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Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGC) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, as they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. While it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we utilized a conditional, inducible transgenic mouse model to fate-map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, 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 to determine seizure frequency and severity. Positive correlations were found between seizure frequency and: 1) the percentage of hilar ectopic DGCs, 2) the amount of mossy fiber sprouting and 3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE, and also support a role for mossy cell loss.
Morphologically abnormal DGCs are a prominent feature of TLE models. Mossy fiber sprouting occurs when DGC axons, termed “mossy fibers,” project into the dentate inner molecular layer and form excitatory connections with the proximal apical dendrites of neighboring DGCs (Tauck and Nadler, 1985; Nadler, 2003). Mossy fiber sprouting has been described in almost all animal models of TLE, and has been consistently identified in humans with the condition (Sutula and Dudek, 2007; de Lanerolle et al., 2012). More recently, DGCs with basal dendrites projecting into the dentate hilus have been observed in numerous 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 by projecting into the dentate hilus these basal processes become targets for mossy fiber innervation. Finally, DGCs with their somata ectopically located in the dentate hilus have been 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 has become 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, are most 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 of recurrent excitatory connections within the dentate (Danzer, 2012), and computational modeling studies support a pro-epileptogenic role for these neurons (Morgan and Soltesz, 2008). Moreover, investigators have found that blocking neurogenesis after an epileptogenic brain injury, thereby reducing the “load” of abnormal newborn cells, reduces the frequency of spontaneous seizures (Jung et al., 2004; Jung et al., 2006). Conversely, increasing the load of abnormal DGCs by deleting the mTOR pathway inhibitor PTEN – which induces abnormal DGC integration – leads to the development of epilepsy in otherwise normal rodents (Pun et al., 2012).
If abnormal integration of newborn DGCs plays a critical role in epileptogenesis then it would be logical for an animal harboring a greater number of these cells to exhibit a more severe phenotype. Here, we tested this hypothesis by determining whether the percentage of newborn DGCs that integrated abnormally was correlated with seizure frequency or duration. Newborn DGCs were labeled using bitransgenic Gli1-CreERT2::GFP reporter mice. 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 assessed because loss of these neurons is implicated in TLE (Jiao and Nadler, 2007).
All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children’s Hospital Research Foundation and conform to NIH guidelines 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 mice homozygous for a CAG-CAT-EGFP (GFP) reporter construct driven by the CMV-B actin promoter (Nakamura et al., 2006). Nine Gli1-CreERT2::GFP reporter bitransgenic offspring from 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 hippocampal subgranular zone. Postnatal tamoxifen treatment of bitransgenic mice activates cre recombinase in these DGC progenitors (Ahn and Joyner, 2005; Murphy et al., 2011), leading to the persistent expression of GFP in the progenitor cells and all of their progeny. Mice were given injections of tamoxifen (250 mg/kg, s.c.) at 3, 5, 7, 9 and 11 weeks of age. At eight 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 monitored behaviorally for seizures and the onset of status epilepticus (defined as continuous tonic-clonic seizures). Following three hours of status epilepticus (SE) mice were given two injections of diazepam ten minutes apart (10 mg/kg, s.c.) to mitigate seizure activity. Mice were given sterile Ringers as needed to maintain pretreatment body weight and housed in an incubator overnight at 32°C. Animals were then returned to their home cages, where they were provided with food and water ad libitum with a 14/10 hour light/dark cycle.
Although non-epileptic animals (no pilocarpine treatment) were not explicitly included in the present study, we have examined GFP-expressing DGCs in more than 100 non-epileptic Gli1-CreERT2::GFP mice with tamoxifen injection protocols ranging from P7 to adulthood and 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 absence of mossy fiber sprouting, and incidences of GFP-expressing DGCs with basal dendrites or ectopic somata’s of roughly 1% or less. This low rate of DGC abnormalities in controls is consistent 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 present study is among epileptic animals with differing seizure frequencies, additional non-epileptic mice were not included.
Tamoxifen-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 at positions 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 above the dura. Additional support was provided by setting two skull screws and the entire assembly was secured with dental cement. Electrode wires fed into a two-lead wireless transmitter (DSI; TA11ETA-F10), which was placed subcutaneously under the back of each animal. 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). Synchronized video/EEG data was collected 24/7 for the next 3–4 weeks. Seizures were identified by an investigator blinded to morphological phenotype using Neuroscore software (DSI, version 3.4.2). Seizures were defined by an initial increase in EEG amplitude (minimum twice baseline) and progressive frequency changes over the course of the event (typically high frequency tonic firing followed by clonic bursting and ending with the appearance of theta activity; Figure 1A). To be scored as a seizure, the event had to have a minimum duration of ten seconds. Video data was also used to assess the behavioral manifestations of each EEG seizure according to the scale developed by Racine (1972). Because of the more limited resolution 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 as stage 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 ascending sucrose series (10, 20, 30%) in PBS and snap-frozen in isopentane at −25°C. Brains were sectioned coronally at 60 μm, and sections were mounted to gelatin coated slides and stored at −80°C.
Slide mounted brain sections (2–4 per slide) from both dorsal and ventral hippocampus were processed 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 secondary antibodies were used (Invitrogen, Eugene, OR). Tissue was dehydrated in alcohol series, cleared in xylenes and coverslips were secured with mounting-media (Krystalon, Harleco, Darmstadt, Germany).
Imaging was conducted using a Leica SP5 confocal system set up on a DMI 6000 inverted microscope equipped with a 63X oil immersion objective (NA 1.4). Images were collected by an investigator blind to seizure score. For each parameter, dentate gyri were analyzed from the left and right hemispheres for dorsal (2 mm posterior to bregma) and ventral (3 mm posterior 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 0.5 μm 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 per animal were imaged. For ventral hippocampus a similar strategy was used; however, due to the larger size, left and right dentate gyri were sampled. Specifically, confocal image stacks through the z-depth were collected from the midpoint of both the upper and lower blades of the dentate cell body layer (4 confocal z-stacks each with a field size 240 × 240 μm). Image stacks were imported into Neurolucida software to determine the percentage of GFP-expressing DGCs that also possessed basal dendrites projecting into the dentate hilus. Basal dendrites were distinguished from axons by their greater diameter and presence of dendritic spines. Basal dendrites were only counted for mature DGCs if their cell bodies were fully contained within the tissue section and correctly located in the DGC layer (normotopic DGCs). Mature DGCs were distinguished by the presence of a spiny apical dendrite projecting through the molecular layer and terminating at the hippocampal fissure and a basal axon extending into the hilus. Only DGCs with robust GFP labeling, such that dendritic structures were clearly revealed, were analyzed. All DGCs meeting selection criteria were scored.
To determine the percentage of newborn DGCs that were ectopic, dentate gyri were screened under epifluorescent illumination. DGCs were scored as ectopic if their cell body was located in the hilus and was at least two cell body diameters [≈20 μm] from the DGC layer-hilar border. Ectopic cells were counted using a variation of the optical dissector method (Howell et al., 2002).
To assess DGC mossy fiber axon sprouting, confocal optical sections of ZnT3-labeling were collected from the midpoint of the upper and lower blades of the dentate gyrus. Images were collected using identical confocal settings for each animal (63X; excitation wavelength 543 nm, 100% power; emission range collected 500–550 nm). To control for antibody penetration gradients, confocal optical sections were collected 3 μm below the surface of the tissue section for all samples. The area of ZnT3-immunoreactive puncta within the dentate inner molecular layer was quantified using Neurolucida software (version 3.4.2). Puncta were defined as ZnT3-immunoreactive regions with a diameter greater than 0.5 μm. The degree 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 of GluR2-labeling. Images stacks were captured from a 240 × 240 μm scanning field placed in the center of the hilus. Image stacks were collected with a 0.5 μm step through 10 μm of tissue, beginning 3 μm below the surface to avoid regions damaged by cryosectioning. The hilar area in each image stack was calculated using Neurolucida software by drawing a contour around the hilar border. Sample volume was determined by multiplying the hilar area examined by the depth of tissue imaged (10 μm). GluR2-positive mossy cells were counted using a variation of the optical dissector method (Howell et al., 2002). Small hilar GluR2-immunoreactive cells (8–12μm diameter) were considered to be ectopic DGCs and were excluded, while larger GluR2-expressing neurons (30–40μm diameter) localized to the hilus 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-expressing hilar mossy cells/mm3 of hilus]. For comparative purposes, mossy cell density was also determined in five C57BL/6 control (non-epileptic) mice.
Statistical analyses were performed using Sigma Stat software (version 12.3). Seizure frequency, seizure duration, Racine score and the number and percentages of abnormal cells were similar between males and females (not shown), so data were pooled for analysis. In the interest of thoroughness, correlations were generated using several different approaches. Firstly, correlations were generated for dorsal and ventral hippocampus separately, and then for dorsal and ventral regions combined. In the first case, the possibility that one region might predominate was explored; and in the second, the goal was to examine the impact of the overall load of abnormal cells throughout the hippocampus. Data were combined by taking the average of dorsal and ventral measures for each animal. In addition, correlations were generated using normalized data, in which the percentage of newborn DGCs that integrated abnormally was determined using the following equations: for ectopic cells [ectopic GFP expressing DGCs per dentate/total GFP expressing DGCs per dentate] and for cells with basal dendrites [normatopic GFP-expressing mature DGCs with basal dendrites per dentate/total normatopic GFP-expressing mature DGCs per dentate]. Since the efficiency of tamoxifen-induced recombination is not 100% (not all newborn cells are labeled), data were normalized to the number of GFP-expressing cells to insure that artifactual changes in tamoxifen-induced recombination efficiency didn’t impact the results (i.e. differences in tamoxifen 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 equal variance, while non-parametric versions of these tests were used for data that violated one or both assumptions. Pearson Product Moment Correlation was used unless otherwise specified in the text. P values < 0.05 were considered significant. Values are presented as means±SEM.
Figures were prepared using Adobe Photoshop (CS5-Extended). Brightness and contrast were adjusted to optimize cellular detail. Identical adjustments were made to all images meant for comparison.
To determine seizure frequency among pilocarpine-treated mice, continuous video/EEG data were recorded from neocortex for three to four weeks, beginning 11 weeks after status epilepticus. Eight of the nine pilocarpine-treated mice exhibited spontaneous recurrent seizures during the recording period (Figure 1A, B & F). Whether the one animal that exhibited no seizures would have developed epilepsy at later time points is not known. In addition, 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 cannot be excluded. All animals had electrodes placed at the same coordinates, however, so comparisons among animals are justified.
A total of 423 electrographic seizures were recorded for all animals combined. Seizure frequency among the eight animals with confirmed epilepsy ranged from 0.1 to 3.0 seizures per day (Figure 1B; mean for all animals combined was 1.8±0.4 seizures/day). Seizure duration was variable among animals, averaging from 15 to 43 seconds per seizure (Figure 1C; mean for all animals combined was 27.5±4.3 seconds/seizure). For the majority of EEG seizures (414), behavioral changes were observed according to the scale developed by Racine (Racine, 1972). The average Racine score for all animals combined was 3.8±0.3 (the range of individual animal averages was 2.0 to 4.4; Figure 1D). Racine score was significantly 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 seizure frequency 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 in seizure frequency (p=0.597, t-test). Seizures did, however, tend to occur in clusters (Figure 1F), consistent with previous studies (Goffin et al., 2007; Bajorat et al., 2011). Clusters were defined as the occurrence of five or more seizures preceded by and followed by at least two or more seizure-free days. Clusters lasted an average of 5.2±0.3 days with a mean of 27.9±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), became moribund in the days thereafter and was killed after three weeks of monitoring for animal welfare considerations. No other animals displayed declining health during the four-week recording period.
DGCs arising from recombined progenitor cells displayed robust GFP expression. Fine neuronal structures such as axons and spines were clearly distinguishable, providing more than ample resolution for the purposes of this study. For dorsal hippocampus, a total of 1710 GFP-expressing newborn DGCs were scored for both hilar basal dendrites and ectopic somata (range = 30 to 308 cells/animal; mean=190±35 cells/animal). For ventral hippocampus, all GFP-expressing DGCs present in the section were counted to determine the 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 ventral hippocampus, however, GFP expressing cells were sampled to determine the percentage with basal dendrites (see methods). A total of 330 normotopic DGCs from ventral hippocampus 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 immature granule cell marker calretinin to assess recombination efficiency. Three months after the last tamoxifen injection, 5.9±1.5% of immature (calretinin-immunoreative) DGCs expressed GFP, indicating that the Gli1-CreERT2 fate-mapping strategy used for the present study provides a reasonable sample of newborn DGCs throughout the experimental period.
The dorsal and ventral regions of the hippocampus are morphologically and functionally distinct (Fanselow and Dong, 2010); however, robust DGC neurogenesis occurs in both regions (Kaplan and Hinds, 1977; Jinno, 2011). To determine whether epileptogenesis differentially affects newborn cells generated in dorsal vs. ventral hippocampus, measures of abnormal 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 nine animals combined (Figure 1, G, I, K, M). In dorsal hippocampus 5.3±1.9% of newborn DGCs 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% of newborn normotopic DGCs had basal dendrites, while 26.3±6.3% of ventral newborn normotopic DGCs possessed basal dendrites (Figure 1I, p=0.963, t-test). The degree of mossy fiber sprouting was also similar between regions (Figure 1K, dorsal, 2.6±0.1% of IML area occupied by MFS; ventral, 3.6±1.0%; p=0.469, t-test). Lastly, the density of surviving hilar mossy cells was similar between regions (Figure 1M, dorsal, 12.5±5.8 cells per mm3; ventral, 15.9±6.4; p=0.756, Mann-Whitney Rank Sum Test). Within animals, most measures for dorsal and ventral parameters were significantly correlated. This was true for the percentages of newborn cells with basal dendrites (Figure 1J, R=0.767; p=0.016), the amount of mossy fiber sprouting (Figure 1L, R=0.726, p=0.027) and density of surviving mossy cells (Figure 1N, R=0.897, p=0.001). The one exception was ectopic cells, where no correlation between regions was found (Figure 1H, R=0.512, p=0.159). Together, these findings suggest that status epilepticus and the development of spontaneous seizures have similar impacts on DGC plasticity and mossy cell death in dorsal and ventral regions of the hippocampus. Although measurements were collected from a subset of sections from the two hippocampal regions, the correspondence between these relatively disparate regions suggests that our measures are likely representative of the entire hippocampus.
The percentage of newborn DGCs that were ectopic was significantly correlated with seizure frequency for dorsal and ventral hippocampus combined (Table 1, Figure 2F). Regionally, dorsal hippocampus did not significantly correlate to the percentage of ectopic DGCs, while ventral hippocampus did reach significance, though the trend was similar in both regions. The ectopic DGC population constituted less than 1% of all newborn DGCs in animals exhibiting the fewest seizures (≤0.1 seizures/day); ectopic DGCs were largely absent from these animals (Figure 2A & C). Conversely, animals with the highest seizure frequencies (>2.8 seizures/day) contained ectopic DGC populations exceeding 10% of all newborn DGCs (Figure 2B & D). There was no significant correlation between the percentage of hilar ectopic DGCs and seizure duration, either for dorsal and ventral hippocampus combined or individually (Figure 2G, Table 1). There was also no correlation between Racine score and the percentage of hilar ectopic DGCs (Table 1).
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 no observed seizures possessed almost no DGCs with basal dendrites (<1%). In the animal exhibiting the greatest frequency of seizures, more than 50% of the GFP-expressing newborn DGCs possessed basal dendrites (Figure 3B & D). Conversely, the animal exhibiting 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 of newborn cells with basal dendrites and seizure frequency for dorsal and ventral hippocampus combined (Figure 3F, Table 1). Similarly, neither region produced significant correlations when examined individually. There was, however, a significant positive correlation between basal dendrites and seizure duration within dorsal hippocampus. This effect was absent from ventral hippocampus, and didn’t quite reach significance for dorsal and ventral combined (Figure 3G, Table 1). There was no correlation between Racine score and the percentage of normotopic DGCs containing basal dendrites (Table 1).
Sprouting of DGC mossy fiber axons into the dentate inner molecular layer was assessed in each animal by ZnT3 immunohistochemistry. ZnT3 labeling reveals the axon terminals of both mature and newborn DGCs, and provides a reliable measure of mossy fiber sprouting (McAuliffe et al., 2011; Murphy et al., 2011). Mice that had few seizures exhibited little to no mossy fiber sprouting (Figure 4A–F), while mice exhibiting high seizure frequencies showed robust mossy fiber sprouting in the inner molecular layer (Figure 4G–L). The degree of mossy fiber sprouting was significantly correlated with seizure frequency for dorsal and ventral hippocampus combined, however only dorsal hippocampus reached significance when analyzed separately (Figure 4N, Table 1). Additionally, mossy fiber sprouting and seizure 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 was evident in ventral hippocampus (Table 1). The average Racine score paralleled the correlations between seizure duration and mossy fiber sprouting (Table 1).
Mossy 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 as a mediator of epileptogenesis (Sloviter et al., 2012) we sought to establish whether the extent of cell loss correlated with seizure frequency. To assess loss of mossy cells from the dentate hilus, sections were immunostained for glutamate receptor 2 (GluR2). GluR2 is expressed by glutamatergic mossy cells, which project into the dentate inner molecular layer. Consistent with previous studies, pilocarpine treatment significantly reduced mossy cell 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, we found a significant, negative correlation between the density of GluR2-expressing mossy cells and seizure frequency (Figure 5D, Table 1). Both dorsal and ventral hippocampus reached 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 with frequent seizures contained few, if any, mossy cells (Figure 5B). Additionally, there was a significant negative correlation between mossy cell density and seizure duration for dorsal and ventral hippocampus combined and individually (Figure 5D, Table 1). The same pattern of correlations was found between Racine score and mossy cell loss (Table 1). Of note, there was also a significant negative correlation between the density of surviving mossy cells and the amount of mossy fiber sprouting (R= −0.743, p=0.022).
The total number of GFP-expressing DGCs/hippocampal section was not significantly correlated with either seizure frequency or duration. This was true for both dorsal and ventral 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 the hippocampal dentate gyrus of epileptic Gli1-CreERT2 mice were DGCs (96.3±1.1%), a small number of cells meeting morphological criteria for protoplasmic astrocytes were found. GFP-expressing astrocytes ranged from two to 17 per hippocampal section. Their percentage as total number of GFP positive cells did not significantly correlate with seizure frequency (p=0.083, R=0.607), duration (p=0.322, R=0.373) or Racine score (p=0.101, R=0.620).
Among the nine animals examined, there were three clear jumps in seizure frequency (Table 2; group 1, 0.0 to 0.1 seizures/day; group 2, 0.1 to 0.64; group 3, 0.64 to >2.0). We took advantage of these jumps to query whether there were any notable changes in pathology within each group between the low and high seizure animals. This exercise produced two observations. Firstly, no single pathology consistently increased in severity in the high seizure animals relative to the low. Indeed, some measures of pathology actually improved with greater seizure frequency in individual animals (e.g. BD’s for animals 6–7 vs. 3, green arrowheads). Secondly, however, in all but one case (animal 7 vs. 3), at least one of the four variables did increase in severity (change of 50% or more; Table 2, red arrowheads). To quantify this effect, the animals were ranked from one to nine in order of increasing severity for each parameter, and the four rank values were added to generate a single “DGC abnormality measure” for each animal. Statistical analysis of this combined measure found that it significantly correlated with seizure frequency (R=0.816, p=0.007). These findings suggest that examination of all four variables together may better account for the differences in seizure frequency among animals than examination of any single variable alone.
Abnormal integration of adult-generated DGCs has been hypothesized to promote the development of TLE (Parent and Lowenstein, 2002). If abnormal DGCs promote epileptogenesis, then animals with a greater number or load of abnormal cells could be predicted to exhibit more frequent seizures. To test this hypothesis, we conducted 24/7 video/EEG monitoring studies in epileptic mice in which newborn DGCs were labeled with GFP using a genetic fate-mapping approach. The present study revealed a significant positive correlation between aberrantly-integrated newborn DGCs and seizure frequency; specifically, animals possessing a substantial percentage of ectopic newborn DGCs and robust mossy fiber sprouting exhibited seizures more often than epileptic animals in which a smaller percentage of newborn cells developed abnormally. While correlative, these findings are consistent with the hypothesis that abnormal DGCs promote temporal lobe epileptogenesis. In addition to the positive correlations between abnormal newborn DGCs and seizure frequency, a negative correlation was found between seizure frequency and the density of remaining hilar mossy cells; such that animals with the lowest density of surviving cells experienced seizures at the highest rate. Therefore, in addition to a role for newborn 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 significantly correlated with seizure duration and behavioral seizure score. These findings are important because they suggest that – in addition to regulating the incidence of seizures – dentate pathology might also regulate seizure severity. Taken together, the present findings provide new correlative evidence suggesting that aberrant neuronal integration, potentially acting in concert with cell loss, are key steps in temporal lobe epileptogenesis.
The present study was designed to assess the key morphological abnormalities found among newborn DGCs in the epileptic brain, as follows; 1) mossy fiber sprouting, 2) aberrant basal dendrites 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 of these neurons has been proposed as a key step in temporal lobe epileptogenesis (Sloviter et al., 2012). While correlations between some of these variables and seizures have been examined 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.
Examination of numerous parameters was made possible by using Gli1-CreERT2::GFP reporter expressing bi-transgenic mice. Treatment of these animals with tamoxifen leads to cre-mediated recombination and persistent GFP-expression in Gli1-expressing DGC progenitors. Gli1 is a transcription factor which is activated by the sonic hedgehog signaling pathway (Shh). Shh is a critical regulator of adult neurogenesis in the dentate (Lai et al., 2003; Pozniak and Pleasure, 2006; Han et al., 2008), so the Gli1-CreERT2 mice are a useful tool for selective labeling of adult-generated cells. Not all granule cell progenitors undergo recombination 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 a sample of the newborn cell population; and the percentage of recombined cells may have been higher at earlier time points. Importantly, studies by Ahn and Joyner (2005) indicate that the recombined cells are representative of the Shh-responding population. Consistent with this interpretation, the types of abnormalities observed in the present study, as well as the proportions of cells showing abnormalities, were similar to previously published studies using 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; Ribak et al., 2012). These observations support the conclusion that the bitransgenic labeling strategy used here provides a reliable measure of aberrant DGC integration rates.
The present study also takes advantage of new technologies that greatly simplify 24/7 seizure monitoring. Monitoring animals 24/7 can be critical, especially for the pilocarpine model, 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 dramatically underestimate seizure frequency; and improved techniques may account for the conflicting findings 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 on the current findings, that animals with intermediate seizure rates will exhibit intermediate levels of abnormal DGC integration. Until additional studies can be conducted with more animals representing intermediate seizure frequencies, however, the present findings should be interpreted with the more limited data set in mind.
In different ways, all four pathologies examined here are hypothesized to promote epileptogenesis. Firstly, ectopic migration of DGCs has been observed in numerous rodent models of TLE (Parent et al., 1997; Scharfman et al., 2000; Dashtipour et al., 2001; Bonde et 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 input compared to normotopic DGCs. Furthermore, these cells have been shown to generate spontaneous epileptiform bursts (Scharfman et al., 2000; Cameron et al., 2011), a phenomenon which is absent from normotopic DGCs. Therefore, it is conceivable that hilar DGCs act as seizure initiating “hub cells,” which have been hypothesized to play a key role in epileptogenesis (Scharfman and Pierce, 2012). Secondly, mossy fiber sprouting has long been associated with the epileptic brain (Tauck and Nadler, 1985). The formation of recurrent excitatory circuits via mossy fiber sprouting has made the phenomena an attractive candidate as a key regulator of dentate hyperexcitability (Golarai and Sutula, 1996). Thirdly, DGC basal dendrites, by projecting aberrantly into the dentate hilus, become targets for innervation by DGC mossy fiber axons, which normally form extensive collaterals in this region. Innervation of basal dendrites by mossy fiber axons creates functional recurrent circuits within the dentate (Ribak et al., 2000; Austin and Buckmaster, 2004; Shapiro and Ribak, 2006; Thind et al., 2008), potentially promoting hyperexcitability (Morgan and Soltesz, 2008). Finally, hilar mossy cells are commonly lost following epileptogenic brain injury. Mossy cells are glutamatergic excitatory neurons located in the dentate hilus that mediate both monosynaptic recurrent excitation and polysynaptic (via inhibitory GABAergic basket cells) recurrent inhibition of DGCs (Scharfman, 1995; Jackson and Scharfman, 1996; Ribak and Shapiro, 2007; Scharfman and Myers, 2013). Loss of these neurons is hypothesized to contribute to epileptogenesis by upsetting the balance of excitation and inhibition (Sloviter et al., 2012).
A key finding of the present study is that no single DGC pathology was able to account for seizure frequency in all animals. Rather, our data suggest that, to varying degrees, all four contribute. Even basal dendrites, which did not produce a significant correlation, still exhibited notable trends. Correlation does not prove causation, however, and one interpretation of these findings is that some (or all) reflect epiphenomena. Perhaps some feature yet to be examined will fully account for epileptogenesis, or only one of the changes examined here is truly epileptogenic, with the others occurring as secondary consequences of the first. Consistent with this latter idea, mossy fiber sprouting may be a consequence of mossy cell loss, rather than a cause of epilepsy (Buckmaster, 2012). Mossy cells innervate the portion of DGC apical dendrites located within the inner molecular layer. Extensive loss of mossy cells in rodent status epilepticus models of epilepsy partially deafferents these dendritic segments, creating an opportunity for innervation by sprouted DGC axons. Accordingly, mossy fiber sprouting has been shown to directly correlate to mossy cell loss following pilocarpine-induced SE (Jiao and Nadler, 2007; but see also Volz et al., 2011). A similar correlation was found in the present study.
It is also possible that multiple pathological changes act in concert to produce the epileptic state, potentially with different patterns of change accounting for the seizure phenotype in each animal. To explore this idea, we tabulated the data for all the animals (Table 2) and queried whether differences among the four parameters – either alone or in combination – could account for the biggest differences in seizure frequency. Interestingly, we found that although 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, these findings are consistent with the idea that seizure frequency in each animal reflects the combined effects of multiple pathological changes, and that these changes can pool in many different ways to produce the seizure phenotype for the animal.
This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and R01NS062806). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. We would like to thank Raymund Pun for his guidance with electrophysiological techniques. We would also like to thank Keri Kaeding for useful comments on earlier versions of this manuscript.
Conflict of Interests: The authors declare no competing financial interests