Altered apical dendritic structure among newly-generated granule cells exposed to status epilepticus
Brain sections from Thy1-GFP expressing control animals and animals that underwent pilocarpine-induced status epilepticus were screened to identify BrdU-labeled, GFP-expressing dentate granule cells. Eighteen cells from seven control animals and 44 cells from seven epileptic animals were identified for reconstruction. Cells were selected from dorsal hippocampus, so findings are limited to this region. BrdU-labeled, GFP-expressing granule cells from control animals exhibited the typical morphology of mature granule cells (Claiborne et al., 1990
), with round cell bodies located in the granule cell layer, fanlike dendritic trees extending to the hippocampal fissure, and an absence of basal dendrites (). By contrast, half of the granule cells born just before status epilepticus exhibited aberrant hilar basal dendrites, consistent with previous studies (Walter et al., 2007
Figure 2 Neurolucida reconstructions of newborn granule cells from Thy1-GFP expressing control animals (top row) and animals treated with pilocarpine to induce status epilepticus. All cells are 12-weeks-old, and cells from epileptic animals were exposed to status (more ...)
Obvious qualitative apical dendrite abnormalities were evident among 27% (12 of 44) of granule cells born just before status. By contrast, gross abnormalities were absent among cells from control animals (0 of 18; P=0.013, Fisher exact test). Abnormalities among cells from epileptic animals did not follow a consistent pattern; rather, they manifested primarily as a failure to develop typical fanlike apical dendritic trees, and were present equally among cells with and without basal dendrites. Cells with obvious abnormalities included three with dendrites that projected obliquely rather than directly into the molecular layer and four with collapsed, rather than spreading, apical dendritic trees. The former abnormality gave the affected cells a “windswept” appearance, while the latter cells resembled a “closed parasol” (). Both abnormalities are reminiscent of pathologies described by Scheibel and Scheibel (1973)
from human epileptic material. The remaining five cells exhibited generally disorganized apical dendritic trees (, atypical DGC) and included abnormalities such as crossing dendrites (, blue arrow), recurrent basal dendrites (, blue arrowheads), a projection which exited the dentate molecular layer (, red arrow) and a cell with a corkscrewed primary apical dendrite (not shown). Finally, although not included among the features scored definitively as abnormal, two cells possessed unusually large somas, giving them a hypertrophied appearance.
To confirm qualitative impressions of altered dendritic trajectory among newborn cells from epileptic animals (e.g. windswept cells), the angle of each apical dendrite initial segment was measured relative to a line drawn perpendicular to the granule cell body layer (such that greater angles reflect more oblique trajectories). Angles were significantly greater for cells from epileptic animals relative to controls (newborn control, 13.4° [0.8–33.5°]; newborn SE, 21.9° [1.0–87.5°]; P=0.034, Mann-Whitney rank sum test [RST]). Cells with the greatest angles appeared windswept.
The reduction in dendritic spread evident among cells with the “closed parasol deformation” would tend to increase the number of self-crossing dendritic branches (defined as dendrites that intersected in two-dimensional neuronal projections, so branches need not make physical contact to be scored as crossing). The number of crossing dendritic branches significantly increased among newborn cells exposed to SE (newborn control, 2.5 crossings/DGC (range=0–7); newborn SE, 4.0/DGC (range=0–17); P=0.032; t-test on data normalized by square root transformation). This finding confirms qualitative impressions that the apical dendrites of newborn granule exposed to SE tend to overlap and project into the same region of neuropil, rather than spreading evenly throughout the molecular layer.
In contrast to these changes, typical measures of apical dendritic structure, including apical dendrite number, total apical dendrite length, apical dendrite length by layer and total number of branch points, revealed similar values for both groups (). Sholl analyses, measure of dendritic nodes by layer and terminations by layer also revealed no significant differences (data not shown). Since dendritic truncation could minimize any real differences in these values, however, we took the additional step of generating estimates (see methods) to compensate for truncation effects. As with the raw data, estimated measures of total dendritic length (; P=0.443, RST) and dendritic length by layer (not shown) were statistically similar between groups. As another means of controlling for truncation effects, we examined several parameters that tend to be less affected by artificial truncation. Specifically, dendritic branches and branch segments (1st through 5th order) with intact terminations were examined. The length (apical dendrite length from the cell body to its natural ending) and terminal branch order for intact dendrites did not differ between groups (). The median length of 2nd and 3rd order dendritic segments, however, was significantly increased for cells exposed to status (). Taken together, these findings suggest a relative preservation of typical measures of dendritic structure, however, there are clearly subtle changes present, and since the impact of dendritic truncation cannot be entirely obviated, the possibility that other subtle changes remain undetected cannot be excluded.
The dendritic morphology of molecular layer border dentate granule cells is only minimally altered by status epilepticus
To determine whether status epilepticus disrupts the dendritic structure of granule cells already mature at the time of the insult, BrdU-negative, GFP-expressing cells located on the granule cell layer molecular layer border were examined. A total of 21 MLB granule cells were selected from 6 control mice, and 20 MLB cells were selected from 6 epileptic mice. In contrast to newborn granule cells exposed to status, MLB cells exposed to status were almost identical to MLB cells from control animals ( and ). None of the gross abnormalities evident among newborn cells were present among MLB cells () and evidence for somatic hypertrophy was absent (). Changes in dendritic trajectory (MLB control, 37.7° [3.9–75.9°]; MLB SE, 39.8° [11.8–89.1°]; P=0.927, Mann-Whitney RST) and crossing branches (MLB control, 3.0 crossings/DGC (range=0–7); MLB SE, 3.0/DGC (range=1–12); P=0.696; t-test on data normalized by square root transformation) were also absent. Curiously, however, apical dendrite length to a natural ending was slightly decreased among MLB SE cells (, P=0.022, rank sum test). A segment analysis revealed that first through fifth order segments were similar in control and epileptic mice, but terminal dendritic segments (which could be of any order), were significantly shorter for MLB SE cells (, P=0.015, rank sum test). Taken together, these findings indicate that granule cells located along the molecular layer border may exhibit a slight reduction in growth at the dendritic tips following status, but overall, their structure is relatively resistant to seizure-induced disruption.
Figure 3 Neurolucida reconstructions of molecular layer border (MLB) granule cells from Thy1-GFP expressing control animals (top row) and animals treated with pilocarpine to induce status epilepticus (bottom row). Apical dendrites are shown in black. Yellow lines (more ...)
Although MLB cells were not birthdated with BrdU, granule cells located along the molecular layer border are among the first generated during development (Schlessinger et al., 1975
; Bayer, 1980a
; Altman and Bayer, 1990a
). It is likely, therefore, that the majority of these cells were already mature at the time of the insult. We further tested this assumption by measuring the distance between the soma of each cell and the hilar border. The outside-in layering pattern of the dentate places the oldest cells close to the molecular layer border, and the youngest cells next to the hilar border. There was almost no positional overlap between the somas of birthdated newborn granule cells and MLB cells (Supplemental tables 1&2
; P<0.001 for both MLB groups vs. both newborn groups, ANOVA). Thus, although the exact age of MLB cells was not determined, they clearly represent a distinct cell population likely generated around embryonic day E15 (Mathews et al., 2010
Newborn granule cells in the epileptic brain have fewer spines
Dendritic spine density was measured along dendritic segments located within the dentate granule cell layer and the inner, middle and outer molecular layers (control, n=16 cells; SE, n=38), providing a means to assess relative numbers of excitatory synaptic inputs to the apical dendritic trees of these newly-generated cells (Trommald and Hulleberg, 1997
). In the case of the epileptic brain, changes in the granule cell layer and the inner molecular layer are of particular interest, as these regions are targeted by sprouted mossy fiber axons. Spine density was significantly lower in all three regions of the molecular layer for granule cells exposed to status epilepticus relative to controls (, ). The density of spines in the granule cell layer was unchanged. Since the dendritic trees of these cells were reconstructed, it was also possible to determine the total number of spines present on a cell in each layer (spine density per layer X apical dendrite length in that layer). Spine number was determined using both raw length data, and estimated length data (compensating for the number and position of dendritic truncations). While counts generated using the former approach did not differ significantly (), significant reductions in spine number were evident in the inner (P=0.035, RST) and middle (P=0.035, RST) molecular layers using the latter approach (). Estimated total spine number was also reduced for cells from epileptic animals relative to controls (; P=0.033, RST). These findings suggest that input to newborn granule cells is reduced in the epileptic brain.
Figure 4 Confocal reconstructions of dentate granule cell layer (DGCL) and inner (IML), middle (MML) and outer (OML) molecular layer dendrites of 12-week-old BrdU-labeled, GFP-expressing granule cells from control and epileptic (SE) animals. Cells from epileptic (more ...)
Figure 5 Estimated spine numbers (spine density X estimated AD length) for BrdU-labeled, GFP-expressing 12-week-old granule cells from control animals (gray boxes) and epileptic animals (SE, blue boxes). Granule cells from epileptic animals exhibited significant (more ...)
Molecular layer border granule cells exhibit reduced spine density after status
To determine whether MLB granule cells exhibit similar or disparate patterns of spine loss in the same animals, inner molecular layer spine density was quantified along dendritic segments belonging to cells from this population. In the inner molecular layer, MLB cells from epileptic animals (n=33) exhibited reductions in spine density relative to MLB cells from control animals (n=20), suggesting that both newborn and mature granule cells respond similarly to status epilepticus with regard to inner molecular layer spine changes (MLB control, 2.2±0.2 spines/μm; MLB SE, 1.4±0.1; P<0.001, t-test).
Spine number among newborn granule cells exposed to status is highly variable
Interestingly, while the majority of newborn granule cells exposed to status displayed reductions in spine density and number, the range was quite large and substantially overlapped the control population (). Gross observations supported this impression; with some newborn cells appearing almost aspiny, while others were coated with spines. This was true even for cells from the same epileptic animal, suggesting that inter-animal differences in seizure severity are unlikely to account for the variability.
Since high spine densities could be indicative of cellular hyperexcitabilty, we were quite intrigued by this subpopulation. Therefore, we queried whether cells with large numbers of spines exhibited other abnormal traits that might suggest a role in epileptogenesis. Qualitative observations were ambiguous. Both morphologically normal and abnormal cells exhibited high spine numbers (see , asterisks denote cells with high spine numbers). A more quantitative approach using a forward stepwise regression was thus conducted to systematically identify traits predictive of high spine density and number. The regression analysis utilized the dendritic measurements that differed between cells from control and epileptic animals, including basal dendrite length, primary dendrite trajectory and number of crossing branches. In addition, soma area was included based on qualitative findings of a somatic hypertrophy. Regression analyses focused on IML spine density and number, as this is the region targeted by sprouted mossy fiber axons. This approach identified basal dendrite length (P=0.025), and soma area (P<0.041) as key variables predicting inner molecular layer spine density, with cells having long basal dendrites and large somas tending towards greater spine density. A similar approach was used to determine which traits were predictive of spine numbers, although in this case crossing branch number was normalized by dividing by total dendrite length (since spine number and crossing branch number are both dependent on dendrite length, the two values would automatically be correlated otherwise). Both soma area (P=0.019) and basal dendrite length (P=0.047) were predictive of inner molecular layer spine numbers (), while only soma area was predictive of total spine numbers (P=0.002; ) among neurons from epileptic animals. In contrast to these findings, none of the traits examined predicted spine density or number for control cells ().
Figure 6 A & B: Three dimensional scatter plots showing relationships between estimated (EST.) inner molecular layer (IML) spine number, soma area and basal dendrite length. For 12-week-old granule cells from control animals (A) regression analyses indicated (more ...)
Given that longer basal dendrites predict greater spine number, we subdivided the cells from epileptic animals into groups with and without basal dendrites and queried whether spine values (generated using length estimates) for the two subgroups differed significantly. Interestingly, cells with basal dendrites had significantly more spines in the granule cell body layer (no BD, 37.5 [12–133]; BD, 64.0 [18–132]; P=0.045). Inner molecular layer values, on the other hand, did not differ significantly (IML, no BD, 213 [28–836]; BD, 287 [98–1504]). We interpreted this negative result cautiously, however, since the regression analyses only found basal dendrite length – not the presence of a basal dendrite per se – to be predictive.
Gli1-CreERT2 X GFP reporter double-transgenic mice
To further explore the association between spine density and basal dendrites, and to confirm and quantify the level of somatic hypertrophy among newborn cells, we utilized a conditional, inducible bi-transgenic mouse model that provides improved yields of newborn granule cells for study. This new approach used Gli1-CreERT2 X GFP reporter mice to fate-map adult-generated granule cells.
Fate-mapped newborn granule cells exhibit dendritic abnormalities and somatic hypertrophy
Gli1-CreERT2 X GFP-expressing bi-transgenic mice were dosed with tamoxifen following pilocarpine (or control) treatment to persistently label Gli1-expressing granule cell progenitors and all their progeny with GFP. While labeled progenitors could conceivably generate new cells throughout the three month survival period, morphologically immature granule cells were excluded from the study (see methods), so the cells examined here were likely born 0–2 months after status. Consistent with this interpretation, cells from Gli1 mice were qualitatively similar to the three-month-old cells labeled in the Thy1-GFP expressing animals. Moreover, while cells from control Gli1 animals possessed normal morphologies (), cells from epileptic animals exhibited all the key abnormalities described in the present and previous works, including ectopic granule cells, granule cells with basal dendrites and granule cells with windswept, collapsed and disorganized dendritic trees (). Numerous striking examples of hypertrophied granule cells, with enlarged somas and thick proximal dendrites, were also observed in epileptic mice (). To quantify this effect, the somatic area of 101 cells from control animals and 94 cells from epileptic animals was determined. Mean areas (±SD) for control and epileptic cells were 63.6±12.5 and 67±17.3 μm2, respectively). For each group, granule cells with soma areas two-standard deviations or greater above the control mean were defined as hypertrophied. In the epileptic group, 14% of cells met this criterion, while only 4% of cells were this large in controls. This difference was significant (P=0.029, Chi-square), confirming qualitative impressions from the BrdU-labeled, Thy1-GFP mice that a subset of newborn cells in epileptic animals are hypertrophied. Finally, although there was no evidence that any neuron types other than granule cells expressed GFP in the hippocampus of Gli1-CreERT2 mice, these hypertrophied cells so dwarfed their neighbors that we co-labeled some of the more impressive examples with the granule cell specific marker Prox1 (), confirming their identity as granule cells.
Figure 7 A–F: Confocal reconstructions of GFP-expressing newborn granule cells from Gli1-CreERT2 X GFP reporter mice. A: Confocal reconstruction of a newly-generated granule cell from a control animal with typical apical dendritic structure. B: Reconstruction (more ...)
Newborn granule cells with long basal dendrites have more spines and receive more recurrent input than adjacent newborn cells without basal dendrites
We next queried whether newborn cells with basal dendrites exhibited greater spine density and number in the inner molecular later relative to adjacent newborn cells lacking basal dendrites. In contrast to studies with Thy1-GFP mice, however, only granule cells with basal dendrites projecting at least halfway across the hilus were included to specifically address the question as to whether cells with long
basal dendrites possess more spines. In addition, spines were assessed to determine whether or not they were apposed to puncta immunoreactive for zinc transporter-3 (ZnT-3). Previous studies have demonstrated that ZnT-3 antibodies label mossy fiber terminals with high specificity (McAuliffe et al., 2010
), and immunostaining of control and epileptic mice confirmed the utility of this antibody for revealing mossy fiber sprouting grossly, and at the level of individual mossy fiber terminals (). Spines apposed to ZnT-3 immunoreactive puncta (), therefore, are likely receiving mossy fiber input.
Figure 8 A: Hippocampus from a control Thy1-GFP expressing mouse co-immunostained for ZnT-3 (A), which reveals mossy fiber terminals, and GFP (A.1 shows merged images). In control animals, ZnT-3 staining is most prominent in the mossy fiber pathway (hilus and (more ...)
Figure 9 Confocal images of GFP-expressing apical dendrite segments from the inner molecular layer. Sections are co-stained for ZnT-3, which reveals mossy fiber sprouting in these epileptic animals. Dendrites pictured belong to granule cells with basal dendrites. (more ...)
In epileptic Gli1 mice, inner molecular layer spine density was significantly lower among newborn granule cells lacking basal dendrites (no BD, n=10) relative to cells from control animals (n=10). Critically, however, cells lacking basal dendrites also differed significantly from neighboring newborn granule cells (BD+, n=10) with basal dendrites (control, 3.3 [2.3–6.6]; no BD, 1.8 [1.2–2.8]; BD+, 3.4 [2.1–8.1]; Kruskal-Wallis ANOVA on ranks, P<0.001). Similarly, inner molecular layer spine number (IML spine density X EST. IML dendrite length) was reduced for cells lacking basal dendrites relative to neighboring cells with basal dendrites and to cells from control animals (; Kruskal-Wallis, P=0.004). These findings confirm the prediction from Thy1-GFP based regression data () that cells with long basal dendrites will have more spines than cells lacking basal dendrites.
Figure 10 A: Estimated (EST.) number of inner molecular layer (IML) spines possessed by GFP-expressing newborn granule cells from control and epileptic Gli1-CreERT2 X GFP expressing mice treated with tamoxifen three months earlier. For epileptic animals (SE), cells (more ...)
Finally, the number of spines apposed to ZnT-3 immunoreactive puncta was determined for each group to assess the degree of recurrent mossy fiber innervation. While ZnT-3 apposed spines were almost non-existent in control animals, nearly half of the spines on newborn cells from epileptic animals were apposed to immunoreactive puncta. Moreover, while the proportion of spines apposed to ZnT-3 immunoreactive puncta was similar for cells from epileptic animals with and without basal dendrites (48 and 45%, respectively), since cells with basal dendrites possessed more than twice the total spines, their net input from ZnT-3 positive puncta was significantly greater (; ANOVA on ranked data with Holm-Sidak post test, P<0.001).