional removal of NeuroD1 reduces the number of Dcx+ ectopic newborn neurons

  • Conditional removal of NeuroD1 does not affect SRS

Acknowledgments

We thank Zane Lybrand, Farrah Tafacory, and Ling Zhang for technical assistance and Jose Cabrera for graphics support. This work was supported by grants from National Institute of Health (NIH) (R01NS081203, R01NS089770, R01NS093992 and K02AG041815 to J.H.), Department of Defense W81XWH-15-1-0399 to J.H., American Heart Association 15GRNT25750034 to J.H., a grant from the Texas Institute for Brain Injury and Repair to J.H. and an NIH pre-doctoral training grant 5T32GM083831-05 to R.B.

Abbreviations

SRS

Spontaneous Recurring Seizures

TLE

Temporal Lobe Epilepsy

GCL

Granule Cell Layer

SGZ

Subgranular Zone

DG

Dentate Gyrus

YFP

Yellow Fluorescent Protein

KA

Kainic Acid

Dcx

Doublecortin

cKO

Conditional knock-out

WT

wildtype

ND1

NeuroD1

Footnotes

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Conflict of Interest: The authors declare no competing financial interests

References

  1. Bielefeld P, Van Vliet EA, Gorter JA, Lucassen PJ, Fitzsimmons CP. Different subsets of newborn granule cells: a possible role in epileptogenesis? European Journal of Neuroscience. 2013;39:1–11. doi: 10.1111/ejn.12387. [DOI] [PubMed] [Google Scholar]
  2. Binder DK, Steinhauser C. Functional Changes in Astroglial Cells in Epilepsy. Glia. 2006;54:358–68. doi: 10.1002/glia.20394. [DOI] [PubMed] [Google Scholar]
  3. Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L. Long-term effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilepsia. 1991;32:778–82. doi: 10.1111/j.1528-1157.1991.tb05533.x. [DOI] [PubMed] [Google Scholar]
  4. Cho K, Lybrand ZR, Ito N, Brulet R, Tafacory F, Zhang L, Good L, Ure K, Kernie SG, Birnbaum SG, Scharfman HE, Eisch AJ, Hsieh J. Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline. Nature Communications. 2015;26 doi: 10.1038/ncomms7606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Covolan L, Mello LE. Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus. Epilepsy Research. 2000;39:133–52. doi: 10.1016/s0920-1211(99)00119-9. [DOI] [PubMed] [Google Scholar]
  6. Curia G, Longo D, Biagini G, Jones RSG, Avoli M. The pilocarpine model of temporal lobe epilepsy. Journal of Neuroscience Methods. 2008;172:143–57. doi: 10.1016/j.jneumeth.2008.04.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C. Newly formed exictitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy. THe Journal of Comparative Neurology. 1999;408:449–60. doi: 10.1002/(sici)1096-9861(19990614)408:4<449::aid-cne1>3.0.co;2-r. [DOI] [PubMed] [Google Scholar]
  8. Gao Z, Ure K, Ables JL, Lagace DC, Nave K, Goebbels S, Eisch AJ, Hsieh J. NeuroDl is essential for the survival and maturation of adult-born neurons. Nature Neuroscience Brief Comm. 2009;12:1090–2. doi: 10.1038/nn.2385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goebbels S, Bode U, Pieper A, Funfschilling U, Schwab MH, Nave KA. Cre/loxP-mediated inactivation of the bHLH transcription factor gene NeuroD/BETA2. Genesis. 2005;42:247–52. doi: 10.1002/gene.20138. [DOI] [PubMed] [Google Scholar]
  10. Guillemot F. Cell fate specification in the mammalian telencephalon. Progress in Neurobiology. 2007;83:37–52. doi: 10.1016/j.pneurobio.2007.02.009. [DOI] [PubMed] [Google Scholar]
  11. Hosford BE, Liska JP, Danzer SC. Ablation of Newly Generated Hippocampal Granule Cells Has Disease-Modifying Effects in Epilepsy. Journal of Neuroscience. 2016;36:11013–23. doi: 10.1523/JNEUROSCI.1371-16.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Houser CR, Esclapez M. Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures. Epilepsy Research. 1996;26:207–18. doi: 10.1016/s0920-1211(96)00054-x. [DOI] [PubMed] [Google Scholar]
  13. Iyengar SS, LaFrancois JJ, Friedman D, Drew LJ, Denny CA, Burghardt NS, Wu MV, Hsieh J, Hen R, Scharfman HE. Supression of adult neurogenesis increases the acute effects of kainic acid. Experimental Neurology. 2015;264:135–49. doi: 10.1016/j.expneurol.2014.11.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jakubs K, Nanobashvili A, Bonde S, Ekdahl CT, Kokaia Z, Kaokaia M, Lindvall O. Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability. Neuron. 2006;52:1047–59. doi: 10.1016/j.neuron.2006.11.004. [DOI] [PubMed] [Google Scholar]
  15. Jessberger S, Parent J. Epilepsy and Adult Neurogenesis. Cold Spring Harbor Perspectives in Biology. 2015;7:1–10. doi: 10.1101/cshperspect.a020677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jessberger S, Römer B, Babu H, Kempermann G. Seizures induce proliferation and dispersion of doublecortin-positive hippcampal progenitor cells. Experimental Neurology. 2005;196:342–51. doi: 10.1016/j.expneurol.2005.08.010. [DOI] [PubMed] [Google Scholar]
  17. Jessberger S, Zhao C, Toni N, Clemenson GD, Jr, Li Y, Gage FH. Seizure-associated, aberrant neurogenesis in adult rats characterized with retrovirus-mediated cell labeling. Journal of Neuroscience. 2007;27:9400–7. doi: 10.1523/JNEUROSCI.2002-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jung KH, Chu K, Kim J, Jeong SW, Song YM, Lee ST, Kim JY, Lee SK, Roh JK. Continuous cytosine-b-D-arabinofuranoside infusion reduces ectopic granule cells in adult rat hippocampus with attenutation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus. European Journal of Neuroscience. 2004;19:3219–26. doi: 10.1111/j.0953-816X.2004.03412.x. [DOI] [PubMed] [Google Scholar]
  19. Jung KH, Chu K, Lee ST, Kim J, Sinn DI, Kim JM, Park DK, Lee JJ, Kim SU, Kim M, Lee SK, Roh JK. Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal neurogenesis with attenuation of spotaneous recurrent seizures following pilocarpine-induced status epilepticus. Neurobiology of Disease. 2006;23:237–46. doi: 10.1016/j.nbd.2006.02.016. [DOI] [PubMed] [Google Scholar]
  20. Kempermann G, Song H, Gage FH. Neurogenesis in the Adult Hippocampus. Cold Spring Harbor Perspectives in Biology. 2015 doi: 10.1101/cshperspect.a018812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kobayashi M, Buckmaster PS. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. Journal of Neuroscience. 2003;6:2440–52. doi: 10.1523/JNEUROSCI.23-06-02440.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kron MM, Zhang H, Parent JM. The developmental stage of dentate granule cells dictates their contribution to seizure-induced plasticity. Journal of Neuroscience. 2010;30:2051–9. doi: 10.1523/JNEUROSCI.5655-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lagace DC, Whitman MC, Noonan MA, Abies JL, DeCarolis NA, Arguello AA, Donovan MH, Fischer SJ, Farnbauch LA, Beech RD, DiLeone RJ, Greer CA, Mandyam CD, Eisch AJ. Dynamic Contribution of Nestin-Expressing Stem Cells to Adult Neurogenesis. Journal of Neuroscience. 2007;27:12623–9. doi: 10.1523/JNEUROSCI.3812-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Levesque M, Avoli M, Bernard C. Animal models of temporal lobe epilepsy following systemic chemoconvulsant administration. Journal of Neuroscience Methods. 2015;260:45–52. doi: 10.1016/j.jneumeth.2015.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morgan RJ, Soltesz I. Nonrandom connectivity of the epileptic dentate gyrus predicts a major role for neuronal hubs in seizures. PNAS. 2008;105:6179–84. doi: 10.1073/pnas.0801372105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Myers CE, Bermudez-Hernandez K, Scharfman HE. The influnence of ectopic migration of granule cells into the hilus on dentate gyrus-CA3 function. PLoS One. 2013;28 doi: 10.1371/journal.pone.0068208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV. Neurogenesis and Epilepsy. Jasper’s Basic Mechanisms of the Epilepsies (National Center for Biotechnology Information. 2012 [PubMed] [Google Scholar]
  28. Parent JM, Elliot RC, Pleasure SJ, Bárbaro NM, Lowenstein DH. Aberrant seizure-induced neurognesis is temporal lobe epilepsy. Annals of neurology. 2006;59:81–91. doi: 10.1002/ana.20699. [DOI] [PubMed] [Google Scholar]
  29. Parent JM, Janumpalli S, McNamara JO, Lowenstein DH. Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neuroscience Letters. 1998;247:9–12. doi: 10.1016/s0304-3940(98)00269-9. [DOI] [PubMed] [Google Scholar]
  30. Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH. Dentate Granule Cell Neurogenesis is Increase by Seizures and Contributes to Aberrant Network Reorganization in the Adult Rat Hippocampus. The Journal of Neuroscience. 1997;17:3727–38. doi: 10.1523/JNEUROSCI.17-10-03727.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pleasure SJ, Collins AE, Lowenstein DH. Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development. Journal of Neuroscience. 2000;20:6095–105. doi: 10.1523/JNEUROSCI.20-16-06095.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pun R, Rolle IJ, LaSarge CL, Hosford BE, Rosen JM, Uhl JD, Schmeltzer SN, Faulkner C, Bronson SL, Murphy BL, Richards DA, Holland KD, Danzer SC. Excessive Activation of mTOR in Postnatally Generated Granule Cells Is Sufficient to Cause Epilepsy. Neuron. 2012;75:1022–34. doi: 10.1016/j.neuron.2012.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Racine RJ. Modification of seizure activity by electrical stimulation II. Motor seizure. Electroencephalography. 1972;32:281–94. doi: 10.1016/0013-4694(72)90177-0. [DOI] [PubMed] [Google Scholar]
  34. Reddy DS, Kuruba R. Experimental Models of Status Epilepticus and Neuronal Injury for Evaluation of Therapeutic Interventions. International Journal of Molecular Sciences. 2013;14:18284–318. doi: 10.3390/ijms140918284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ribak CE, Tran PH, Spigelman I, Okazaki MM, Nadler JV. Status epilepticus induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry. Journal of Computational Neurology. 2000;428:240–53. doi: 10.1002/1096-9861(20001211)428:2<240::aid-cne4>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  36. Sarkisian MA. Overview of the Current Animal Models for Human Seizure and Epileptic Disorders. Epilepsy & Behavior. 2001;2:201–16. doi: 10.1006/ebeh.2001.0193. [DOI] [PubMed] [Google Scholar]
  37. Scharfman HE. Epilepsy as an example of neural plasticity. Neuroscientist. 2002;8:154–73. doi: 10.1177/107385840200800211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scharfman HE, McCloskey DP. Postnatal neurognesis as a therapeutic target in temporal lobe epilepsy. Epilepsy Research. 2009;85:150–61. doi: 10.1016/j.eplepsyres.2009.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shapiro LA, Korn MJ, Ribak CE. Newly generated dentate granule cells from epileptic rats exhibit elongated hilar basal dendrites that align along GFAP-immunolabeled processes. Neuroscience. 2005;136:823–31. doi: 10.1016/j.neuroscience.2005.03.059. [DOI] [PubMed] [Google Scholar]
  40. Walter C, Murphy BL, Pun RY, Speiles-Engemann AL, Danzer SC. Pilocarpine-induced seizures cause selective time-dependant changes to adult-generated hippocampal dentate granule cells. Journal of Neuroscience. 2007a;11:75. doi: 10.1523/JNEUROSCI.0431-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Walter C, Murphy BL, Pun RY, Spieles-Engemann AL, Danzer SC. Pilocarpine-induced seizures cause selective time-depedant changes to adult-generated hippocampal dentate granule cells. Journal of Neuroscience. 2007b;11:7541–52. doi: 10.1523/JNEUROSCI.0431-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Williamson A, Patrylo PR, Spencer DD. Decrease in inhibition in dentate granule cells from patients with medial temporal lobe epilepsy. Annals of neurology. 1999;45:92–9. doi: 10.1002/1531-8249(199901)45:1<92::aid-art15>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]

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