newly generated granule cells show rapid neuroplastic changes in the adult rat dentate gyrus during...

Newly generated granule cells show rapid neuroplasticchanges in the adult rat dentate gyrus during the first fivedays following pilocarpine-induced seizures

Lee A. Shapiro, Sergio Figueroa-Aragon and Charles E. RibakDepartment of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA 92697–1275

Keywords: adult neurogenesis, doublecortin immunocytochemistry, epilepsy, hippocampus, subgranular zone

Abstract

Long-term neuroplastic changes to dentate granule cells have been reported after seizures and were shown to contribute to recurrentexcitatory circuitry. These changes include increased numbers of newborn granule cells, sprouted mossy fibers, granule cell layerdispersion, increased hilar ectopic granule cells and formation of hilar basal dendrites on granule cells. The goal of the current studywas to determine the acute progression of neuroplastic changes involving newly generated granule cells after pilocarpine-inducedseizures. Doublecortin (DCX) immunocytochemical preparations were used to examine the newly generated granule cells 1–5 daysafter seizures were induced. The results showed that there are rapid neuroplastic changes to the DCX-labeled cells. At 1 day afterseizures were induced, there were significant increases in the percentage of DCX-labeled cells with hilar basal dendrites and in theprogenitor cell population. At 2 days after seizures were induced, an increase in the thickness of the layer of DCX-labeled cellsoccurred. At 3 days after seizures were induced, the number of DCX-labeled cells was significantly increased. At 4 days afterseizures were induced, developing synapses were observed on DCX-labeled hilar basal dendrites. Thus, newly generated granulecells in the adult dentate gyrus display neuroplastic changes by 1 day after pilocarpine-induced seizures and further changes occur tothis population of cells in the subsequent 4 days. The presence of synapses, albeit developing ones, on hilar basal dendrites duringthis period indicates that newly generated granule cells become rapidly incorporated into dentate gyrus circuitry following seizures.

Introduction

Long-term neuroplastic changes have been reported for granule cellsin the dentate gyrus following spontaneous seizures in the pilocarpinerodent model of temporal lobe epilepsy (TLE). These changes includeincreased granule cell neurogenesis (Parent et al., 1997; Parent &Lowenstein, 2002), sprouted mossy fibers (Mello et al., 1993), granulecell layer dispersion (Mello et al., 1993), an increase in hilar ectopicgranule cells (Parent et al., 1997; Scharfman et al., 2000; Pierce et al.,2005), and the formation of hilar basal dendrites with synapses(Spigelman et al., 1998; Ribak et al., 2000). Some of these long-termchanges were observed for newly generated granule cells 30 days afterpilocarpine-induced seizures (Shapiro et al., 2005; Shapiro & Ribak,2006). It has also been reported that there is an increase in thepopulation of type-3 neural progenitor cells (Jessberger et al., 2006),which are mitotic cells that express an immature neuronal phenotype(Seri et al., 2004). Increased adult neurogenesis was also reported afterkainic acid induced-seizures (Gray & Sundstrom, 1998) and the rapidelectrical amygdala kindling (Smith et al., 2006).

Because previous studies primarily examined newly generatedneurons 3–4 weeks after pilocarpine-induced seizures, it is unclearwhether these neuroplastic changes occur during the acute timepointsimmediately after pilocarpine-induced seizures are initiated. Thus, thisstudy used immunocytochemical preparations of doublecortin (DCX)-labeled newly born granule cells (Brown et al., 2003; Rao & Shetty,

2004; Ribak et al., 2004) to test, during the first 5 days afterpilocarpine-induced seizures, four ideas: (i) to determine whether thereis an increase in the number of newly generated neurons; (ii) toanalyse the temporal growth pattern of DCX-labeled hilar basaldendrites; (iii) to determine whether DCX-labeled hilar basal dendritesare targeted for aberrant synaptogenesis; and (iv) to determine whetherthe number of type-3 neuronal precursor cells is altered followingseizures, by counting cells double-labeled for DCX and Ki-67.Together, these experiments demonstrate that newly generated neuronsdisplay several rapid neuroplastic changes after seizures and that thesechanges may provide an anatomical substrate for aberrant circuitformation in the epileptic dentate gyrus.

Materials and methods

Animals and seizure induction

Adult male Sprague–Dawley rats (200–300 g; Charles River Labor-atories, Wilmington, MA, USA) were used in this study. Allexperiments were carried out in accordance with the NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals and with the approval of the Institutional Animal Care andUse Committee at the University of California, Irvine. To induceseizures, the animals were first injected with scopolamine (1 mg ⁄ kg)and, 30 min later, pilocarpine (300-320 mg ⁄ kg; i.p.). Animals weremonitored for 2 h to confirm that they demonstrated at least stage 5seizures (Racine, 1972). Animals that did not reach stage 5 seizureswere omitted from the analysis. At 2 h after seizure induction, the rats

Correspondence: Dr Charles E. Ribak, as above.E-mail: [email protected]

Received 3 August 2006, revised 17 May 2007, accepted 29 May 2007

European Journal of Neuroscience, Vol. 26, pp. 583–592, 2007 doi:10.1111/j.1460-9568.2007.05662.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

were injected with Nembutal to stop seizure activity. Control rats(n ¼ 5) were given all of the same injections except for pilocarpine,which was substituted with an equal quantity of 0.9% saline. Duringthe recovery stage, the animals were placed in individual cages andprovided with food and water ad libitum. The rats with stage 5seizures were allowed to survive for 1, 2, 3, 4 or 5 days (n ¼ 4 pertimepoint; total n ¼ 20) after the initiation of seizures. Only one groupof control rats was compared to the groups from these five timepoints(n ¼ 5).

Perfusion and tissue preparation

Animals were anaesthetized with an overdose of euthasol (23 mg ⁄ kgpentobarbitol sodium and 3 mg ⁄ kg phenytoin sodium), and perfusedintracardially with 200 mL of saline followed by 150 mL of 4%paraformaldehyde in 0.1 m phosphate buffer. The brains were allowedto fix within the skull for 48 h, and then extracted and placed into 4%paraformaldehyde for 48 h. Coronal sections were cut at 50 lm with avibratome (Pelco 1000 Plus), and serial sections were collected into12-well culture dishes containing 0.01 m PBS.Sections to be used for immunocytochemistry were blocked with

0.5% H2O2 for 30 min, followed by 60 min in 1% H2O2, and thenagain for 30 min in 0.5% H2O2. Sections were then rinsed with PBSand incubated in anti-DCX (1 : 500; Santa Cruz Biotechnology Inc.,Santa Cruz, CA, USA) diluted in PBS and 5% normal horse serum,rotating for 48 h, at room temperature. The sections were then rinsedfor 25 min in PBS and incubated in biotinylated antigoat IgG (1 : 200;Vector Laboratories, Burlingame, CA, USA), rotating for 1 h. Sectionswere rinsed again in PBS, and incubated for 75 min in ABC solution(Vector Laboratories) in PBS. Following incubation, sections wererinsed with PBS for 20 min and were developed by incubating indiaminobenzidine (0.025%) and H2O2 (0.002%) solution. Finally,sections were rinsed with PBS for 30 min, mounted onto glass slidesand dehydrated in graded alcohol baths; coverslips were applied usingPermount.Serial sections of the ones used above were stained for double-

immunofluorescence analysis of the DCX ⁄ Ki-67-labeled cells. Thetissue was blocked in peroxide as described above, followed byincubation for 48 h in a mixture of DCX (1 : 500) and Ki-67 (1 : 200;Vector Laboratories) antibodies in 3% normal horse serum, with0.05% Triton X-100 in PBS. The tissue was next rinsed in PBS andincubated for 2 h in fluorescent-tagged secondary antibodies directedagainst goat (for DCX) or rabbit (for Ki-67) antibodies. The tissue wasrinsed in PBS and mounted onto clean glass slides using distilled water.The tissue was allowed to dry overnight and coverslips were appliedusing Fluoromount-G (Southern Biotech, Birmingham, AL, USA).

Analysis of DCX-labeled cells and their hilar basal dendrites

Sections containing DCX-labeled cells and their labeled dendriteswere viewed using either a Zeiss Axioplan light microscope or aNikon Eclipse E600 light microscope. Images were captured usingeither a Zeiss Axio-Vision camera or a MTI CCD-300T IFG camera.The number of DCX-labeled cells was quantified by using the StereoInvestigator program (MicroBrightfield Inc., Burlington, VT, USA).Briefly, every 12th hippocampal section was analysed from eachanimal in the study (five controls and 20 with pilocarpine-inducedseizures). This was done by drawing a boundary around the granulecell layer of the dentate gyrus using a cursor and then having theprogram select random portions of this area for counting the numberof DCX-labeled cell bodies within a 250-lm2 area. The counts were

made by an individual who was blinded to the identity of theanimal’s treatment and survival time following pilocarpine-inducedseizures.The depth of the DCX-labeled cell body layer was also analysed for

rats at each of the 1- to 5-day timepoints and for the controls todetermine whether there was a thickening of this layer after pilocar-pine-induced seizures. Measurements were taken using a calibratedline that was drawn at 10 points along the suprapyramidal andinfrapyramidal blades where DCX-labeled cell bodies were observed(Image Pro program; Media Cybernetics, Inc., Bethesda, MD, USA).The apex of the dentate gyrus was not included in this analysis.Analysis of the hilar basal dendrites was done using the Zeiss

Axioplan light microscope with a 40· (NA ¼ 0.75) objective lens andthe Axio-Vision camera for video-assisted analysis. Hilar basaldendrites were defined as a DCX-labeled process emanating fromthe basal part of the DCX-labeled cell body and extending into thehilus. Recurrent basal dendrites were not included in the analysisbecause they do not enter the hilus but instead course along the base ofthe granule cell layer for varying distances before entering this layer.The percentage of DCX-labeled cells with hilar basal dendrites wascalculated by counting the number of DCX-labeled cells within a250 · 250 lm counting frame, and then counting which of theseDCX-labeled cells had hilar basal dendrites.For these three parameters (no. of DCX-labeled cells, thickness of

the layer of DCX-labeled cells and percentage of DCX-labeled cellswith basal dendrites), descriptive statistics were calculated to examinethe variation in means for the group of control rats at baseline (n ¼ 5)and in the groups measured at successive 24-h periods (n ¼ 4 each)for the first 5 days following pilocarpine-induced seizures. One-wayanova was used to test for an overall difference between groupmeans. Dunnett’s method (1955), with a significance level of 0.05,was applied to test for pair-wise differences between the mean for thecontrol group vs. those of the five experimental groups (1-, 2-, 3-,4- and 5-day) and to compare the mean value obtained at day 5 vs. themeans for the other four experimental groups (1-, 2-, 3- and 4-day).For pair-wise tests, the adjusted P-values are presented. Linearregression analysis was applied to model the mean percentage ofDCX-labeled cells with basal dendrites from the number of days afterpilocarpine-induced seizures.To analyse whether the DCX-labeled hilar basal dendrites were

longer at each successive day, confocal Z-stack images of the dentategyrus were first captured (60·, NA ¼ 1.40) with a Bio-Rad Radiance2100 laser scanning confocal microscope using lambda-strobing toavoid nonspecific cross-excitation or detection. A grid with equallyspaced 10-lm points was placed over the merged confocal Z-stackimages containing DCX-labeled basal dendrites. The grid was alignedwith the base of the granule cell layer. Only those processes whichcould be traced back to a DCX-labeled cell body were counted. Thiswas done to avoid the possibility that the process was in fact an apicaldendrite from a DCX-labeled cell in the hilus. It is pertinent to notethat the thickness of the tissue sections was 50 lm and serial sectionreconstructions were not performed. Therefore, these measurementsprobably underestimate the full extent of these dendrites. Using thesecriteria, the number of dendritic endings was counted within each levelof grid squares. This allowed quantification of the frequency of thedendrites at each 10-lm increment (from 10 to 100 lm) from the baseof the granule cell layer, as well as analysis of the maximum distancewithin the hilus. Five random sampling points, each with an area of137 lm2, were examined from both the infra- and supra-pyramidalblades, for a total of 10 sampling points per animal. The total numberof dendritic endings over all sections was recorded for each distance(10–100 lm). Descriptive statistics were calculated and box plots

584 L. A. Shapiro et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 26, 583–592

were generated to examine the variation in mean dendritic endings inthe group of control rats at baseline (n ¼ 5) and in groups measured atsuccessive 24-h periods (n ¼ 4 per timepoint), for the first 5 daysfollowing pilocarpine-induced seizures. For analysis of mean numberof dendritic endings at the different distances from the granule celllayer, the generalized estimating equation (GEE) method was appliedwith two factors, group and distance into the hilus, an exchangeableworking correlation matrix and the assumption of an underlyingnormal probability distribution. Similarly, for each outcome measure,to test for differences between pairs of group means, the Bonferroni–Dunn multiple comparisons procedure was applied to control thetype I error rate at 0.05. Nominal P-values for pair-wise comparisonsare presented and significance at the overall 0.05 level is noted.

Analysis of hilar basal dendrites for synapses

To determine whether more synapses occurred on DCX-labeled basaldendrites at different days after the pilocarpine injections, a modifiedversion of a previous protocol was used (Shapiro & Ribak, 2006). Thismethod allowed us to estimate the numbers of axodendritic synapseson DCX-labeled basal dendrites in the hilus. A light micrograph wascaptured from semithin sections containing DCX-labeled basaldendrites from 10 separate basal dendrites per animal, for each ofthe timepoints. Once the image was captured, a grid with equallyspaced points was placed over the low-magnification image of theDCX-labeled basal dendrites to be used as sampling points. Fivesampling points from each basal dendrite were randomly selected to beexamined for synapses. In some cases, only a dendritic profile wasobserved in the thin section. In these cases, the profile was often toosmall for five grids to be selected for analysis, and the entire dendritewas examined for synapses. If the fixation and reaction product in thinsections appeared insufficient to allow us to resolve membranes onDCX-labeled basal dendrites, a new block was made and a new imagewas captured from the resulting semithin sections. At each analysispoint selected, an area of � 1 lm2 was photographed on three serialsections to serve as the reference and look-up sections. A Gatan digitalcamera was used to capture the images from each of three serialsections per Formvar-coated slot grid. Initially, a low-magnificationelectron micrograph was captured; this was followed by successiveimages of increasing magnification. The identification of axodendriticsynapses on these DCX-labeled basal dendrites used the followingcriteria: a membrane-bound profile that was apposed to the DCX-labeled basal dendrite and contained three or more synaptic vesicles inthe presynaptic terminal with at least one of these vesicles locatedadjacent to a synaptic cleft with parallel pre- and postsynapticmembranes (Murray & Goldberger, 1986). Synapses were marked onthe reference section, and were then compared with the look-upsection. Synapses that were observed in the reference but were notobserved in the look-up section were counted. Then, the reference andlook-up sections were switched, and the process was repeated. Thesedata provided an estimate of the prevalence of axodendritic synapseson DCX-labeled basal dendrites in the dentate gyrus. Because nosynapses were found on basal dendrites from the control animals, orfrom the day 1–3 timepoints, and very few from days 4 and 5, nostatistics were performed. Instead, we have presented the data as aratio of the number of synapses to the number of dendrites examined.

Analysis of Ki-67-labeled and Ki-67 ⁄ DCX double-labeled cells

Ki-67 (single)- and Ki-67 ⁄ DCX (double)-labeled cells were quantifiedby counting them in every 12th hippocampal section with the Bio-Rad

confocal microscope. To confirm that the Ki-67 labeling wascontained within a DCX-labeled cell, two-photon lasers were scannedthrough the z-axis of 10% of randomly selected double-labeled cells.Orthogonal views were used to verify that the Ki-67-labeled nucleuswas contained within the DCX-labeled cell body. A one-way anova

was used to compare the data, followed by planned comparisons usinga Bonferroni–Dunn test.

Results

Description of DCX-labeled cells at 1–5 daysafter pilocarpine-induced seizures

The morphology and distribution of the DCX-labeled cells wereexamined in the controls and at each of the timepoints followingpilocarpine-induced seizures. The control rats had a distribution ofDCX-labeled cells similar to that previously reported (Fig. 1A). Only asmall number of the DCX-labeled cells had DCX-labeled basaldendrites in the control rats (Fig. 1A). By 2 days after pilocarpine-induced seizures, the layer of DCX-labeled cells had noticeablyexpanded at the base of the granule cell layer (Fig. 1B). At thistimepoint, more DCX-labeled cells had hilar basal dendrites (Fig. 1B).By the 3- and 4-day timepoints, an increased layer of DCX-labeledcells was apparent (Fig. 1C), and by the 5-day timepoint the dispersionof this layer was so great that one could observe an abundance of hilarectopic granule cells (Fig. 1D).

Quantitative analysis of DCX-labeled cells at 1–5 daysfollowing pilocarpine-induced seizures

Three parameters were examined for each day following pilocarpine-induced seizures. These parameters include the number of DCX-labeled cells, the thickness of the layer of DCX-labeled cells and thepercentage of DCX-labeled cells with basal dendrites. Only significantdifferences in each of these parameters are reported for each day.These results are summarized in Fig. 2.At 1 day after pilocarpine-induced seizures, the percentage of DCX-

labeled cells with basal dendrites was significantly increased (nominalP ¼ 0.001 for control vs. day 1).At 2 days after seizures, there was a significant increase in the

thickness of the DCX-labeled cell layer compared to the control(P < 0.01). At an overall significance level of 0.05, themean percentageof DCX-labeled cells with hilar basal dendrites at this timepoint wassignificantly increased relative to controls (P < 0.0001).At 3 days after seizures, there was a significant increase in the

number of DCX-labeled cells compared to the control group(P < 0.05). The analysis also showed that there was a significantincrease in the thickness of the DCX-labeled cell layer compared tothe control (P < 0.001). There was also a significant increase in thethickness of the DCX-labeled cell layer from day 1 to day 3(P < 0.05). At an overall significance level of 0.05, the meanpercentage of DCX-labeled cells with hilar basal dendrites for thistimepoint was significantly greater than for the controls (P < 0.0001).At 4 days after seizures, the analysis showed that there was a

significant increase in the thickness of the DCX-labeled cell layer fromthe control (P < 0.001) and from day 1 (P < 0.002). At an overallsignificance level of 0.05, the mean percentage of DCX-labeled cellswith hilar basal dendrites at 4 days was significantly increased ascompared to controls (P < 0.0001). Moreover, there was a significantincrease in the mean percentage of DCX-labeled cells with basaldendrites compared to days 1 and 3 after seizures (nominalP < 0.0001 for each).

Rapid changes for newborn neurons after seizures 585


Fig. 1. Confocal micrographs of DCX-labeled cells and their labeled processes in the dentate gyrus of control and pilocarpine-treated rats. (A) DCX-labeled cellsare shown from a control rat at the border between the hilus (H) and the base of the granule cell layer (GL). Note that many of these DCX-labeled cellshave apical dendrites (arrowheads) that extend through the GL. (B and C) DCX-labeled cells in the dentate gyrus from rats at (B) 1 and (C) 3 days, afterpilocarpine-induced seizures, are observed to accumulate at the border between the hilus and the granule cell layer. Note that by day 1 in B, DCX-labeledbasal dendrites (arrowheads) are already observed in the hilus (H) and by day 3 in C there is a robust plexus of DCX-labeled hilar basal dendrites (arrowheads).(D) DCX-labeled cells from rats at 5 days after pilocarpine-induced seizures are found more dispersed throughout the hilus (H) and the granule cell layer (GL). Alsonote the dense plexus of DCX-labeled hilar basal dendrites (arrowheads) that extend ‡ 100 lm into the hilus by this timepoint. Scale bars, 25 lm (A and D), 20 lm(B and C).



At 5 days after seizures there was a significant increase in thenumber of DCX-labeled cells compared to the control group(P < 0.01). There were also significantly more cells than at the

1-day (P < 0.05) and 2-day (P < 0.01) timepoints. The analysis alsoshowed that there was a significant increase in the thickness of theDCX-labeled cell layer compared to the control (P < 0.001) and day 1(P < 0.01). At an overall significance level of 0.05, the meanpercentage of DCX-labeled cells with hilar basal dendrites wassignificantly increased compared to the controls (P < 0.001).

Hilar basal dendrite sprouting from newly born granule cells

The results of the basal dendrite length analysis showed that at eachsuccessive day after pilocarpine-induced seizures the hilar basaldendrites elongated further into the hilus. Analysis of the length ofthe hilar basal dendrites at each of the timepoints revealedsignificantly greater numbers of hilar basal dendrite endings inthe experimental groups at the 30- (P < 0.001), 40- (P < 0.05),50- (P < 0.05), 60- (P < 0.01) and 70-lm (P < 0.01) distances thanin the control group. Quantitative analysis also revealed that days 4and 5 had significantly greater numbers of hilar basal dendritesendings that were > 50 lm than did the 1-day timepoint (P < 0.05;see Fig. 1 for micrographs). The variation in mean dendritic endingsat baseline and at successive 24-h periods for the first 5 daysfollowing pilocarpine-induced seizures are illustrated by box plotsshown in Fig. 3.

Regression analysis of DCX-labeled cells with hilar basaldendrites

The estimated mean ± SEM percentage of DCX-labeled cells withhilar basal dendrites for controls and from 1 to 5 days afterpilocarpine-induced seizures, adjusted for variability between sec-tions, were as follows: 32.0 ± 1.29% for controls, 41.6 ± 2.512% at1 day after seizures, 46.7 ± 3.036% at 2 days after seizures,45.6 ± 0.384% at 3 days after seizures, 55.3 ± 1.280% at 4 daysafter seizures and 51.5 ± 2.84% at 5 days after seizures. The linearregression of mean percentage of DCX-labeled cells with hilar basaldendrites on number of days after seizures was statisticallysignificant (P < 0.0001). The model coefficient of variation was0.708, indicating that �71% of the variation in mean percentage ofDCX-labeled cells with hilar basal dendrites can be explained by theregression of percentage of cells on number of days after pilocar-pine-induced seizures. For each day (24-h period), on average, therewas an absolute increase of 4% in mean percentage of DCX-labeledcells with hilar basal dendrites.

Synapses on hilar basal dendrites first appeared at 4 daysafter seizures

The DCX-labeled basal dendrites in the hilus were examined forsynapses at each of the five timepoints following pilocarpine-inducedseizures. At 1 and 2 days following pilocarpine-induced seizures, nosynapses were found on DCX-labeled hilar basal dendrites in thehilus. At the 3-day timepoint, small profiles apposed the DCX-labeled basal dendrites in the hilus but no synapses were found eventhough parallel membranes were observed for DCX-labeled dendritesand their apposed unlabeled axon terminals. The first synapses onDCX-labeled hilar basal dendrites were observed at 4 days afterpilocarpine-induced seizures. A total of three such synapses wereobserved from the 40 basal dendrite segments examined at thistimepoint. These synapses were characterized by the presence of asingle synaptic vesicle apposed to the presynaptic membrane, andthickened and parallel pre- and postsynaptic membranes at a small

DCX-labeled cells per 250 µm2

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Fig. 2. Histograms of the mean number of DCX-labeled cells, DCX-labeledcell layer thickness and percentage of DCX-labeled cells with DCX-labeled hilarbasal dendrites. (A) The mean number of DCX-labeled cells is shown for each ofthe timepoints examined. The data show significantly more cells at day 3(*P < 0.05) and day 5 (**P < 0.005) than in the control rats. There were alsosignificantly more cells at the 5-day timepoint (�) than at the 1-day (P < 0.05)and 2-day (P < 0.01) timepoints. (B) Mean thickness of the DCX-labeled celllayer for all of the groups. There was a significant increase from the control today 2 (**P < 0.005), and to days 3–5 (***P < 0.001), as well as a significantincrease (**) from day 1 to day 3 (�P < 0.05), day 4 (��P < 0.005) and day 5(��P < 0.005). (C) Mean percentage of DCX-labeled cells with DCX-labeledhilar basal dendrites. At an overall significance level of 0.05, the mean valuefor controls was significantly lower than for measurements made at succes-sive days after seizures: for control vs. day 1 (nominal *P ¼ 0.001); and forcontrol vs. day 2 to day 5 (**P < 0.0001) . Mean values calculated frommeasurements made at 1 and 3 days after seizures were significantly lower thanthose made at 4 days after seizures (nominal #P < 0.0001 for each). All errorbars are SEM.



active zone (Fig. 4A–C). At the 5-day timepoint, developingsynapses onto DCX-labeled hilar basal dendrites were also observed.One such synapse was observed onto a stubby spine of a DCX-labeled hilar basal dendrite (Fig. 4D–F). At this timepoint, four ofthe 40 basal dendrite segments examined had these synapses on them(see Fig. 4G–I for another example).

Type-3 progenitor cell population increased immediately afterseizures

To determine the number of type-3 neuronal progenitor cells at thesetimepoints, Ki-67 ⁄ DCX double-immunolabeling was used. Theresults showed that Ki-67 ⁄ DCX double-labeled cells were found at

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Fig. 3. The variation in mean dendritic endings of DCX-labeled hilar basal dendrites at baseline and at successive 24-h periods for the first 5 days followingpilocarpine-induced seizures is illustrated by box plots. The box stretches from the 25th to the 75th percentile. The median is shown with a line across the box, and themean with an asterisk. The whiskers indicate 1.5· the interquartile range above the third and below the first quartiles, or the upper or lower extreme values, whichever iscloser to the 25th or 75th percentiles. Note that most of the basal dendrites in control rats only extend 10–20 lm, only one is 50 lm and none extend further than that. At1 day after pilocarpine-induced seizures, not only were more DCX-labeled hilar basal dendrites observed, but many more extended 50 lm or beyond. At 2 days afterpilocarpine-induced seizures, there were significantly more hilar basal dendrites than in controls at 20–50 lm distances. At days 4 and 5 after pilocarpine-inducedseizures, there were significantly more hilar basal dendrites at the longest distances than at the 1-day timepoint. See text for significant differences at each timepoint.



the border between the hilus and the base of the granule cell layer, inthe hilus and in the granule cell layer (Fig. 5A and B). Quantitativeanalysis of Ki-67-labeled cells revealed significantly more Ki-67-labeled cells in the experimental groups than the control group(F ¼ 10.275, P < 0.001). Post hoc analysis of each day using aBonferroni correction factor showed that each timepoint had signifi-cantly more Ki-67-labeled cells than the control group (days 1 and 4,P < 0.05; days 2 and 3, P < 0.001; day 5, P < 0.005). Analysis ofthe number of Ki-67 ⁄ DCX double-labeled cells also showed signi-ficant increases in the experimental groups (F ¼ 14.535, P < 0.001).Post hoc analysis using the Bonferroni correction revealed that each of

the timepoints had significantly more of these cells than the controlrats (day 1, P < 0.005; days 2, 3 and 5, P < 0.001; day 4, P < 0.05).These results are summarized in Fig. 5A.It is pertinent to note that, although there were no increases in the ratio

of DCX to DCX ⁄ Ki-67-labeled cells from 1 to 5 days after seizures, thelocation and appearance of these cells did change (Fig. 5B–D). Byday 4,they were frequently found in clusters of neuroblasts with multiplenuclei and continuous DCX-labeled perikaryal cytoplasm. Within theseclusters, there were several labeled and unlabeled nuclei and it wasnot possible to delineate individual cells down to the finest resolutionof the confocal microscope (0.15 lm; Fig. 5D). When individual

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Fig. 4. Electron micrographs of DCX-labeled hilar basal dendrites at 4 and 5 days after pilocarpine-induced seizures. (A) A cross-sectioned DCX-labeled hilarbasal dendrite (D) is shown at 4 days after seizures in the hilus. Note the myelinated axons (arrows) in close proximity to the labeled dendrite, indicative of the hilarregion. (B and C) This dendrite is enlarged in B to show the orientation and the boxed area is enlarged in C to show the parallel membranes of the pre- and post(arrow)-synaptic profiles. Note that a single synaptic vesicle is present in the presynaptic axon terminal. (Panel D) A longitudinally sectioned DCX-labeled hilar basaldendrite (D) is shown from a rat at 5 days after pilocarpine-induced seizures. This dendrite (D) is enlarged in (E) and (F) to show a developing synapse onto astubby spine of the DCX-labeled hilar basal dendrite (arrow). (G) Another DCX-labeled hilar basal dendrite (D) is shown at 5 days after pilocarpine-inducedseizures. Note that this dendrite is emanating from a cell body with the nucleus (n) visible in the upper left corner of the micrograph. The boxed area is enlarged in(H) and (I) to show the parallel membranes of the pre- and postsynaptic profiles, and the vesicle (arrow) within the presynaptic terminal. It should be noted thatother synaptic vesicles were observed within this presynaptic terminal in serial sections. Scale bars, 1 lm (A and G), 0.5 lm (B, C, D, E, H and I), 0.2 lm (F).



DCX ⁄ Ki-67+ cells were discernible, they were often observed in thehilus or deeper in the granule cell layer. This is distinctly different fromthe situation in the controls, where only one Ki-67 ⁄ DCX+ cell typicallyoccurred in clusters of other DCX+ cells (Fig. 5B).

Discussion

The current study demonstrates several rapid neuroplastic changesinvolving newly generated granule cells during the first 5 days afterpilocarpine-induced seizures. The earliest change observed from theparameters analysed indicate that, at 1 day after pilocarpine-inducedseizures, there was an increase in the percentage of DCX-labeled cellswith basal dendrites and an increase in the progenitor cell population.At 2 days after seizures, the thickness of the DCX-labeled cell layerwas increased. At 3 days after seizures, the number of DCX-labeledcells was increased and at 4 days after seizures synapses were firstobserved on DCX-labeled hilar basal dendrites. Many of thesechanges persisted through the 5-day period, once they appeared.

Although some of these changes were previously reported at latertimepoints after pilocarpine-induced seizures, the current study is thefirst to demonstrate neuroplastic changes to newly born granule cells atsuch early timepoints following seizures.

Rapid response of DCX-labeled cells to pilocarpine-inducedseizures

The fact that there was a rapid increase in the number of DCX-labeledcells found after seizures could be partially due to the increase in theprogenitor cell population that occurs by 1 day after seizures. Thesedata using pilocarpine-induced seizures are consistent with the resultsfrom a previous study showing that DCX-labeled cells may becomeproliferative in response to kainate-induced seizures (Jessberger et al.,2005). The fact that many of the type-3 progenitor cells were found indense clusters beyond the hilar border by 5 days after seizuressuggests that they may also contribute to the thickening of the newlyborn granule cell layer, because they are generating cells beyond thehilar border region. This neuroplastic change is analogous to humanand primate TLE and rodent models of TLE, where granule cell layerdispersion and hilar ectopic granule cells are prominent features of theepileptic hippocampus (Houser, 1990; Parent et al., 1997; Ribak et al.,1998; Scharfman et al., 2000; Parent et al., 2006). Although ectopicgranule cells have been shown to become functionally integrated intogranule cell circuitry (Scharfman et al., 2000), it is unclear whether thenewly born granule cells in the current study also become functionallyintegrated during the first 5 days following pilocarpine-inducedseizures.

DCX-labeled cells rapidly sprout hilar basal dendritesthat elongate after seizures and are targeted for aberrantsynaptogenesis

A previous study showed that hilar basal dendrites are observed ongranule cells by 7 days after pilocarpine-induced seizures (Dashtipour

et al., 2003). The current study expands upon these findings to showthat the increase in the length of hilar basal dendrites is seen by only2 days after seizures. In addition, the data show that hilar basaldendrites continue to elongate into the hilus during the subsequentdays after seizures. This is distinctly different from normal adult rats,where the basal dendrite was shown to be a transient feature of thenewly born granule cells and typically retracted beginning 3 days afterthe birth of the granule cell (Jones et al., 2003; Shapiro et al., 2007).The increase in the number and length of DCX-labeled hilar basal

dendrites after seizures could be due to the fact that these cells areexposed to aberrant neurotrophic cues. This hypothesis is supportedby data showing that after seizures there is an increase in growth factorexpression (Binder et al., 2001; Hagihara et al., 2005) that has beendirectly shown to correspond to alterations to newly generated granulecells, including the sprouting of basal dendrites (Danzer et al., 2002;Shetty et al., 2005).The developing axodendritic synapses on DCX-labeled hilar basal

dendrites beginning at 4 days after seizures may be the forerunners ofthe axo-spinous synapses found at later timepoints after seizures(Jessberger et al., 2006). The observation of a developing synapseonto a stubby spine of a DCX-labeled hilar basal dendrite supports thisnotion. Previous studies have shown that mossy fibre synapses ontohilar basal dendrites constitute both a morphological and a functionalrecurrent excitatory circuitry (Ribak et al., 2000; Austin & Buckmas-ter, 2004). The fact that synapses onto DCX-labeled hilar basaldendrites appear at 4–5 days following seizures and coincide with thetime at which mossy fibers sprout into the inner molecular layer of thedentate gyrus (Mello et al., 1993) suggests that mossy fibre axonsmight be targeting hilar basal dendrites shortly after seizures. In fact,some of the DCX-labeled hilar basal dendrites that were examinedusing the electron microscope were found to have their membranesparallel with what appeared to be mossy fibre axon terminals (data notshown). However, using only anatomical criteria it is not possible todefinitively conclude that these axon terminals are mossy fibers.Future studies will be needed to determine whether the axon terminalsthat synapse with the hilar basal dendrites beginning at 4 days afterseizures are in fact mossy fibers, and could thus provide the basis for afunctional recurrent circuitry.

Conclusion

The results from this study show that several neuroplastic changesinvolving DCX-labeled newly generated granule cells occur rapidlyafter pilocarpine-induced seizures (within the first 5 days afterseizures). Some of these changes may be the forerunners of severalwell-known morphological changes, such as granule cell layerdispersion, hilar ectopic granule cells and aberrant synaptogenesis,all of which have been reported in the hippocampus of rodents withspontaneous seizures and in that of humans with TLE. The rapidchanges associated with these newly generated granule cells afterseizures may provide an anatomical substrate for aberrant circuitformation in the epileptic dentate gyrus. Future studies will beneeded to determine whether these rapid changes to newly born

Fig. 5. Ki-67- (green) and DCX- (red) labeled cells in the dentate gyrus of pilocarpine-treated rats. (A) Histogram showing the mean numbers of Ki-67 single- andKi-67 ⁄ DCX double-labeled cells in control rats and rats given pilocarpine-induced seizures. At each of the timepoints following pilocarpine-induced seizuressignificantly more Ki-67-labeled and Ki-67 ⁄ DCX double-labeled cells were observed compared with controls (*P < 0.05, **P < 0.001, ***P < 0.005, �P < 0.005,��P < 0.001, ��P < 0.05). The error bars are SEM. (B) Example of a single Ki-67 ⁄ DCX double-labeled cell (arrow) found within a cluster of DCX-labeled (red)cells from the control rat. (C) At 1 day after pilocarpine-induced seizures, several double-labeled cells (arrows) are seen within the expanding population of DCX-labeled cells. (D) Double-labeled cells are observed in the hilus (arrow), base of the granule cell layer (boxed area) and within the area of the granule cell layer (GL)where mature granule cells are found (arrowhead). (E) The boxed area is enlarged in a merged Z-stack image and orthogonal views are provided to show that the Ki-67-labeled nucleus is contained within the DCX-labeled cytoplasm. Scale bars, 10 lm (B and E), 30 lm (C), 40 lm (D).



granule cells after seizures result in increased excitability ofhippocampal neurons.

Acknowledgements

The authors wish to recognize the contributions of Dr Zhiyin Shan and MandiRuch for their technical expertise, Kim Stecker for stereological analysis andWen-Pin Chen, MS, and Christine McLaren, PhD, for assistance with thestatistical analyses. We also acknowledge support from NIH grant R01-NS38331 (to C.E.R.) and NIH training grant T32-NS45540 (for L.A.S.).

Abbreviations

DCX, doublecortin; TLE, temporal lobe epilepsy.

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