michael s. hester nih public access steve c. danzer author

Accumulation of abnormal adult-generated hippocampal granulecells predicts seizure frequency and severity

Michael S. Hester1,3 and Steve C. Danzer1,2,3

1Department of Anesthesia, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 452292Departments of Anesthesia and Pediatrics, University of Cincinnati, Cincinnati, OH, 452673Molecular and Developmental Biology Graduate Program, Cincinnati Children’s Hospital MedicalCenter, Cincinnati, OH, 45229

AbstractAccumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGC) ishypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have longbeen implicated in TLE, as they regulate excitatory signaling through the hippocampus and exhibitneuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they arecontinually generated throughout life, with aberrant integration of new cells underlying themajority of restructuring in the dentate during epileptogenesis. While it is known that theseabnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to beestablished whether they directly contribute to seizure generation. If abnormal DGCs docontribute, a reasonable prediction would be that the severity of epilepsy will be correlated withthe number or load of abnormal DGCs. To test this prediction, we utilized a conditional, inducibletransgenic mouse model to fate-map adult-generated DGCs. Mossy cell loss, also implicated inepileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored 24/7 by video/EEG for four weeks todetermine seizure frequency and severity. Positive correlations were found between seizurefrequency and: 1) the percentage of hilar ectopic DGCs, 2) the amount of mossy fiber sproutingand 3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death werecorrelated with seizure severity. These studies provide correlative evidence in support of thehypothesis that abnormal DGCs contribute to the development of TLE, and also support a role formossy cell loss.

IntroductionMorphologically abnormal DGCs are a prominent feature of TLE models. Mossy fibersprouting occurs when DGC axons, termed “mossy fibers,” project into the dentate innermolecular layer and form excitatory connections with the proximal apical dendrites ofneighboring DGCs (Tauck and Nadler, 1985; Nadler, 2003). Mossy fiber sprouting has beendescribed in almost all animal models of TLE, and has been consistently identified inhumans with the condition (Sutula and Dudek, 2007; de Lanerolle et al., 2012). Morerecently, DGCs with basal dendrites projecting into the dentate hilus have been observed innumerous rodent TLE models (Spigelman et al., 1998; Ribak et al., 2000; Murphy et al.,2012; Sanchez et al., 2012). In rodents, DGCs normally lack basal dendrites, and byprojecting into the dentate hilus these basal processes become targets for mossy fiber

Corresponding author: (Laboratory of Origin), Dr. Steve C. Danzer, 3333 Burnet Avenue, ML 2001, Cincinnati, Ohio 45229-3039,(513) 636-4526 (phone), (513) 636-7337 (fax), [email protected].

Conflict of Interests: The authors declare no competing financial interests

NIH Public AccessAuthor ManuscriptJ Neurosci. Author manuscript; available in PMC 2013 November 22.

Published in final edited form as:J Neurosci. 2013 May 22; 33(21): 8926–8936. doi:10.1523/JNEUROSCI.5161-12.2013.

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innervation. Finally, DGCs with their somata ectopically located in the dentate hilus havebeen identified in both animals (Scharfman et al., 2000) and humans (Parent et al., 2006)with TLE. These ectopic cells are hypothesized to drive seizures (Scharfman et al., 2000;Cameron et al., 2011).

Unlike many neurons, DGCs are generated throughout life, and in recent years it hasbecome clear that the majority of abnormal cells in epilepsy models are newly-generated.Both cells less than five weeks old at the time of an insult and cells born after an insult, aremost vulnerable (Jessberger et al., 2007; Walter et al., 2007; Kuruba et al., 2009; Kron et al.,2010; Murphy et al., 2011; Santos et al., 2011). Abnormal DGCs mediate the formation ofrecurrent excitatory connections within the dentate (Danzer, 2012), and computationalmodeling studies support a pro-epileptogenic role for these neurons (Morgan and Soltesz,2008). Moreover, investigators have found that blocking neurogenesis after an epileptogenicbrain injury, thereby reducing the “load” of abnormal newborn cells, reduces the frequencyof spontaneous seizures (Jung et al., 2004; Jung et al., 2006). Conversely, increasing theload of abnormal DGCs by deleting the mTOR pathway inhibitor PTEN – which inducesabnormal DGC integration – leads to the development of epilepsy in otherwise normalrodents (Pun et al., 2012).

If abnormal integration of newborn DGCs plays a critical role in epileptogenesis then itwould be logical for an animal harboring a greater number of these cells to exhibit a moresevere phenotype. Here, we tested this hypothesis by determining whether the percentage ofnewborn DGCs that integrated abnormally was correlated with seizure frequency orduration. Newborn DGCs were labeled using bitransgenic Gli1-CreERT2::GFP reportermice. Seizure frequency and severity were determined by 24/7 video/EEG monitoring.Although not directly related to neurogenesis, death of hilar mossy cells was also assessedbecause loss of these neurons is implicated in TLE (Jiao and Nadler, 2007).

MethodsAnimals

All procedures involving animals were approved by the Institutional Animal Care and UseCommittee of the Cincinnati Children’s Hospital Research Foundation and conform to NIHguidelines for the care and use of animals. To generate animals for the present study,hemizygous Gli1-CreERT2 mice (Ahn and Joyner, 2004, 2005) were crossed to micehomozygous for a CAG-CAT-EGFP (GFP) reporter construct driven by the CMV-B actinpromoter (Nakamura et al., 2006). Nine Gli1-CreERT2::GFP reporter bitransgenic offspringfrom this cross were used for experiments. All animals were on a C57BL/6 background.

The Gli1 promoter drives CreERT2 expression among progenitor cells in the hippocampalsubgranular zone. Postnatal tamoxifen treatment of bitransgenic mice activates crerecombinase in these DGC progenitors (Ahn and Joyner, 2005; Murphy et al., 2011), leadingto the persistent expression of GFP in the progenitor cells and all of their progeny. Micewere given injections of tamoxifen (250 mg/kg, s.c.) at 3, 5, 7, 9 and 11 weeks of age. Ateight weeks of age, mice received methyl scopolamine nitrate in sterile saline (1 mg/kg, s.c.)followed by pilocarpine (420 mg/kg, s.c.) 15 minutes later. Animals were monitoredbehaviorally for seizures and the onset of status epilepticus (defined as continuous tonic-clonic seizures). Following three hours of status epilepticus (SE) mice were given twoinjections of diazepam ten minutes apart (10 mg/kg, s.c.) to mitigate seizure activity. Micewere given sterile Ringers as needed to maintain pretreatment body weight and housed in anincubator overnight at 32°C. Animals were then returned to their home cages, where theywere provided with food and water ad libitum with a 14/10 hour light/dark cycle.

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Although non-epileptic animals (no pilocarpine treatment) were not explicitly included inthe present study, we have examined GFP-expressing DGCs in more than 100 non-epilepticGli1-CreERT2::GFP mice with tamoxifen injection protocols ranging from P7 to adulthoodand survival periods from one week to eight months (Murphy et al., 2011; Pun et al., 2012;unpublished observations). In all of these animals, we have consistently observed an absenceof mossy fiber sprouting, and incidences of GFP-expressing DGCs with basal dendrites orectopic somata’s of roughly 1% or less. This low rate of DGC abnormalities in controls isconsistent with the published literature (Buckmaster and Dudek, 1999; Scharfman et al.,2000; Ribak et al., 2000; McClosky et al., 2006; Jiao and Nadler, 2007; Walter et al., 2007;Jessberger et al., 2007; Buckmaster, 2012), and since the primary comparison in the presentstudy is among epileptic animals with differing seizure frequencies, additional non-epilepticmice were not included.

Video-EEG MonitoringTamoxifen-treated Gli1-CreERT2::GFP mice that developed SE (n=9; 5 male, 4 female)were implanted with EEG electrodes at 18 weeks of age in accord with established protocols(Castro et al., 2012). Briefly, animals were anesthetized with isoflurane (induction at 3.5%,maintainance at 1.5%); the skull was exposed; and 1 mm diameter holes were drilled atpositions 1.5 mm anterior to lambda and 1.5 mm lateral to midline over each hemisphere.The dura was left intact. A single wire electrode was then positioned in each hole just abovethe dura. Additional support was provided by setting two skull screws and the entireassembly was secured with dental cement. Electrode wires fed into a two-lead wirelesstransmitter (DSI; TA11ETA-F10), which was placed subcutaneously under the back of eachanimal. Animals were allowed to recover for one week, and then housed in(12″×6.5″×5.5″) cages placed on top of the wireless EEG receiver plates (DSI, RPC1).Animal behavior was monitored by video (Axis 221, resolution 640×480). Synchronizedvideo/EEG data was collected 24/7 for the next 3–4 weeks. Seizures were identified by aninvestigator blinded to morphological phenotype using Neuroscore software (DSI, version3.4.2). Seizures were defined by an initial increase in EEG amplitude (minimum twicebaseline) and progressive frequency changes over the course of the event (typically highfrequency tonic firing followed by clonic bursting and ending with the appearance of thetaactivity; Figure 1A). To be scored as a seizure, the event had to have a minimum duration often seconds. Video data was also used to assess the behavioral manifestations of each EEGseizure according to the scale developed by Racine (1972). Because of the more limitedresolution of the video and sometimes poor viewing angle, however, behavioral stage 1(mouth and facial movements) and stage 2 (head nodding) seizures were both scored asstage 1.5. At 23 weeks of age mice were anesthetized with pentobarbital (100 mg/kg, i.p.)and perfused with 1U/ml heparin, 2.5% paraformaldehyde and 4% sucrose in PBS (pH 7.4).Brains were removed, post-fixed overnight in the same fixative, cryoprotected in ascendingsucrose series (10, 20, 30%) in PBS and snap-frozen in isopentane at −25°C. Brains weresectioned coronally at 60 μm, and sections were mounted to gelatin coated slides and storedat −80°C.

ImmunohistochemistrySlide mounted brain sections (2–4 per slide) from both dorsal and ventral hippocampus wereprocessed for histological studies. Sections were double-immunostained with chicken anti-GFP (1:500, Abcam, Boston, MA) and either rabbit anti-zinc transporter 3 (ZnT3) (1:3000,Synaptic Systems, Gottingen, Germany), rabbit anti-GluR2 (1:200, Millipore, Chicago, IL)or mouse anti-calretinin (1:1000, Millipore, Temecula, CA). AlexaFluor488 goat anti-chicken, AlexaFluor594 goat anti-rabbit and AlexaFluor594 goat anti-mouse secondaryantibodies were used (Invitrogen, Eugene, OR). Tissue was dehydrated in alcohol series,

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cleared in xylenes and coverslips were secured with mounting-media (Krystalon, Harleco,Darmstadt, Germany).

Confocal Microscopy and Histological AnalysesImaging was conducted using a Leica SP5 confocal system set up on a DMI 6000 invertedmicroscope equipped with a 63X oil immersion objective (NA 1.4). Images were collectedby an investigator blind to seizure score. For each parameter, dentate gyri were analyzedfrom the left and right hemispheres for dorsal (2 mm posterior to bregma) and ventral (3 mmposterior to bregma; Paxinos and Franklin, 1997) hippocampus (4 dentate gyri per animal).

To assess basal dendrite frequency, images of GFP-expressing cells were collected at 0.5μm increments through the 60 μm z-depth of the tissue section to generate three-dimensional confocal “z-stacks.” For dorsal hippocampus, two complete dentate gyri peranimal were imaged. For ventral hippocampus a similar strategy was used; however, due tothe larger size, left and right dentate gyri were sampled. Specifically, confocal image stacksthrough the z-depth were collected from the midpoint of both the upper and lower blades ofthe dentate cell body layer (4 confocal z-stacks each with a field size 240 × 240 μm). Imagestacks were imported into Neurolucida software to determine the percentage of GFP-expressing DGCs that also possessed basal dendrites projecting into the dentate hilus. Basaldendrites were distinguished from axons by their greater diameter and presence of dendriticspines. Basal dendrites were only counted for mature DGCs if their cell bodies were fullycontained within the tissue section and correctly located in the DGC layer (normotopicDGCs). Mature DGCs were distinguished by the presence of a spiny apical dendriteprojecting through the molecular layer and terminating at the hippocampal fissure and abasal axon extending into the hilus. Only DGCs with robust GFP labeling, such thatdendritic structures were clearly revealed, were analyzed. All DGCs meeting selectioncriteria were scored.

To determine the percentage of newborn DGCs that were ectopic, dentate gyri werescreened under epifluorescent illumination. DGCs were scored as ectopic if their cell bodywas located in the hilus and was at least two cell body diameters [≈20 μm] from the DGClayer-hilar border. Ectopic cells were counted using a variation of the optical dissectormethod (Howell et al., 2002).

To assess DGC mossy fiber axon sprouting, confocal optical sections of ZnT3-labeling werecollected from the midpoint of the upper and lower blades of the dentate gyrus. Images werecollected using identical confocal settings for each animal (63X; excitation wavelength 543nm, 100% power; emission range collected 500–550 nm). To control for antibodypenetration gradients, confocal optical sections were collected 3 μm below the surface of thetissue section for all samples. The area of ZnT3-immunoreactive puncta within the dentateinner molecular layer was quantified using Neurolucida software (version 3.4.2). Punctawere defined as ZnT3-immunoreactive regions with a diameter greater than 0.5 μm. Thedegree of mossy fiber sprouting was defined as [(MFS area/total IML area) × 100].

The density of mossy cells in the hilus was determined from confocal image stacks ofGluR2-labeling. Images stacks were captured from a 240 × 240 μm scanning field placed inthe center of the hilus. Image stacks were collected with a 0.5 μm step through 10 μm oftissue, beginning 3 μm below the surface to avoid regions damaged by cryosectioning. Thehilar area in each image stack was calculated using Neurolucida software by drawing acontour around the hilar border. Sample volume was determined by multiplying the hilararea examined by the depth of tissue imaged (10 μm). GluR2-positive mossy cells werecounted using a variation of the optical dissector method (Howell et al., 2002). Small hilarGluR2-immunoreactive cells (8–12μm diameter) were considered to be ectopic DGCs and

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were excluded, while larger GluR2-expressing neurons (30–40μm diameter) localized to thehilus were counted as mossy cells (Fujise and Kosaka, 1999; Jiao and Nadler, 2007;Scharfman and Myers, 2013). Mossy cell hilar density is presented as [GluR2-expressinghilar mossy cells/mm3 of hilus]. For comparative purposes, mossy cell density was alsodetermined in five C57BL/6 control (non-epileptic) mice.

StatisticsStatistical analyses were performed using Sigma Stat software (version 12.3). Seizurefrequency, seizure duration, Racine score and the number and percentages of abnormal cellswere similar between males and females (not shown), so data were pooled for analysis. Inthe interest of thoroughness, correlations were generated using several different approaches.Firstly, correlations were generated for dorsal and ventral hippocampus separately, and thenfor dorsal and ventral regions combined. In the first case, the possibility that one regionmight predominate was explored; and in the second, the goal was to examine the impact ofthe overall load of abnormal cells throughout the hippocampus. Data were combined bytaking the average of dorsal and ventral measures for each animal. In addition, correlationswere generated using normalized data, in which the percentage of newborn DGCs thatintegrated abnormally was determined using the following equations: for ectopic cells[ectopic GFP expressing DGCs per dentate/total GFP expressing DGCs per dentate] and forcells with basal dendrites [normatopic GFP-expressing mature DGCs with basal dendritesper dentate/total normatopic GFP-expressing mature DGCs per dentate]. Since the efficiencyof tamoxifen-induced recombination is not 100% (not all newborn cells are labeled), datawere normalized to the number of GFP-expressing cells to insure that artifactual changes intamoxifen-induced recombination efficiency didn’t impact the results (i.e. differences intamoxifen absorption/distribution due to animal adipose tissue content or health; Lien et al.,1991). Parametric tests were used for data that met assumptions of normality and equalvariance, while non-parametric versions of these tests were used for data that violated one orboth assumptions. Pearson Product Moment Correlation was used unless otherwise specifiedin the text. P values < 0.05 were considered significant. Values are presented as means±SEM.

Figure preparationFigures were prepared using Adobe Photoshop (CS5-Extended). Brightness and contrastwere adjusted to optimize cellular detail. Identical adjustments were made to all imagesmeant for comparison.

ResultsSeizure frequency and duration following pilocarpine-induced status epilepticus

To determine seizure frequency among pilocarpine-treated mice, continuous video/EEG datawere recorded from neocortex for three to four weeks, beginning 11 weeks after statusepilepticus. Eight of the nine pilocarpine-treated mice exhibited spontaneous recurrentseizures during the recording period (Figure 1A, B & F). Whether the one animal thatexhibited no seizures would have developed epilepsy at later time points is not known. Inaddition, since EEG measures are limited to the brain regions in proximity to the electrodes(neocortex), the possibility that seizures occurring in other brain regions were missed cannotbe excluded. All animals had electrodes placed at the same coordinates, however, socomparisons among animals are justified.

A total of 423 electrographic seizures were recorded for all animals combined. Seizurefrequency among the eight animals with confirmed epilepsy ranged from 0.1 to 3.0 seizuresper day (Figure 1B; mean for all animals combined was 1.8±0.4 seizures/day). Seizure

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duration was variable among animals, averaging from 15 to 43 seconds per seizure (Figure1C; mean for all animals combined was 27.5±4.3 seconds/seizure). For the majority of EEGseizures (414), behavioral changes were observed according to the scale developed byRacine (Racine, 1972). The average Racine score for all animals combined was 3.8±0.3 (therange of individual animal averages was 2.0 to 4.4; Figure 1D). Racine score wassignificantly correlated to both seizure frequency (p=0.017, R=0.803) and seizure duration(p=0.027, R=0.764). Similarly, a significant correlation was found between high seizurefrequency and longer seizure duration (Figure 1E, p=0.018, R=0.760).

There were no overt changes in seizure frequency (Figure 1F) or duration (not shown)within animals during the recording period. There were also no day/night differences inseizure frequency (p=0.597, t-test). Seizures did, however, tend to occur in clusters (Figure1F), consistent with previous studies (Goffin et al., 2007; Bajorat et al., 2011). Clusters weredefined as the occurrence of five or more seizures preceded by and followed by at least twoor more seizure-free days. Clusters lasted an average of 5.2±0.3 days with a mean of27.9±3.3 seizures/cluster. The mean seizure-free period between clusters was 6.7±0.8 days.One animal (#8), which exhibited the largest seizure cluster (52 seizures in 5 days), becamemoribund in the days thereafter and was killed after three weeks of monitoring for animalwelfare considerations. No other animals displayed declining health during the four-weekrecording period.

GFP-labeling of newborn DGCsDGCs arising from recombined progenitor cells displayed robust GFP expression. Fineneuronal structures such as axons and spines were clearly distinguishable, providing morethan ample resolution for the purposes of this study. For dorsal hippocampus, a total of 1710GFP-expressing newborn DGCs were scored for both hilar basal dendrites and ectopicsomata (range = 30 to 308 cells/animal; mean=190±35 cells/animal). For ventralhippocampus, all GFP-expressing DGCs present in the section were counted to determinethe percentage of ectopic cells, as for dorsal hippocampus, yielding a total of 2718 DGCs(range = 119 to 575 cells/animal; mean = 302±53). Due the larger size of ventralhippocampus, however, GFP expressing cells were sampled to determine the percentagewith basal dendrites (see methods). A total of 330 normotopic DGCs from ventralhippocampus were scored for hilar basal dendrites (range = 18 to 53 cells/animal; mean =37±4 cells/animal).

Sections from a subset of animals (n=3) were immunostained with GFP and the immaturegranule cell marker calretinin to assess recombination efficiency. Three months after the lasttamoxifen injection, 5.9±1.5% of immature (calretinin-immunoreative) DGCs expressedGFP, indicating that the Gli1-CreERT2 fate-mapping strategy used for the present studyprovides a reasonable sample of newborn DGCs throughout the experimental period.

Dorsal versus ventral hippocampusThe dorsal and ventral regions of the hippocampus are morphologically and functionallydistinct (Fanselow and Dong, 2010); however, robust DGC neurogenesis occurs in bothregions (Kaplan and Hinds, 1977; Jinno, 2011). To determine whether epileptogenesisdifferentially affects newborn cells generated in dorsal vs. ventral hippocampus, measures ofabnormal DGC integration were compared across regions and correlated within animals.Overall, there were no differences in dorsal vs. ventral means for any parameter for all nineanimals combined (Figure 1, G, I, K, M). In dorsal hippocampus 5.3±1.9% of newbornDGCs were ectopically located in the hilus, while 3.1±0.9% of ventral cells were ectopic(Figure 1G, p=0.508, Mann-Whitney Rank Sum Test). In dorsal hippocampus, 26.7±5.4% ofnewborn normotopic DGCs had basal dendrites, while 26.3±6.3% of ventral newborn

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normotopic DGCs possessed basal dendrites (Figure 1I, p=0.963, t-test). The degree ofmossy fiber sprouting was also similar between regions (Figure 1K, dorsal, 2.6±0.1% ofIML area occupied by MFS; ventral, 3.6±1.0%; p=0.469, t-test). Lastly, the density ofsurviving hilar mossy cells was similar between regions (Figure 1M, dorsal, 12.5±5.8 cellsper mm3; ventral, 15.9±6.4; p=0.756, Mann-Whitney Rank Sum Test). Within animals, mostmeasures for dorsal and ventral parameters were significantly correlated. This was true forthe percentages of newborn cells with basal dendrites (Figure 1J, R=0.767; p=0.016), theamount of mossy fiber sprouting (Figure 1L, R=0.726, p=0.027) and density of survivingmossy cells (Figure 1N, R=0.897, p=0.001). The one exception was ectopic cells, where nocorrelation between regions was found (Figure 1H, R=0.512, p=0.159). Together, thesefindings suggest that status epilepticus and the development of spontaneous seizures havesimilar impacts on DGC plasticity and mossy cell death in dorsal and ventral regions of thehippocampus. Although measurements were collected from a subset of sections from thetwo hippocampal regions, the correspondence between these relatively disparate regionssuggests that our measures are likely representative of the entire hippocampus.

Ectopic DGCs significantly correlate with seizure frequency, but not seizure durationThe percentage of newborn DGCs that were ectopic was significantly correlated withseizure frequency for dorsal and ventral hippocampus combined (Table 1, Figure 2F).Regionally, dorsal hippocampus did not significantly correlate to the percentage of ectopicDGCs, while ventral hippocampus did reach significance, though the trend was similar inboth regions. The ectopic DGC population constituted less than 1% of all newborn DGCs inanimals exhibiting the fewest seizures (≤0.1 seizures/day); ectopic DGCs were largelyabsent from these animals (Figure 2A & C). Conversely, animals with the highest seizurefrequencies (>2.8 seizures/day) contained ectopic DGC populations exceeding 10% of allnewborn DGCs (Figure 2B & D). There was no significant correlation between thepercentage of hilar ectopic DGCs and seizure duration, either for dorsal and ventralhippocampus combined or individually (Figure 2G, Table 1). There was also no correlationbetween Racine score and the percentage of hilar ectopic DGCs (Table 1).

The percentage of normotopic DGCs with basal dendrites does not correlate with seizurefrequency, but may be linked with seizure duration

Basal dendrites were present on a substantial portion of the newborn DGC population(>10%) in all eight animals exhibiting at least one seizure, while the single animal with noobserved seizures possessed almost no DGCs with basal dendrites (<1%). In the animalexhibiting the greatest frequency of seizures, more than 50% of the GFP-expressingnewborn DGCs possessed basal dendrites (Figure 3B & D). Conversely, the animalexhibiting only 0.1 seizures per day was comparatively free of basal dendrites (Figure 3A &C). Despite these trends, there was no significant correlation between the percentage ofnewborn cells with basal dendrites and seizure frequency for dorsal and ventralhippocampus combined (Figure 3F, Table 1). Similarly, neither region produced significantcorrelations when examined individually. There was, however, a significant positivecorrelation between basal dendrites and seizure duration within dorsal hippocampus. Thiseffect was absent from ventral hippocampus, and didn’t quite reach significance for dorsaland ventral combined (Figure 3G, Table 1). There was no correlation between Racine scoreand the percentage of normotopic DGCs containing basal dendrites (Table 1).

The intensity of mossy fiber sprouting significantly correlates with seizure frequency andseizure duration

Sprouting of DGC mossy fiber axons into the dentate inner molecular layer was assessed ineach animal by ZnT3 immunohistochemistry. ZnT3 labeling reveals the axon terminals ofboth mature and newborn DGCs, and provides a reliable measure of mossy fiber sprouting

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(McAuliffe et al., 2011; Murphy et al., 2011). Mice that had few seizures exhibited little tono mossy fiber sprouting (Figure 4A–F), while mice exhibiting high seizure frequenciesshowed robust mossy fiber sprouting in the inner molecular layer (Figure 4G–L). The degreeof mossy fiber sprouting was significantly correlated with seizure frequency for dorsal andventral hippocampus combined, however only dorsal hippocampus reached significancewhen analyzed separately (Figure 4N, Table 1). Additionally, mossy fiber sprouting andseizure duration were significantly correlated when both regions were combined (Figure 4O,Table 1). The correlation was most robust in dorsal hippocampus, while only a trend wasevident in ventral hippocampus (Table 1). The average Racine score paralleled thecorrelations between seizure duration and mossy fiber sprouting (Table 1).

Mossy cell loss significantly correlates with seizure frequency and seizure durationMossy cells are extremely vulnerable to seizure-induced cell death (Sloviter, 1987;Scharfman et al., 2001; Danzer et al., 2010). Since loss of mossy cells has been proposed asa mediator of epileptogenesis (Sloviter et al., 2012) we sought to establish whether theextent of cell loss correlated with seizure frequency. To assess loss of mossy cells from thedentate hilus, sections were immunostained for glutamate receptor 2 (GluR2). GluR2 isexpressed by glutamatergic mossy cells, which project into the dentate inner molecularlayer. Consistent with previous studies, pilocarpine treatment significantly reduced mossycell density relative to control (non-epileptic) mice (control, n=5, 57.3±4.7; epileptic, n=9,14.2±6.0; p=0.005, Mann-Whitney Rank Sum Test). Within pilocarpine-treated animals, wefound a significant, negative correlation between the density of GluR2-expressing mossycells and seizure frequency (Figure 5D, Table 1). Both dorsal and ventral hippocampusreached significance individually. Mice exhibiting the fewest seizures (≤ 0.1 seizures/day)contained a dense network of mossy cells in the hilar area (Figure 5A), while mice withfrequent seizures contained few, if any, mossy cells (Figure 5B). Additionally, there was asignificant negative correlation between mossy cell density and seizure duration for dorsaland ventral hippocampus combined and individually (Figure 5D, Table 1). The same patternof correlations was found between Racine score and mossy cell loss (Table 1). Of note, therewas also a significant negative correlation between the density of surviving mossy cells andthe amount of mossy fiber sprouting (R= −0.743, p=0.022).

Seizure frequency is not correlated with the number of GFP-expressing neurons or thepercentage of newborn cells that developed as astrocytes

The total number of GFP-expressing DGCs/hippocampal section was not significantlycorrelated with either seizure frequency or duration. This was true for both dorsal andventral hippocampus (dorsal, seizure frequency, R=−0.058, p=0.882; seizure duration, R=−0.144, p=0.711; ventral, seizure frequency, R=0.309, p=0.419; seizure duration R=0.061,p=0.877). Finally, while the overwhelming majority of GFP-expressing cells within thehippocampal dentate gyrus of epileptic Gli1-CreERT2 mice were DGCs (96.3±1.1%), asmall number of cells meeting morphological criteria for protoplasmic astrocytes werefound. GFP-expressing astrocytes ranged from two to 17 per hippocampal section. Theirpercentage as total number of GFP positive cells did not significantly correlate with seizurefrequency (p=0.083, R=0.607), duration (p=0.322, R=0.373) or Racine score (p=0.101,R=0.620).

Examination of all four variables combined predicts seizure frequency bestAmong the nine animals examined, there were three clear jumps in seizure frequency (Table2; group 1, 0.0 to 0.1 seizures/day; group 2, 0.1 to 0.64; group 3, 0.64 to >2.0). We tookadvantage of these jumps to query whether there were any notable changes in pathologywithin each group between the low and high seizure animals. This exercise produced twoobservations. Firstly, no single pathology consistently increased in severity in the high

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seizure animals relative to the low. Indeed, some measures of pathology actually improvedwith greater seizure frequency in individual animals (e.g. BD’s for animals 6–7 vs. 3, greenarrowheads). Secondly, however, in all but one case (animal 7 vs. 3), at least one of the fourvariables did increase in severity (change of 50% or more; Table 2, red arrowheads). Toquantify this effect, the animals were ranked from one to nine in order of increasing severityfor each parameter, and the four rank values were added to generate a single “DGCabnormality measure” for each animal. Statistical analysis of this combined measure foundthat it significantly correlated with seizure frequency (R=0.816, p=0.007). These findingssuggest that examination of all four variables together may better account for the differencesin seizure frequency among animals than examination of any single variable alone.

DiscussionAbnormal integration of adult-generated DGCs has been hypothesized to promote thedevelopment of TLE (Parent and Lowenstein, 2002). If abnormal DGCs promoteepileptogenesis, then animals with a greater number or load of abnormal cells could bepredicted to exhibit more frequent seizures. To test this hypothesis, we conducted 24/7video/EEG monitoring studies in epileptic mice in which newborn DGCs were labeled withGFP using a genetic fate-mapping approach. The present study revealed a significantpositive correlation between aberrantly-integrated newborn DGCs and seizure frequency;specifically, animals possessing a substantial percentage of ectopic newborn DGCs androbust mossy fiber sprouting exhibited seizures more often than epileptic animals in which asmaller percentage of newborn cells developed abnormally. While correlative, these findingsare consistent with the hypothesis that abnormal DGCs promote temporal lobeepileptogenesis. In addition to the positive correlations between abnormal newborn DGCsand seizure frequency, a negative correlation was found between seizure frequency and thedensity of remaining hilar mossy cells; such that animals with the lowest density ofsurviving cells experienced seizures at the highest rate. Therefore, in addition to a role fornewborn DGCs, the present findings support a role for mossy cell death in epileptogenesis.Finally, robust mossy fiber sprouting and extensive mossy cell death were both significantlycorrelated with seizure duration and behavioral seizure score. These findings are importantbecause they suggest that – in addition to regulating the incidence of seizures – dentatepathology might also regulate seizure severity. Taken together, the present findings providenew correlative evidence suggesting that aberrant neuronal integration, potentially acting inconcert with cell loss, are key steps in temporal lobe epileptogenesis.

Relationship to prior studiesThe present study was designed to assess the key morphological abnormalities found amongnewborn DGCs in the epileptic brain, as follows; 1) mossy fiber sprouting, 2) aberrant basaldendrites and 3) hilar ectopic DGCs (Parent et al., 2006; Walter et al., 2007; Kron et al.,2010; Murphy et al., 2011). In addition, loss of hilar mossy cells was assessed, as death ofthese neurons has been proposed as a key step in temporal lobe epileptogenesis (Sloviter etal., 2012). While correlations between some of these variables and seizures have beenexamined in prior studies (Mathern et al., 1997; Gorter et al., 2001; McCloskey et al., 2006),to our knowledge this is the first study to examine all of these changes in the same animals.

Technical aspects of the present studyExamination of numerous parameters was made possible by using Gli1-CreERT2::GFPreporter expressing bi-transgenic mice. Treatment of these animals with tamoxifen leads tocre-mediated recombination and persistent GFP-expression in Gli1-expressing DGCprogenitors. Gli1 is a transcription factor which is activated by the sonic hedgehog signalingpathway (Shh). Shh is a critical regulator of adult neurogenesis in the dentate (Lai et al.,

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2003; Pozniak and Pleasure, 2006; Han et al., 2008), so the Gli1-CreERT2 mice are a usefultool for selective labeling of adult-generated cells. Not all granule cell progenitors undergorecombination following tamoxifen treatment in these animals; only 6% of recently-generated cells in our study expressed GFP three months after the last tamoxifen injection(calretinin-expressing cells are ≈2 weeks old). Nonetheless, this is sufficient to provide asample of the newborn cell population; and the percentage of recombined cells may havebeen higher at earlier time points. Importantly, studies by Ahn and Joyner (2005) indicatethat the recombined cells are representative of the Shh-responding population. Consistentwith this interpretation, the types of abnormalities observed in the present study, as well asthe proportions of cells showing abnormalities, were similar to previously published studiesusing a variety of different approaches (Buckmaster and Dudek, 1999; Jessberger et al.,2007; Walter et al., 2007; Kron et al., 2010; Murphy et al., 2011; Santos et al., 2011; Ribaket al., 2012). These observations support the conclusion that the bitransgenic labelingstrategy used here provides a reliable measure of aberrant DGC integration rates.

The present study also takes advantage of new technologies that greatly simplify 24/7seizure monitoring. Monitoring animals 24/7 can be critical, especially for the pilocarpinemodel, since seizures can occur in clusters (Goffin et al., 2007; Bajorat et al., 2011; Fig. 1F)- a phenomenon also seen in epileptic patients (Haut et al., 2002; Haut et al., 2005).Intermittent monitoring that misses even a single seizure cluster could dramaticallyunderestimate seizure frequency; and improved techniques may account for the conflictingfindings between the present work and past studies which did not utilize continuous EEG-monitoring (Cronin and Dudek, 1988), or monitored for only brief periods (Pitkanen et al.,2000; Nissinen et al., 2001).

Seizure frequency in the pilocarpine model is unpredictable, and in the present study,animals tended to exhibit either high or low rates (Figure 1B). We would predict, based onthe current findings, that animals with intermediate seizure rates will exhibit intermediatelevels of abnormal DGC integration. Until additional studies can be conducted with moreanimals representing intermediate seizure frequencies, however, the present findings shouldbe interpreted with the more limited data set in mind.

Significance of correlations between dentate pathology and seizure frequencyIn different ways, all four pathologies examined here are hypothesized to promoteepileptogenesis. Firstly, ectopic migration of DGCs has been observed in numerous rodentmodels of TLE (Parent et al., 1997; Scharfman et al., 2000; Dashtipour et al., 2001; Bondeet al., 2006; Fournier et al., 2010), as well as in tissue samples from epileptic humans(Parent et al., 2006). These cells receive a disproportionate amount of excitatory inputcompared to normotopic DGCs. Furthermore, these cells have been shown to generatespontaneous epileptiform bursts (Scharfman et al., 2000; Cameron et al., 2011), aphenomenon which is absent from normotopic DGCs. Therefore, it is conceivable that hilarDGCs act as seizure initiating “hub cells,” which have been hypothesized to play a key rolein epileptogenesis (Scharfman and Pierce, 2012). Secondly, mossy fiber sprouting has longbeen associated with the epileptic brain (Tauck and Nadler, 1985). The formation ofrecurrent excitatory circuits via mossy fiber sprouting has made the phenomena an attractivecandidate as a key regulator of dentate hyperexcitability (Golarai and Sutula, 1996). Thirdly,DGC basal dendrites, by projecting aberrantly into the dentate hilus, become targets forinnervation by DGC mossy fiber axons, which normally form extensive collaterals in thisregion. Innervation of basal dendrites by mossy fiber axons creates functional recurrentcircuits within the dentate (Ribak et al., 2000; Austin and Buckmaster, 2004; Shapiro andRibak, 2006; Thind et al., 2008), potentially promoting hyperexcitability (Morgan andSoltesz, 2008). Finally, hilar mossy cells are commonly lost following epileptogenic braininjury. Mossy cells are glutamatergic excitatory neurons located in the dentate hilus that

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mediate both monosynaptic recurrent excitation and polysynaptic (via inhibitoryGABAergic basket cells) recurrent inhibition of DGCs (Scharfman, 1995; Jackson andScharfman, 1996; Ribak and Shapiro, 2007; Scharfman and Myers, 2013). Loss of theseneurons is hypothesized to contribute to epileptogenesis by upsetting the balance ofexcitation and inhibition (Sloviter et al., 2012).

A key finding of the present study is that no single DGC pathology was able to account forseizure frequency in all animals. Rather, our data suggest that, to varying degrees, all fourcontribute. Even basal dendrites, which did not produce a significant correlation, stillexhibited notable trends. Correlation does not prove causation, however, and oneinterpretation of these findings is that some (or all) reflect epiphenomena. Perhaps somefeature yet to be examined will fully account for epileptogenesis, or only one of the changesexamined here is truly epileptogenic, with the others occurring as secondary consequencesof the first. Consistent with this latter idea, mossy fiber sprouting may be a consequence ofmossy cell loss, rather than a cause of epilepsy (Buckmaster, 2012). Mossy cells innervatethe portion of DGC apical dendrites located within the inner molecular layer. Extensive lossof mossy cells in rodent status epilepticus models of epilepsy partially deafferents thesedendritic segments, creating an opportunity for innervation by sprouted DGC axons.Accordingly, mossy fiber sprouting has been shown to directly correlate to mossy cell lossfollowing pilocarpine-induced SE (Jiao and Nadler, 2007; but see also Volz et al., 2011). Asimilar correlation was found in the present study.

It is also possible that multiple pathological changes act in concert to produce the epilepticstate, potentially with different patterns of change accounting for the seizure phenotype ineach animal. To explore this idea, we tabulated the data for all the animals (Table 2) andqueried whether differences among the four parameters – either alone or in combination –could account for the biggest differences in seizure frequency. Interestingly, we found thatalthough no single variable increased from animal to animal as seizure frequency increased,in almost all cases at least one of the four variables increased. Although speculative, thesefindings are consistent with the idea that seizure frequency in each animal reflects thecombined effects of multiple pathological changes, and that these changes can pool in manydifferent ways to produce the seizure phenotype for the animal.

AcknowledgmentsThis work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Award NumbersR01NS065020 and R01NS062806). The content is solely the responsibility of the authors and does not necessarilyrepresent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutesof Health. We would like to thank Raymund Pun for his guidance with electrophysiological techniques. We wouldalso like to thank Keri Kaeding for useful comments on earlier versions of this manuscript.

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Figure 1. EEG and hippocampal abnormalities in pilocarpine treated animalsCortical EEG recording showing a spontaneous seizure (A). Seizure frequencies in animalsexhibiting pilocarpine-induced status epilepticus ranged from 0 to 3 seizures per day (B).The average spontaneous seizure duration between animals ranged from 15 to 43 secondsper seizure (C), with higher seizure frequencies being positively correlated with longerseizure durations (E). The average behavioral seizure score for each animal ranged from 2.0to 4.4 (D). Four weeks of 24 hour video/EEG monitoring revealed the typical “seizureclustering” phenomenon associated with the pilocarpine model (F). No significantdifferences were found between dorsal and ventral hippocampus for the percentage ofectopic DGCs (G), the percentage of DGCs harboring basal dendrites (bd; I), the percentincrease of mossy fiber sprouting (mfs; K) or hilar mossy cell density (M). Dorsal andventral hippocampus were significantly correlated for the percentage of newborn DGCs withbasal dendrites (J), mossy fiber sprouting (L) and density of surviving mossy cells (N), butnot for ectopic DGCs (H). Bars are means ± SEM.

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Figure 2. Ectopically located newborn granule cells correlate with seizure frequencyImages are confocal maximum projections showing GFP-expressing newborn granule cellsin the dorsal hippocampus of bitransgenic mice. Mice experienced 0.1 (A & C) and 3.0 (B &D) seizures per day. Panels C & D are higher resolution images of the boxed regions inpanels A & B, respectively. The dentate hilus in each image is located between the dottedlines. Note the large number of ectopic granule cells in D, and the absence of such cells in C.E: Schematics showing the sample regions in red for ectopic cell measures for dorsal andventral hippocampus (the entire hilus). F, G: The percentage of newborn granule cellsectopically located in the dentate hilus was significantly correlated with seizure frequency,but not seizure duration (regression line is in red). Scale bar for A and B = 250 μm; C and D= 20 μm.

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Figure 3. Newborn granule cells’ basal dendrites and seizuresImages are confocal maximum projections from dorsal hippocampus showing GFP-expressing newborn granule cells with (B & D) and without (A & C) basal dendrites. Imageshave been processed using a depth filter, which assigns different colors to processes locatedat different depths within the tissue. Mice had mean seizure frequencies of 0.1 (A & C) and3.0 (B & D) seizures per day. Panels C & D are high magnification images of the boxedregions in panels A & B, respectively. The entire dentate was scored for dorsal hippocampus(E, region in red in the left schematic), while ventral hippocampus was sampled in the upperand lower blades of both hemispheres (E, right schematic). F, G: Scatterplots display a non-significant trend between the percentage of normotopic DGCs harboring basal dendrites andseizure frequency and duration (regression line is in red). Scale bar for A and B = 20 μm; Cand D = 10 μm.

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Figure 4. Mossy fiber sprouting correlates with seizure frequency and durationImages are confocal maximum projections showing ZnT3 and GFP immunostaining indorsal hippocampus of mice that exhibited means of 0.1 (A–F) and 2.1 (G–L) seizures perday. Arrowheads (I) highlight sprouted granule cell mossy fiber axons within the dentateinner molecular layer (IML). M: Schematics showing the sample regions in red for mossyfiber sprouting (MFS) measures for dorsal and ventral hippocampus. Greater mossy fibersprouting density was significantly correlated with increased seizure frequency (N) andseizure duration (O) [regression line is in red]. Granule cell layer (gcl); hilus (h). Scale barfor A–C and G–I = 250 μm; D–F and J–L = 50 μm.

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Figure 5. Mossy cells density negatively correlates with seizure frequency and durationImages are confocal maximum projections of GluR2 immunoreactivity in ventralhippocampus of mice that exhibited means of 0.1 (A) and 3.0 (B) seizures per day. Arrowsdenote presumptive mossy cells, while arrowheads denote presumptive hilar ectopic granulecells. Note the almost complete loss of mossy cell labeling in B. C: Schematics showing thesample regions in red for mossy cell counts for dorsal and ventral hippocampus. D, E:Scatterplots show low hilar mossy cell density in mice exhibiting high seizure frequency andduration (regression line is in red). Green dots denote mossy cell densities of age-matchedcontrol mice that did not undergo SE (not included for statistical analysis of correlations).Scale bar = 25μm.

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ach

mor

phol

ogic

al p

aram

eter

vs.

sei

zure

fre

quen

cy, s

eizu

re d

urat

ion

and

Rac

ine

scor

e in

dors

al (

D)

and

vent

ral (

V)

hipp

ocam

pus,

and

bot

h co

mbi

ned

(D+

V).

D+V

(R

)D

+V (

p)D

(R

)D

(p)

V (

R)

V (

p)

% D

GC

s E

ctop

icF

requ

ency

0.75

10.

020*

0.61

90.

076

0.76

30.

017*

Dur

atio

n0.

529

0.14

30.

386

0.30

50.

641

0.06

3

Rac

ine

0.46

70.

243

0.32

40.

434

0.58

30.

129

% D

GC

s w

ith

BD

sF

requ

ency

0.48

90.

182

0.61

70.

077

0.32

60.

392

Dur

atio

n0.

657

0.05

40.

763

0.01

7*0.

494

0.17

6

Rac

ine

0.56

20.

147

0.59

50.

120

0.44

80.

265

% I

ML

wit

h M

FS

Fre

quen

cy0.

673

0.04

7*0.

774

0.01

4*0.

527

0.14

5

Dur

atio

n0.

713

0.03

1*0.

746

0.02

1*0.

609

0.08

2

Rac

ine

0.80

60.

016*

0.84

60.

008*

0.66

40.

072

Mos

sy C

ell D

ensi

tyF

requ

ency

−0.

793

0.01

1*−

0.85

70.

003*

−0.

695

0.03

8*

Dur

atio

n−

0.84

50.

004*

−0.

879

0.00

2*−

0.77

30.

015*

Rac

ine

−0.

922

0.00

1*−

0.93

20.

001*

−0.

810.

015*


NIH


NIH


NIH


Hester and Danzer

Tabl

e 2

Raw

dat

a ra

nked

by

incr

easi

ng s

eizu

re f

requ

ency

Thr

ee g

roup

s ar

e pr

esen

ted

com

pari

ng a

nim

als

with

larg

e di

ffer

ence

s in

sei

zure

fre

quen

cy (

Gro

up 1

, ani

mal

2 v

s. 1

[0.

1 se

izur

es/d

ay v

s. 0

]; G

roup

2,

anim

al 3

vs.

2 [

0.64

sei

zure

s/da

y vs

. 0.1

]; G

roup

3, a

nim

als

4–9

vs. 3

[>

2 se

izur

es/d

ay v

s. 0

.64]

). G

reen

arr

owhe

ads

deno

te p

aram

eter

s th

at im

prov

edw

ith in

crea

sing

sei

zure

fre

quen

cy (

50%

dec

reas

e in

sev

erity

or

mor

e) a

nd r

ed a

rrow

head

s de

note

par

amet

ers

that

bec

ame

mor

e se

vere

with

incr

easi

ngse

izur

e fr

eque

ncy

(50%

incr

ease

in s

ever

ity o

r m

ore)

.

Ani

mal

#Se

izur

e D

urat

ion

Seiz

ure

Fre

quen

cy%

DG

Cs

Ect

opic

% D

GC

s w

ith

BD

s%

IM

L w

ith

MF

SM

ossy

Cel

ls/m

m ^

3 (×

1000

)

Gro

up 1

10.

000.

000.

550.

470.

0451

.11

214

.67

0.10

0.00

11.4

30.

1237

.00

Gro

up 2

214

.67

0.10

0.00

11.4

30.

1237

.00

332

.11

0.64

3.54

42.3

32.

767.

23

Gro

up 3

332

.11

0.64

3.54

42.3

32.

767.

23

433

.36

2.11

2.73

43.5

44.

664.

42

533

.49

2.45

4.11

23.9

27.

350.

00

625

.46

2.46

2.97

20.2

91.

291.

30

743

.24

2.54

3.65

18.5

53.

4413

.71

831

.71

2.89

10.2

426

.65

2.98

11.1

3

933

.77

3.00

10.0

650

.95

5.21

1.83


michael s. hester nih public access steve c. danzer author

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